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Retroviral Superantigens

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Retroviral Superantigens
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Torres, Barbara Aurea, 1956-
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ix, 96 leaves : ill. ; 29 cm.

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Antibodies ( jstor )
Antigens ( jstor )
B lymphocytes ( jstor )
Cultured cells ( jstor )
Enterotoxins ( jstor )
Histocompatibility antigens class II ( jstor )
HIV ( jstor )
HIV 1 ( jstor )
Seas ( jstor )
Superantigens ( jstor )
Dissertations, Academic -- Microbiology and Cell Science -- UF
HIV infections ( lcsh )
Microbiology and Cell Science thesis, Ph. D
Retroviruses ( lcsh )
Superantigens ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1995.
Bibliography:
Includes bibliographical references (leaves 86-95).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Barbara Aurea Torres.

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RETROVIRAL SUPERANTIGENS













By


BARBARA AUREA TORRES












A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA 1995













ACKNOWLEDGMENTS


I would like to thank my mentor, Dr. Howard M. Johnson, for his unflagging support, insight, and wisdom. All of these qualities have helped guide me, not just a in scientific sense, but in finding my way through the quagmire of academic science. On a scientific level, Howard has always been unfailingly logical and knowledgeable, which makes him a wonderful mentor.

I would also like to thank the other members of my committee. Dr. Shanmugam and Dr. Hoffmann have been very supportive throughout this venture. Dr. Yamamoto has given me excellent advice on steering my HIV work towards clinical relevance. I give special thanks to Dr. Blalock, who was a tough, but caring, committee member and confidante.

Many thanks must go to my fellow labmates who have always been willing to lend a hand, ear, or a shoulder to cry on, whichever was needed at the time. I thank Jeanne, Brian, Prem, and Taishi for generously sharing their past experiences of graduate school. I also am grateful for my fellow graduate school mates, Amy, George, and Mustafa, for their understanding and help. And I thank Aaron, Laurie, and Tim for their warmth and humor.

Finally, I wish to thank my family for their unwavering support through my graduate school tenure. My parents, Manuel and Antonia Torres, have stood by with understanding and a warm hug whenever needed. Although far away, my sister, Marisol Beaton, has cheered me on to complete my doctoral work. I owe a great debt of gratitude to my wonderful family.


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TABLE OF CONTENTS

ACKNOWLEDGMENTS ...................................................... .................... ii

LIST O F TA B LE S ......................................................................... ..................vi

LIST OF FIGURES ...................................... vii

A B S T R A C T ............................................................................. ...................... viii

CHAPTERS
I INTRODUCTION ............................................................. .................. 1

O verview ................................................................................................. 1
Bacterial Superantigens ........................................ .............................. 6
Retroviruses ............................................................ 8
Mouse Mammary Tumor Virus Superantigen........................... 10
Evidence for a Superantigen Associated with
Human Immunodeficiency Virus ........................................ 12

II MMTV SUPERANTIGEN BINDING TO MHC CLASS II
ANTIG ENS ...................................................... ......... .......... 16

Introduction ....................................................................................... 16
M aterials and M ethods ..................................... .............................. 17
Synthetic Peptides .......................................... ................................ 17
Cell Lines and Reagents ............................................ 17
Radioiodinations .................................................... 1 9
Binding Studies .................................................... 19
Radioim m unoassay .......................................... .............................. 20
R esu lts ............................................................................ ..................... 2 1
Competition of vSAg Peptides with Radiolabeled SEA for
M HC Binding .................................... . ....... . ........ 21
vSAg(76-119) Peptide Competition Is Dose-Dependent ..........21
Direct Binding of vSAg(76-119) Peptide to A20 Cells ............... 24
Evidence that vSAg(76-119) Binds Specifically to MHC
Class II Antigens .................................... . ............ 26
D iscussion.............................................................................................. 2 6





Ill










III IDENTIFICATION OF AN HIV-1 NEF PEPTIDE THAT BINDS TO
MHC CLASS II ANTIGENS ...................................... ..... 32
Introduction .............................................................32
Materials and Methods .................................... ............. 33
Synthetic Peptides .......................................... ................................ 33
Cells and Reagents ............................................ ...... 33
Radioiodinations ................................... ........ ....... 35
Binding Studies .................................................... 35
R esults ............................................................................ .....................3 5
Competition of Nef Peptides with Radiolabeled SEA for
MHC Binding .................................................... 36
Nef(123-160) Peptide Competition Is Dose-Dependent ....... 39
Ability of Nef Peptides to Compete with Ses for Binding to
R aji C ells ............................................................. ......................... 39
Direct Binding of Nef(123-160) Peptide to Raji Cells ............ 39
MAbs to MHC Class II Antigens Block Nef(123-160) Peptide
Binding to Raji Cells ............................................... 42
D iscussion ........................................................................................ 42

IV ACTIVATION OF CD4 T CELLS BY THE NEF PROTEIN FROM
HIV-1
Introductio n ....................................................................... .................. 4 5
Materials and Methods .................................................45
Nef Protein.................................................... ........... ...........45
R eagents .................................................................................. 48
Proliferation Assays .......................................... .............................. 48
Purification of T Cells from PBMC ........................................ 49
Autologous Antigen-Presenting Cells (APC) ................... 49
Synthetic Peptides and Antibodies to Nef Peptides ................49
Cytokine Production and Assays ...................................................50
Studies on V-Specific T Cell Expansion and Induction
of A nergy ...................................................... ......... .......... 51
R e sults ............................................................................ ..................... 5 1
Nef Proliferative Response............................ ..... ...... 51
Antigen-Presenting Cells (APC) Are Required for Nef Activity .53
Nef(123-160) Peptide Specifically Blocks Proliferation of
PBMC Induced by Nef and SEA ...................................... 58
Induction of T Cell Cytokines by Nef................................. ...60
D iscu ss io n ................................................................................................. 6 4

V HIV ENCODES FOR ITS OWN CD4 T CELL MITOGEN.............. 68
Intro d uctio n ....................................................................... .................. 6 8



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Materials and Methods ................................................. 69
PBMC Cultures for HIV Infectivity Studies ..................................... 69
PBMC Cultures for Proliferation Studies ..................................69
Reverse Transcriptase (RT) Assay ...................................... 70
ELISA for HIV p24 Antigen ........................................................... 70
Assay for Infectious Virus from PBMC Cultures ................ 70
Fluorescent Antibody Cell Sorter (FACS) Analysis of
Nef-Activated Cells ............................................................ 71
Studies on Proliferation of PBMC Induced by Autlogous
HIV-Infected Cells ........................................... ...... 71
Anti-Nef Peptide Antibodies ....................................... ....... 71
R e su lts ............................................................................ .....................7 2
HIV Replication in Nef-Stimulated PBMC Cultures ..................72
Infectious Virus Is Produced by Nef-Stimulated Cultures...........72
Nef-Stimulated PBMC Express T Cell Activation Markers.........75 Expression of Nef by HIV-Infected Cells ....................................75
D iscussion ................................................................ ........................... 7 8

VI A MODEL FOR THE ROLE OF NEF IN HIV PATHOGENESIS .........82

LIST OF REFERENCES .................................................... 86

BIOGRAPHICAL SKETCH ..................................................100


























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LIST OF TABLES

Table page

I. List of bacterial superantigens implicated in disease ...................... 3
II. List of viral superantigens implicated in disease .............................. 5
II. Amino acid sequences of vSAg peptides ........................................... 18
IV. Amino acid sequences of Nef peptides ...............................................34
V. Ability of anti-peptide antibodies to block Nef-induced
p rolife ration ...................................................................... ................ 5 1
VI. IL 2 production induced by Nef ..........................................................61
VII. IFNy is induced by Nef ..................................... ........... 63
VIII. Maximal proliferation, RT, and p24 values from stimulated
cultures .......................................................................... .................... 7 3
IX. Nef-induced activation of T cells as assessed by FACS ..............77
X. Ability of anti-Nef antibodies to block proliferation induced by
autologous HIV-infected cells............... ................................... 79





























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LIST OF FIGURES

Figure page

1. Ability of vSAG peptides to compete with 1251-SEA for binding
to A20 cells ............................................................22
2. Ability of vSAG(76-119) to compete with 1251-SEA for binding
to A20 cells in dose response studeis ...................................... 23
3. Ability of SEA, SEB, and TSST-1 to compete with 1251-vSAg(76-119)
for binding to A20 cells ................................................. 25
4. Evidence that vSAg(76-119) peptide binds to MHC class II
antigens, and does not bind to class I antigens ..............................27
5. Binding of 1251-vSAg(76-119) to MHC class II peptides .......................28
6. Blockage of 1251-SEA binding to Raji and DR1-transfected
L cells by Nef peptides ................................................ 37
7. Relative abilities of Nef(123-160), purified Nef protein, and SEA to
compete with 1251-SEA for binding to Raji cells ..............................38
8. Blockage of 1251-SE binding to Raji cells by Nef peptides ................40
9. Direct binding of 1251-Nef(123-160) to Raji cells ..................................41
10. Blockage of 1251-Nef(123-160) binding to Raji cells by
antibodies to MHC class I and class II antigens ..............................43
11. Comparison of the mitogenic activities of Nef protein
preparations .............................................................................. 52
12. Lack of proliferation by PBMC in response to the Nef fusion
partner M BP ............................................... ........ .......... .......... 54
13. Nef-induced proliferative responses from a representative
sam pling of donors ................................................... 56
14. Nef-induced activation of T cells requires APC but does
not require prcessing of Nef...................................................................57
15. Nef(123-160) peptide specifically blocks proliferation
of PBMC induced by Nef and SEA ..................................... ...59
16. Kinetics of IFN production induced by Nef .................................... 62
17. Infectivity assay on supernatants from Nef-stimulated
HIV-infected cultures .................................... . ............. 76
18. Model for the role of Nef in the pathogenesis of HIV.......................85











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Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

RETROVIRAL SUPERANTIGENS

By

Barbara Aurea Torres

December 1995




Chairman: Howard M. Johnson
Major Department: Microbiology and Cell Science


Superantigens (SAg) are potent inducers of T cell activation that cause proliferation and massive cytokine release. The receptors for superantigens on antigen-presenting cells (APC) are major histocompatibility (MHC) class II molecules. Both Nef protein of human immunodeficiency virus (HIV) and mouse mammary tumor virus (MMTV) SAgs are encoded in genes that overlap the terminal repeat at the 3' end. Using synthetic peptides, a region was identifed on the MMTV-1 SAg, corresponding to residues 76-119, that specifically binds to mouse MHC class II antigens. MMTVSAg(76-119) also bound to and competed with bacterial superantigens for binding to MHC class II peptides, suggesting similar binding regions on class II. These studies are the first demonstration that retroviral superantigens bind to MHC class II antigens.




vim







A peptide corresponding to an internal region of HIV Nef protein was identified that specifically binds to to human class II antigens. Nef protein induced proliferation of human peripheral blood mononuclear cells (PBMC) in 85-90% of the HIV-negative donors tested. Nef stimulation required APC, and did not require processing. Interleukin 2 and gamma interferon were produced in Nef-stimulated cultures. These results strongly suggest that Nef acts like a retroviral SAg that stimulates CD4+ T cells.
Activated T cells are required for HIV replication. The role of Nef in HIV pathogenesis was investigated by treating PBMC with Nef prior to in vitro infection with HIV. Significant levels of infection were found in these cultures, as compared to unstimulated controls. Nef-stimulated T cells were found to express T cell activation markers. Inactivated HIV-infected cells were capable of inducing proliferation in autologous fresh PBMC, and proliferation was significantly reduced by anti-Nef antibodies. These results indicate that Nef is expressed on the surface of infected T cells, possibly in the context of class II antigens, and as such, Nef can activate a cellular reservoir in an paracrine fashion for continual viral replication. Thus, Nef is an HIV-encoded SAg-like mitogen that promotes HIV replication in T cells. As such, Nef is probabaly an HIV virulence factor.
















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CHAPTER I
INTRODUCTION


Overview

A novel class of antigens called superantigens has recently been characterized. Unlike conventional antigens, which stimulate approximately 1 in 10,000 cells, superantigens can induce the proliferation and activation of as many as 1 in 5 T cells (Johnson et al., 1991). The staphylococcal enterotoxins, prototypic of this class of antigens, are among the most potent inducers of T cell proliferation, and are effective at concentrations as low as 10-16 M (Langford et al., 1978a).
Major differences exist between conventional antigens and superantigens. Superantigens exert their effects as whole molecules and do not require processing by antigen presenting cells for T cell recognition. Although superantigens require MHC class II molecules, they bind at site(s) outside the peptide antigen binding groove (Russell et al., 1990; Dellabonna et al., 1990). Superantigens presented in the context of MHC class II molecules are recognized by the variable region of the 3 chain (V3) of the T cell receptor. Only T cells that express T cell receptors with specific V3 regions will be activated by a given superantigen. For example, toxic shock syndrome toxin-1 activates only V32-bearing human T cells (Abe et al., 1992) and staphylococcal enterotoxin A activates several Vps on human T cells, including V35 and V312, (Kappler et al., 1989). Thus, T cell responses to superantigens are said to be Vp-specific.



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T cell activation by superantigens results in massive proliferation of VPspecific T cells. Concomitant with proliferation is prodigious production of cytokines, in particular interleukin 2 and gamma interferon (IFNy). Such responses can be detrimental to the host, as is the case with food poisoning which occurs upon ingestion of staphylococcal enterotoxin-laden food (Bergdoll, 1985). As an eventual consequence of superantigen activation, T cells can become unresponsive to further stimulation (anergy) or undergo programmed cell death (apoptosis) (Kawabe and Ochi, 1990, 1991; Rellahan et al., 1990). Thus, the fate of T cells activated by superantigen differs from that of cells activated by conventional antigens, with the possible consequence of loss of immune function.

Superantigens are produced by a number of microorganisms, ranging from bacteria to viruses. The prototypic bacterial superantigens are the enterotoxins produced by Staphylococcus aureus (Table I). Staphylococcal enterotoxin A (SEA) is the most common cause of food poisoning, and its superantigen effects on immunocytes have been implicated in the syndrome of short-term nausea, fever, and diarrhea that are symptomatic of food poisoning (Bergdoll, 1985; Johnson and Magazine, 1988). Another staphylococcal enterotoxin, toxic shock syndrome toxin-1 (TSST-1), is produced during infection and its superantigenic effects result in toxic shock syndrome (Bergdoll, 1985). Other bacterial sources of superantigens are the group A streptococci, which produce pyrogenic exotoxins that have been implicated in psoriasis (Kotzin et al., 1993) and rheumatic heart disease (Lewis et al., 1993). Superantigens may also be involved in tuberculosis (Ohmen et al., 1994) and Reiter's syndrome (Uchiyama et al., 1993). One species of mycoplasma, M. arthiditis, has been shown to produce a superantigen that may be involved in arthritis (Cole and Griffiths, 1993).





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Table I.
List of bacterial superantigens implicated in disease.



Superantigenic Organism Protein Disease/Effecta


Prototype: Staphylococcal enterotoxins

Staphylococcus aureus Enterotoxins Food poisoning Toxic shock Kawasaki's disease (?)b Multiple sclerosis

(?)
Group A streptococci Pyrogenic exotoxins Psoriasis (?) Rheumatic heart disease (?) Mycoplasma arthitidis T-cell mitogen Arthritis (?) Mycobacterium tuberculosis Not identified Tuberculosis (?) Yersinia pestis Not identified Reiter's syndrome Reactive arthritis Clostridium perfringens Exotoxin Sudden infant death syndrome (?)

aJohnson et al., 1995. bDenotes diseases in which superantigens have been implicated, but no direct evidence is available.





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Like their bacterial counterparts, viruses produce superantigens that have been implicated in pathogenic processes (Table II). The prototypic viral superantigen is that produced by the oncornavirus, mouse mammary tumor virus (MMTV) (Marrack et al., 1991; Frankel et al., 1991; Woodland et al., 1991; Dyson et al., 1991). The Gag protein of mouse leukemia virus, the causative agent of mouse acquired immunodeficiency syndrome (MAIDS), has been shown to possess superantigenic properties (Hugin et al., 1991). A superantigen has been implicated in the pathogenesis of rabies virus (Lafon et al., 1992) and recently the superantigenic viral component has been identified as the nucleocapsid protein (Lafon et al., 1994). It has been suggested that human immunodeficiency virus (HIV) encodes for a superantigen that may, at least in part, be responsible for the immunopathogenesis associated with infection (Laurence et al., 1992). Recent evidence points to a superantigen encoded by human cytomegalovirus (CMV) that expands specific V-bearing T cells, thereby enhancing HIV replication in CMV/HIV-infected individuals (Dobrescu et al., 1995). Although several reports suggest that other viruses, such as Epstein-Barr virus and human foamy virus, encode for superantigens, to date no hard evidence has been obtained.
The possible harmful consequences of activation by superantigens make this class of molecules relevant to human disease. The prodigious expansion of T cells having diverse specificities may be important in the induction and establishment of autoimmunity. Concomitant cytokine production may also have deleterious effects. The potential for loss of immune function via mechansisms such as anergy or apoptosis can also result in immunodeficiency diseases. Superantigens have been shown to be encoded by retroviruses, suggesting that they may play a role in the syndrome associated with human





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Table II.
List of viral superantigens implicated in disease.



Virus Superantigenic

(Family or Subfamily) Protein Disease/Effecta


Prototype: Superantigen from Mouse Mammary Tumor Virus

Mouse mammary tumor virus vsag gene product Mammary tumors
(oncornavirus)
Mouse leukemia virus Gag protein Murine AIDS
(oncornavirus)
Human immunodeficiency virus Not identified AIDS
(lentivirus)
Human foamy virus be/3 gene product Grave's disease
(?)b

(spumavirus)
Rabies Nucleocapsid Rabies
(rhabdovirus)
Epstein-Barr virus Not identified B cell lymphoma

(?)
(herpesvirus) Chronic fatigue syndrome (?)
Cytomegalovirus Not identified Enhanced HIV
(herpesvirus) replication aJohnson et al. 1995.
bDenotes diseases in which superantigens have been implicated, but no direct evidence is available.





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immunodeficiency virus. Thus, superantigens may be involved in disease processes and, as such, can be classified as virulence factors.



Bacterial Superantigens

In the 1970s, staphylococcal enterotoxins were recognized as inducers of lymphocyte proliferation (Peavy et al., 1970). Responses to staphylococcal enterotoxin A (SEA) stimulation were shown to be T cell specific (Johnson and Bukovic, 1975), with high levels of gamma interferon (IFNy) produced by this lymphocyte fraction (Langford et al., 1978a). SEA was shown to have mitogenic effects on human lymphocytes at extremely low concentrations, making it one of the most potent T cell activators known (Langford et al., 1978a). The ability to produce large quantities of IFNy using SEA paved the way for the characterization of the key immunomodulatory properties of this cytokine (Johnson, 1985). Interestingly, intravenous administration of SEA to mice resulted in the induction of suppressor cell activity that was present in spleen as early as 24 hours after injection (Johnson, 1981; Torres et al., 1982). SEAinduced suppressor cells were capable of blocking naive spleen cells from responding to SEA stimulation. Thus, SEA was shown to stimulate T cells to proliferate and produce cytokines, and to induce suppressor cell activity.
Further investigations on the interaction of bacterial enterotoxins with human lymphocytes revealed that antigen-presenting cells are required for proliferative effects and that binding to major histocompatibility (MHC) class II antigens occurred (Carlsson et al., 1988; Fleischer and Schrezenmeier, 1988; Mollick et al., 1989). Unlike most antigens which interact with MHC class II molecules as peptides, the staphylococal enterotoxins were shown to bind in an unprocessed form (Fleischer and Schrezenmeier, 1988). The site of





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interaction of enterotoxins on the MHC class II molecule was shown to be distinct from conventional antigens in that binding occurs distal to the peptide antigen binding groove (Russell et al., 1990; Dellabonna et al., 1990). Thus, this class of antigens differs significantly from classic antigens and were given the designation of superantigens (Marrack and Kappler, 1990).
The study of staphylococcal enterotoxins has led to a wealth of information on the interaction of superantigens with MHC class II molecules. Through the use of synthetic peptides, regions of interaction of SEA and MHC class II antigens have been identified. Regions on the SEA molecule that are involved in binding to MHC class II molecules on antigen presenting cells include the N-terminus and three internal sequences (Griggs et al., 1992). A peptide corresponding to the N-terminus of SEA, SEA(1-45), blocked proliferation of cells in response to whole native SEA (Pontzer et al., 1990). Interestingly, a peptide corresponding to another of these MHC class II-binding regions, SEA(121-149), has agonist properties in that it induces cytokine production (Pontzer et al., 1993) and proliferation (A.C. Hobeika, personal communication). Conversely, sites on MHC class II molecules which bind superantigens have been identified. Regions on mouse MHC class II molecules that interact with SEA have been identified and include the a-helical region of the 3 chain associated with the peptide binding groove encompassed by amino acids 65-85 (Russell et al., 1991). A similar site has been found on human MHC class II antigens (Herman et al., 1991). A superantigen-binding region on the a helical region of the a chain associated with the peptide binding groove of MHC class II has also been defined (Russell et al., 1991). Both of these binding sites reside outside of the peptide binding groove and are required for SEA-induced mitogenesis. Thus the two outside faces of the a helices of the peptide binding groove are involved in binding of SEA.





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Presentation of superantigen in the context of MHC class II molecules is required for interaction with T cell receptor (TCR). For this reason superantigen sites that interact with TCR have been more difficult to elucidate. However, through the use of synthetic peptides, a TCR site that interacts with SEA has been identified. A peptide corresponding to residues 57-77 of mouse V33 blocked SEA-induced proliferation and IFNy production (Pontzer et al., 1992). VB3-bearing mouse T cells are known to be stimulated by SEA. Future studies may help determine the sequences involved in the formation of the ternary complex of superantigen:MHC:TCR. Such studies are needed to determine how superantigens activate T cells bearing specific Vps and induce anergy and deletion of these VO subsets.



Retroviruses

Retroviruses belong to a family of viruses that are characterized by the use of a unique RNA-dependent DNA polymerase, reverse transcriptase. Reverse transcriptase, in conjunction with another virally-encoded enzyme, RNAse H, transcribes the single-stranded RNA genome into a double-stranded linear DNA provirus. This DNA intermediate is then capable of integrating into the host genome. Retroviruses were first described in studies in which cell-free filtered extracts were shown to transmit sarcoma to chickens (Rous 1910, 1911). Since that time, retroviruses have been found in several vertebrates including mice, cats, and primates. The first human retrovirus to be discovered was human foamy virus, which has been speculated to cause disease (Achong et al., 1971), although definitive proof of pathogenicity is lacking. In 1980, the causative agent of adult T-cell leukemia was discovered, human T-cell





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leukemia virus (Poiesz et al., 1980; Yoshida et al., 1982), and established this family of viruses as human pathogens.
The retrovirus family consists of three subgroups based on similar pathogenicities: oncornaviruses, lentiviruses, and spumaviruses. Spumaviruses or foamy viruses are the least characterized of the three subfamilies of retroviruses, and have been grouped according to their ability to cause vacuolation of infected cells in vitro, which gives the cells a "foamy" appearance. Spumaviruses have been found in primates and humans, and are generally considered benign, although data suggest these viruses may play a role in human disease. Oncornaviruses are tumor-causing retroviruses, and members of this subfamily include the avian leukosis-sarcoma viruses, mouse mammary tumor virus, feline leukemia virus, and human T-cell leukemia virus. As a result of proviral integration into the host genome, tumors arise by upregulation of host genes that encode for growth factors or by retroviralencoded oncogenes.
Viruses belonging to the lentiviral subfamily include the "slow" viruses maedi/visna and equine infectious anemia virus. More recently, the viruses that cause human and feline acquired immundeofiency syndromes have been classified as lentiviruses, based on several parameters including genomic complexity and virion morphology. Unlike oncornaviruses, lentiviruses have not been implicated directly in causing neoplastic disease. Members of the lentiviral subfamily cause long-term disease characterized by autoimmunity, encephalopathy, immunodeficiency, or a combination thereof. Lentiviruses are considerably more complex than some of the other retroviral subfamilies, in that the level of gene regulation is much greater.





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Mouse Mammary Tumor Virus Superantigen

The first viral superantigens to be described are the proteins encoded by the open reading frame that overlaps the 3' long terminal repeat of the oncornavirus, mouse mammary tumor virus (MMTV) (Marrack et al., 1991; Frankel et al., 1991; Woodland et al., 1991; Dyson et al., 1991). Although MMTV superantigens were recognized in the 1990s, they were originally described by Festenstein in the early 1970s as minor lymphocyte stimulating (mls) antigens (Festenstein, 1973). The ability of T cells from certain mouse strains to be stimulated by lymphocytes from MHC-identical strains was ascribed by Festenstein to the presence of mis antigens. More recently, mis antigens were found to be endogenous superantigens from germline-encoded MMTV provirus (Choi et al., 1991; Acha-Orbea et al., 1991). Like their bacterial counterparts, MMTV superantigens are thought to be presented in the context of MHC class II antigens. MMTV superantigens are known to stimulate T cells in a Vp-specific fashion (Acha-Orbea and Palmer, 1991). MMTV superantigens show MHC preference, with more efficient presentation occurring in the context of I-E as opposed to I-A, although presentation by I-A does occur (MacDonald et al., 1989). Interestingly, this MHC preference corresponds with the greater infectivity of B cells bearing I-E (Held et al., 1994b).
MMTV is a type B retrovirus that was determined to be the causative agent for the induction and transmission of mammary carcinomas in mice (Heston et al., 1945). It has been known for several years that infectivity by MMTV requires an intact immune system (Tsubura et al., 1988), and only recently has this paradox been explained (Held et al., 1993, 1994a). Transmission of MMTV to offspring can occur via infectious virions in mother's milk or can occur vertically as endogenous provirus. Most mouse strains have





11

been shown to contain one or more copies of endogenous MMTV, and many contain distinct MMTV strains (Salmons and Gunzburg, 1987). Upon passage into the gut of the host, virus enters the gut-associated immune tissue and infects B cells. B cells then express the MMTV-encoded superantigen, presumably in the context of class II antigens, causing V3-specific T cell stimulation. Although investigators have been able to show that actual infection of T cells occurs, it is thought that T cell stimulation and subsequent cytokine production by these cells indirectly enhances the further infection of B cells (Held et al., 1994a). Infected B cells migrate to the main site of viral infection, the mammary gland. Epithelial cells then become infected and are the source of infectious virions that are transmitted in milk (Ringold, 1983). Proviral integration can occur during infection of mammary epithelial cells, resulting in tumors.
Germline-encoded MMTV serves an important protective mechanism for the host. It is known that bacterial superantigens can cause anergy and/or deletion of V-specific T cells (Johnson et al., 1992). Similarly, expression of MMTV superantigen early in the ontogeny of the immune system induces the eventual deletion of T cells bearing Vps specific for that particular strain of MMTV superantigen. In this manner, T cells that would otherwise be stimulated are lost and the host is protected against subsequent infection by MMTV strains that stimulate those V-specific T cell populations. This has been shown in mice transgenic for a MMTV superantigen that stimulates Vp14+ T cells (Golovkina et al., 1992; Acha-Orbea, 1991a). Transgenic mice showed partial to complete deletion of Vpl4+ T cells, depending on the level of superantigen expression. Those mice in which V314+ T cells were deleted were subsequently protected from infection upon challenge with the same MMTV strain.





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Evidence for a Superantigen Associated with Human Immundeficiency Virus

In the early 1980s, unusual incidences of young homosexual males stricken with Pneumocystis carinii pneumonia and Kaposi's sarcoma were reported to the Centers fo( Disease Control. These reports led to the first description of acquired immunodeficiency syndrome (AIDS). Subsequently, similar immunodeficiency-associated illnesses were reported among hemophiliacs, recipients of blood and blood components, and intravenous drug users and their heterosexual partners. The incidence of AIDS in these distinct populations was suggestive of an infectious agent, and this was confirmed when, in 1984, human immunodeficiency virus (HIV) was isolated from the blood of an AIDS patient (Klatzmann et al., 1984; Gallo et al., 1984). With the advent of diagnostic assays, the number of infected individuals was found to exceed the number of patients afflicted with AIDS, indicative of a serious epidemic. The number of deaths is expected to increase, as asymptomatic HIVinfected individuals progress to AIDS.
HIV is a member of the lentivirus subfamily of retroviruses. Infection with HIV is associated with a debilitating and eventually fatal immunodeficiency. In addition to immunologic impairment, neurological dysfunctions can occur, including encephalopathies, sleep disorders, and dementia (the latter is also referred to as HIV-associated cognitive/motor complex).
The size of the HIV genome is approximately 9.8 kbases. The genome encodes for a total of 16 proteins, some of which are post-translationally cleaved by either cellular or viral proteases. The primary HIV transcript is gagpol mRNA, which is translated to yield Gag (group antigen) and Pol (polymerase). Synthesis of Gag-Pol occurs at a ratio of 20:1 (Oroszlan and Luftig, 1990). Gag is proteolytically cleaved by a viral-encoded protease,





13

yielding 5 distinct proteins. Cleavage of Pol results in 2 proteins, one of which is reverse transcriptase. Two envelope (Env) proteins are the cleavage products from an initial Env precursor. In this case, the precursor is cleaved by a cellular protease. Two regulatory proteins, Tat (transactivator) and Rev (regulator for viral expression) are translated from multiply-spliced mRNA transcripts.
One putative regulatory protein, Nef (negative factor) is encoded by a gene that overlaps the terminal repeat at the 3' end of the HIV genome. It was initially named based on its apparent negative effects on viral transcription and replication in vitro (Terwillegar et al, 1986). However, subsequent reports have been conflicting, and have suggested that Nef has no effect (Hammes et al., 1989) or a positive effect (DeRonde et al., 1992) on HIV transcription. Such widely disparate Nef effects may, in part, be explained by the fact that several immortalized human T cell lines and primary human lymphocytes were used in these studies. These data may reflect inherent differences in primary cells and cell lines, such as activation signals. Data from studies on Nef-transfected T cells suggest that Nef may play a role in down-regulation of the CD4 molecule (Garcia and Miller, 1991; Mariani and Skowronski, 1993).
HIV-1 Nef is a 25-27K protein and is myristylated at the N-terminus. Myristylation is thought to be a mechanism by which Nef associates with the cytoplasmic membrane (Franchini et al., 1986; Guy et al., 1987). Nef primarily localizes to the cytoplasm (Franchini et al., 1986) but has been shown to be present on the surface of infected cells (Fujii et al., 1993). Antibodies to Nef appear early in HIV-infected individuals (Allan et al., 1985) and cytotoxic T lymphocyte activity has been detected against cells presenting Nef peptides (Bahraoui et al., 1990). These data confirm that Nef is expressed during active HIV infection.





14


A hallmark of infection with HIV is the alteration of CD4/CD8 T cell ratios due to loss of CD4+ T helper cells. Such losses ultimately result in the inability of an infected individual to mount effective immune responses against opportunistic infections, thus resulting in death. Several possible mechanisms by which CD4 loss occurs have been postulated, including direct cytolysis by HIV (Lemaitre et al., 1990), HIV-induced syncitia formation (Lifson et al., 1986; Sodroski et al., 1986) and/or cytolytic T cell activity against infected CD4 cells (Zinkernagel and Hentgartner, 1994). In addition to skewing of the CD4/CD8 ratios, other immunologic alterations seen in HIV-infected individuals include polyclonal activation of B cells with increased immunoglobulin production and reduced antigen and mitogen responses (Edelman and Zolla-Pazner, 1989). Initial increased natural killer cell activity is observed in asymptomatic HIVinfected individuals, but these activities decrease during disease progression (Edelman and Zolla-Pazner, 1989).
Because of the known induction of T cell anergy and/or deletion by superantigens (Johnson et al., 1992), it has been speculated that the HIV genome encodes for a superantigen that may cause some of the immunopathologies observed with AIDS. Upon interaction with MHC class II antigens and TCR, Vp-specific T cell populations would be activated and expand, eventually leading to functional (anergy) or actual (deletion) loss of T cells. Several points of evidence suggest a role for an HIV-derived superantigen. Although initial studies suggested that AIDS patients had deletions in V3 T cell populations (Imberti et al., 1991), such deletions could not be found by others (Laurence et al., 1992). VI312+ T cells were shown to support enhanced replication of HIV compared to other V3 subsets and proliferated in response to cells from HIV+ patients, suggesting the presence of a superantigen (Laurence et al., 1992). Asymptomatic patients exhibit altered





15

Vp profiles (Dalgleish et al., 1992) as well as skewed T cell V1 usage upon stimulation with staphylococcal enterotoxins in vitro (Bisset et al., 1993). Interestingly, one study found skewed V3 T cell profiles in monozygotic twins that were discordant for HIV infection, with perturbations in several Vps (Rebai et al., 1994). This last study is of particular importance in that the altered VI profiles in infected individuals cannot be ascribed to differences in MHC, since only identical twins were used. Thus, these studies hint at the presence of a superantigen encoded by HIV that may play a role in HIV pathogenesis by augmenting a population of T cells, resulting in a reservoir for virus replication, with eventual deletion of those T cells.












CHAPTER II
MMTV SUPERANTIGEN BINDING TO MHC CLASS II ANTIGENS


Introduction

SEA is one of the most potent T-cell mitogens known, and has been classified as a bacterial superantigen based on its ability to stimulate V5specific T-cell subsets (Johnson et al., 1991). Concurrent with studies on SEA, minor lymphocyte stimulating (mls) antigens have recently been shown to be products of MMTV (Choi et al., 1991; Acha-Orbea et al., 1991). Two exogenous strains of MMTV encode for retroviral superantigens in genes that overlap the terminal repeat at the 3' end of the viral genome (Pullen et al., 1992; Choi et al., 1992). These genes have been designated vsag, denoting that they encode for viral superantigens (Marrack and Kappler, 1990). No direct binding of the putative MMTV superantigen to either MHC antigens or TCR has been shown. To determine sites that interact with class II MHC antigens, overlapping synthetic peptides were synthesized that encompass the putative extracellular domain of MMTV-1 superantigen. The data presented here indicate that a site that is encompassed by amino acid residues 76-119 of MMTV-1 superantigen competes with SEA for binding to class II MHC antigens. Further, direct binding studies show that this region of the MMTV superantigen binds directly to class II MHC antigens. These data indicate that SEA and MMTV superantigen share at least one common binding region on class II MHC molecules.







16





17

Materials and Methods

Synthetic Peptides
Overlapping peptides corresponding to the putative extracellular domain of the MMTV-1 vSAg protein (Pullen et al., 1992) were synthesized with a Biosearch 9500AT automated peptide synthesizer using N-(9flurenyl)methoxycarbonyl chemistry (Griggs et al., 1992). Peptides were synthesized based on a surface profile which uses a composite of three parameters: 1) HPLC hydophilicity; 2) accessibility; and 3) segmental mobility (B value) (Parker et al., 1986). The sequences of the peptides are listed in Table IIl. Peptides were cleaved from the resins using trifluoroacetic acid/ ethanedithiol/ thioanisole/ anisole at a ratio of 90:3:5:2. The cleaved peptides were then extracted in ether and ethyl acetate and subsequently dissolved in water and lyophilized. Peptides were extensively dialyzed against water to remove the remaining cleavage products. Amino acid analysis of the peptides showed that the amino acid contents and molecular weights corresponded closely to theoretical values. Peptides were not purified further since reverse phase HPLC analysis of crude peptides indicated one major peak in each profile.


Cell Lines and Reagents
The A20 cell line (ATCC, Gaithersburg, MD) was used for MMTV superantigen binding studies. A20 cells are a BALB/c B lymphoma line that expresses la. Highly purified SEA and other staphylococcal enterotoxins were obtained from Toxin Technology (Sarasota, FL). Several monoclonal antibodies (mAb) were used in this study. MAbs were purchased from Accurate Chemicals, Westbury, NY. MAb Kd (34-1-2S) is specific for m31 determinant of Kd (Ozato et al., 1982). MAb IAd (MK-D6) is specific for the 1i








Table IIIl.
Amino acid sequences of vSAg peptides




vSAg Peptide Sequence


vSAg( 76-119) DSFNNSSVQDYNLNDSENSTFLLGQGPQPTSSYKPHRLCPSEIE vSAg(1 17-147) EIEIRMLAKNYIFTNETNPIGRLLIMMLRNE vSAg(142-172) MMLRNESLSFSTIFTQIQRLEMGIENRKRRS vSAg(166-195) ENRKRRSTSVEEQVQGLRASGLEVKRGKRS vSAg(1 90-222) KRGKRSALVKIGDRWWQPGTYRGPYIYRPTDAP vSAg(220-250) DAPLPYTGRYDLNFDRWVTVNGYKVLYRSLP vSAg(245-276) LYRSLPFRERLARARPPWCVLSQEEKDDMKQQ vSAg(274-313) KQQVHDYIYLGTGMIHWKVFYNSREEAKRHIIEHIKALP vSAg(76-119)
(scrambled) PNSNEGLSQQSTDPSPHNFILSNENSYPCYSLLGDVQREDSTKF

The vSAg(76-119) scrambled peptide sequence was generated using the sequence edit program (Devereax et al., 1984).





19


helix of I-Ad (Lee and Watts, 1990). All mAb were used at a final concentration of 10 gg/ml. MAbs used in this study had similar potencies as determined by the Hybridoma Core Facility, Interdisciplinary Center for Biotechnology Research, University of Florida. Polyclonal antibodies were used at a final dilution of 1:100. Poly IAd (Cat.Y1-9-04-26-01) is specific for the mll determinant of I-Ad and poly la.7 (Cat.Y1-7-02-02-01) is specific for the m7 determinant of I-Ed. Both antibodies were obtained from NIAID, Rockville, MD. Poly I-Ak was obtained from Accurate Chemicals, Westbury, NY.


Radioiodinations
SEA (2.5 gg) and vSAg peptide (10 gg) were radiolabeled using chloramine T as described (Griggs et al., 1992). Briefly, ligands were labeled with 500 gCi of Na1251 (15 mCi/Cg, Amersham Corp., Arlington Heights, IL) in 25 gI of 0.5 M potassium phosphate buffer, pH 7.4, and 10 gC of chloramine T (5 mg/ml) for 2 min. After neutralization of the reaction with 10 p1 each of sodium bisulfite (10 mg/ml), potassium iodide (70 mg/ml), bovine serum albumin (BSA; 20 mg/ml), and 15 Cl of NaCI (4 M), the preparation was sieved on a 5 ml Sepharose G-10 column. The two fractions with the highest radioactivity in the first eluted peak were pooled and used in the radiolabeled binding assays. The specific activities of the SEs and vSAg peptide ranged from 130-150 RLCi/gg and 30-60 gCi/jg.


Binding Studies
In binding studies using A20 cells, unlabeled competitors (SEs and vSAg peptides) were added in 50 il volumes (in PBS with 1% BSA) at indicated final concentrations to 50 jIl of cells in "Eppendorf" tubes. Competitors were incubated with cells at room temperature for 45 minutes,





20


followed by the addition of radiolabeled SEs or vSAg peptide. After 45 minutes, the cells were washed three times and the radioactivity associated with the cell pellets was quantified using a gamma counter. L cells were used as an MHC class II-negative control cell line.
L cells were grown to confluence in the wells of microtiter plates and were subjected to the same incubation times and volumes of competitors and labeled ligands as used for A20 cells. After three washes, the cells were solubilized in 1% SDS, the liquid was absorbed by cotton-tipped applicators, and radioactivity was quantified using a gamma counter.


Radioimmunoassay
Two MHC peptides I-Apb(30-60) and I-Apb(60-90) were used to for binding of SEA and ORF(76-119) to class II antigens. The I-Apb(60-90) site was previously shown to be involved in SEA binding to MHC class II antigens (Russell et al., 1990). MHC peptides were dissolved in PBS at a concentration of 25 gg/ml. Peptide solutions were pipetted into polystyrene plastic tubes and tubes were placed at 10C for 4 hours to allow adherence of the peptides. The tubes were washed three times with PBS. Nonspecific sites on the plastic were blocked using 2 ml of PBS containing 1% BSA at 100C overnight. After the tubes were washed three times of PBS containing 1% BSA, unlabeled competitors (SEA and vSAg peptide) were added in 0.1 ml and allowed to bind at room temperature for 3 hours. The tubes were washed three times with 2 ml of PBS/1% BSA prior to addition of 0.1 ml of either radiolabeled SEA or radiolabeled vSAg(76-119)peptide, at final concentrations of 2 nm and 5 nm, respectively. Radiolabeled ligands were allowed to bind to MHC peptides at room temperature for 4 hours. After washing three times with 2 ml of PBS containing 1% BSA, the tubes were placed in a gamma counter and bound





21

radioactivity was quantified. Experiments were performed at least four times each using replicates of three.


Results


Competition of vSAg Peptides with Radiolabeled SEA for MHC Binding
Overlapping peptides (referred to as vSAg peptides) corresponding to the predicted extracellular domain of the MMTV-1 superantigen were synthesized and their sequences are listed in Table II. The vSAg peptides were initially tested at a concentration of 200 gM for their relative abilities to compete with 1251-SEA for binding to A20 cells, which express I-Ad and I-Ed (which are MHC class II antigens of the d haplotype, A isotype and E isotype, respectively). The vSAg(76-119) peptide reduced 1251-SEA binding to A20 cells by 63%, while the other peptides had no effect (Figure 1). Thus, only one of the overlapping vSAg peptides, vSAg(76-119), significantly blocked the binding of 1251-SEA binding to A20 cells.


vSAg(76-119) Peptide Competition Is Dose-Dependent
Dose response studies were performed on A20 cells with several vSAg peptides, including vSAg(76-119). The vSAg(76-119) peptide reduced 1251SEA binding by 50% at a concentration of 20 pM (Figure 2). Further, vSAg(76119) peptide consistently competed with 1251-SEA in a manner similar to unlabeled SEA, although SEA was 20 times more effective. Unlabeled SEA at a concentration of 1 gIM reduced 1251-SEA binding to A20 cells by 50%. These data are consistent with the reported Kd for SEA binding to I-Ed of approximately 10-6 M (Lee and Watts, 1990). The peptide corresponding to a





22







160140120100Io



604020



0
76-119 117-147 142-172 166-195 190-222 220-250 245-276 274-313 vSAg peptides Figure 1. Ability of vSAg peptides to compete with 1251-SEA for binding to A20 cells. Binding of 1251-SEA in the absence of competitors was 6384�400 CPM.
The data presented represent the mean of three individual experiments each performed in duplicate. Each bar represents the mean percent of SEA control
binding in the presence of vSAg peptides � SD.





23





140
-SEA

120 --- vSAg(76-119)
- - vSAg(245-276) 100 _ vSAg(274-313)


80


60


40


20


0 I...I.. .......I.......II......I....... I
0.01 0.10 1.00 10.00 100.00 1000.00 Competitor (jgM)







Figure 2. Ability of vSAg(76-119) to compete with 1251-SEA for binding to A20 cells in dose response studies. 1251-SEA was used at 2.5 nM. Binding of 1251SEA to A20 cells in the absence of competitors was 5184�380 CPM. The data presented represent the mean of three individual experiments, each performed in duplicate. Each point represents the mean percent of SEA control binding in
the presence of competitors � SD.





24


C-terminal region of the MMTV superantigen, vSAg(245-276), was not a consistent competitor for SEA binding. The peptide corresponding to the Cterminal tail, vSAg(274-313), did not compete. A peptide having the same amino acid content as vSAg(76-119) but in a scrambled sequence (see Table III for sequence) did not compete, thus indicating that vSAg(76-119) peptide competition was sequence specific. No direct binding of 1251-vSAg(76-119) to SEA or to related superantigens was detected (data not shown). Thus, the vSAg(76-119) peptide binds specifically to A20 cells, and competes for SEA binding in a dose-dependent manner.


Direct Binding of vSAg(76-119) Peptide to A20 Cells
In direct binding studies, 1251-vSAg(76-119) peptide bound to A20 cells and was effectively inhibited by both unlabeled SEA and unlabeled vSAg(76119) peptide (Figure 3). Unlabeled SEA and unlabeled vSAg(76-119) peptide competed with 1251-vSAg(76-119) in a similar manner, although SEA was a more potent competitor. SEA reduced 1251-vSAg(76-119) binding by 50% at a concentration of 1.8 gIM as compared to 25 gM for unlabeled vSAg(76-119) peptide. Neither vSAg(76-119) scrambled peptide nor the C-terminal vSAg peptide competed, thereby showing that vSAg(76-119) binding was sequence and region specific. TSST-1 did not compete with 1251-vSAg(76-119) peptide for binding to A20 cells, while SEB competed less effectively than SEA, which is consistent with their relative abilities to compete with SEA for MHC class II binding (Fraser, 1989; Pontzer et al., 1991). Thus these results indicate that binding of vSAg(76-119) peptide occurs to murine MHC class II antigens. Binding of vSAg(76-119) may occur at a region(s) to which SEA also binds or to a neighboring site which interferes with the binding of SEA.





25












120



100



80



U 60
6------- SEA

S vSAg(76-119)
40
40- A TSST-1

A SEB
20
20 - vSAg(76-119) scrambled

vSAg(274-313)
0 ....... 1 ' 1 ........ . i ........ * I .
0.01 0.10 1.00 10.00 100.00 1000.00

Competitor (pM)






Figure 3. Ability of SEA, SEB, and TSST-1 to compete with 1251-vSAg(76-119) for binding to A20 cells. Experimental conditions were the same as those described in Figure 1. 1251-vSAg(76-119) was used at a final concentration of 2.3 nM. Binding of 1251-vSAg(76-119) in the absence of competitors was 2278�224 CPM. The data presented represent the mean of three individual experiments, each performed in duplicate. Each point represents the mean percent of vSAg control binding in the presence of competitors � SD.





26

Evidence that vSAg(76-119) Binds Specifically to MHC Class II Antigens
Two further pieces of evidence indicate that vSAg(76-119) binds to class II MHC antigens. Binding of vSAg(76-119) to class II negative mouse L cells was insignificant as compared to mouse A20 cells even at concentrations as high as 5 nM (Figure 4A). Monoclonal antibodies to class I MHC antigens had no effect on 1251-vSAg(76-119) binding to A20 cells (Figure 4B). Polyclonal anti-lAd and anti-I-Ed significantly blocked binding and, in combination, these antibodies reduced binding to A20 cells by 73%. These data suggest that vSAg(76-119), like SEA (Lee and Watts, 1990), binds to both I-A and I-E. An IAd pl helix-specific monoclonal antibody reduced binding by approximately 30%, suggesting that this is a region on I-A to which vSAg(76-119) binds. Polyclonal antibodies to I-Ak had minor effects on vSAg(76-119) binding. Thus, these data indicate that vSAg(76-119) binds to class II MHC antigens.
In order to directly determine if vSAg(76-119) binds to the pl helix of I-A, a competitive radioimmunoassay was performed in which 1251-SEA and 1251vSAg(76-119) were tested for their relative abilities to bind to I-Apb(60-90) peptide. SEA and vSAg(76-119), but not scrambled VSAG(76-119), competed with both 1251-SEA and 1251-vSAg(76-119) for binding to I-Apb(60-90) in a manner similar to the competition seen on whole cells (Figure 5A). As previously shown for SEA (Russell et al., 1990, 1991), vSAg(76-119) did not directly bind to I-Apb(30-60) (Figure 5B). Thus, SEA and vSAg(76-119) bind to a similar region on the p chain of the class II MHC molecule.

Discussion
To date, no information has been available on the ability of MMTV vSAg protein to bind to MHC antigens and, in fact, this ability has been questioned (Marrack and Kappler, 1990; Acha-Orbea and Palmer, 1991; Acha-Orbea et al.,





27


6000 A
6 A20 cells 5000
O] L cells 4000

3000

2000

1000

0
0 200 Concentration of ORF(76-119)
(RM)


160
U MAb Kd M Poly IAd El Ia.7 B
140
] Combination j MAb IAd 0 Poly IAk
120- (poly lAd + Ia.7)

-100- A A

80
) v
60

40- v

20


Antisera


Figure 4. Evidence that vSAg(76-119) peptide binds to MHC class II antigens,
and does not bind to class I antigens.
Panel A: Binding of 1251-vSAg(76-119) to class II-positive A20 cells and class II-negative L cells. 1251-vSAg(76-119) was used at a final concentration of 5
nM.
Panel B: Blockage of 1251-vSAg(76-119) binding to A20 cells by antibodies to class I and class II MHC antigens. 1251-vSAg(76-119) was used at a final
concentration of 5 nM. MAbs were used at a final dilution of 1:30.





28




---- SEA vs. radiolabeled SEA A 120
----- vSAg(76-119) vs. radiolabeled SEA
--J--- SEA vs. radiolabeled vSAg(76-199) 100
--0-- vSAg(76-119) vs. radiolabeled vSAg(76-119)

80


so60


40


20



0.1 1.0 10.0 100.0 1000.0 Concentration (tiM)

2000
E1 No competitor 1500
150 � vSAg(76-119)

S1000500


0
60-90 30-60 MHC Peptide


Figure 5. Binding of 1251-vSAg(76-119) to MHC class II peptides.
Panel A: Binding of 1251-SEA and 1251-vSAg(76-119) to I-Apb(60-90) and inhibition by vSAg(76-119). Each point represents the mean percent reduction of control binding in the presence of competitors � SD. Binding of 1251-SEA and 1251-vSAg(76-119) in the absence of competitor was 2656�92 CPM and
1547�33 CPM, respectively.
Panel B: Relative ability of vSAg(76-119) to bind I-Ab(60-90) and I-Apb(60-90) in the presence and absence of vSAg(76-119). Unlabeled and 1251-vSAg(76119) were used at final concentrations of 5 nM and 300 uM, respectively.





29

1991). Such binding studies are hampered by the difficulties inherent in expressing the MMTV superantigen. A model has recently been proposed that suggests that MMTV superantigens may act in the same or similar fashion to bacterial superantigens by bridging MHC antigens and TCR on the appropriate cell types (Acha-Orbea and Palmer, 1991). MMTV superantigens, presented in the context of MHC class II antigens, may act by stimulating and expanding Vpspecific T cell subsets, thereby indirectly enhancing the further infection of B cells (Held et al., 1994a). The results presented in this chapter suggest that MMTV vSAg protein binds to MHC antigens, thereby strengthening the argument that bacterial and retroviral superantigens act in a similar manner.
It has been reported that MMTV superantigen is a 45 kd Type II integral membrane protein with an intracellular N-terminus, an essential hydrophobic transmembrane region near the N-terminus (residues 45-64), and a glycosylated extracellular C-terminus (Choi et al., 1992). Although no direct evidence exists, the variability of the C-terminal residues of vSAg proteins of various MMTV strains seems to correlate with their differences in V3 specificity, lending support to the concept that this region binds TCR (Pullen et al., 1992). Truncated versions of the vsag gene were transfected into MHC class II-bearing cells and tested for superantigen activity. Complete loss of superantigen activity occurred when the MMTV vsag gene was N-terminally truncated to the third methionine (residue 122) and beyond (Choi et al., 1992). The authors concluded that a hydrophobic region, which was missing in this N-terminally truncated version, acts as a transmembrane region and is essential for superantigen activity. However, our binding data suggest that loss of superantigen activity may have been due, at least in part, to the deletion of the MHC-binding domain which is encompassed by residues 76-119. In fact, the





30

superantigen activity of MMTV vSAg protein was lost in truncations that did not contain the region encompassed by residues 76-119.
Subsequent studies by the same group of investigators suggest that MMTV-7 superantigen is synthesized as a precursor molecule, is proteolytically cleaved at an internal site (- residue 164) and is expressed as an 18.5 kd surface protein consisting of the C-terminal residues (Winslow et al., 1992). Membrane association of this truncated form may involve "tethering" to MHC class II antigens or to the N-terminal portion of the precursor molecule. As a cautionary note, the transfected cell line used to characterize this truncated form of the MMTV-7 superantigen was shown to have high expression of the protein but low activity. Conversely, a transfected cell line with moderate superantigen expression and high activity was not used for characterization.
Recently, studies were reported on the binding of truncated forms of MMTV-7 superantigen to human MHC class II antigens. Binding studies were performed with 28K and 18K versions of the superantigen and showed that both molecules bind to human MHC class II antigens (Mottershead et al., 1995). Thus, studies by our laboratory indicate one site for binding to mouse MHC class II antigens, whereas studies performed by others suggest that two sites on MMTV superantigen are involved in binding to human MHC class II antigens.

Despite the diverse origins of SEA and MMTV vSAg protein, it is likely that these two proteins exert superantigen activity by a similar mechanism. Two different regions of the MMTV superantigen are probably involved in MHC binding and Vp specificity. The variability of the C-terminal 30 residues between Vp-specific MMTV superantigens is indirect evidence that the Cterminus is responsible for V3 specificity (Pullen et al., 1992). Data presented here on competitive and direct binding of vSAg peptides to A20 cells suggest that the region encompassed by residues 76-119 is involved in MHC binding.





31

This region would be part of the extracellular domain based on a proposed Type II membrane protein model for MMTV vSAg protein (Choi et al., 1992). Thus, both the C-terminal tail and an N-terminal region may be required for ternary complex formation with MHC and TCR by MMTV superantigen. Conversely, SEA contains several N-terminal domains that bind MHC (Griggs et al., 1992) and an internal domain that may be involved in binding to TCR, so that the sites of interaction of these superantigens with MHC and TCR may not be completely analogous. Although little sequence homology exists between MMTV vSAg protein and SEA, circular dichroism analysis indicates that both SEA and vSAg(76-119) have significant P structure, suggesting that similar structural motifs may be important for MHC binding by superantigens.
Finally, results presented herein indicate that a peptide corresponding to residues 76-119 of the MMTV superantigen binds directly to MHC class II antigens. Further, competition studies indicate that SEA and vSAg(76-119) peptide bind to at least one common region on mouse MHC antigens. Future studies using synthetic peptides and peptide analogues may help further elucidate the site(s) on MHC for which SEA and MMTV superantigen compete.












CHAPTER III
IDENTIFICATION OF AN HIV-1 NEF PEPTIDE THAT BINDS TO MHC CLASS II ANTIGENS

Introduction

In Chapter Two, a site was identified on the MMTV-1 superantigen that binds to class II MHC antigens, suggesting that retroviral and bacterial superantigens exert their effects similarly. MMTV superantigen is encoded in a gene that overlaps the terminal repeat at the 3' end of the viral genome. The genome of human spumavirus also contains a gene (bel3) that overlaps the terminal repeat at the 3' end, the product of which may have superantigenic properties (Fluegel, 1993). Recently, it has been suggested that human immunodeficiency virus (HIV) may possess superantigen activity, although no specific superantigenic protein has been identified (Imberti et al., 1991; Laurence et al., 1992; Dalgleish et al., 1992; Bisset et al., 1993). The HIV genome also contains a gene that overlaps the terminal repeat at the 3' end, the product of which is called Nef. Although Nef is one of the early proteins produced during the replication of primate lentiviruses, the role of Nef in HIV pathogenesis has yet to be established.
To determine if Nef had binding characteristics similar to superantigens, a study was undertaken to determine its ability to bind to MHC class II antigens, at sites that are known to bind superantigens. Using overlapping peptides corresponding to the entire length of Nef (HIVLAv), we have identified a region which binds to MHC class II antigens, and which competes for binding with known bacterial superantigens.


32





33

Materials and Methods


Synthetic Peptides
Overlapping peptides corresponding to the entire length of HIVLAV Nef (Wain-Hobson et al., 1985) were synthesized with a Biosearch 9500AT automated peptide synthesizer using N-(9-flurenyl)methoxycarbonyl chemistry as described in chapter II of this text. Peptides were synthesized based on a surface profile as described. The amino acid sequences of the peptides are presented in Table IV.


Cells and Reagents
Two cell lines were used for the binding studies. Raji cells are EBVtransformed B cells that express DR3, Dwl0, DQwl, and DQw2 (Merryman et al., 1989). DR1-transfected L cells were kindly provided by Dr. Eric O. Long and are described elsewhere (Lechler et al., 1988). SEs were obtained from Toxin Technology (Sarasota, FL). Several mAb were used in this study. AntiHLA-DR clone L243 reacts with a nonpolymorphic DR epitope and does not cross-react with DP or DQ (Robbins et al., 1987). Anti-HLA-DP clone B7/21 reacts with a monomorphic epitope present on DP1, DP2, DP3, DP4, and DP5 (Robbins et al., 1987). Anti-HLA-DQ clone SK10 reacts with a common polymorphic epitope present on cells expressing DQwl and DQw3 (associated with DR1. DR2, DR4, DR5, w8, w9, and w10) (Brodsky, 1984). Anti-HLA-DR clone L227 reacts with a nonpolymorphic region of DR (Bamstable et al., 1978). Clone W6/32 reacts with a monomorphic epitope on HLA-A, B, and C (Barnstable et al., 1978). Clones L243, B7/21, and SK10 were obtained from Becton-Dickinson (Mountain View, CA) and clones L227 and W6/32 were kindly provided by Dr. Robert Rich. All mAbs were used at a final concentration





34

Table IV.
Amino acid sequences of Nef peptides.



Nef Peptide Sequence


1-38 MGGKWSKSSVVGWPTVRERMRRAEPAADGVGAASRDLE 31-65 GAASRDLEKHGAITSSNTAATNAACAWLEAQEEEE 62-99 EEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEG 93-132 EKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPG 123-160 DWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGE 156-186 NKGENTSLLHPVSLHGMDDPEREVLEWRFD 182-206 EWRFDSRLAFHHVARELHPEYFKNC





35


of 10 ig/ml. Purified recombinant Nef protein was purchased from Repligen, Cambridge, MA.


Radioiodinations

SEs (2.5 gLg) and Nef peptide (10 jg) were radioiodinated using chloramine T as described in chapter II of this text. The specific activities of the SEs and Nef peptide ranged from 70-120 gCi/lgg and 30-40 gCi/gg, respectively.


Binding Studies
For binding studies on Raji cells, unlabeled competitors (SEs and Nef peptides) were added in 50 i1l (in PBS with 1% BSA) to 50 1l of cells in 1.5 ml "Eppendorf" tubes. Cells and competitors were incubated at room temperature for 45 min., followed by the addition of radiolabeled SEs or Nef peptide. After 45 min., the cells were washed three times and the bottoms of the tubes were cut off. Radioactivity was quantified using a gamma counter. Similarly, DR-1 transfected L cells, which were grown to confluence in the wells of microtiter plates, were subjected to the same incubation times and volumes of competitors and labeled ligands as used for the Raji cells. After three washes, the cells were solubilized in 1% SDS, and the liquid was absorbed by cottontipped applicators and radioactivity was quantified using a gamma counter.




Results
Competition of Nef Peptides with Radiolabeled SEA for MHC Binding
Overlapping peptides corresponding to the entire length of the Nef sequence (HIVLAV) were synthesized (Table IV). The Nef peptides were





36


initially tested at a concentration of 300 gM for their relative abilities to compete with 1251-SEA for binding to Raji cells, which express HLA-DR3 and HLADRwlO, and DR1-transfected L cells (Figure 6). Nef(123-160) reduced 1251SEA binding to Raji cells by 41% which was significant at p<0.002. This degree of inhibition was consistent in repeated experiments. The N-terminal peptide, Nef(1-38), had a marginal but insignificant effect (21% reduction, p>0.05) on 1251-SEA binding to Raji cells in the same experiment. This slight inhibition by Nef(1-38) was not seen in repeated experiments. The other peptides did not affect SEA binding to Raji cells. A similar pattern was seen in competitive binding studies performed on DR1-transfected L cells. Nef(123160) blocked 1251-SEA binding to DR1 cells by 35% (p<0.008), whereas Nef(138) had only a slight effect (p>0.8). Thus, only one of the overlapping Nef peptides, Nef(123-160), significantly and consistently blocked 1251-SEA binding to both Raji cells and DR1-transfected L cells.


Nef(123-160) Peptide Competition Is Dose-Dependent
The relative abilities of Nef(123-160), purified recombinant Nef protein (Repligen, Cambridge, MA), and SEA to block SEA binding to MHC class II were tested in dose response studies (Figure 7). Nef(123-160) reduced 1251SEA binding to Raji cells by 40% at the highest concentration tested (300 RiM), and competed with SEA in a dose-dependent manner. Two other Nef peptides, Nef (1-38) and Nef(31-65), did not block SEA in a dose-dependent manner. Unlabeled SEA reduced 1251-SEA binding by 50% at a concentration of 0.2 AM, which is consistent with the reported Kd of SEA for human MHC class II antigens (Chintagumpala et al., 1991). Thus, Nef protein and the internal Nef sequence, Nef(123-160), competed for SEA binding to Raji cells in a dosedependent manner.





37





140
* Raji cells
120 -] DRI cells


100


80


60


40


20


0
1-38 31-65 62-99 93-132 123-160 156-186 182-206

NeF Peptide






Figure 6. Blockage of 1251-SEA binding to Raji and DR1-transfected L cells by Nef peptides. Nef peptides were used at a final concentration of 300 M. 1251.
SEA was used at a final concentration of 2 nM. 105 Raji or DR1-transfected L cells were used per tube. Binding of 1251-SEA to Raji and DR1-transfected L cells in the absence of competitors was 31,469 � 2292 and 5,708 � 41 CPM, respectively. Data represent the mean percent of control of three individual experiments, each performed in duplicate. Bars represent binding to Raji and
DR1-transfected L cells in the presence of Nef peptides � SD.





38









125
- NeF(123-160) NeF(1-38)

NeF(31-65) SEA 100 - Nef protein




75




50




25
0.01 0.10 1.00 10.00 100.00 1000.00 Concentration (pAM)





Figure 7. Relative abilities of Nef(123-160), purified Nef protein, and SEA to compete with 1251-SEA for binding to Raji cells. Experimental conditions were the same as those described in Figure 1. Binding of 1251-SEA to Raji cells in the absence of competitors was 30,406 � 2851 CPM. The data presented represent the mean of three individual experiments, each performed in duplicate. Each point represents the mean percent of SEA control binding in the presence of competitors � SD.





39


Ability of Nef Peptides to Compete with SEs for Binding to Raii Cells

Nef peptides were also tested for their relative abilities to compete with other staphylococcal enterotoxins (SEs) for binding to Raji cells. As shown in Figure 8, Nef(123-160) significantly blocked binding of 1251-SEE by 42% (p<0.02) and 1251-SEC1 by 26% (p<0.03), but only blocked binding of 1251. SEB by 10% (p>0.1). Thus, Nef(123-160) significantly inhibited the binding of two highly homologous SEs, SEA and SEE, while it was less effective against SEB and SEC1.


Direct Binding of Nef(123-160) Peptide to Rai Cells
In direct binding studies, 1251-Nef(123-160) bound to Raji cells and was effectively inhibited by unlabeled Nef(123-160) and SEs (Figure 9). Unlabeled Nef(123-160) reduced 1251-Nef(123-160) binding by 50% at a concentration of 30 pgM. SEE was a better competitor and reduced 1251-Nef(123-160) binding by 50% at a concentration of 2 gM. Both SEE and unlabeled Nef(123-160) competed in a similar manner, but SEE was a more potent competitor. SEA did not inhibit 1251-Nef(123-160) binding as well as SEE, but it was more effective than SEC1 or SEB. This pattern of inhibition is reflective of the ability of Nef(123-160) to block SE binding to MHC class II antigens. Although SEs compete for similar sites on MHC class II antigens, our results suggest that Nef(123-160) competes for sites that are more closely associated with SEE than the other SEs tested. Nef(123-160) binding was not blocked by other Nef peptides, such as Nef(1-38) and Nef (31-65), at 300 gM. These results suggest that Nef(123-160) binds directly to Raji cells and competes for a site on MHC class II antigens to which SEs bind.





40








160 - O SEE U SEB 140
0 SECI 120

.100

80

60

40

20


1-38 31-65 62-99 93-132 123-160 156-186 182-206 NeF Peptide






Figure 8. Blockage of 1251-SE binding to Raji cells by Nef peptides. Nef peptides were used at a final concentration of 300 gM. 1251-SEs were used at 2 nM. All other experimental conditions were the same as those described in Figure 7. Binding of 1251-SEE, 1251-SEB, and 1251-SEC1 in the absence of competitor was 5,917�612 CPM, 6,438�134 CPM, and 4,493�425 CPM, respectively. Data represent the mean of three individual experiments, each performed in duplicate. Each bar represents the mean percent of the appropriate SE control binding to Raji cells in the presence of Nef peptides +
SD.





41






120


100


80


60


40
Nef(123-160) - SEA
20 - A SEE 0 SEC1

0- SEB
0 I I....... I
0.1 1.0 10.0 100.0 1000.0

Concentration (giM)



Figure 9. Direct binding of Nef(123-160) peptide to Raji cells. 1251-Nef(123160) was used at a final concentration of 3 nM. All experimental conditions were the same as those described in Figure 7. Binding of 1251-Nef(123-160) in the absence of competitor was 1956 � 150 CPM. The data presented represent the mean of three individual experiments, each performed in duplicate. Each point represents the mean percent of 1251-Nef(123-160) binding relative to control in the presence of competitors � SD.





42

MAbs to MHC Class II Antigens Block Nef(123-160) Peptide Binding to Rai Cells
To ascertain the receptor to which 1251-Nef(123-160) bound on Raji cells, binding was performed in the presence of monoclonal antibodies (mAb) to class II and class I antigens (Figure 10). Clone L243, a mAb specific for HLADR, reduced 1251-Nef(123-160) binding by 35%. Another mAb specific for HLA-DR, clone L227, also reduced Nef(123-160) binding, but was not as effective as clone L243. This is consistent with reports that L227 mAb is not very effective at blocking SEA binding to Raji cells (Chintagumpala et al., 1991). MAb to other class II antigens (HLA-DP and DQ) had no effect on 1251-Nef(123160) binding, as was also the case for a mAb to class I antigens (clone W6/32). Thus, our studies indicate that Nef(123-160) binds to class II MHC antigens, the known receptors for superantigens on APC.


Discussion


Data presented here show that binding of bacterial superantigens to MHC class II antigens can be blocked by a peptide corresponding to an internal Nef region. Anti-HLA-DR mAb blocked Nef(123-160) binding to Raji cells, suggesting that binding to class II molecules occurred. Binding of Nef(123-160) to HLA-DR probably occurs outside the antigen binding groove, since Nef(123160) was able to block binding of the superantigens SEE and SEA. A recent study has shown that HLA-DR is present on HIV-1 and SIV, and that it is selectively incorporated into the virus membrane over HLA-DP or HLA-DQ (Arthur et al., 1992). Antibodies to HLA-DR, but not antibodies to HLA-DP or HLA-DQ, effectively inhibited viral infection of cells in vitro and these antibodies target the HLA-DR antigens present on the virus particles and not those present





43









120


10080 60


4020 0
B7/21 SK10O W6/32 L243 L227
(Anti-DP) (Anti-DQ) (Anti-class I) (Anti-DR) (Anti-DR) Antibody






Figure 10. Blockage of 1251-Nef(123-160) binding to Raji cells by antibodies to MHC class I and class II antigens. 1251-Nef(123-160) was used at a final concentration of 3 nM. MAbs were used at a final concentration of 10 gg/ml.





44


on the cells. The ability of these antibodies to neutralize HIV suggest that HLADR may play a role in infectivity. Our results suggest that Nef may bind to HLADR as it is being expressed on the surface of an infected cell in a manner analogous to the putative expression of MMTV superantigen (Choi et al., 1992). Further, Nef has been shown to be present on the surface of infected cells, as assessed using anti-Nef antibodies labeled with fluorescein (Fujii et al., 1993). This is in contrast to the situation with MMTV superantigen in that no one has been able to show that cells from MMTV-infected animals express superantigen, although superantigen functional activity is observed (Choi et al., 1991). Transgenic mice expressing MMTV superantigen delete Vp14+ T cells, probably during the ontogeny of the immune system, and these mice are immune to infection by exogenous MMTV infection (Acha-Orbea and Palmer, 1991). Thus it is possible that Nef interacts with class II MHC antigens in a manner somewhat similar to MMTV superantigen, suggesting a possible superantigen function for Nef in HIV pathogenesis.













CHAPTER IV
ACTIVATION OF CD4 T CELLS BY THE NEF PROTEIN FROM HIV-1


Introduction

In the previous chapter, data was presented which showed that Nef binds specifically to class II-bearing Raji cells at a site(s) involved in staphylococcal enterotoxin binding. This binding was similar to that shown for MMTV superantigen. These data suggest that Nef may have characteristics of superantigens. The data presented in this chapter show that Nef induces proliferation of human peripheral blood mononuclear cells. Further, the proliferative response is T cell specific. The data presented in this chapter indicate that Nef activates T cells and induces T cell cytokine production in a manner reminiscent of staphylococcal superantigens.




Materials and Methods

Nef Protein

Nef protein was expressed and purified using a fusion protein and purification system. The HIV-1 nef gene (HIV-1 IIIB, R & D Systems, Cambridge, MA) was amplified by polymerase chain reaction (PCR), using the following primer set :
5' ATG GGT GGC AAG TGG TCA AAA AGT (+) 5' GCC AAG CTT GAT GTC AGC AGT TCT (-)





45





46

The extra A added to the 3' end of the amplified nef gene fragment as a result of PCR was removed by treatment with the Klenow fragment of DNA polymerase. This DNA was ligated into the Xmnl/Hindlll cloning site of the prokaryotic expression vector pMALTM-c2 (New England BioLabs, Beverley, MA). The sequence of the fusion construct was verified by the DNA Sequencing Laboratory, Interdisciplinary Center for Biotechnology Research, University of Florida. The fusion construct was engineered such that the N terminus of Nef was immediately downstream from the Factor Xa cleavage site at the C terminus of maltose binding protein (MBP). Thus, cleavage with Factor Xa was predicted not to add any vector-derived amino acids. E. coi strain TB1 was transformed with the vector containing mbp/nef. Cultured E. coli TB1 were suspended in column buffer consisting of 20 mM Tris-HCI (pH 7.4), 200 mM NaCI, and 1 mM ethylenediaminetetraacetic acid (EDTA). The cells were sonicated and the supernatants were recovered after centrifugation. Protease inhibitors (2 mM phenylmethylsulfonyl fluoride (PMSF), pepstatin A, leupeptin, and aprotinin) were added to extracts. The protein concentration of the extracts was adjusted to 2.5 mg protein/ml prior to loading onto an amylose affinity column containing a bed volume of 50 ml. The column was washed with 350 ml of column buffer. The fusion protein was eluted with 200 ml of column buffer containing 10 mM maltose. Fractions (7 ml) were collected. The fusion protein generally eluted within the first 70 ml. After cleaving the fusion protein with factor Xa (New England BioLabs, Beverley, MA), the cleavage mixture was loaded onto a hydroxyapatite column containing a bed volume of 35 ml. After extensive washing of the column with 180 ml of 10 mM potassium phosphate (pH 7.2) containing 2 mM PMSF and 1 mM benzamidine, fractions were eluted using a linear gradient from 10 mM to 400 mM potassium phosphate buffer (pH 7.2) containing 2 mM PMSF. Fractions (5 ml) were collected. Nef eluted from





47


the column between 100-150 mM potassium phosphate, and was generally contained within 7 fractions. The fraction containing Nef protein was dialyzed against affinity column buffer containing 150 mM NaCI. The dialyzed material was loaded onto a second amylose affinity column for removal of MBP. The flow-through containing Nef protein was collected and loaded onto an High-Q ion-exchange cartridge (BioRad Laboratories, Richmond, CA). The High-Q cartridge was washed with 100 ml of 20 mM Tris buffer (pH 8.0) containing 150 mM NaCI. Fractions were eluted with a linear gradient from 150-400 mM NaCI in the same buffer. The fractions containing Nef protein, which eluted at 180 mM NaCI, were collected and dialyzed overnight against PBS and stored at
-700C. The purity of Nef protein was assessed by SDS-PAGE, followed by staining with silver. Upon staining with silver, a single band was found, and a corresponding band was detected by Western blotting with monoclonal anti-Nef antibody (Repligen, Cambridge, MA). MBP purified by this method and used at the same concentrations as that used for the purified Nef preparation, did not have proliferative activity on human peripheral blood mononuclear cells.
Nef proliferative activity was confirmed using recombinant Nef proteins obtained from two other sources and which were produced using different expression systems. The following reagent was obtained through the AIDS Research and Reference Reagent Program, AIDS Program, NIAID, NIH: HIV-1 LAV Nef from the Division of AIDS, NIAID. The HIV-1 nef gene (LAV) used to produce this Nef preparation was isolated from pBENN 6 and cloned into the bacterial expression vector pPD-YN-61. The protein was produced in E. coli strain Sf930 and isolated as inclusion bodies. Nef protein was also obtained from Repligen (Cambridge, MA). In this case, the HIV-1 nef gene (LAV) was cloned into the bacterial vector pD10, which adds a hexahistidine tag for protein purification using nickel chelate chromatography. The protein was produced in





48

E. coli strain MC10611POL.1. All Nef preparations were negative for endotoxin as assessed by the limulus amoebocyte lysate assay.


Reagents
Staphylococcal enterotoxin A (SEA) was purchased from Toxin Technology (Sarasota, FL). Concanavalin A (Con A), anti-CD3, and anti-lgM were purchased from Sigma Chemical Co. (St. Louis, MO).


Proliferation Assays
Proliferation studies on human peripheral blood mononuclear cells (PBMC) were performed as described previously (Pontzer et al., 1992). Peripheral blood from healthy adult blood bank donors was obtained from Civitan Regional Blood Center (Gainesville, FL) for use in these studies. All donors were negative for cytomegalovirus, hepatitis B virus, and HIV. PBMC were isolated from peripheral blood using ficoll hypaque gradient centrifugation. After extensive washing, PBMC were plated into wells of microtiter plates at 2.8 x 106 cells/ml, followed by addition of activators. Final volumes were adjusted to 150 [l/well with RPMI 1640 tissue culture medium containing 5% fetal bovine serum (FBS) and penicillin/streptomycin. 3Hthymidine (l~Ci/well; Amersham Corporation, Indianapolis, IN) was added at 90 h after initiation of cultures and the cells were incubated for an additional 6 h prior to harvest onto filter paper. Filter paper was placed in liquid scintillation fluid and radioactivity was quantified using a 13 scintillation counter. Stimulation index (S.I.) was determined by dividing the experimental CPM by the CPM obtained from control (unstimulated) cultures.





49

Purification of T Cells from PBMC

Two methods were used to isolate purified T cell populations. One method involved passing PBMC over T Cellect columns (Biotex, Edmunton, Alberta, Canada). A second method involved a double rosetting technique using neuraminidase-treated sheep red blood cells (NA-SRBC) (Spawski and Lipsky, 1992). PBMC (5 ml at 107/ml) were mixed with 2.5 ml each of NASRBC and FBS. PBMC were incubated for 10 min at 370C, centrifuged at 900 rpm to gently pellet the cells, and incubated for 90 min at 370C. Rosetted T cells were separated from non-rosetted cells by ficoll hypaque centrifugation. NASRBC were removed from the rosetted fraction by lysis with ammonium chloride. The procedure was repeated on the rosetted fraction to insure purity. Autologous rosetted T cells were plated at 2.8 x 106 cells/mi. Cells were plated into wells of microtiter plates at 2.8 x 106 cells/mI. To insure that these cultures were depleted of APC, T cells were stimulated with staphylococcal enterotoxins, which stimulate T cells only in the presence of APC.


Autologous Antigen-Presenting Cells (APC)
For proliferation studies, APC consisted of PBMC that were inactivated with 0.8% paraformaldehyde. For cytokine studies, APC consisted of cells that did not form rosettes after two treatments with NA-SRBC. APC were plated at 2.8 x 106 cells/ml. To insure inactivation with paraformaldehyde was complete, APC were tested for possible proliferation using anti-lgM and Con A.


Synthetic Nef Peptides and Antibodies to Nef Peptides
The sequences of the peptides used are listed in Table Ill. Peptides were purified by reverse phase high performance liquid chromatography. Polyclonal antibodies to Nef peptides were generated in rabbits. Anti-peptide





50

antibodies were tested for their relative abilities to neutralize Nef mitogenic activity by incubating Nef with antibodies at 370C for 60 minutes prior to addition of PBMC. Antibodies to both Nef(123-160) and Nef(182-206) showed strong reactivity to whole recombinant Nef protein, and these combined antisera had significant neutralizing activity against Nef. Nef was treated with preimmune sera as control.


Cytokine Production and Assays
PBMC were plated at 106 cell/ml into wells of 24-well plates (Corning Corp., Corning, NY). Activators were added and final volumes were adjusted to 300 pl/well. Cultures were incubated at 370C over a 96 h period and samples (100 p1) were obtained at 24 h intervals. Cell cultures were replenished with RPMI/5% FBS (100 pl) after obtaining samples. Culture supernatants were tested for interleukin 2 (IL 2) using the IL 2-dependent HT-2 cell line (Ho et al., 1987). The amounts of IL 2 present in culture supernatants were determined using recombinant human IL 2 (Genzyme, Cambridge, MA) as a standard. Samples were tested for interferon (IFN) activity on human WISH cells by a microplaque reduction method (Langford, et al., 1978b), using approximately 40 plaque-forming units (PFU) of vesicular stomatitis virus (VSV) per well. In our studies, 1 U/ml of IFN is defined as the concentration required to decrease the number of PFU per well by 50%.
IFN activity was typed by neutralization reactions with specific antisera, as described (Johnson et al., 1982). Briefly, samples were pretreated with 1000 neutralizing units of either anti-human IFNa (Lee Biomolecular, San Diego, CA) or anti-human IFNy (Genzyme, Cambridge, MA). Controls were sham-treated with EMEM/2% FBS. The neutralizing activity of anti-IFN antisera was confirmed using HulFNa (Lee Biomolecular, San Diego, CA) and HulFNy





51


(Genzyme, Cambridge, MA) as positive controls. Samples were incubated at 370C for 1 h prior to transfer to confluent human WISH cells. Residual IFN activity in sham- and antiserum-treated samples was measured as described above.


Studies on Vl-Specific T Cell Expansion and Induction of Anergy
PBMC cultured in the presence of Nef, TSST-1, and ConA were tested by flow cytometry for specific Vp expansion as previously described (Soos and Johnson, 1994) using a panel of anti-V3 antibodies. Induction of anergy in vitro by Nef, TSST-1, and ConA was also assessed with anti-V3 antibodies as described (Schiffenbauer et al., 1993). Antibodies to Vp5a, Vp5b, V35c, Vl6a, Vp8a, and Vp12 were obtained from T Cell Sciences, Inc., Cambridge, MA. Antibodies to VB2, VI3, Vpl3, Vpl 7, Vpl18, V121, and Vp22 were purchased from Immunotech, Marseilles, France.



Results

Nef Proliferative Response
Recombinant Nef proteins from two sources were tested for proliferative activity. One Nef preparation (referred to as Nef 1) was purified and kindly provided by Dr. Taishi Tanabe in the laboratory of Dr. Howard M. Johnson. This Nef preparation was compared to purified Nef from Repligen (Nef 2). Both Nef protein preparations, which are derived from the HIVLAV sequence, induced similar levels of proliferation (Figure 11). Nef proteins from both sources were pure and neither preparation contained detectable levels of endotoxin. Thus, Nef proteins from both sources had similar proliferative activity, and the proliferation observed was significant. Nef from these two sources were





52






15
Nef 1

0 Nef 2

* 105





1000 100 10 1 Concentration (ng/ml)








Figure 11. Comparison of the mitogenic activities of Nef protein preparations. Two Nef preparations,Nef 1 and Nef 2, were compared for proliferative activities on human PBMC. Data are from a representative experiment, performed in triplicate, and are expressed as mean stimulation index + S.D.. Comparison of the proliferation of PBMC cultured for 4 days in the presence of either Nef 1 or Nef 2. The mean value for 3H-thymidine incorporation by unstimulated cultures was 900�67.





53

subsequently used in these studies. The possibility was raised that the proliferative activity ascribed to Nef was due to a contaminant in the recombinant preparation. As a control, the proliferative activity of the Nef fusion partner, MBP, was also assessed. No proliferation was observed with MBP (Figure 12). Further, polyclonal antisera generated against Nef synthetic peptides were tested for neutralizing activity. As shown in Table V, anti-Nef antibodies significantly reduced Nef-induced proliferative responses. Antibody neutralization of Nef activity occurred in a dose-dependent manner. These data confirm that the proliferative activity seen in these studies is due to Nef protein, and not to a vector-derived contaminant.
Nef was also tested for the ability to induce proliferation of PBMC from fifteen donors. Results from a representative sample of ten donors are shown in Figure 13. Nef induced significant proliferation in 90% of the donors tested. Variation in the Nef response was seen, similar to the responses to the potent staphylococcal enterotoxin, SEA. It is unlikely that these donors were sensitized to Nef, since they tested negative for HIV-1. Similar results were obtained using purified recombinant Nef protein preparations from two different sources, and which used different expression systems (see Materials and Methods in this chapter). The high number of donors that responded to Nef indicates Nef protein has similar mitogenic activity to SEA for PBMC from a wide sampling of donors.


Antigen-Presenting Cells (APC) Are Required for Nef Activity
The question arose as to whether Nef, like SEA, requires APC in order to stimulate lymphocyte proliferation. Nef induced significant proliferation in PBMC (Figure 14). Purified T cell cultures did not proliferate in response to Nef or to SEA. Purified APC, which contained monocytes and B cells, did not





54





15
Expt. 1 E] Expt. 2 = 10




3; 5




0
Nef MBP PBMC stimulated with Nef or E. coli MBP:








Figure 12. Lack of proliferation by PBMC in response to the Nef fusion partner MBP. MBP and Nef were used at 3 lig/ml. Data are expressed as mean stimulation index � SD. The mean values for 3H-thymidine incorporation by unstimulated cultures in eperiments 1 and 2 were 852�39 CPM and 1093�136 CPM, respectively.





55

Table V.
Ability of anti-peptide antibodies to block Nef-induced proliferation




PBMC cultured Anti-Nef Stimulation Indexc in the presence ofa: antibodiesb (Mean � SD) p value


Nef 4.7 + 0.2

Nef + 1.3 + 0.1 <0.001

SEA 59.7 � 2.6

SEA + 62.0 + 0.6 >0.15 + 0.9 � 0.4



aNef (3 lgg/ml) and SEA (0.3 gg/ml) were incubated with anti-Nef antibodies for 60 minutes prior to addition of PBMC. bA mixture of antisera to Nef(123-160) peptide and Nef(182-206) peptide were used, each at a final dilution of 1:1000. Preimmune sera had no effect. Both anti-Nef(123-160) and anti-Nef(182-206) had strong reactivity to Nef protein by ELISA.
cData are from a representative experiment performed in triplicate.





56






1000
U SEA O Nef a 100S 10




1
1 2 3 4 5 6 7 8 9 10 Donor






Figure 13. Nef-induced proliferative responses from a representative sampling of donors. PBMC of blood bank donors were tested for proliferation induced by SEA or Nef. SEA and Nef were used at 300 ng/ml. Mean values for 3Hthymidine incorporation by unstimulated cultures ranged from 352+86 CPM to 1983�274 CPM.





57











25

T Nef




20



5


0
Unfractionated HPMC APC T cells APC + T cells

Cells



Figure 14. Nef-induced activation of T cells requires APC but does not require processing of Nef. Unfractionated PBMC, purified APC alone, purified T cells alone, and purified T cells in the presence of APC were tested for proliferation in response to Nef, SEA, and Con A. Data are from a representative experiment, performed in triplicate, and are expressed as mean stimulation index � S.D. Purified T cells were reconstituted with paraformaldehydeinactivated APC. Con A was used at 10 gg/ml. Nef and SEA were both used at 100 ng/ml.





58


respond to stimulation by either Nef or SEA. Upon reconstitution with paraformaldehyde-inactivated autologous APC, significant Nef-induced proliferation of T cells occurred, with essentially complete reconstitution of the response. Similar proliferative responses were obtained using cells from other donors. Thus, T cells responded to Nef stimulation only in the presence of autologous APC, and Nef does not require processing to be presented by APC. These results indicate that Nef-induced proliferation required APC in a manner reminiscent of SEA.


Nef(123-160) Peptide Specifically Blocks Proliferation of PBMC Induced by Nef and SEA
As shown in Chapter 3, a synthetic peptide corresponding to an internal Nef sequence, Nef(123-160), blocked binding of Nef and SEA to Raji cells. It was important to determine if this peptide could also block proliferation induced by Nef and SEA. The results of this study are presented in Figure 15. Nef(123160) blocked both Nef-induced and SEA induced proliferation, consistent with its ability to block binding of Nef and SEA to Raji cells. Further, the blocking was specific in that proliferation induced by the T cell mitogens Con A and antiCD3, or the B cell mitogen anti-lgM, were not blocked by Nef(123-160). Nef(157-186), whose sequence slightly overlaps that of Nef(123-160), had no effect on the proliferative effects of either Nef, SEA, or Con A. These results confirm that the proliferative responses observed in PBMC cultures were specific for Nef, and were not due to a contaminant. Thus, proliferative responses to Nef and SEA were specifically blocked by a peptide corresponding to the region on Nef that binds to class II antigens and which blocks binding of Nef and the superantigen SEA.





59




No Peptide
60
[ + Nef(123-160)
" [ + Nef(157-186)

40



S20



0
SEA Nef Anti-IgM Anti-CD3

Mitogen





Figure 15. Nef(123-160) peptide specifically blocks proliferation of PBMC induced by Nef and SEA. PBMC cultures were stimulated for 96 h with mitogens in the absence of peptides or in the presence of either Nef(123-160) peptide or Nef(157-186) peptide. Data are from a representative experiment, performed in triplicate, and are expressed as mean stimulation index � SD. Nef and SEA were used at 300 ng/ml. Anti-lgM and anti-CD3 were used at 10 gg/ml. Peptides were used at a final concentration of 100 gM. The mean value for 3H-thymidine incorporation by unstimulated cultures was 1005�37 CPM.





60


Induction of T Cell Cytokines by Nef
T cell activation by the staphylococcal superantigens results in the prodigious production of T cell cytokines, such as IL 2 and IFNy. Because of the significant T cell proliferation induced by Nef, it was determined if Nef induced Thelper cell cytokines. The results of a representative experiment are presented in Table VI. Consistent with proliferation data, Nef induced significant levels of IL 2, although IL 2 levels were lower than those induced by SEA. The question arose as to the ability of Nef to also induce another important T helper cell cytokine, IFNy. Samples were removed from cultures of cells over the course of 96 h and tested for IFN antiviral activity. Nef induced high levels of IFN activity, with peak production occurring by 96 h (Figure 16, Panel A). IFN levels were lower than those induced by SEA, but were similar than those induced by Con A. Purified T cells did not produce IFN upon stimulation with either Nef or SEA (Figure 16, Panel B). However, purified T cell cultures were capable of producing IFN upon Con A stimulation. No IFN activity was produced in cultures of APC (consisting of B cells) stimulated with either Nef, SEA, or Con A. Purified T cell cultures reconstituted with purified B cells for antigen presentation produced significant levels of IFN upon either Nef or SEA stimulation (Figure 16, Panel C). These results are consistent with T cell mitogen activity for both Nef and SEA.
The type of IFN activity induced by Nef was determined by neutralization reactions with specific antisera. Treatment of Nef-induced IFN with anti-IFNy resulted in a significant reduction of activity (Table VII). Antisera to IFNa did not affect the IFN activity in these samples. Consistent with previous data (Langford et al., 1978a; Johnson et al., 1982), the IFN activity induced by SEA was IFNy. Similar to the results found with unfractionated PBMC, the IFN produced





61

Table VI.
IL 2 production induced by Nef.


IL 2 (U/ml) produced atb: Mitogena 48 h 96 h


Nef 14.7 + 0.1 16.9 + 1.1 SEA 17.2 � 0.7 24.7 + 1.8
None <3 <3

aSamples were obtained from PBMC cultures which had been stimulated with Nef and SEA at 300 ng/ml and 500 ng/ml, respectively.
blL 2 in culture samples were determined using IL 2-dependent HT-2 cells. The data presented are from a representative experiment performed in triplicate.





62





10000----- 0PBMC + Nef 100
Z---- PBMC + Con A
10 --- - PBMC + media

1 I I I I
24 48 72 96 Hours


10000
1000 T cells + SEA

S- T cells + Nef
100
---- T cells + Con A

- 10 - - T cells + media



24 48 72 96 Hours


10000

1000- T+APC+SEA

4- T+APC+Nef 100
z---- T+APC+ConA
10 - 4- T+APC+media

1I ' I I I
24 48 72 96 Hours



Figure 16. Kinetics of IFN production induced by Nef. Cultures of PBMC (Panel A), purified T cells (Panel B), and reconstituted cultures of purified APC and purified T cells (Panel C) were tested for IFN production at 24, 48, 72, and 96 h. Cultures were stimulated with either Nef at 100 ng/ml, SEA at 100 ng/ml, or Con A at 10 gg/ml, or were left unstimulated. Cultures of purified APC produced <10 U/ml IFN at all timepoints.





63

Table VII.
IFNy is induced by Nef.


Samples from PBMC IFN titer (U/ml) after treatment with: cultures stimulated with: EMEM anti-IFNa anti-IFNy



Nef 300 300 20 SEA 300 300 30 Con A 300 300 100


IFNa Control 300 <3 300 IFNy Control 30 30 <3





64


by T cell cultures reconstituted with B cells in response to Nef was IFNy. Thus, Nef induced high levels of IFNy in cultures of PBMC, indicating that Nef activates T cells to produce the cytokines IL 2 and IFNy, lymphokines that are products of activated T helper 1 cells.
PBMC cultured in the presence of Nef were tested for specific V3 expansion and anergy in vitro. No expansion of specific VP populations was observed with the anti-V3 antibodies used (data not shown). Unlike pretreatment with TSST-1, which anergized the response to anti-VP2, Nef did not significantly anergize the responses of T cells to the tested anti-V3 antibodies (data not shown). Thus, no specific Vp expansion or anergy was detected using the available human Vp reagents.

Discussion

Nef protein induced significant levels of proliferation in unfractionated PBMC from a number of donors. The observed Nef responses were significant, with proliferation induced in cells from a large majority (85-90%) of the donors tested. It is unlikely that prior sensitization to Nef is responsible for the proliferative response, since these donors were negative for HIV. The following evidence points to Nef as the inducer of the observed proliferation: 1) proliferation was observed using recombinant Nef from three different expression systems; 2) antibodies to synthetic Nef peptides neutralized the proliferative activity of Nef; 3) the Nef fusion partner, MBP, did not induce proliferation; and 4) a synthetic peptide, Nef(123-160), previously shown to block binding of superantigens to MHC class II, blocked the Nef-induced proliferative responses. Nef induced proliferation was generally lower than that induced by SEA. This is not unusual in that SEA is extremely potent, causing proliferation at concentrations as low as 10-16 M (Langford et al., 1978a).





65


Proliferation induced by Nef occurred only in the presence of APC. Cultures of purified APC or purified T cells did not respond to Nef. T cells cultured in the presence of inactivated autologous APC proliferated to a similar extent to that seen in unfractionated cultures. These data suggest that Nef is presented to T cells by APC in an unprocessed form. The production of the T helper 1 lymphokines IL 2 and IFNy from Nef-induced cultures is evidence that Nef activates T helper 1 cells.
In Chapter III, data was presented showing that Nef binds to Raji cells at a site(s) involved in SEA binding. A peptide corresponding to an internal region of Nef, Nef(123-160), specifically bound to DR 1-transfected L cells and blocked proliferation induced by Nef and SEA, evidence that Nef binds to MHC class II antigens. Con A-induced proliferation was not affected by Nef(123160). This region of Nef was shown to be involved in binding of Nef to Raji cells. These functional data suggest that binding to MHC class II antigens is required for Nef proliferative activity. Binding of Nef to MHC class II occurs on APC, since Nef proliferative activity also requires presentation, but not processing, by APC. However, these data do not preclude Nef presentation by T cells expressing HLA-DR, a T cell activation marker.
Superantigenic activity of Nef was not observed using the anti-V3 mAb currently available. Neither V-specific expansion or anergy were observed. Further, preliminary results using reverse transcriptase-polymerase chain reaction (RT-PCR), which quantifies the amount of mRNA produced, did not show a Vp preference in Nef-activated cells. These results raise a question on the nature of Nef receptor on T cells, and it is tempting to speculate on possible candidates such as TCR or CD4.
Nef induces human B cells to differentiate into immunoglobulin secreting cells (Chirmule et al., 1994). HLA-DR and adhesion molecules seem to be





66


involved in Nef-induced B cell differentiation, since monoclonal antibodies to these surface proteins abrogated Nef-induced responses. Consistent with the findings on T cell activation by Nef, B cell differentiation induced by Nef required T cells. In addition to activating T cells, staphylococcal superantigens have also been shown to induce B cell differentiation (Stohl et al., 1994). Thus, additional parallels can be drawn between the functional activities of staphylococcal superantigens and Nef.
It has been shown that HIV requires activated T cells in which to replicate. Specific antiviral immune responses may not be sufficient to activate large numbers of T cells. For this reason, the identification of an HIV protein that induces T cell proliferation is of considerable interest and may, in part, explain the role of Nef in HIV pathogenesis. Activation of T cells may result from interaction with Nef, either in soluble form released from lysed infected cells or as a cell-associated complex with HLA-DR on the surface of infected T cells. T cell activation by Nef could result in a stable cellular reservoir for virus production as a result of continuous stimulation. In fact, hyperimmunization against Nef has been proposed as a means of reducing viral load, either prophylactically or therapeutically (Montagnier, 1995).
The T cell expansion observed in response to Nef may not be the only mechanism of polyclonal activation of CD4+ T cells for HIV replication. Recent evidence points to a superantigen encoded by the human herpesvirus, cytomegalovirus (CMV), that expands Vp12-bearing T cells, thereby enhancing HIV replication in CMV-infected individuals (Dobrescu et al., 1995). It is not surprising that no expansion of Vpl2-bearing T cells was observed in response to Nef, since the donors used in the studies discussed in this chapter were negative for CMV. This versatility in polyclonal CD4+ T cell expansion via the endogenous mitogen Nef and exogenous superantigens such as that of CMV





67

probably play an important role in HIV pathogenesis. Clearly, the control of the mitogenic activity of these substances should help reduce the viral load in HIVinfected individuals.












CHAPTER V
HIV ENCODES FOR ITS OWN CD4 T CELL MITOGEN



Introduction

Nef protein is encoded in the genomes of both HIV-1 and the related primate virus simian immunodeficiency virus (SIV). The function that Nef plays in the pathogenesis of these viruses is uncertain, although its importance is reflected in studies in which a nef-deleted SIV mutant did not cause disease and protected against infection with the pathogenic wildtype strain (Daniel et al., 1992). Further, the viral load in SIV mutant-infected animals was considerably lower than in animals infected with the wildtype strain. However, challenge of neonatal macaques with the same attenuated SIV strain caused disease and, in some cases, death (Baba et al., 1995). The different outcomes of these two studies may involve differences in the immune system development between neonates and adults.

In Chapter IV, data was presented on the ability of Nef protein to induce proliferation of human peripheral blood mononuclear cells (PBMC) from a wide sampling of HIV-negative donors. Proliferative responses were T cell specific and were accompanied by production of cytokines such as interleukin 2 (IL 2) and gamma interferon (IFNy), indicative of CD4 T cell activation. Nef-induced T cell proliferation and activation required the presence of antigen-presenting cells. These results are interesting in that T cell activation has been shown to be required for active HIV replication. Further, although resting or quiescent T cells can be infected, evidence suggests that HIV replication only occurs upon


68





69

subsequent cellular activation. Herein, work is described that indicates that Nef activation of T cells is sufficient for HIV replication. These results show that Nef is an HIV-encoded mitogen that helps, at least in part, in establishing a cellular reservoir for virus replication.


Materials and Methods

PBMC Cultures for HIV Infectivity Studies
PBMC were isolated from peripheral blood of healthy adult donors as described in Chapter IV. PBMC were resuspended in RPMI 1640 medium containing 5% FBS and cultured in 25 cm2 flasks (Sarstedt Inc., Newton, NC) at a concentration of 2 x 106 cells/ml in a final volume of 4 ml (or 8 x 106 cells/flask). Mitogens were added at the initiation of cultures. Nef, SEA, and PHA were used at 3 gg/ml, 0.1 gg/ml, and 10 jgg/ml, respectively. After four days, PBMC were washed extensively in PBS, resuspended in RPMI 1640 medium containing 10% FBS, 10-6 M 2-mercaptoethanol, and 10 U rHulL 2/mi. IL 2 is rapidly depleted in these cultures, and thus rHulL 2 was added to medium. Cells were counted and all cultures were adjusted to 8 x 106 cells/flask. Cultures were infected with HIV at a final reverse transcriptase titer of 20,000 CPM/ml. Culture supernatants were harvested and cells were fed with fresh medium every third day for 12 days.


PBMC Cultures for Proliferation Assays
Concomitant with infectivity studies, PBMC were cultured in microtiter plates to monitor proliferation in response to Nef and mitogens. The culture conditions were the same as those described in Chapter IV. Nef and mitogens were used at the same concentrations as described above. Stimulation indices were calculated as described in Chapter IV.





70

Reverse Transcriptase (RT) Assay

Harvested culture supernatants (1 ml) were placed in 1.5 ml "Eppendorf" tubes and centrifuged at 17,000 rpm for 70 minutes. A cocktail containing Mg++ as the divalent cation, poly(rA)oligo(dT12-18) as template primer, and 2.5 RCi of
(3H)TTP per sample was used to resupend the virus. Tubes were incubated at 370C for 60 minutes, after which time samples were blotted onto filter paper discs. Discs were allowed to air-dry and were washed extensively in sequential baths. Specifically, discs were washed twice in 10% trichloroacetic acid (TCA) for 15 and 5 minutes, respectively. Discs were then placed in 5% TCA containing 0.1% SDS for 5 minutes. Finally, the discs were washed in 95% ethanol, after which they were allowed to air-dry. Discs were placed in vials containing scintillation fluid and radioactivity was quantified on a p-scintillation counter.


ELISA for HIV p24 Antigen
Levels of p24 antigen in infected culture supernatants were assessed using a sandwich ELISA (DuPont, Boston, MA). Levels of p24 (in ng/ml) were quantified according to the Manufacturer's recommendations based on a standard curve using purified p24 antigen (provided by Manufacturer).


Assay for Infectious Virus from PBMC Cultures
Supematants from PBMC cultures infected for 6 days with HIV were used to assess the titer of infectious virus produced in these cultures. Supernatants were adjusted to achieve RT titers of 12,000 CPM/ml and added to SEAstimulated PBMC cultures. Culture conditions were the same as those described above. RT activity at Day 9 of cultures was assessed as described above.





71


Fluorescent Antibody Cell Sorter (FACS) Analysis of Nef-Activated Cells

PBMC were stimulated for 4 days with either Nef (3gg/ml) or SEA (0.1 gg/ml). PBMC were washed and resuspended in FACS buffer containing 0.5% BSA and 10 mM sodium azide. Cells were incubated with fluorescein-labeled mAb to HLA-DR for 45 minutes and washed with FACS buffer. Cells were then incubated with phycoerythrin-labeled mAbs to either CD4 or IL 2 receptor (IL 2R) for 45 minutes. Cells were washed and analyzed on a FACScan (BectonDickinson, Mountain View, CA) at 10,000 events/sample. All mAbs used were obtained from Becton-Dickinson, San Jose, CA.


Studies on Proliferation of PBMC Induced by Autologous HIV-Infected Cells
PBMC cultures were stimulated as described above. PBMC were infected with HIV for 6 days, at which time the cells were washed and inactivated by overnight treatment with 2% paraformaldehyde. Inactivated HIVinfected cells were washed extensively to remove excess paraformaldehyde. Fresh autologous PBMC were cultured in microtiter plates in the presence of inactivated HIV-infected cells at a ratio of 3:1. After 4 days, 3H-thymidine incorporation was assessed as described above.


Anti-Nef Peptide Antibodies
A mixture of antibodies to Nef(123-160) peptide and Nef(182-206) peptide were used to block proliferation in response to autologous HIV-infected cells. Antibodies were each used at a final dilution of 1:1000, as described in Chapter IV. Inactivated HIV-infected cells were treated with antibodies for 60 minutes prior to addition of fresh autologous PBMC. Preimmune sera were used and had no effect on proliferation induced by HIV-infected cells.





72

Results

HIV Replication in Nef-Stimulated PBMC Cultures
Since T cell activation occurs with exogenous Nef protein, the question arose as to whether Nef stimulation of PBMC was sufficient to induce HIV-1 replication. To this end, PBMC of several HIV-negative donors were tested for proliferation induced by Nef and other T cell mitogens (Table VIII). Consistent with previous data, PBMC from most of the donors proliferated significantly in response to Nef protein, as well as to the T cell mitogens SEA and phytohemagglutinin A (PHA).

Unstimulated and mitogen-stimulated PBMC were infected with HIV-1 and cultures were monitored over a 12-day period for signs of viral replication. Supernatants from unstimulated PBMC showed only marginal reverse transcriptase (RT) activity and p24 antigen production (Table VIII). RT and p24 antigen levels were high in cells stimulated with T cell mitogens, in particular SEA. Interestingly, Nef-stimulated cells produced moderate to high levels of RT and p24 antigen, indicating that these cells were capable of sustaining virus replication. In an attempt to quantify the number of infected cells in these cultures, PCR analysis of serially-diluted DNA was performed using nested primers for the gag gene. Results indicated 10- to 30-fold increases in the number of cells harboring virus in Nef-stimulated cultures, as compared to unstimulated controls. These data indicate that Nef stimulation resulted in a large number of infected cells and a concomitant increase in viral load.


Infectious Virus is Produced by Nef-Stimulated Cultures
As a means of determining whether the virus produced by Nef-stimulated was infective, supernatants from HIV-infected cultures were tested for reinfectivity. SEA-stimulated PBMC were infected with virus from 6-day







Table VIII
Maximal proliferation, RT, and p24 antigen values from stimulated cultures


Donor Stimulation Maximal Proliferation Maximal RT value Maximal p24 antigen value Mean + SD Fold increase Mean + SD Fold increase Mean � SD Fold increase


204 Nef 8108 � 238 8.0 19014 � 7141 10.9 256 � 24 10240 SEA 43684 � 4316 43.3 29680 + 3509 17.1 1024 � 402 40960 PHA 54874 + 4711 54.4 35817 � 5233 20.6 1814 + 80 72560

None 1009 + 199 - 1740 + 41 - 0.025 � 0.01 205 Nef 12363� 864 10.1 26614 3119 7.1 73+ 2 1197 SEA 73243 � 10333 60.6 125515 + 16634 33.5 1523 + 332 24967 PHA 57267 � 455 47.6 91313 + 11325 24.4 1668 � 49 27344

None 1228 � 583 - 3732 + 947 - 0.061 + 0.052 206 Nef 18753 + 1711 15.3 9823 + 3709 18.8 30.6 � 0.7 124 SEA 124742 + 10411 96.0 218412 +49382 412.9 819 + 153 3316 PHA N.D. - 12006 � 4132 22.7 38.1 + 6.7 154

None 1313 + 356 529 + 35 - 0.247�0.03







Table VII --continued


Donor Stimulation Maximal Proliferation Maximal RT value Maximal p24 antigen value Mean � SD Fold increase Mean � SD Fold increase Mean � SD Fold increase


207 Nef 14480 + 707 7.8 19229 + 3284 8.7 1408 + 38 14667 SEA 44040 + 2424 19.4 26700 + 3117 12.0 2541 + 178 26469

PHA N.D. - N.D. - N.D.

None 2389 � 690 - 2118 � 162 - 0.096 � 0.01 208 Nef 19473 + 3713 17.7 13133 � 2083 19.7 434 � 172 4667 SEA 38033 � 7971 33.6 14424 � 318 21.7 520 � 61 5591

PHA N.D. - N.D. - N.D.

None 1167 + 58 - 1009 � 249 - 0.093 + 0.01

209 Nef 3314 � 830 4.3 1310 � 291 1.3 51 + 25 70 SEA 20244 + 1204 21.2 4558 + 360 4.5 226 + 81 310

PHA N.D. - N.D. - N.D.

None 1004 + 185 666 + 130 - 0.73 � 0.1





75


supernatants of cultures stimulated with Nef, SEA, and PHA. All supernatants were adjusted to achieve equivalent RT units per culture. The results of two representative experiments are shown in Figure 17. Supernatants from Nefstimulated cultures had high titers of infectious virus similar to supernatants from cultures stimulated with SEA and PHA. These results indicate that virus replication in Nef-stimulated cultures, like the replication in SEA- and PHAstimulated cultures, yielded infectious virus.


Nef-Stimulated PBMC Express T Cell Activation Markers
It has been shown that HIV requires activated T cells for virus replication. Cellular activation was investigated by double-staining FACS analysis of Nefstimulated cells using antibodies to CD4 and HLA-DR (Table IX). Increased levels of dually-stained cells were observed in cultures of PBMC stimulated with Nef protein, as compared to unstimulated cultures. HLA-DR expression on CD4+ human T cells has been shown to correlate with an activated T cell state. No increase was seen in cells that stained for CD8, either alone or in conjunction with HLA-DR. These results are consistent with our previous studies showing production of T helper cytokines upon stimulation with Nef. Thus, stimulation of PBMC with Nef protein induces activation of T helper cells.


Expression of Nef by HIV-Infected Cells
Culture supernatants of infected cells were tested for soluble Nef and none could be detected, suggesting that release of soluble Nef may not be occurring in these cultures. However, it has been shown that Nef is expressed on the surface of infected cells (Fujii et al., 1993). This leads to the question of whether Nef, in association with the extracellular membrane of infected cells, plays a role in the activation event(s) required for continuous HIV replication.





76





40000

0 Unstimulated Nef
30000 - SEA II PHA 20000




10000




0
Expt 1 Expt 2




Figure 17. Infectivity assay on supernatants from Nef-stimulated HIV-infected cultures.





77

Table IX
Nef-induced activation of T cells as assessed by FACS


% Total of dually-stained populations Cultures from: CD4 + HLA-DR CD4 + IL 2 R


Donor 206

Unstimulated 3.17 N.D.c

Nef-stimulated 7.39 N.D.

SEA-stimulated 29.46 N.D.


Donor 213

Unstimulated 6.65 N.D.

Nef-stimulated 16.74 N.D.


Donor 214

Unstimulated 3.45 N.D.

Nef-stimulated 11.15 N.D.


Donor 215

Unstimulated N.D. 7.75

Nef-stimulated N.D. 10.55


Donor 216

Unstimulated 1.52 4.66 Nef-stimulated 9.02 7.68





78

Thus, we investigated the possibility that HIV-infected cells could activate autologous PBMC, similar to the activation seen with exogenously added Nef. To achieve maximal infection, PBMC were stimulated with mitogen prior to infection with HIV. After 5 days, HIV-infected cells were washed and inactivated with paraformaldehyde. Fresh autologous PBMC were cultured in the presence of infected cells and monitored for proliferation. Proliferation was observed in co-cultures of fresh and infected autologous cells (Table X). Uninfected stimulated cells served as controls, and no proliferation was observed in these co-cultures. Proliferative responses were significantly reduced by the addition of polyclonal anti-Nef antibodies, while control anti-SEA antibodies were without effect. Anti-Nef inhibition of proliferation was dose dependent, with lower concentrations of antibodies having less inhibitory activity. Similar to the results observed on HIV-infected cells, anti-Nef antibodies were capable of reducing proliferation in response to Nef, but not to SEA. Thus, a significant portion of the observed mitogenic activity of HIV-infected cells is due to Nef protein.


Discussion
It is interesting that HIV-infected cells were more potent at inducing T cell proliferation than was soluble Nef. Given that Nef interacts with MHC class II antigens, it is conceivable that optimal conditions for Nef association with MHC are achieved in HIV-infected cells, analogous to the expression of the mouse mammary tumor virus superantigen in infected B cells. These data suggest that proliferation occurred as a result of Nef expressed on the surface of HIVinfected cells, thereby indicating that Nef is a virally-encoded mitogen. Thus, the genome of HIV encodes for its own T cell mitogen, which would amplify the





79


Table X.
Ability of anti-Nef peptide antibodies to block proliferation induced by autologous HIV-infected cells




Fresh PBMC cultured Anti-Nef Proliferation
in the presence of: antibodies CPM Fold increase p value (Mean � SD)


HIV-infected PBMC - 137187 � 1923 162.2 HIV-infected PBMC + 79740 � 8764 94.3 <0.003 + 1973� 880 2.3
846+ 271 -





80


replication of virus in the host. As such, Nef could be classified as a virulence factor for HIV.
It has been demonstrated that activated T cells are required for replication of HIV-1. On the other hand, the ability of HIV-1 to infect quiescent or resting cells is still questioned. In two studies using quiescent cells, HIV-1 replication in vitro was blocked at the level of virus entry (Gowda et al., 1989; Tang and Levy, 1990). Others have shown that blockage of replication occurred at an intracellular event, possibly at reverse transcription (Zack et al., 1990) or proviral integration (Stevenson et al., 1990; Bukrinsky et al., 1991). Upon entry into quiescent cells, replication could be induced by subsequent T cell activation. Consistent with data from in vitro studies are reports that high numbers of quiescent CD4+ cells from asymptomatic HIV-infected patients harbor unintegrated viral DNA capable of integration subsequent to PHA stimulation. Further, AIDS patients have increased numbers of cells containing integrated virus, corresponding with greater production of virus at this stage of the disease. These findings indicate that HIV-1 may infect quiescent cells, but such infection is nonproductive until cell activation occurs.
Studies suggest that Nef plays an important role in HIV pathogenesis. Infection of adult rhesus monkey with a nef-deleted mutant of SIVmac239 resulted in low viral burden and did not cause disease, although infected animals were persistently seropositive (Daniel et al., 1992). Recently, genomic analysis of an HIV strain from a longterm survivor revealed a 118 bp deletion in the nef gene which resulted in an out-of-frame shift for downstream sequences (Kirchhoff et al., 1995). These studies hint at the importance of Nef in HIV pathogenesis in that repeated attempts to isolate virus from this patient with nonprogressive disease were unsuccessful. Mutations in nef may result in altered mitogenic activity of the protein, with concomitant reduction or loss of the





81

ability to activate T cells. Possible consequences of nef mutations, then, would be a reduction both in the cellular reservoir for virus replication and in the viral load in the infected host. Other investigators have not found alterations in the nef gene in strains isolated from other longterm survivors (Cao et al., 1 P95). It would be interesting to determine the T cell reactivity of these individuals to Nef, as we have found that 10-15% of individuals that we tested did not respond to Nef. It is possible that nonresponsiveness to Nef may play an important role in longterm survival in some HIV-infected individuals. Thus, Nef may aid in the establishment and maintenance of infection, suggesting its role as a virulence factor for HIV.












CHAPTER VI
A MODEL FOR THE ROLE OF NEF IN THE PATHOGENESIS OF HIV

As shown in Chapters II-V, Nef binds to MHC class II antigens at a site(s) involved in bacterial superantigen binding. Like MMTV superantigen, Nef protein is encoded in the 3' long terminal repeat of the genome of lentiviruses such as HIV-1 and simian immunodeficiency virus (SIV). Nef induced significant levels of proliferation in PBMC from a wide sampling of donors (8590%), although these responses are lower than those induced by SEA. Further, Nef stimulation resulted in the production of the T helper cell cytokines, IL 2 and interferon gamma (IFNy). Proliferation in response to Nef was observed in reconstituted cultures consisting of T cells and inactivated APCs, which is compelling evidence for Nef superantigen activity. Further, Nef did not induce purified T cells to proliferate in the absence of APCs, which is consistent with Nef superantigen activity. However, no apparent V3 expansions were found, although such data do not preclude the V3 specificity of Nef activation. Thus, Nef has superantigen-like characteristics in that it binds to class II antigens, does not require processing by APC, and activates T cells to proliferate and release cytokines such as IL 2 and IFN-y.

The functional role that Nef plays in the lentiviral pathogenesis is uncertain, although its importance is reflected in vaccine studies in which a nefdeleted SIV mutant did not cause disease and protected animals upon challenge with the pathogenic wildtype strain (Daniel et al. 1992). The viral load in SIV mutant-infected animals was considerably lower than in animals challenged with wildtype SIV. Within this context, the ability of Nef to activate T


82





83


cells is interesting in that active HIV replication requires T cell activation. Further, although resting or quiescent T cells can be infected, evidence suggests that HIV replication only occurs upon subsequent cellular activation.
Nef stimulation of PBMC was sufficient to induce HIV-1 replication. Virus replication in Nef-stimulated cultures yielded infectious virus, rather than defective particles. Flow cytometric analysis showed that increased percentages of cells that stained for both CD4 and HLA-DR were present in Nef-stimulated cultures. Further, increased percentages of cells that stained for both CD4 and IL 2R were observed in Nef-stimulated cultures. These results are consistent with the production of T helper cytokines upon stimulation with Nef. Thus, stimulation of PBMC with Nef protein induces T cell activation and such activation is sufficient for HIV replication.

Proliferation of fresh autologous PBMC cultured in the presence of infected cells was observed. Further, proliferative responses were significantly reduced by the addition of polyclonal anti-Nef antibodies. These data suggest that proliferation occurred as a result of Nef present on the surface of HIVinfected cells, thereby indicating that Nef may be a virally-encoded mitogen.
Nef has been shown to induce differentiation of human B cells to immunoglobulin secreting cells (Chirmule et al., 1994). Monoclonal antibodies to HLA-DR and adhesion molecules abrogated Nef-induced differentiation. B cell stimulation required T cells and monocytes, the latter producing IL 6 upon Nef stimulation. Interestingly, staphylococcal superantigens have been shown to induce B cell differentiation analogous to that described for Nef (Stohl et al., 1994). Thus, Nef can stimulate both T and B cells in a manner similar to the staphylococcal enterotoxin superantigens.
This leads to the question of the activation event(s) that occurs during HIV-1 exposure and that ultimately lead to the establishment of infection. Our





84


results suggest a model in which Nef acts as a HIV-encoded T cell mitogen (Figure 18). Nef may interact with T cells either as an integral part of the membranes of infected T cells or in a soluble form released as the result of lysis of infected cells. Nef is present on the membranes of infected T cells (Fujii et al., 1993), which is consistent with this model. Nef expression may occur in the context of the T cell activation marker, HLA-DR, and the Nef/HLA-DR complex would stimulate uninfected T cells, as well as B cells. Such stimulation would allow for the expansion of a cellular reservoir for replication of the virus, eventually leading to T cell anergy and/or apoptosis. B cell activation and differentiation could ultimately lead to hypergammaglobulinemia. Thus, the HIV genome may encode for its own T cell mitogen, which would induce the amplification of virus replication in the host, with ultimate deleterious effects on the immune system. As such, Nef could be classified as a virulence factor for HIV.





85







Nef receptor
TCR ?
~HL A.CD4?


RLA-DR HIV+
T
Ne

Uninfected
T





Nef binding to HLA-DR Presentation to uninfected T cells
Nef-induced
cellular Cellular proliferation
effects T cell activation
Cytokine production
B cell proliferation and differentiation


Increased viral load
Immunosuppression via T cell Potential anergy/apoptosis
Nef-related Autoimmunity via activation of B and T
pathogenesis cells
Hypergammaglobulinemia



Figure 18. Model for the role of Nef in the pathogenesis of HIV. Activation of uninfected T cells occurs by Nef interaction in either of two forms: as soluble Nef released from lysed cells, presented in the context of MHC class II antigens on APC, or as an integral part of the membranes of infected T cells, complexed to HLA-DR. Binding to uninfected T cells may occur via TCR or CD4. T cell activation results in proliferation and release of cytokines such as IFNy and IL 2, thereby creating a cellular reservoir for virus and increasing viral load in the host. T cell activation results in depletion of T cells via virus production, anergy and/or apoptosis. B cell differentiation, possibly mediated by T cell cytokine release, results in hypergammaglobulinemia. Autoimmune-like sequelae may result from B cell differentiation into Ig-secreting cells, and activation of T cells.











LIST OF REFERENCES


Abe, J., B. L. Kotzin, K. Jujo, M. E. Melish, M. P. Glode, T. Kohsaka, and D. Y. M. Leung. 1992. Selective expansion of T cells expressing T-cell receptor variable region Vp2 and Vp8 in Kawasaki disease. Proc. Natl. Acad. Sci. USA 89:4066.

Acha--Orbea, H. 1992. Retroviral superantigens. In B. Fleischer, Ed. Ciological Significance of Superantigens. Basel: Karger, pp65-86.

Acha--Orbea, H. and E. Palmer. 1991. MIs - a retrovirus exploits the immune system. Immunol. Today 12:356.

Acha--Orbea, H., A. N. Shakhov, L. Scarpellino, E. Kolb, V. Muller, A. Vessaz-Shaw, R. Fuchs, K. Blochinger, P. Rollini, J. Billotte, M. Sarafidou, H. R. MacDonald, and H. Diggelmann. 1991. Clonal deletion of Vp14-bearing T cells in mice transgenic for mammary tumor viurs. Nature 350:207.

Achong, B. G., P. W. A. Mansell, M. A. Epstein, and P. Clifford. 1971. An unusual virus in cultures from a human nasopharyngeal carcinoma. J. Nat. Cancer Inst. 46:299.

Allan, J. S., J. E. Coligan, T. --H. Lee, M. F. McLane, P. J. Kanki, J. E.
Groopman, and M. Essex. 1985. A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients. Science 230:810.

Arthur, L. O., J. W. Bess, R. C. Sowder, R. E. Benveniste, D. L. Mann, J. C.
Chermann, and L. E. Henderson. 1992. Cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines. Science 258:1935.

Baba, T. W., Y. S. Jeong, D. Penninck, R. Bronson, M. F. Greene, and R. M. Ruprecht. 1995. Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science 267:1820.

Bahraoui, E., M. Yagello, J. --N. Billaud, J. --M. Sabatier, B. Guy, E.
Muchmore, M. Girard, and J. -C. Gluckman. 1990. Immunogenicity of the human immunodeficiency virus (HIV) recombinant nef gene product. Mapping of T-cell and B-cell epitopes in immunized chimpanzees. AIDS Res. Hum. Retroviruses 6:1087.

Barnstable, C. J., W. F. Bodmer, G. Brown, G. Galfre, C. Milstein, A. F. Williams, and A. Ziegler. 1978. Production of monoclonal antibodies to group A erythrocytes, HLA, and other human cell surface antigens - new tools for genetic analysis. Cell 14:9.


86





87

Bergdoll, M. S. 1985. The staphylococcal enterotoxins-an update. In J. Jeljaszewics, Ed. The Staphylococci. New York: Gustav Fisher Verlag, pp247266.

Bisset, L. R., M. Opravil, E. Ludwig, and W. Fierz. 1993. T cell response to staphylococcal superantigens by asymptomatic HIV-infected individuals exhibits selective changes in T cell receptor V beta-chain usage. AIDS Res. Hum. Retroviruses 9:241.

Brodsky, F.M. 1984. A matrix approach to human class II histocompatibility antigens: reactions of four monoclonal antibodies with the products of nine haplotypes. Immunogenetics 19:179.

Buhrinsky, M. I., T. L. Stanwick, M. P. Dempsey, and M. Stevenson. 1991. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection. Science 254:423.

Cao, Y., L. Qin, L. Zhang, J. Safrit, and D. D. Ho. 1995. Virological and immunological characterization of long-term survivors of huma immunodeficiency virus type 1 infection. New Engl. J. Med. 332:233.

Carlsson, R., H. Fischer, and H. O. Sjogren. 1988. Binding of staphylococcal enterotoxin A to accessory cells is a requirement for its ability to activate human T cells. J. Immunol. 140:2484.

Chinatgumpala, M. M., J. A. Mollick, and R. R. Rich. 1991. Staphylococcal toxins bind to different sites on HLA-DR. J. Immunol. 147:3876.

Chirmule, N., N. Oyaizu, C. Saxinger, and S. Pahwa. 1994. Nef protein of HIV-1 has B-cell stimulatory activity. AIDS 8:733.

Choi, Y., J. Kappler, and P. Marrack. 1991. A superantigen encoded in the open reading frame of the 3' long terminal repeat of mouse mammary tumor virus. Nature 350:203.

Choi, Y., J. Kappler, and P. Marrack. 1992. Structural analysis of a mouse mammary tumor virus superantigen. J. Exp. Med. 175:847.

Cole, B. C. and M. M. Griffiths. 1993. Triggering and exacerbation of autoimmune arthritis by the Mycoplasma arthriditis superantigen MAM. Arth. Rheum. 36:994.

Dalgleish, A. G., S. Wilson, M. Gompels, C. Ludlam, B. Gazzard, A. M. Coates, and J. Habeshaw. 1992. T-cell receptor variable gene products and early HIV-1 infection. Lancet 339:824.

Daniel, M. D., F. Kirchhoff, S. C. Czajak, K. Sehgal, and R. C. Desrosiers. 1992. Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science 258:1938.





88

Dellabonna, P. J. Peccoud, J. Kappler, P. Marrack, C. Benoit, and D. Mathis. 1990. Superantigens interact with MHC class II molecules outside of the antigen binding groove. Cell 62:1115.

deRonde, A., B. Klaver, W. Keulen, L. Smit, and J. Goudsmit. 1992. Natural HIV-1 Nef accelerates virus replication in primary human lymphocytes. Virology 188:391.

Devereax, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucl. Acid Res. 12:387.

Dobrescu, D., B. Ursea, M. Pope, A.S. Asch, and D.N. Posnett. 1995. Enhanced HIV-1 replication in Vp12 due to human cytomegalovirus in monocytes: evidence for a putative herpesvirus superantigen. Cell 82:753.

Dyson, P. J., A. M. Knight, S. Fairchild, E. Simpson, and K. Tomonari. 1991. Genes encoding ligands for deletion of Vpl11 T cells cosegregate with mammary tumor virus genomes. Nature 349:531.

Edelman, A. S. and S. Zolla-Pazner. 1989. AIDS: a syndrome of immune dysregulation, dysfunction, and deficiency. FASEB J. 3:22.

Festenstein, H. 1973. Immunogenetic and biological aspects of in vitro lymphocyte allotransformation (MLR) in the mouse. Transplant. Rev. 15:62.

Fischer, H., M. Dohlsten, M. Lindvall, H. O. Sjogren, and R. Carlsson. 1989. Binding of staphylococcal enterotoxin A to HLA-DR on B cell lines. J. Immunol. 142:3151.

Fleischer, B. and H. Schrezenmeier. 1988. T cell stimulation by staphylococcal enterotoxins: Clonally variable response and requirement for major histocompatibility complex class II molecules on accessory or target cells. J. Exp. Med. 167:1697.

Fluegel, R. M. 1993. The molecular biology of the human spumavirus. In B. R. Cullen, Ed. Human Retroviruses. Oxford: IRL Press, pp193-214.

Franchini, G., M. Robert-Guroff, J. Ghrayeb, N. T. Chang, and F. WongStaal. 1986. Cytoplasmic localization of the HTLV-III 3' orf protein in cultured T cells. Virology 155:593.

Frankel, W. N., C. Rudy, J. M. Coffin, and B. T. Huber. 1991. Linkage of MIs genes to endogenous mammary tumor virus. Nature 349:525.

Fraser, J.D. 1989. High-affinity binding of staphylococcal enterotoxins A and B to HLA-DR. Nature 339:221.





89

Fujii, Y., Y. Nishino, T. Nakaya, K. Tokunaga, and K. Ikuta. 1993. Expression of human immunodeficiency virus type 1 Nef antigen on the surface of acutely and persistently infected human T cells. Vaccine 11:1240.

Gallo, R.C., Salahuddin, S.Z., M. Popovic, G. M. Shearer, M. Kaplan, B. F. Haynes, T. J. Palker, R. Redfield, J. Oleske, and B. Safai. 1984. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224:500.

Garcia, J. V. and A. D. Miller. 1991. Serine-phosphorylationindependent downregulation of cell-surface CD4 by nef. Nature 350:508.

Golovkina, T. V., A. Chervonsky, J. P. Dudley, and S. R. Ross. 1992. Transgenic mouse mammary tumor virus superantigen prevents viral infection. Cell 69:637.

Gowda, S. D., B. S. Stein, N. Mohagheghpour, C. J. Benike, and E. G. Engleman. 1989. Evidence that T cell activation is required for HIV-1 entry in CD4+ lymphocytes. J. Immunol. 142:773.

Griggs, N. D., C. H. Pontzer, M. A. Jarpe, and H. M. Johnson. 1992, Mapping of multiple binding domains of the superantigen staphylococcal enterotoxin A for HLA. J. Immunol. 148:2516.

Guy, B., M. P. Kierny, Y. Riviere, C. Le Peuch, K. Dott, M. Girard, L. Montagnier, and J. --P. Lecocq. 1987. HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature 330:266.

Hammes, S. R., E. P. Dixon, M. H. Malim, B. R. Cullen, and W. C. Greene.
1989. Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor. Proc. Natl. Acad. Sci. USA 86:9549.

Held, W., H. Acha--Orbea, H. R. MacDonald, and G. A. Waanders. 1994a. Superantigens and retroviral infections: insights from mouse mammary tumor virus. Immunol. Today 15:184.

Held, W., G. A. Waanders, H. R. MacDonald, and H. Acha--Orbea. 1994b. MHC class II hierarchy of superantigen presentation predicts efficiency of infection with mouse mammary tumor virus. Int. Immunol. 6:1403.

Held, W., G. A. Waanders, A. N. Shakhov, L. Scarpellino, H. Acha-Orbea, and H. R. MacDonald. 1993. Superantigen-induced immune stimulation amplifies mouse mammary tumor virus infection and allows transmission. Cell 74:529.

Herman, A., N. Labrecque, J. Thibodeau, P. Marrack, and R. R. Rich. 1991. Identification of the staphylococcal enterotoxin A superantigen binding site in the beta 1 domain of the human histocompatibility antigen HLA-DR. Proc. Natl. Acad. Sci. USA 88:9954.





90

Heston, W. E., M. K. Deringer, and H. B. Andervont. 1945. Gene-milk agent relationship in mammary tumor development. J. Natl. Cancer Inst. 5:289.

Ho, S. N., R. T. Abraham, S. Gillis, and D. J. Mckean. 1987. Differential bioassay of interleukin 2 and interleukin 4. J. Immunol. Methods. 98:99.

Hugin, A. W., M. S. Vacchio, and H. C. Morse III. 1991. A virus-encoded "superantigen" in a retrovirus-induced immunodefieciency syndrome of mice. Science 252:424.

Imberti, L., A. Sottini, A. Bettinardi, M. Puoti, and D. Primi. 1991. Selective depletion in HIV infection of T cells that bear specific T cell receptor Vp sequences. Science 254:860.

Johnson, H. M. 1981. Cellular regulation of immune interferon production. Antiviral Res. 1:37.

Johnson, H. M. 1985 mechanism of Interferon-y production and assessment of immunoregulatory properties. In E. Pick and M. Landy, Eds. Lymphokines Vol. 11. New York: Academic Press,pp 33-53.

Johnson, H. M. and J. A. Bukovic. 1975. Staphylococcal entertoxin A inhibition of the primary in vitro antibody response to thymus-dependent antigen. IRCS Med. Sci. 3:398.

Johnson, H. M., M. P. Langford, B. Lakhchaura, T. -S. Chan, and G. J. Stanton. 1982. Neutralization of native human gamma interferon (HulFNy) bt antibodies to a synthetic peptide encoded by the 5' end of HulFNy cDNA. J. Immunol. 129:2357.

Johnson, H. M. and H. I. Magazine. 1988. Potent mitogenic activity of staphylococcal enterotoxin A requires induction of interleukin 2. Int. Arch. Allergy Appl. Immunol. 87:87.

Johnson, H. M., J. K. Russell, and C. H. Pontzer. 1991. Staphylococcal enterotoxin superantigens. P.S.E.B.M. 198:765

Johnson, H. M., J. K. Russell, and C. H. Pontzer. 1992. Role of superantigens in human disease. Sci. Am. 266(4):92.

Johnson, H. M., B. A. Torres, and J. M. Soos. 1995. Superantigens : Structure and Relevance to Human Disease. Proc. Soc. Exp. Biol. Med. In Press.

Kappler, J., B. Kotzin, L. Herron, E. W. Gelfand, R. D. Bigler, A. Boylston, S. Carrel, D. N. Posnett, Y. Choi, and P. Marrack. 1989. Vl-specific stimulation of human T cells by staphylococcal toxins. Science 244:811.





91


Kawabe,Y. and A. Ochi. 1990. Selective anergy of VB8+ T cells in staphylococcus enterotoxin B-primed mice. J. Exp. Med. 172:1065.

Kawabe,Y. and A. Ochi. 1991. Programmed cell death and extrathymic reduction of VB8+ CD4+ T cells in mice tolerant to Staphylococcus aureus enterotoxin B. Nature 349:245.

Kirchhoff, F., T. C. Greenough, D. B. Brettler, J. L. Sullivan, and R. C. Desrosiers. 1995. Absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. New Engl. J. Med. 332:228.

Klatzmann, D., F. Barre-Sinoussi, M. T. Nugeyre, C. Danquet, E. Vilmer, C. Griscelli, F. Brun-Veziret, C. Rouzioux, J. C. Gluckman, J. C. Chermann, and L. Montagnier. 1984. Selective tropism of lymphadenopathy associated virus (LAV) for helper-inducer T lymphocytes. Science 225:59.

Kotzin, B. L/, D. Y. M. Leung, J. Kappler, and P. Marrack. 1993. Superantigens and their potential role in human disease. Adv. Immunol, 54:99.

Lafon, M., M. Lafage, A. Martinez-Arends, R. Ramirez, F. Vuiller, D. Charron, V. Lotteau, and D. Scott-Algara. 1992. Edidence for a viral superantigen in humans. Nature 358:507.

Lafon, M., D. Scott-Algara, P. N. Marche, P. A Cazenave. and E. JouvinMarche. 1994. Neonatal deletion and selctive expansion of mouse T cells by exposure to rabies virus nuceocapsid superantigen. J. Exp. Med. 180:1207.

Langford, M. P., G. J. Stanton, and H. M. Johnson. 1978a. Biological effects of staphylococcal enterotoxin A on human peripheral lymphocytes. Infect. Immun. 22:62.

Langford, M. P., D. A. Weigent, G. J. Stanton, and S. Baron. 1978b. Virus plaque-reduction assay for interferon. Microplaqye and regular macroplaque reduction assays. Methods Enzymol. 78:339.

Laurence, J., A. S. Hodstev, and D. N. Posnett. 1992. Superantigen implicated in dependence of HIV-1 replication in T cells on TCR V3 expression. Nature 358:255.

Lechler, R. I., V. Bal, J. B. Rothbard, R. N. Germain, R. Sekaly, E. O. Long, and J. Lamb. 1988. Structural and functional studies of HLA-DR restricted antigen recognition by human helper T lymphocyte clones by using transfected murine cell lines. J. Immuno. 141:3003.

Lee, J. M. and T. H. Watts. 1990. Binding of staphylococcal enterotoxin A to purified murine MHC class II molecules in supported lipid bilayers. J. Immunol. 145:3360.




Full Text
94
Robbins, P. A., E. L. Evans, A. H. Ding, N. L. Warner, and F. M. Brodsky.
1987. Monoclonal antibodies that distinguish between class II antigens (HLA-
DP, DQ, and DR) in 14 haplotypes. Hum. Immunol. 18:301.
Rous, P. 1910. A transmissible avian neoplasm (sarcoma of common
fowl). J. Exp. Med. 12:696.
Rous, P. 1911. Transmission of a malignant new growth by means of a
cell-free filtrate. J. Amer. Med. Assoc. 56:198.
Russell, J. K., C. H. Pontzer, and H. M. Johnson. 1990. The l-Apb region
(65-85) is a binding site for the superantigen, staphylococcal entertoxin A.
Biochem. Biophys. Res. Comm. 168:696.
Russell, J. K., C. H. Pontzer, and H. M. Johnson. 1991. Both a-helices
along the major histocompatibility complex binding cleft are required for
staphylococcal enterotoxin A function. Proc. Natl. Acad. Sci. USA 88:7228.
Salmons, B. and W. H. Gunzburg. 1987. Current perspectives in the
biology of mouse mammary tumor virus. Virus Res. 8:81.
Schiffenbauer, J., H. M. Johnson, E. J. Butfilowski, L. Wegrzyn, and J. M.
Soos. 1993. Staphylococcal enterotoxins can reactivate experimental allergic
encephalomyelitis. Proc. Natl. Acad. Sci. USA 90:8543.
Sodrowski, J., W. C. Goh, C. Rosen, K. Campbell, and W. A. Haseltine.
1986. Role of the HTLV-III/LAV envelope in synctium formation and
cytopathicity. Nature 322:470.
Soos, J. M. and H. M. Johnson. 1994. Type I interferon inhibition of
superantigen stimulation: implications for treatment of superantigen-associated
disease. J. Interferon and Cytokine Res. 15:39.
Spawski, J. B. and P. E. Lipsky. 1992. Isolation of B cell populations. In
J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober,
Eds. Protocols in Immunology. New York: John Wiley and Sons, p7.5.1.
Stevenson, M., S. Haggerty, C. Lamonica, A. M. Mann, C. Meier, and A.
Wasiak. 1990. Cloning and characterization of human immunodeficiency virus
type 1 variants diminished in the ability to induce syncytium-independent
cytloysis. J. Virol. 64:3792.
Stohl, W., J. E. Elliott, and P. S. Linsley. 1994. Human T-cell dependent
B cell differentiation induced by staphylococcal superantigens. J. Immunol.
153:117.
Tang, S. and J. A. Levy. 1990. Parameters involved in the cell fusion
induced by HIV. AIDS 4:409.


ACKNOWLEDGMENTS
I would like to thank my mentor, Dr. Howard M. Johnson, for his
unflagging support, insight, and wisdom. All of these qualities have helped
guide me, not just a in scientific sense, but in finding my way through the
quagmire of academic science. On a scientific level, Howard has always
been unfailingly logical and knowledgeable, which makes him a wonderful
mentor.
I would also like to thank the other members of my committee. Dr.
Shanmugam and Dr. Hoffmann have been very supportive throughout this
venture. Dr. Yamamoto has given me excellent advice on steering my HIV
work towards clinical relevance. I give special thanks to Dr. Blalock, who
was a tough, but caring, committee member and confidante.
Many thanks must go to my fellow labmates who have always been
willing to lend a hand, ear, or a shoulder to cry on, whichever was needed at
the time. I thank Jeanne, Brian, Prem, and Taishi for generously sharing
their past experiences of graduate school. I also am grateful for my fellow
graduate school mates, Amy, George, and Mustafa, for their understanding
and help. And I thank Aaron, Laurie, and Tim for their warmth and humor.
Finally, I wish to thank my family for their unwavering support through
my graduate school tenure. My parents, Manuel and Antonia Torres, have
stood by with understanding and a warm hug whenever needed. Although
far away, my sister, Marisol Beaton, has cheered me on to complete my
doctoral work. I owe a great debt of gratitude to my wonderful family.
n


47
the column between 100-150 mM potassium phosphate, and was generally
contained within 7 fractions. The fraction containing Nef protein was dialyzed
against affinity column buffer containing 150 mM NaCI. The dialyzed material
was loaded onto a second amylose affinity column for removal of MBP. The
flow-through containing Nef protein was collected and loaded onto an High-Q
ion-exchange cartridge (BioRad Laboratories, Richmond, CA). The High-Q
cartridge was washed with 100 ml of 20 mM Tris buffer (pH 8.0) containing 150
mM NaCI. Fractions were eluted with a linear gradient from 150-400 mM NaCI
in the same buffer. The fractions containing Nef protein, which eluted at 180
mM NaCI, were collected and dialyzed overnight against PBS and stored at
-70C. The purity of Nef protein was assessed by SDS-PAGE, followed by
staining with silver. Upon staining with silver, a single band was found, and a
corresponding band was detected by Western blotting with monoclonal anti-Nef
antibody (Repligen, Cambridge, MA). MBP purified by this method and used at
the same concentrations as that used for the purified Nef preparation, did not
have proliferative activity on human peripheral blood mononuclear cells.
Nef proliferative activity was confirmed using recombinant Nef proteins
obtained from two other sources and which were produced using different
expression systems. The following reagent was obtained through the AIDS
Research and Reference Reagent Program, AIDS Program, NIAID, NIH: HIV-1
LAV Nef from the Division of AIDS, NIAID. The HIV-1 nef gene (LAV) used to
produce this Nef preparation was isolated from pBENN 6 and cloned into the
bacterial expression vector pPD-YN-61. The protein was produced in E. coli
strain Sf930 and isolated as inclusion bodies. Nef protein was also obtained
from Repligen (Cambridge, MA). In this case, the HIV-1 nef gene (LAV) was
cloned into the bacterial vector pD10, which adds a hexahistidine tag for protein
purification using nickel chelate chromatography. The protein was produced in


10
Mouse Mammary Tumor Virus Superantiqen
The first viral superantigens to be described are the proteins encoded by
the open reading frame that overlaps the 3' long terminal repeat of the
oncornavirus, mouse mammary tumor virus (MMTV) (Marrack et al., 1991;
Frankel et al., 1991; Woodland et al., 1991; Dyson et al., 1991). Although
MMTV superantigens were recognized in the 1990s, they were originally
described by Festenstein in the early 1970s as minor lymphocyte stimulating
(mis) antigens (Festenstein, 1973). The ability of T cells from certain mouse
strains to be stimulated by lymphocytes from MHC-identical strains was
ascribed by Festenstein to the presence of mis antigens. More recently, mis
antigens were found to be endogenous superantigens from germline-encoded
MMTV provirus (Choi et al., 1991; Acha-Orbea et al., 1991). Like their bacterial
counterparts, MMTV superantigens are thought to be presented in the context of
MHC class II antigens. MMTV superantigens are known to stimulate T cells in a
Vp-specific fashion (Acha-Orbea and Palmer, 1991). MMTV superantigens
show MHC preference, with more efficient presentation occurring in the context
of l-E as opposed to l-A, although presentation by l-A does occur (MacDonald et
al., 1989). Interestingly, this MHC preference corresponds with the greater
infectivity of B cells bearing l-E (Held et al., 1994b).
MMTV is a type B retrovirus that was determined to be the causative
agent for the induction and transmission of mammary carcinomas in mice
(Heston et al., 1945). It has been known for several years that infectivity by
MMTV requires an intact immune system (Tsubura et al., 1988), and only
recently has this paradox been explained (Held et al., 1993, 1994a).
Transmission of MMTV to offspring can occur via infectious virions in mother's
milk or can occur vertically as endogenous provirus. Most mouse strains have


% Control
40
1-38 31-65 62-99 93-132 123-160 156-186 182-206
NeF Peptide
Figure 8. Blockage of 125I-SE binding to Raji cells by Nef peptides. Nef
peptides were used at a final concentration of 300 pM. 125l-SEs were used at 2
nM. All other experimental conditions were the same as those described in
Figure 7. Binding of 125I-SEE, 125I-SEB, and 125I-SEC1 in the absence of
competitor was 5,917612 CPM, 6,438134 CPM, and 4,493425 CPM,
respectively. Data represent the mean of three individual experiments, each
performed in duplicate. Each bar represents the mean percent of the
appropriate SE control binding to Raji cells in the presence of Nef peptides
SD.


46
The extra A added to the 3' end of the amplified nef gene fragment as a result
of PCR was removed by treatment with the Klenow fragment of DNA
polymerase. This DNA was ligated into the Xmn\/Hind\\\ cloning site of the
prokaryotic expression vector pMAL-c2 (New England BioLabs, Beverley,
MA). The sequence of the fusion construct was verified by the DNA
Sequencing Laboratory, Interdisciplinary Center for Biotechnology Research,
University of Florida. The fusion construct was engineered such that the N
terminus of Nef was immediately downstream from the Factor Xa cleavage site
at the C terminus of maltose binding protein (MBP). Thus, cleavage with Factor
Xa was predicted not to add any vector-derived amino acids. E. coli strain TB1
was transformed with the vector containing mbp/nef. Cultured E. coli TB1 were
suspended in column buffer consisting of 20 mM Tris-HCI (pH 7.4), 200 mM
NaCI, and 1 mM ethylenediaminetetraacetic acid (EDTA). The cells were
sonicated and the supernatants were recovered after centrifugation. Protease
inhibitors (2 mM phenylmethylsulfonyl fluoride (PMSF), pepstatin A, leupeptin,
and aprotinin) were added to extracts. The protein concentration of the extracts
was adjusted to 2.5 mg protein/ml prior to loading onto an amylose affinity
column containing a bed volume of 50 ml. The column was washed with 350
ml of column buffer. The fusion protein was eluted with 200 ml of column buffer
containing 10 mM maltose. Fractions (7 ml) were collected. The fusion protein
generally eluted within the first 70 ml. After cleaving the fusion protein with
factor Xa (New England BioLabs, Beverley, MA), the cleavage mixture was
loaded onto a hydroxyapatite column containing a bed volume of 35 ml. After
extensive washing of the column with 180 ml of 10 mM potassium phosphate
(pH 7.2) containing 2 mM PMSF and 1 mM benzamidine, fractions were eluted
using a linear gradient from 10 mM to 400 mM potassium phosphate buffer (pH
7.2) containing 2 mM PMSF. Fractions (5 ml) were collected. Nef eluted from


% Control
23
Competitor (|iM)
Figure 2. Ability of vSAg(76-119) to compete with 125I-SEA for binding to A20
cells in dose response studies. 125I-SEA was used at 2.5 nM. Binding of ^SI
SEA to A20 cells in the absence of competitors was 5184380 CPM. The data
presented represent the mean of three individual experiments, each performed
in duplicate. Each point represents the mean percent of SEA control binding in
the presence of competitors SD.


48
E. coli strain MC10611 P0L.1. All Net preparations were negative for endotoxin
as assessed by the limulus amoebocyte lysate assay.
Reagents
Staphylococcal enterotoxin A (SEA) was purchased from Toxin
Technology (Sarasota, FL). Concanavalin A (Con A), anti-CD3, and anti-IgM
were purchased from Sigma Chemical Co. (St. Louis, MO).
Proliferation Assays
Proliferation studies on human peripheral blood mononuclear cells
(PBMC) were performed as described previously (Pontzer et al., 1992).
Peripheral blood from healthy adult blood bank donors was obtained from
Civitan Regional Blood Center (Gainesville, FL) for use in these studies. All
donors were negative for cytomegalovirus, hepatitis B virus, and HIV. PBMC
were isolated from peripheral blood using ficoll hypaque gradient
centrifugation. After extensive washing, PBMC were plated into wells of
microtiter plates at 2.8 x 106 cells/ml, followed by addition of activators. Final
volumes were adjusted to 150 pl/well with RPMI 1640 tissue culture medium
containing 5% fetal bovine serum (FBS) and penicillin/streptomycin. 3h-
thymidine (IpCi/well; Amersham Corporation, Indianapolis, IN) was added at
90 h after initiation of cultures and the cells were incubated for an additional 6 h
prior to harvest onto filter paper. Filter paper was placed in liquid scintillation
fluid and radioactivity was quantified using a p scintillation counter. Stimulation
index (S.l.) was determined by dividing the experimental CPM by the CPM
obtained from control (unstimulated) cultures.


CHAPTER VI
A MODEL FOR THE ROLE OF NEF IN THE PATHOGENESIS OF HIV
As shown in Chapters ll-V, Net binds to MHC class II antigens at a site(s)
involved in bacterial superantigen binding. Like MMTV superantigen, Net
protein is encoded in the 3' long terminal repeat of the genome of lentiviruses
such as HIV-1 and simian immunodeficiency virus (SIV). Nef induced
significant levels of proliferation in PBMC from a wide sampling of donors (85-
90%), although these responses are lower than those induced by SEA.
Further, Nef stimulation resulted in the production of the T helper cell cytokines,
IL 2 and interferon gamma (IFNy). Proliferation in response to Nef was
observed in reconstituted cultures consisting of T cells and inactivated APCs,
which is compelling evidence for Nef superantigen activity. Further, Nef did not
induce purified T cells to proliferate in the absence of APCs, which is consistent
with Nef superantigen activity. However, no apparent Vp expansions were
found, although such data do not preclude the Vp specificity of Nef activation.
Thus, Nef has superantigen-like characteristics in that it binds to class II
antigens, does not require processing by APC, and activates T cells to
proliferate and release cytokines such as IL 2 and IFNy.
The functional role that Nef plays in the lentiviral pathogenesis is
uncertain, although its importance is reflected in vaccine studies in which a nef-
deleted SIV mutant did not cause disease and protected animals upon
challenge with the pathogenic wildtype strain (Daniel et al. 1992). The viral
load in SIV mutant-infected animals was considerably lower than in animals
challenged with wildtype SIV. Within this context, the ability of Nef to activate T
82


75
supernatants of cultures stimulated with Nef, SEA, and PHA. All supernatants
were adjusted to achieve equivalent RT units per culture. The results of two
representative experiments are shown in Figure 17. Supernatants from Nef-
stimulated cultures had high titers of infectious virus similar to supernatants
from cultures stimulated with SEA and PHA. These results indicate that virus
replication in Nef-stimulated cultures, like the replication in SEA- and PHA-
stimulated cultures, yielded infectious virus.
Nef-Stimulated PBMC Express T Cell Activation Markers
It has been shown that HIV requires activated T cells for virus replication.
Cellular activation was investigated by double-staining FACS analysis of Nef-
stimulated cells using antibodies to CD4 and HLA-DR (Table IX). Increased
levels of dually-stained cells were observed in cultures of PBMC stimulated with
Nef protein, as compared to unstimulated cultures. HLA-DR expression on
CD4+ human T cells has been shown to correlate with an activated T cell state.
No increase was seen in cells that stained for CD8, either alone or in
conjunction with HLA-DR. These results are consistent with our previous
studies showing production of T helper cytokines upon stimulation with Nef.
Thus, stimulation of PBMC with Nef protein induces activation of T helper cells.
Expression of Nef bv HIV-Infected Cells
Culture supernatants of infected cells were tested for soluble Nef and none
could be detected, suggesting that release of soluble Nef may not be occurring
in these cultures. However, it has been shown that Nef is expressed on the
surface of infected cells (Fujii et al., 1993). This leads to the question of
whether Nef, in association with the extracellular membrane of infected cells,
plays a role in the activation event(s) required for continuous HIV replication.


65
Proliferation induced by Nef occurred only in the presence of APC.
Cultures of purified APC or purified T cells did not respond to Nef. T cells
cultured in the presence of inactivated autologous APC proliferated to a similar
extent to that seen in unfractionated cultures. These data suggest that Nef is
presented to T cells by APC in an unprocessed form. The production of the T
helper 1 lymphokines IL 2 and IFNy from Nef-induced cultures is evidence that
Nef activates T helper 1 cells.
In Chapter III, data was presented showing that Nef binds to Raji cells at
a site(s) involved in SEA binding. A peptide corresponding to an internal
region of Nef, Nef(123-160), specifically bound to DR 1-transfected L cells and
blocked proliferation induced by Nef and SEA, evidence that Nef binds to MHC
class II antigens. Con A-induced proliferation was not affected by Nef(123-
160). This region of Nef was shown to be involved in binding of Nef to Raji
cells. These functional data suggest that binding to MHC class II antigens is
required for Nef proliferative activity. Binding of Nef to MHC class II occurs on
APC, since Nef proliferative activity also requires presentation, but not
processing, by APC. However, these data do not preclude Nef presentation by
T cells expressing HLA-DR, a T cell activation marker.
Superantigenic activity of Nef was not observed using the anti-Vp mAb
currently available. Neither Vp-specific expansion or anergy were observed.
Further, preliminary results using reverse transcriptase-polymerase chain
reaction (RT-PCR), which quantifies the amount of mRNA produced, did not
show a Vp preference in Nef-activated cells. These results raise a question on
the nature of Nef receptor on T cells, and it is tempting to speculate on possible
candidates such as TCR or CD4.
Nef induces human B cells to differentiate into immunoglobulin secreting
cells (Chirmule et al., 1994). HLA-DR and adhesion molecules seem to be


37
140
1-38 31-65 62-99 93-132 123-160 156-186 182-206
NeF Peptide
Figure 6. Blockage of 125I-SEA binding to Raji and DR 1-transfected L cells by
Nef peptides. Nef peptides were used at a final concentration of 300 pM. 125l-
SEA was used at a final concentration of 2 nM. 105 Raji or DR 1-transfected L
cells were used per tube. Binding of 125I-SEA to Raji and DR1-transfected L
cells in the absence of competitors was 31,469 2292 and 5,708 41 CPM,
respectively. Data represent the mean percent of control of three individual
experiments, each performed in duplicate. Bars represent binding to Raji and
DR1-transfected L cells in the presence of Nef peptides SD.


55
Table V.
Ability of anti-peptide antibodies to block Nef-induced proliferation
PBMC cultured
in the presence ofa:
Anti-Nef
antibodies'3
Stimulation Index0
(Mean SD)
p value
Nef
-
4.7 0.2
Nef
+
1.3 0.1
<0.001
SEA
-
59.7 2.6
SEA
+
62.0 0.6
>0.15
-
+
0.9 0.4
-

-
aNef (3 pg/ml) and SEA (0.3 pg/ml) were incubated with anti-Nef antibodies for
60 minutes prior to addition of PBMC.
^A mixture of antisera to Nef(123-160) peptide and Nef( 182-206) peptide were
used, each at a final dilution of 1:1000. Preimmune sera had no effect. Both
anti-Nef(123-160) and anti-Nef(182-206) had strong reactivity to Nef protein by
ELISA.
cData are from a representative experiment performed in triplicate.


70
Reverse Transcriptase (RT1 Assay
Harvested culture supernatants (1 ml) were placed in 1.5 ml "Eppendorf"
tubes and centrifuged at 17,000 rpm for 70 minutes. A cocktail containing Mg++
as the divalent cation, poly(rA)oligo(dTi2-i8) as template primer, and 2.5 pCi of
(3H)TTP per sample was used to resupend the virus. Tubes were incubated at
37C for 60 minutes, after which time samples were blotted onto filter paper
discs. Discs were allowed to air-dry and were washed extensively in sequential
baths. Specifically, discs were washed twice in 10% trichloroacetic acid (TCA)
for 15 and 5 minutes, respectively. Discs were then placed in 5% TCA
containing 0.1% SDS for 5 minutes. Finally, the discs were washed in 95%
ethanol, after which they were allowed to air-dry. Discs were placed in vials
containing scintillation fluid and radioactivity was quantified on a (3-scintillation
counter.
ELISA for HIV p24 Antigen
Levels of p24 antigen in infected culture supernatants were assessed
using a sandwich ELISA (DuPont, Boston, MA). Levels of p24 (in ng/ml) were
quantified according to the Manufacturer's recommendations based on a
standard curve using purified p24 antigen (provided by Manufacturer).
Assay for Infectious Virus from PBMC Cultures
Supernatants from PBMC cultures infected for 6 days with HIV were used
to assess the titer of infectious virus produced in these cultures. Supernatants
were adjusted to achieve RT titers of 12,000 CPM/ml and added to SEA-
stimulated PBMC cultures. Culture conditions were the same as those
described above. RT activity at Day 9 of cultures was assessed as described
above.


CHAPTER III
IDENTIFICATION OF AN HIV-1 NEF PEPTIDE THAT BINDS TO
MHC CLASS II ANTIGENS
Introduction
In Chapter Two, a site was identified on the MMTV-1 superantigen that
binds to class II MHC antigens, suggesting that retroviral and bacterial
superantigens exert their effects similarly. MMTV superantigen is encoded in a
gene that overlaps the terminal repeat at the 3' end of the viral genome. The
genome of human spumavirus also contains a gene {bel3) that overlaps the
terminal repeat at the 3' end, the product of which may have superantigenic
properties (Fluegel, 1993). Recently, it has been suggested that human
immunodeficiency virus (HIV) may possess superantigen activity, although no
specific superantigenic protein has been identified (Imberti et al., 1991;
Laurence et al., 1992; Dalgleish et al., 1992; Bisset et al., 1993). The HIV
genome also contains a gene that overlaps the terminal repeat at the 3' end,
the product of which is called Nef. Although Nef is one of the early proteins
produced during the replication of primate lentiviruses, the role of Nef in HIV
pathogenesis has yet to be established.
To determine if Nef had binding characteristics similar to superantigens,
a study was undertaken to determine its ability to bind to MHC class II antigens,
at sites that are known to bind superantigens. Using overlapping peptides
corresponding to the entire length of Nef (HIVlav). we have identified a region
which binds to MHC class II antigens, and which competes for binding with
known bacterial superantigens.
32


9
leukemia virus (Poiesz et al., 1980; Yoshida et al 1982), and established this
family of viruses as human pathogens.
The retrovirus family consists of three subgroups based on similar
pathogenicities: oncornaviruses, lentiviruses, and spumaviruses.
Spumaviruses or foamy viruses are the least characterized of the three
subfamilies of retroviruses, and have been grouped according to their ability to
cause vacuolation of infected cells in vitro, which gives the cells a foamy"
appearance. Spumaviruses have been found in primates and humans, and are
generally considered benign, although data suggest these viruses may play a
role in human disease. Oncornaviruses are tumor-causing retroviruses, and
members of this subfamily include the avian leukosis-sarcoma viruses, mouse
mammary tumor virus, feline leukemia virus, and human T-cell leukemia virus.
As a result of proviral integration into the host genome, tumors arise by
upregulation of host genes that encode for growth factors or by retroviral-
encoded oncogenes.
Viruses belonging to the lentiviral subfamily include the "slow" viruses
maedi/visna and equine infectious anemia virus. More recently, the viruses that
cause human and feline acquired immundeofiency syndromes have been
classified as lentiviruses, based on several parameters including genomic
complexity and virion morphology. Unlike oncornaviruses, lentiviruses have
not been implicated directly in causing neoplastic disease. Members of the
lentiviral subfamily cause long-term disease characterized by autoimmunity,
encephalopathy, immunodeficiency, or a combination thereof. Lentiviruses are
considerably more complex than some of the other retroviral subfamilies, in that
the level of gene regulation is much greater.


59
x
TJ
C
s
.o

3
E
5Z5
No Peptide
SEA Nef Anti-IgM Anti-CD3
Mitogen
Figure 15. Nef(123-160) peptide specifically blocks proliferation of PBMC
induced by Nef and SEA. PBMC cultures were stimulated for 96 h with
mitogens in the absence of peptides or in the presence of either Nef(123-160)
peptide or Nef(157-186) peptide. Data are from a representative experiment,
performed in triplicate, and are expressed as mean stimulation index SD. Nef
and SEA were used at 300 ng/ml. Anti-IgM and anti-CD3 were used at 10
pg/ml. Peptides were used at a final concentration of 100 pM. The mean value
for ^H-thymidine incorporation by unstimulated cultures was 1005+37 CPM.


13
yielding 5 distinct proteins. Cleavage of Pol results in 2 proteins, one of which is
reverse transcriptase. Two envelope (Env) proteins are the cleavage products
from an initial Env precursor. In this case, the precursor is cleaved by a cellular
protease. Two regulatory proteins, Tat (transactivator) and Rev (regulator for
viral expression) are translated from multiply-spliced mRNA transcripts.
One putative regulatory protein, Nef (negative factor) is encoded by a
gene that overlaps the terminal repeat at the 3' end of the HIV genome. It was
initially named based on its apparent negative effects on viral transcription and
replication in vitro (Terwillegar et al, 1986). However, subsequent reports have
been conflicting, and have suggested that Nef has no effect (Hammes et al.,
1989) or a positive effect (DeRonde et al., 1992) on HIV transcription. Such
widely disparate Nef effects may, in part, be explained by the fact that several
immortalized human T cell lines and primary human lymphocytes were used in
these studies. These data may reflect inherent differences in primary cells and
cell lines, such as activation signals. Data from studies on Nef-transfected T
cells suggest that Nef may play a role in down-regulation of the CD4 molecule
(Garcia and Miller, 1991; Mariani and Skowronski, 1993).
HIV-1 Nef is a 25-27K protein and is myristylated at the N-terminus.
Myristylation is thought to be a mechanism by which Nef associates with the
cytoplasmic membrane (Franchini et al., 1986; Guy et al., 1987). Nef primarily
localizes to the cytoplasm (Franchini et al., 1986) but has been shown to be
present on the surface of infected cells (Fujii et al., 1993). Antibodies to Nef
appear early in HIV-infected individuals (Allan et al., 1985) and cytotoxic T
lymphocyte activity has been detected against cells presenting Nef peptides
(Bahraoui et al., 1990). These data confirm that Nef is expressed during active
HIV infection.


61
Table VI.
IL 2 production induced by Nef.
IL 2 (U/ml) produced atb:
Mitogen3
48 h
96 h
Nef
14.7 0.1
16.9 1.1
SEA
17.2 0.7
24.7 1.8
None
<3
<3
aSamples were obtained from PBMC cultures which had been
stimulated with Nef and SEA at 300 ng/ml and 500 ng/ml,
respectively.
b|L 2 in culture samples were determined using IL 2-dependent
HT-2 cells. The data presented are from a representative
experiment performed in triplicate.


19
helix of l-Ad (Lee and Watts, 1990). All mAb were used at a final concentration
of 10 [ig/ml. MAbs used in this study had similar potencies as determined by
the Hybridoma Core Facility, Interdisciplinary Center for Biotechnology
Research, University of Florida. Polyclonal antibodies were used at a final
dilution of 1:100. Poly IAd (Cat.Y1-9-04-26-01) is specific for the m11
determinant of l-Ad and poly la.7 (Cat.YI-7-02-02-01) is specific for the m7
determinant of l-Ed. Both antibodies were obtained from NIAID, Rockville, MD.
Poly l-A^ was obtained from Accurate Chemicals, Westbury, NY.
Radioiodinations
SEA (2.5 jig) and vSAg peptide (10 pig) were radiolabeled using
chloramine T as described (Griggs et al., 1992). Briefly, ligands were labeled
with 500 pCi of Na125l (15 mCi/pg, Amersham Corp., Arlington Heights, IL) in
25 pi of 0.5 M potassium phosphate buffer, pH 7.4, and 10 pi of chloramine T
(5 mg/ml) for 2 min. After neutralization of the reaction with 10 pi each of
sodium bisulfite (10 mg/ml), potassium iodide (70 mg/ml), bovine serum
albumin (BSA; 20 mg/ml), and 15 pi of NaCI (4 M), the preparation was sieved
on a 5 ml Sepharose G-10 column. The two fractions with the highest
radioactivity in the first eluted peak were pooled and used in the radiolabeled
binding assays. The specific activities of the SEs and vSAg peptide ranged
from 130-150 pCi/pg and 30-60 pCi/pg.
Binding Studies
In binding studies using A20 cells, unlabeled competitors (SEs and
vSAg peptides) were added in 50 pi volumes (in PBS with 1% BSA) at
indicated final concentrations to 50 pi of cells in "Eppendorf" tubes.
Competitors were incubated with cells at room temperature for 45 minutes,


35
of 10 pg/ml. Purified recombinant Nef protein was purchased from Repligen,
Cambridge, MA.
Radioiodinations
SEs (2.5 pg) and Nef peptide (10 pg) were radioiodinated using
chloramine T as described in chapter II of this text. The specific activities of the
SEs and Nef peptide ranged from 70-120 pCi/pg and 30-40 pCi/pg,
respectively.
Binding Studies
For binding studies on Raji cells, unlabeled competitors (SEs and Nef
peptides) were added in 50 pi (in PBS with 1% BSA) to 50 pi of cells in 1.5 ml
"Eppendorf" tubes. Cells and competitors were incubated at room temperature
for 45 min., followed by the addition of radiolabeled SEs or Nef peptide. After
45 min., the cells were washed three times and the bottoms of the tubes were
cut off. Radioactivity was quantified using a gamma counter. Similarly, DR-1
transfected L cells, which were grown to confluence in the wells of microtiter
plates, were subjected to the same incubation times and volumes of
competitors and labeled ligands as used for the Raji cells. After three washes,
the cells were solubilized in 1% SDS, and the liquid was absorbed by cotton-
tipped applicators and radioactivity was quantified using a gamma counter.
Results
Competition of Nef Peptides with Radiolabeled SEA for MHC Binding
Overlapping peptides corresponding to the entire length of the Nef
sequence (HIVlav) were synthesized (Table IV). The Nef peptides were


50
antibodies were tested for their relative abilities to neutralize Nef mitogenic
activity by incubating Nef with antibodies at 37C for 60 minutes prior to
addition of PBMC. Antibodies to both Nef(123-160) and Nef(182-206) showed
strong reactivity to whole recombinant Nef protein, and these combined
antisera had significant neutralizing activity against Nef. Nef was treated with
preimmune sera as control.
Cytokine Production and Assays
PBMC were plated at 106 cell/ml into wells of 24-well plates (Corning
Corp., Corning, NY). Activators were added and final volumes were adjusted to
300 pl/well. Cultures were incubated at 37C over a 96 h period and samples
(100 pi) were obtained at 24 h intervals. Cell cultures were replenished with
RPMI/5% FBS (100 pi) after obtaining samples. Culture supernatants were
tested for interleukin 2 (IL 2) using the IL 2-dependent HT-2 cell line (Ho et al.,
1987). The amounts of IL 2 present in culture supernatants were determined
using recombinant human IL 2 (Genzyme, Cambridge, MA) as a standard.
Samples were tested for interferon (IFN) activity on human WISH cells by a
microplaque reduction method (Langford, et al., 1978b), using approximately
40 plaque-forming units (PFU) of vesicular stomatitis virus (VSV) per well. In
our studies, 1 U/ml of IFN is defined as the concentration required to decrease
the number of PFU per well by 50%.
IFN activity was typed by neutralization reactions with specific antisera,
as described (Johnson et al., 1982). Briefly, samples were pretreated with 1000
neutralizing units of either anti-human IFNa (Lee Biomolecular, San Diego, CA)
or anti-human IFNy (Genzyme, Cambridge, MA). Controls were sham-treated
with EMEM/2% FBS. The neutralizing activity of anti-IFN antisera was
confirmed using HuIFNa (Lee Biomolecular, San Diego, CA) and HuIFNy


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Xdwin Blalock
Professor of Physiology and
Biophysics
University of Alabama
This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate School and was accepted as partial fulfillment
of the requirements for the degree of Doctor of Philosophy.
December, 1995
Dean, College of Agriculture
Dean, Graduate School


Table VIII
Maximal proliferation, RT, and p24 antigen values from stimulated cultures
Donor
Stimulation
Maximal Proliferation
Mean 1 SD Fold increase
Maximal RT value
Mean 1 SD Fold increase
Maximal p24 antigen value
Mean 1 SD Fold increase
204
Nef
8108 238
8.0
1901417141
10.9
256 1 24
10240
SEA
4368414316
43.3
29680 1 3509
17.1
10241402
40960
PHA
54874 4711
54.4
3581715233
20.6
1814180
72560
None
1009+199
-
1740141
-
0.025 1 0.01
-
205
Nef
123631864
10.1
2661413119
7.1
731 2
1197
SEA
732431 10333
60.6
125515116634
33.5
15231332
24967
PHA
57267 1 455
47.6
913131 11325
24.4
1668149
27344
None
12281583
-
3732 1 947
-
0.061 1 0.052
-
206
Nef
187531 1711
15.3
9823 1 3709
18.8
30.610.7
124
SEA
1247421 10411
96.0
218412149382
412.9
8191153
3316
PHA
N.D.
-
1200614132
22.7
38.1 6.7
154
None
13131356
-
529 1 35
-
0.24710.03



66
involved in Nef-induced B cell differentiation, since monoclonal antibodies to
these surface proteins abrogated Nef-induced responses. Consistent with the
findings on T cell activation by Nef, B cell differentiation induced by Net
required T cells. In addition to activating T cells, staphylococcal superantigens
have also been shown to induce B cell differentiation (Stohl et al., 1994). Thus,
additional parallels can be drawn between the functional activities of
staphylococcal superantigens and Nef.
It has been shown that HIV requires activated T cells in which to
replicate. Specific antiviral immune responses may not be sufficient to activate
large numbers of T cells. For this reason, the identification of an HIV protein
that induces T cell proliferation is of considerable interest and may, in part,
explain the role of Nef in HIV pathogenesis. Activation of T cells may result from
interaction with Nef, either in soluble form released from lysed infected cells or
as a cell-associated complex with HLA-DR on the surface of infected T cells. T
cell activation by Nef could result in a stable cellular reservoir for virus
production as a result of continuous stimulation. In fact, hyperimmunization
against Nef has been proposed as a means of reducing viral load, either
prophylactically or therapeutically (Montagnier, 1995).
The T cell expansion observed in response to Nef may not be the only
mechanism of polyclonal activation of CD4+ T cells for HIV replication. Recent
evidence points to a superantigen encoded by the human herpesvirus,
cytomegalovirus (CMV), that expands Vpl2-bearing T cells, thereby enhancing
HIV replication in CMV-infected individuals (Dobrescu et al., 1995). It is not
surprising that no expansion of Vpl 2-bearing T cells was observed in response
to Nef, since the donors used in the studies discussed in this chapter were
negative for CMV. This versatility in polyclonal CD4+ T cell expansion via the
endogenous mitogen Nef and exogenous superantigens such as that of CMV


CHAPTER I
INTRODUCTION
Overview
A novel class of antigens called superantigens has recently been
characterized. Unlike conventional antigens, which stimulate approximately 1
in 10,000 cells, superantigens can induce the proliferation and activation of as
many as 1 in 5 T cells (Johnson et al., 1991). The staphylococcal enterotoxins,
prototypic of this class of antigens, are among the most potent inducers of T cell
proliferation, and are effective at concentrations as low as 10'16 M (Langford et
al., 1978a).
Major differences exist between conventional antigens and
superantigens. Superantigens exert their effects as whole molecules and do
not require processing by antigen presenting cells for T cell recognition.
Although superantigens require MHC class II molecules, they bind at site(s)
outside the peptide antigen binding groove (Russell et al., 1990; Dellabonna et
al., 1990). Superantigens presented in the context of MHC class II molecules
are recognized by the variable region of the p chain (Vp) of the T cell receptor.
Only T cells that express T cell receptors with specific Vp regions will be
activated by a given superantigen. For example, toxic shock syndrome toxin-1
activates only Vp2-bearing human T cells (Abe et al., 1992) and staphylococcal
enterotoxin A activates several Vps on human T cells, including Vp5 and Vpl2,
(Kappler et al., 1989). Thus, T cell responses to superantigens are said to be
Vp-specific.
1


Table VII --continued
Donor
Stimulation
Maximal Proliferation
Mean SD Fold increase
Maximal RT value
Mean + SD Fold increase
Maximal p24 antigen value
Mean SD Fold increase
207
Net
14480 707
7.8
19229 + 3284
8.7
1408 38
14667
SEA
44040 2424
19.4
26700 3117
12.0
2541 178
26469
PHA
N.D.
-
N.D.
-
N.D.
-
None
2389 690
-
2118 162
-
0.096 0.01
-
208
Net
19473 3713
17.7
13133 2083
19.7
434172
4667
SEA
38033 7971
33.6
14424 318
21.7
520 61
5591
PHA
N.D.
-
N.D.
-
N.D.
-
None
1167 + 58
-
1009 249
-
0.093 0.01
-
209
Net
3314 + 830
4.3
1310 291
1.3
51 25
70
SEA
202441204
21.2
4558 360
4.5
226 81
310
PHA
N.D.
-
N.D.
-
N.D.
-
None
1004185
-
666130
-
0.73 0.1
-


90
Heston, W. E., M. K. Deringer, and H. B. Andervont. 1945. Gene-milk
agent relationship in mammary tumor development. J. Natl. Cancer Inst. 5:289.
Ho, S. N., R. T. Abraham, S. Gillis, and D. J. Mckean. 1987. Differential
bioassay of interleukin 2 and interleukin 4. J. Immunol. Methods. 98:99.
Hugin, A. W., M. S. Vacchio, and H. C. Morse III. 1991. A virus-encoded
"superantigen" in a retrovirus-induced immunodefieciency syndrome of mice.
Science 252:424.
Imberti, L, A. Sottini, A. Bettinardi, M. Puoti, and D. Primi. 1991.
Selective depletion in HIV infection of T cells that bear specific T cell receptor
Vp sequences. Science 254:860.
Johnson, H. M. 1981. Cellular regulation of immune interferon
production. Antiviral Res. 1:37.
Johnson, H. M. 1985 mechanism of lnterferon-y production and
assessment of immunoregulatory properties. In E. Pick and M. Landy, Eds.
Lymphokines Vol. 11. New York: Academic Press,pp 33-53.
Johnson, H. M. and J. A. Bukovic. 1975. Staphylococcal entertoxin A
inhibition of the primary in vitro antibody response to thymus-dependent
antigen. IRCS Med. Sci. 3:398.
Johnson, H. M., M. P. Langford, B. Lakhchaura, T. -S. Chan, and G. J.
Stanton. 1982. Neutralization of native human gamma interferon (HuIFNy) bt
antibodies to a synthetic peptide encoded by the 5' end of HuIFNy cDNA. J.
Immunol. 129:2357.
Johnson, H. M. and H. I. Magazine. 1988. Potent mitogenic activity of
staphylococcal enterotoxin A requires induction of interleukin 2. Int. Arch.
Allergy Appl. Immunol. 87:87.
Johnson, H. M., J. K. Russell, and C. H. Pontzer. 1991. Staphylococcal
enterotoxin superantigens. P.S.E.B.M. 198:765
Johnson, H. M., J. K. Russell, and C. H. Pontzer. 1992. Role of
superantigens in human disease. Sci. Am. 266(4):92.
Johnson, H. M., B. A. Torres, and J. M. Soos. 1995. Superantigens :
Structure and Relevance to Human Disease. Proc. Soc. Exp. Biol. Med. In
Press.
Kappler, J., B. Kotzin, L. Herron, E. W. Gelfand, R. D. Bigler, A. Boylston,
S. Carrel, D. N. Posnett, Y. Choi, and P. Marrack. 1989. Vp-specific stimulation
of human T cells by staphylococcal toxins. Science 244:811.


BIOGRAPHICAL SKETCH
Barbara Aurea Torres, a first-generation American, was born on June 4,
1956 at Hialeah Hospital in South Florida. Her parents, Manuel and Antonia
Torres, and sister, Marisol Torres Beaton, were born in Chile and emigrated to
the United States in 1950. She was blessed with parents who intentionally
gave her a first name that allowed her to "blend" into the American melting pot.
Her father's family in Chile has had a long-time connection to medicine and the
sciences. Barbara's grandfather was a physician in a rural community in Chile,
and her uncle is still a general practitioner in Ecuador. In 1989, Barbara's father
gave her a monograph on the atomic theory written by her great-grandfather,
who was an instructor at the Sorbonne University in Paris, France. This
monograph made a lasting impression on Barbara on the nature of scientific
inquiry. Barbara graduated in 1978 with a Bachelor of Arts degree from
Randolph-Macon Woman's College in Lynchburg, Virginia. In 1981, Barbara
did graduate work in the laboratory of Dr. Howard M. Johnson and received a
Master of Science degree from the Department of Microbiology at the University
of Texas Medical Branch in Galveston, Texas. Barbara continued working with
Dr. Johnson for several years, both in Galveston and at the University of Florida.
After a mundane two-year stint at Bio-Rad Laboratories in Richmond, California,
Barbara decided to return to Gainesville to work towards attaining her doctorate,
once again in the laboratory of Dr. Johnson. After graduation, Barbara plans on
continuing her research on HIV, as a postdoctoral fellow in the laboratory of Dr.
Johnson at the University of Florida.
96


6
immunodeficiency virus. Thus, superantigens may be involved in disease
processes and, as such, can be classified as virulence factors.
Bacterial Superantiaens
In the 1970s, staphylococcal enterotoxins were recognized as inducers
of lymphocyte proliferation (Peavy et al., 1970). Responses to staphylococcal
enterotoxin A (SEA) stimulation were shown to be T cell specific (Johnson and
Bukovic, 1975), with high levels of gamma interferon (IFNy) produced by this
lymphocyte fraction (Langford et al., 1978a). SEA was shown to have
mitogenic effects on human lymphocytes at extremely low concentrations,
making it one of the most potent T cell activators known (Langford et al., 1978a).
The ability to produce large quantities of IFNy using SEA paved the way for the
characterization of the key immunomodulatory properties of this cytokine
(Johnson, 1985). Interestingly, intravenous administration of SEA to mice
resulted in the induction of suppressor cell activity that was present in spleen as
early as 24 hours after injection (Johnson, 1981; Torres et al., 1982). SEA-
induced suppressor cells were capable of blocking naive spleen cells from
responding to SEA stimulation. Thus, SEA was shown to stimulate T cells to
proliferate and produce cytokines, and to induce suppressor cell activity.
Further investigations on the interaction of bacterial enterotoxins with
human lymphocytes revealed that antigen-presenting cells are required for
proliferative effects and that binding to major histocompatibility (MHC) class II
antigens occurred (Carlsson et al., 1988; Fleischer and Schrezenmeier, 1988;
Mollick et al., 1989). Unlike most antigens which interact with MHC class II
molecules as peptides, the staphylococal enterotoxins were shown to bind in
an unprocessed form (Fleischer and Schrezenmeier, 1988). The site of


80
replication of virus in the host. As such, Nef could be classified as a virulence
factor for HIV.
It has been demonstrated that activated T cells are required for
replication of HIV-1. On the other hand, the ability of HIV-1 to infect quiescent or
resting cells is still questioned. In two studies using quiescent cells, HIV-1
replication in vitro was blocked at the level of virus entry (Gowda et al., 1989;
Tang and Levy, 1990). Others have shown that blockage of replication
occurred at an intracellular event, possibly at reverse transcription (Zack et al.,
1990) or proviral integration (Stevenson et al., 1990; Bukrinsky et al., 1991).
Upon entry into quiescent cells, replication could be induced by subsequent T
cell activation. Consistent with data from in vitro studies are reports that high
numbers of quiescent CD4+ cells from asymptomatic HIV-infected patients
harbor unintegrated viral DNA capable of integration subsequent to PHA
stimulation. Further, AIDS patients have increased numbers of cells containing
integrated virus, corresponding with greater production of virus at this stage of
the disease. These findings indicate that HIV-1 may infect quiescent cells, but
such infection is nonproductive until cell activation occurs.
Studies suggest that Nef plays an important role in HIV pathogenesis.
Infection of adult rhesus monkey with a neAdeleted mutant of SIVmac239
resulted in low viral burden and did not cause disease, although infected
animals were persistently seropositive (Daniel et al., 1992). Recently, genomic
analysis of an HIV strain from a longterm survivor revealed a 118 bp deletion in
the nef gene which resulted in an out-of-frame shift for downstream sequences
(Kirchhoff et al., 1995). These studies hint at the importance of Nef in HIV
pathogenesis in that repeated attempts to isolate virus from this patient with
nonprogressive disease were unsuccessful. Mutations in nef may result in
altered mitogenic activity of the protein, with concomitant reduction or loss of the


93
Ozato, K, N. M. Mayer, and D. H. Sachs. 1982. Monoclonal antibodies to
mouse major histocompatibility complex antigens. Transplantation 34:113.
Parker, J. M. R., D. Guo, and R. S. Hodges. 1986. New hydrophilicity
scale derived from high-performance liquid chromatography peptide retention
data: correlation of predicted surface residues with antigenicity and x-ray-
derived accessible sites. Biochem. 25:5425.
Peavy, D. L, W. H. Adler, and R. T. Smith. 1970. The mitogenic effects of
endotoxin and staphylococcal enterotoxin B on mouse spleen cells and human
peripheral lymphocytes. J. Immunol. 105:1453.
Poiesz, B. J., F. W. Ruscetti, A. F. Gazdar, P. A. Bunn, J. D. Minna, and R.
C. Gallo. 1980. Detection and isolation of type C retrovirus particles from fresh
cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. natl.
Acad. Sci. USA 77:7415.
Pontzer, C. H., N. D. Griggs, and H. M. Johnson. 1993. Agonist
properties of a microbial superantigen peptide. J. Immunol. 193:191.
Pontzer, C. H., M. J. Irwin, N. R. J. Gascoigne, and H. M. Johnson. 1992.
T cell antigen receptor binding sites for the microbial superantigen
staphylococcal enterotoxin A. Proc Natl. Acad. Sci. USA 89:7727.
Pontzer C. H., J. K. Russell, and H. M. Johnson HM. 1991. Site of non-
restrictive binding of SEA to class II MHC antigens. Int. Arch. Allergy Appl.
Immunol. 93:107.
Pontzer, C. H., J. K. Russell, and H. M. Johnson. 1990. Site of
nonrestrictive binding of SEA to class II MHC antigens. Int. Arch. Allergy Appl.
Immunol. 93:107.
Pullen, A. M., Y. Choi, E. Kushnir, J. Kappler, and P. Marrack. 1992. The
open reading frames inthe 3' long terminal repeats of several mouse mammary
tumor virus integrants encode Vp3-specific superantigens. J. Exp. Med. 175:41.
Rebai, N, G. Pantaleo, J. F. Demarest, C. Ciurli, H. Soudeyns, J. W.
Adelsberger, M. Vaccarezza, R. E. Walker, R. P. Sekaly, and A. S. Fauci. 1994.
Analysis of the T-cell receptor beta-chain variable-region (Vp) repertoire in
monozygotic twins discordant for human immunodeficiency virus: evidence for
perturbations of specific V beta segments in CD4+ T cells of the virus-positive
twins. Proc. Natl. Acad. Sci. USA 91:1529.
Rellahan, B. L, L. A. Jones, A. M. Kruisbeek, A. M. Fry, and L. A. Mathis.
1990. In vivo induction of anergy in peripheral Vp8+ T cells by staphylococcal
enterotoxin B. J. Exp. Med. 172:1091.
Ringold, G.M. 1983. Regulation of mouse mammary tumor virus gene
expression by glucocorticoid hormones. Curr. Top. Microbiol. Immunol. 106:79.


78
Thus, we investigated the possibility that HIV-infected cells could activate
autologous PBMC, similar to the activation seen with exogenously added Net.
To achieve maximal infection, PBMC were stimulated with mitogen prior to
infection with HIV. After 5 days, HIV-infected cells were washed and inactivated
with paraformaldehyde. Fresh autologous PBMC were cultured in the presence
of infected cells and monitored for proliferation. Proliferation was observed in
co-cultures of fresh and infected autologous cells (Table X). Uninfected
stimulated cells served as controls, and no proliferation was observed in these
co-cultures. Proliferative responses were significantly reduced by the addition
of polyclonal anti-Nef antibodies, while control anti-SEA antibodies were
without effect. Anti-Nef inhibition of proliferation was dose dependent, with
lower concentrations of antibodies having less inhibitory activity. Similar to the
results observed on HIV-infected cells, anti-Nef antibodies were capable of
reducing proliferation in response to Nef, but not to SEA. Thus, a significant
portion of the observed mitogenic activity of HIV-infected cells is due to Nef
protein.
Discussion
It is interesting that HIV-infected cells were more potent at inducing T cell
proliferation than was soluble Nef. Given that Nef interacts with MHC class II
antigens, it is conceivable that optimal conditions for Nef association with MHC
are achieved in HIV-infected cells, analogous to the expression of the mouse
mammary tumor virus superantigen in infected B cells. These data suggest that
proliferation occurred as a result of Nef expressed on the surface of HIV-
infected cells, thereby indicating that Nef is a virally-encoded mitogen. Thus,
the genome of HIV encodes for its own T cell mitogen, which would amplify the


% Control
41
Concentration (p.M)
Figure 9. Direct binding of Nef(123-160) peptide to Raji cells. 125l-Nef(123-
160) was used at a final concentration of 3 nM. All experimental conditions
were the same as those described in Figure 7. Binding of 125l-Nef(123-160) in
the absence of competitor was 1956 150 CPM. The data presented represent
the mean of three individual experiments, each performed in duplicate. Each
point represents the mean percent of 125l-Nef(123-160) binding relative to
control in the presence of competitors SD.


91
Kawabe.Y. and A. Ochi. 1990. Selective anergy of Vp8+ T cells in
staphylococcus enterotoxin B-primed mice. J. Exp. Med. 172:1065.
Kawabe.Y. and A. Ochi. 1991. Programmed cell death and extrathymic
reduction of Vp8+ CD4+ T cells in mice tolerant to Staphylococcus aureus
enterotoxin B. Nature 349:245.
Kirchhoff, F., T. C. Greenough, D. B. Brettler, J. L. Sullivan, and R. C.
Desrosiers. 1995. Absence of intact nef sequences in a long-term survivor with
nonprogressive HIV-1 infection. New Engl. J. Med. 332:228.
Klatzmann, D., F. Barre-Sinoussi, M. T. Nugeyre, C. Danquet, E. Vilmer,
C. Griscelli, F. Brun-Veziret, C. Rouzioux, J. C. Gluckman, J. C. Chermann, and
L. Montagnier. 1984. Selective tropism of lymphadenopathy associated virus
(LAV) for helper-inducer T lymphocytes. Science 225:59.
Kotzin, B. \J, D. Y. M. Leung, J. Kappler, and P. Marrack. 1993.
Superantigens and their potential role in human disease. Adv. Immunol, 54:99.
Lafon, M., M. Lafage, A. Martinez-Arends, R. Ramirez, F. Vuiller, D.
Charron, V. Lotteau, and D. Scott-Algara. 1992. Edidence for a viral
superantigen in humans. Nature 358:507.
Lafon, M., D. Scott-Algara, P. N. Marche, P. A Cazenave. and E. Jouvin-
Marche. 1994. Neonatal deletion and selctive expansion of mouse T cells by
exposure to rabies virus nuceocapsid superantigen. J. Exp. Med. 180:1207.
Langford, M. P., G. J. Stanton, and H. M. Johnson. 1978a. Biological
effects of staphylococcal enterotoxin A on human peripheral lymphocytes.
Infect. Immun. 22:62.
Langford, M. P., D. A. Weigent, G. J. Stanton, and S. Baron. 1978b.
Virus plaque-reduction assay for interferon. Microplaqye and regular
macroplaque reduction assays. Methods Enzymol. 78:339.
Laurence, J., A. S. Hodstev, and D. N. Posnett. 1992. Superantigen
implicated in dependence of HIV-1 replication in T cells on TCR Vp expression.
Nature 358:255.
Lechler, R. I., V. Bal, J. B. Rothbard, R. N. Germain, R. Sekaly, E. O. Long,
and J. Lamb. 1988. Structural and functional studies of HLA-DR restricted
antigen recognition by human helper T lymphocyte clones by using transfected
murine cell lines. J. Immuno. 141:3003.
Lee, J. M. and T. H. Watts. 1990. Binding of staphylococcal enterotoxin
A to purified murine MHC class II molecules in supported lipid bilayers. J.
Immunol. 145:3360.


14
A hallmark of infection with HIV is the alteration of CD4/CD8 T cell ratios
due to loss of CD4+ T helper cells. Such losses ultimately result in the inability
of an infected individual to mount effective immune responses against
opportunistic infections, thus resulting in death. Several possible mechanisms
by which CD4 loss occurs have been postulated, including direct cytolysis by
HIV (Lemaitre et al., 1990), HIV-induced syncitia formation (Lifson et al., 1986;
Sodroski et al., 1986) and/or cytolytic T cell activity against infected CD4 cells
(Zinkernagel and Hentgartner, 1994). In addition to skewing of the CD4/CD8
ratios, other immunologic alterations seen in HIV-infected individuals include
polyclonal activation of B cells with increased immunoglobulin production and
reduced antigen and mitogen responses (Edelman and Zolla-Pazner, 1989).
Initial increased natural killer cell activity is observed in asymptomatic HIV-
infected individuals, but these activities decrease during disease progression
(Edelman and Zolla-Pazner, 1989).
Because of the known induction of T cell anergy and/or deletion by
superantigens (Johnson et al., 1992), it has been speculated that the HIV
genome encodes for a superantigen that may cause some of the
immunopathologies observed with AIDS. Upon interaction with MHC class II
antigens and TCR, Vp-specific T cell populations would be activated and
expand, eventually leading to functional (anergy) or actual (deletion) loss of T
cells. Several points of evidence suggest a role for an HIV-derived
superantigen. Although initial studies suggested that AIDS patients had
deletions in Vp T cell populations (Imberti et al., 1991), such deletions could not
be found by others (Laurence et al., 1992). Vpl2+ T cells were shown to
support enhanced replication of HIV compared to other Vp subsets and
proliferated in response to cells from HIV+ patients, suggesting the presence of
a superantigen (Laurence et al., 1992). Asymptomatic patients exhibit altered


>
CHAPTER II
MMTV SUPERANTIGEN BINDING TO MHC CLASS II ANTIGENS
Introduction
SEA is one of the most potent T-cell mitogens known, and has been
classified as a bacterial superantigen based on its ability to stimulate Vp-
specific T-cell subsets (Johnson et al., 1991). Concurrent with studies on SEA,
minor lymphocyte stimulating (mis) antigens have recently been shown to be
products of MMTV (Choi et al., 1991; Acha-Orbea et al., 1991). Two exogenous
strains of MMTV encode for retroviral superantigens in genes that overlap the
terminal repeat at the 3' end of the viral genome (Pullen et al., 1992; Choi et al.,
1992). These genes have been designated vsag, denoting that they encode for
viral superantigens (Marrack and Kappler, 1990). No direct binding of the
putative MMTV superantigen to either MHC antigens or TCR has been shown.
To determine sites that interact with class II MHC antigens, overlapping
synthetic peptides were synthesized that encompass the putative extracellular
domain of MMTV-1 superantigen. The data presented here indicate that a site
that is encompassed by amino acid residues 76-119 of MMTV-1 superantigen
competes with SEA for binding to class II MHC antigens. Further, direct binding
studies show that this region of the MMTV superantigen binds directly to class II
MHC antigens. These data indicate that SEA and MMTV superantigen share at
least one common binding region on class II MHC molecules.
16


Table III.
Amino acid sequences of vSAg peptides
vSAg Peptide
Sequence
vSAg( 76-119)
DSFNNSSVQDYNLNDSENSTFLLGQGPQPTSSYKPHRLCPSEIE
vSAg(117-147)
EIEIRMLAKNYIFTNETNPIGRLLIMMLRNE
vSAg(142-172)
MMLRNESLSFSTIFTQIQRLEMGIENRKRRS
vSAg(166-195)
ENRKRRSTSVEEQVQGLRASGLEVKRGKRS
vSAg(190-222)
KRGKRSALVKIGDRWWQPGTYRGPYIYRPTDAP
vSAg(220-250)
DAPLPYTGRYDLNFDRWVTVNGYKVLYRSLP
vSAg(245-276)
LYRSLPFRERLARARPPWCVLSQEEKDDMKQQ
vSAg(274-313)
KQQVHDYIYLGTGMIHWKVFYNSREEAKRHIIEHIKALP
vSAg(76-119)
(scrambled)
PNSNEGLSQQSTDPSPHNFILSNENSYPCYSLLGDVQREDSTKF
The vSAg(76-119) scrambled peptide sequence was generated using the sequence edit
program (Devereax et al., 1984).


58
respond to stimulation by either Net or SEA. Upon reconstitution with
paraformaldehyde-inactivated autologous APC, significant Nef-induced
proliferation of T cells occurred, with essentially complete reconstitution of the
response. Similar proliferative responses were obtained using cells from other
donors. Thus, T cells responded to Nef stimulation only in the presence of
autologous APC, and Nef does not require processing to be presented by APC.
These results indicate that Nef-induced proliferation required APC in a manner
reminiscent of SEA.
Nef(123-160) Peptide Specifically Blocks Proliferation of PBMC Induced by Nef
and SEA
As shown in Chapter 3, a synthetic peptide corresponding to an internal
Nef sequence, Nef(123-160), blocked binding of Nef and SEA to Raji cells. It
was important to determine if this peptide could also block proliferation induced
by Nef and SEA. The results of this study are presented in Figure 15. Nef(123-
160) blocked both Nef-induced and SEA induced proliferation, consistent with
its ability to block binding of Nef and SEA to Raji cells. Further, the blocking
was specific in that proliferation induced by the T cell mitogens Con A and anti-
CD3, or the B cell mitogen anti-IgM, were not blocked by Nef(123-160).
Nef(157-186), whose sequence slightly overlaps that of Nef(123-160), had no
effect on the proliferative effects of either Nef, SEA, or Con A. These results
confirm that the proliferative responses observed in PBMC cultures were
specific for Nef, and were not due to a contaminant. Thus, proliferative
responses to Nef and SEA were specifically blocked by a peptide
corresponding to the region on Nef that binds to class II antigens and which
blocks binding of Nef and the superantigen SEA.


5
Table II.
List of viral superantigens implicated in disease.
Virus
(Family or Subfamily)
Superantigenic
Protein
Disease/Effect3
Prototype: Superantigen from Mouse Mammary Tumor Virus
Mouse mammary tumor virus
vsag gene product
Mammary tumors
(oncornavirus)
Mouse leukemia virus
Gag protein
Murine AIDS
(oncornavirus)
Human immunodeficiency virus
Not identified
AIDS
(lentivirus)
Human foamy virus
bel3 gene product
Grave's disease
(?>b
(spumavirus)
Rabies
Nucleocapsid
Rabies
(rhabdovirus)
Epstein-Barr virus
Not identified
B cell lymphoma
(?)
(herpesvirus)
Chronic fatigue
Cytomegalovirus
Not identified
syndrome (?)
Enhanced HIV
(herpesvirus)
replication
aJohnson et al. 1995.
^Denotes diseases in which superantigens have been implicated, but no direct
evidence is available.


A peptide corresponding to an internal region of HIV Nef protein was
identified that specifically binds to to human class II antigens. Nef protein
induced proliferation of human peripheral blood mononuclear cells (PBMC) in
85-90% of the HIV-negative donors tested. Nef stimulation required APC, and
did not require processing. Interleukin 2 and gamma interferon were produced
in Nef-stimulated cultures. These results strongly suggest that Nef acts like a
retroviral SAg that stimulates CD4+ T cells.
Activated T cells are required for HIV replication. The role of Nef in HIV
pathogenesis was investigated by treating PBMC with Nef prior to in vitro
infection with HIV. Significant levels of infection were found in these cultures,
as compared to unstimulated controls. Nef-stimulated T cells were found to
express T cell activation markers. Inactivated HIV-infected cells were capable
of inducing proliferation in autologous fresh PBMC, and proliferation was
significantly reduced by anti-Nef antibodies. These results indicate that Nef is
expressed on the surface of infected T cells, possibly in the context of class II
antigens, and as such, Nef can activate a cellular reservoir in an paracrine
fashion for continual viral replication. Thus, Nef is an HIV-encoded SAg-like
mitogen that promotes HIV replication in T cells. As such, Nef is probabaly an
HIV virulence factor.
IX


2
T cell activation by superantigens results in massive proliferation of Vp-
specific T cells. Concomitant with proliferation is prodigious production of
cytokines, in particular interleukin 2 and gamma interferon (IFNy). Such
responses can be detrimental to the host, as is the case with food poisoning
which occurs upon ingestion of staphylococcal enterotoxin-laden food
(Bergdoll, 1985). As an eventual consequence of superantigen activation, T
cells can become unresponsive to further stimulation (anergy) or undergo
programmed cell death (apoptosis) (Kawabe and Ochi, 1990, 1991; Rellahan et
al., 1990). Thus, the fate of T cells activated by superantigen differs from that of
cells activated by conventional antigens, with the possible consequence of loss
of immune function.
Superantigens are produced by a number of microorganisms, ranging from
bacteria to viruses. The prototypic bacterial superantigens are the enterotoxins
produced by Staphylococcus aureus (Table I). Staphylococcal enterotoxin A
(SEA) is the most common cause of food poisoning, and its superantigen
effects on immunocytes have been implicated in the syndrome of short-term
nausea, fever, and diarrhea that are symptomatic of food poisoning (Bergdoll,
1985; Johnson and Magazine, 1988). Another staphylococcal enterotoxin, toxic
shock syndrome toxin-1 (TSST-1), is produced during infection and its
superantigenic effects result in toxic shock syndrome (Bergdoll, 1985). Other
bacterial sources of superantigens are the group A streptococci, which produce
pyrogenic exotoxins that have been implicated in psoriasis (Kotzin et al., 1993)
and rheumatic heart disease (Lewis et al., 1993). Superantigens may also be
involved in tuberculosis (Ohmen et al., 1994) and Reiter's syndrome (Uchiyama
et al., 1993). One species of mycoplasma, M. arthiditis, has been shown to
produce a superantigen that may be involved in arthritis (Cole and Griffiths,
1993).


24
C-terminal region of the MMTV superantigen, vSAg(245-276), was not a
consistent competitor for SEA binding. The peptide corresponding to the C-
terminal tail, vSAg(274-313), did not compete. A peptide having the same
amino acid content as vSAg(76-119) but in a scrambled sequence (see Table
III for sequence) did not compete, thus indicating that vSAg(76-119) peptide
competition was sequence specific. No direct binding of 125l-vSAg(76-119) to
SEA or to related superantigens was detected (data not shown). Thus, the
vSAg(76-119) peptide binds specifically to A20 cells, and competes for SEA
binding in a dose-dependent manner.
Direct Binding of vSAa(76-119) Peptide to A20 Cells
In direct binding studies, 125l-vSAg(76-119) peptide bound to A20 cells
and was effectively inhibited by both unlabeled SEA and unlabeled vSAg(76-
119) peptide (Figure 3). Unlabeled SEA and unlabeled vSAg(76-119) peptide
competed with 125l-vSAg(76-119) in a similar manner, although SEA was a
more potent competitor. SEA reduced 125l-vSAg(76-119) binding by 50% at a
concentration of 1.8 pM as compared to 25 pM for unlabeled vSAg(76-119)
peptide. Neither vSAg(76-119) scrambled peptide nor the C-terminal vSAg
peptide competed, thereby showing that vSAg(76-119) binding was sequence
and region specific. TSST-1 did not compete with 125l-vSAg(76-119) peptide
for binding to A20 cells, while SEB competed less effectively than SEA, which is
consistent with their relative abilities to compete with SEA for MHC class II
binding (Fraser, 1989; Pontzer et al., 1991). Thus these results indicate that
binding of vSAg(76-119) peptide occurs to murine MHC class II antigens.
Binding of vSAg(76-119) may occur at a region(s) to which SEA also binds or to
a neighboring site which interferes with the binding of SEA.


CHAPTER IV
ACTIVATION OF CD4 T CELLS BY THE NEF PROTEIN FROM HIV-1
Introduction
In the previous chapter, data was presented which showed that Net
binds specifically to class ll-bearing Raji cells at a site(s) involved in
staphylococcal enterotoxin binding. This binding was similar to that shown for
MMTV superantigen. These data suggest that Nef may have characteristics of
superantigens. The data presented in this chapter show that Nef induces
proliferation of human peripheral blood mononuclear cells. Further, the
proliferative response is T cell specific. The data presented in this chapter
indicate that Nef activates T cells and induces T cell cytokine production in a
manner reminiscent of staphylococcal superantigens.
Materials and Methods
Nef Protein
Nef protein was expressed and purified using a fusion protein and
purification system. The HIV-1 nef gene (HIV-1 IIIb, R & D Systems, Cambridge,
MA) was amplified by polymerase chain reaction (PCR), using the following
primer set:
5' ATG GGT GGC AAG TGG TCA AAA AGT (+)
51 GCC AAG CTT GAT GTC AGC AGT TCT (-)
45


IFN (U/ml) IFN (u/m|) IFN (U/ml)
62

PBMC + SEA
PBMC + Nef
PBMC + Con A
PBMC + media
T cells + SEA
T cells + Nef
T cells + Con A
T cells + media
10000
1000
100
10
1
24 48 72 96
Hours
T+APC+SEA
T+APC+Nef
T+APC+ConA
T+APC+media
Figure 16. Kinetics of IFN production induced by Nef. Cultures of PBMC (Panel
A), purified T cells (Panel B), and reconstituted cultures of purified APC and
purified T cells (Panel C) were tested for IFN production at 24, 48, 72, and 96 h.
Cultures were stimulated with either Nef at 100 ng/ml, SEA at 100 ng/ml, or Con
A at 10 pg/ml, or were left unstimulated. Cultures of purified APC produced <10
U/ml IFN at all timepoints.


56
1000
Figure 13. Nef-induced proliferative responses from a representative sampling
of donors. PBMC of blood bank donors were tested for proliferation induced by
SEA or Nef. SEA and Nef were used at 300 ng/ml. Mean values for 3H-
thymidine incorporation by unstimulated cultures ranged from 35286 CPM to
1983+274 CPM.


26
Evidence that vSAa(76-1191 Binds Specifically to MHC Class II Antigens
Two further pieces of evidence indicate that vSAg(76-119) binds to class II
MHC antigens. Binding of vSAg(76-119) to class II negative mouse L cells was
insignificant as compared to mouse A20 cells even at concentrations as high as
5 nM (Figure 4A). Monoclonal antibodies to class I MHC antigens had no
effect on 125l-vSAg(76-119) binding to A20 cells (Figure 4B). Polyclonal anti-l-
A^ and anti-l-Eb significantly blocked binding and, in combination, these
antibodies reduced binding to A20 cells by 73%. These data suggest that
vSAg(76-119), like SEA (Lee and Watts, 1990), binds to both l-A and l-E. An I-
Ab p1 helix-specific monoclonal antibody reduced binding by approximately
30%, suggesting that this is a region on l-A to which vSAg(76-119) binds.
Polyclonal antibodies to l-A^ had minor effects on vSAg(76-119) binding. Thus,
these data indicate that vSAg(76-119) binds to class II MHC antigens.
In order to directly determine if vSAg(76-119) binds to the pi helix of l-A,
a competitive radioimmunoassay was performed in which 125I-SEA and 125l-
vSAg(76-119) were tested for their relative abilities to bind to l-Apb(60-90)
peptide. SEA and vSAg(76-119), but not scrambled VSAG(76-119), competed
with both 125I-SEA and 125l-vSAg(76-119) for binding to l-Apb(60-90) in a
manner similar to the competition seen on whole cells (Figure 5A). As
previously shown for SEA (Russell et al., 1990, 1991), vSAg(76-119) did not
directly bind to l-Apb(30-60) (Figure 5B). Thus, SEA and vSAg(76-119) bind to
a similar region on the p chain of the class II MHC molecule.
Discussion
To date, no information has been available on the ability of MMTV vSAg
protein to bind to MHC antigens and, in fact, this ability has been questioned
(Marrack and Kappler, 1990; Acha-Orbea and Palmer, 1991; Acha-Orbea et al.,


RT (in CPM)
76
40000
30000 -
20000
10000 -
Expt 1
Expt 2
Figure 17. Infectivity assay on supernatants from Nef-stimulated HIV-infected
cultures.


LIST OF REFERENCES
Abe, J., B. L. Kotzin, K. Jujo, M. E. Melish, M. P. Glode, T. Kohsaka, and
D. Y. M. Leung. 1992. Selective expansion of T cells expressing T-cell
receptor variable region Vp2 and Vp8 in Kawasaki disease. Proc. Natl. Acad.
Sci. USA 89:4066.
Acha--Orbea, H. 1992. Retroviral superantigens. In B. Fleischer, Ed.
Ciological Significance of Superantigens. Basel: Karger, pp65-86.
Acha--Orbea, H. and E. Palmer. 1991. Mis a retrovirus exploits the
immune system. Immunol. Today 12:356.
Acha--Orbea, H., A. N. Shakhov, L. Scarpellino, E. Kolb, V. Muller, A.
Vessaz-Shaw, R. Fuchs, K. Blochinger, P. Rollini, J. Billotte, M. Sarafidou, H. R.
MacDonald, and H. Diggelmann. 1991. Clonal deletion of Vpl4-bearing T cells
in mice transgenic for mammary tumor viurs. Nature 350:207.
Achong, B. G., P. W. A. Mansell, M. A. Epstein, and P. Clifford. 1971. An
unusual virus in cultures from a human nasopharyngeal carcinoma. J. Nat.
Cancer Inst. 46:299.
Allan, J. S., J. E. Coligan, T. --H. Lee, M. F. McLane, P. J. Kanki, J. E.
Groopman, and M. Essex. 1985. A new HTLV-III/LAV encoded antigen
detected by antibodies from AIDS patients. Science 230:810.
Arthur, L. O., J. W. Bess, R. C. Sowder, R. E. Benveniste, D. L. Mann, J. C.
Chermann, and L. E. Henderson. 1992. Cellular proteins bound to
immunodeficiency viruses: implications for pathogenesis and vaccines.
Science 258:1935.
Baba, T. W., Y. S. Jeong, D. Penninck, R. Bronson, M. F. Greene, and R.
M. Ruprecht. 1995. Pathogenicity of live, attenuated SIV after mucosal
infection of neonatal macaques. Science 267:1820.
Bahraoui, E., M. Yagello, J. --N. Billaud, J. --M. Sabatier, B. Guy, E.
Muchmore, M. Girard, and J. -C. Gluckman. 1990. Immunogenicity of the
human immunodeficiency virus (HIV) recombinant nef gene product. Mapping
of T-cell and B-cell epitopes in immunized chimpanzees. AIDS Res. Hum.
Retroviruses 6:1087.
Barnstable, C. J., W. F. Bodmer, G. Brown, G. Galfre, C. Milstein, A. F.
Williams, and A. Ziegler. 1978. Production of monoclonal antibodies to group
A erythrocytes, HLA, and other human cell surface antigens new tools for
genetic analysis. Cell 14:9.
86


88
Dellabonna, P. J. Peccoud, J. Kappler, P. Marrack, C. Benoit, and D.
Mathis. 1990. Superantigens interact with MHC class II molecules outside of
the antigen binding groove. Cell 62:1115.
deRonde, A., B. Klaver, W. Keulen, L. Smit, and J. Goudsmit. 1992.
Natural HIV-1 Nef accelerates virus replication in primary human lymphocytes.
Virology 188:391.
Devereax, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set
of sequence analysis programs for the VAX. Nucl. Acid Res. 12:387.
Dobrescu, D., B. Ursea, M. Pope, A.S. Asch, and D.N. Posnett. 1995.
Enhanced HIV-1 replication in V(312 due to human cytomegalovirus in
monocytes: evidence for a putative herpesvirus superantigen. Cell 82:753.
Dyson, P. J., A. M. Knight, S. Fairchild, E. Simpson, and K. Tomonari.
1991. Genes encoding ligands for deletion of Vpl1 T cells cosegregate with
mammary tumor virus genomes. Nature 349:531.
Edelman, A. S. and S. Zolla-Pazner. 1989. AIDS: a syndrome of
immune dysregulation, dysfunction, and deficiency. FASEB J. 3:22.
Festenstein, H. 1973. Immunogenetic and biological aspects of in vitro
lymphocyte allotransformation (MLR) in the mouse. Transplant. Rev. 15:62.
Fischer, H., M. Dohlsten, M. Lindvall, H. O. Sjogren, and R. Carlsson.
1989. Binding of staphylococcal enterotoxin A to HLA-DR on B cell lines. J.
Immunol. 142:3151.
Fleischer, B. and H. Schrezenmeier. 1988. T cell stimulation by
staphylococcal enterotoxins: Clonally variable response and requirement for
major histocompatibility complex class II molecules on accessory or target cells.
J. Exp. Med. 167:1697.
Fluegel, R. M. 1993. The molecular biology of the human spumavirus.
In B. R. Cullen, Ed. Human Retroviruses. Oxford: IRL Press, pp193-214.
Franchini, G., M. Robert-Guroff, J. Ghrayeb, N. T. Chang, and F. Wong-
Staal. 1986. Cytoplasmic localization of the HTLV-III 3' orf protein in cultured T
cells. Virology 155:593.
Frankel, W. N., C. Rudy, J. M. Coffin, and B. T. Huber. 1991. Linkage of
Mis genes to endogenous mammary tumor virus. Nature 349:525.
Fraser, J.D. 1989. High-affinity binding of staphylococcal enterotoxins A
and B to HLA-DR. Nature 339:221.


RETROVIRAL SUPERANTIGENS
By
BARBARA AUREA TORRES
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995


30
superantigen activity of MMTV vSAg protein was lost in truncations that did not
contain the region encompassed by residues 76-119.
Subsequent studies by the same group of investigators suggest that
MMTV-7 superantigen is synthesized as a precursor molecule, is proteolytically
cleaved at an internal site (- residue 164) and is expressed as an 18.5 kd
surface protein consisting of the C-terminal residues (Winslow et al., 1992).
Membrane association of this truncated form may involve "tethering" to MHC
class II antigens or to the N-terminal portion of the precursor molecule. As a
cautionary note, the transfected cell line used to characterize this truncated form
of the MMTV-7 superantigen was shown to have high expression of the protein
but low activity. Conversely, a transfected cell line with moderate superantigen
expression and high activity was not used for characterization.
Recently, studies were reported on the binding of truncated forms of
MMTV-7 superantigen to human MHC class II antigens. Binding studies were
performed with 28K and 18K versions of the superantigen and showed that
both molecules bind to human MHC class II antigens (Mottershead et al., 1995).
Thus, studies by our laboratory indicate one site for binding to mouse MHC
class II antigens, whereas studies performed by others suggest that two sites on
MMTV superantigen are involved in binding to human MHC class II antigens.
Despite the diverse origins of SEA and MMTV vSAg protein, it is likely
that these two proteins exert superantigen activity by a similar mechanism. Two
different regions of the MMTV superantigen are probably involved in MHC
binding and Vp specificity. The variability of the C-terminal 30 residues
between Vp-specific MMTV superantigens is indirect evidence that the C-
terminus is responsible for Vp specificity (Pullen et al., 1992). Data presented
here on competitive and direct binding of vSAg peptides to A20 cells suggest
that the region encompassed by residues 76-119 is involved in MHC binding.


15
Vp profiles (Dalgleish et al., 1992) as well as skewed T cell Vp usage upon
stimulation with staphylococcal enterotoxins in vitro (Bisset et al., 1993).
Interestingly, one study found skewed Vp T cell profiles in monozygotic twins
that were discordant for HIV infection, with perturbations in several Vps (Rebai
et al., 1994). This last study is of particular importance in that the altered Vp
profiles in infected individuals cannot be ascribed to differences in MHC, since
only identical twins were used. Thus, these studies hint at the presence of a
superantigen encoded by HIV that may play a role in HIV pathogenesis by
augmenting a population of T cells, resulting in a reservoir for virus replication,
with eventual deletion of those T cells.


27
6000
0 200
Concentration of ORF(76-119)
OiM)
Antisera
Figure 4. Evidence that vSAg(76-119) peptide binds to MHC class II antigens,
and does not bind to class I antigens.
Panel A: Binding of 125l-vSAg(76-119) to class ll-positive A20 cells and class
ll-negative L cells. 125l-vSAg(76-119) was used at a final concentration of 5
nM.
Panel B: Blockage of 125l-vSAg(76-119) binding to A20 cells by antibodies to
class I and class II MHC antigens. 125l-vSAg(76-119) was used at a final
concentration of 5 nM. MAbs were used at a final dilution of 1:30.


CHAPTER V
HIV ENCODES FOR ITS OWN CD4 T CELL MITOGEN
Introduction
Net protein is encoded in the genomes of both HIV-1 and the related
primate virus simian immunodeficiency virus (SIV). The function that Nef plays
in the pathogenesis of these viruses is uncertain, although its importance is
reflected in studies in which a nef-deleted SIV mutant did not cause disease
and protected against infection with the pathogenic wildtype strain (Daniel et
al., 1992). Further, the viral load in SIV mutant-infected animals was
considerably lower than in animals infected with the wildtype strain. However,
challenge of neonatal macaques with the same attenuated SIV strain caused
disease and, in some cases, death (Baba et al., 1995). The different outcomes
of these two studies may involve differences in the immune system
development between neonates and adults.
In Chapter IV, data was presented on the ability of Nef protein to induce
proliferation of human peripheral blood mononuclear cells (PBMC) from a wide
sampling of HIV-negative donors. Proliferative responses were T cell specific
and were accompanied by production of cytokines such as interleukin 2 (IL 2)
and gamma interferon (IFNy), indicative of CD4 T cell activation. Nef-induced T
cell proliferation and activation required the presence of antigen-presenting
cells. These results are interesting in that T cell activation has been shown to
be required for active HIV replication. Further, although resting or quiescent T
cells can be infected, evidence suggests that HIV replication only occurs upon
68


53
subsequently used in these studies. The possibility was raised that the
proliferative activity ascribed to Net was due to a contaminant in the
recombinant preparation. As a control, the proliferative activity of the Nef fusion
partner, MBP, was also assessed. No proliferation was observed with MBP
(Figure 12). Further, polyclonal antisera generated against Nef synthetic
peptides were tested for neutralizing activity. As shown in Table V, anti-Nef
antibodies significantly reduced Nef-induced proliferative responses. Antibody
neutralization of Nef activity occurred in a dose-dependent manner. These data
confirm that the proliferative activity seen in these studies is due to Nef protein,
and not to a vector-derived contaminant.
Nef was also tested for the ability to induce proliferation of PBMC from
fifteen donors. Results from a representative sample of ten donors are shown in
Figure 13. Nef induced significant proliferation in 90% of the donors tested.
Variation in the Nef response was seen, similar to the responses to the potent
staphylococcal enterotoxin, SEA. It is unlikely that these donors were
sensitized to Nef, since they tested negative for HIV-1. Similar results were
obtained using purified recombinant Nef protein preparations from two different
sources, and which used different expression systems (see Materials and
Methods in this chapter). The high number of donors that responded to Nef
indicates Nef protein has similar mitogenic activity to SEA for PBMC from a
wide sampling of donors.
Antigen-Presenting Cells (APC1 Are Required for Nef Activity
The question arose as to whether Nef, like SEA, requires APC in order to
stimulate lymphocyte proliferation. Nef induced significant proliferation in
PBMC (Figure 14). Purified T cell cultures did not proliferate in response to Nef
or to SEA. Purified APC, which contained monocytes and B cells, did not


71
Fluorescent Antibody Cell Sorter (FACS1 Analysis of Nef-Activated Cells
PBMC were stimulated for 4 days with either Nef (3|ig/ml) or SEA (0.1
|ig/ml). PBMC were washed and resuspended in FACS buffer containing 0.5%
BSA and 10 mM sodium azide. Cells were incubated with fluorescein-labeled
mAb to HLA-DR for 45 minutes and washed with FACS buffer. Cells were then
incubated with phycoerythrin-labeled mAbs to either CD4 or IL 2 receptor (IL
2R) for 45 minutes. Cells were washed and analyzed on a FACScan (Becton-
Dickinson, Mountain View, CA) at 10,000 events/sample. All mAbs used were
obtained from Becton-Dickinson, San Jose, CA.
Studies on Proliferation of PBMC Induced bv Autologous HIV-Infected Cells
PBMC cultures were stimulated as described above. PBMC were
infected with HIV for 6 days, at which time the cells were washed and
inactivated by overnight treatment with 2% paraformaldehyde. Inactivated HIV-
infected cells were washed extensively to remove excess paraformaldehyde.
Fresh autologous PBMC were cultured in microtiter plates in the presence of
inactivated HIV-infected cells at a ratio of 3:1. After 4 days, 3H-thymidine
incorporation was assessed as described above.
Anti-Nef Peptide Antibodies
A mixture of antibodies to Nef(123-160) peptide and Nef(182-206)
peptide were used to block proliferation in response to autologous HIV-infected
cells. Antibodies were each used at a final dilution of 1:1000, as described in
Chapter IV. Inactivated HIV-infected cells were treated with antibodies for 60
minutes prior to addition of fresh autologous PBMC. Preimmune sera were
used and had no effect on proliferation induced by HIV-infected cells.


69
subsequent cellular activation. Herein, work is described that indicates that Net
activation of T cells is sufficient for HIV replication. These results show that Net
is an HIV-encoded mitogen that helps, at least in part, in establishing a cellular
reservoir for virus replication.
Materials and Methods
PBMC Cultures for HIV Infectivitv Studies
PBMC were isolated from peripheral blood of healthy adult donors as
described in Chapter IV. PBMC were resuspended in RPMI 1640 medium
containing 5% FBS and cultured in 25 cm2 flasks (Sarstedt Inc., Newton, NC) at
a concentration of 2 x 10 cells/ml in a final volume of 4 ml (or 8 x 10
cells/flask). Mitogens were added at the initiation of cultures. Nef, SEA, and
PHA were used at 3 pg/ml, 0.1 pg/ml, and 10 pg/ml, respectively. After four
days, PBMC were washed extensively in PBS, resuspended in RPMI 1640
medium containing 10% FBS, 106 M 2-mercaptoethanol, and 10 U rHulL 2/ml.
IL 2 is rapidly depleted in these cultures, and thus rHulL 2 was added to
medium. Cells were counted and all cultures were adjusted to 8 x 106
cells/flask. Cultures were infected with HIV at a final reverse transcriptase titer
of 20,000 CPM/ml. Culture supernatants were harvested and cells were fed
with fresh medium every third day for 12 days.
PBMC Cultures for Proliferation Assays
Concomitant with infectivity studies, PBMC were cultured in microtiter
plates to monitor proliferation in response to Nef and mitogens. The culture
conditions were the same as those described in Chapter IV. Nef and mitogens
were used at the same concentrations as described above. Stimulation indices
were calculated as described in Chapter IV.


28
Figure 5. Binding of 125l-vSAg(76-119) to MHC class II peptides.
Panel A: Binding of 125I-SEA and 125l-vSAg(76-119) to l-Apb(60-90) and
inhibition by vSAg(76-119). Each point represents the mean percent reduction
of control binding in the presence of competitors SD. Binding of 125I-SEA
and 125l-vSAg(76-119) in the absence of competitor was 265692 CPM and
154733 CPM, respectively.
Panel B: Relative ability of vSAg(76-119) to bind l-Ab(60-90) and l-Apb(60-90)
in the presence and absence of vSAg(76-119). Unlabeled and 125l-vSAg(76-
119) were used at final concentrations of 5 nM and 300 pM, respectively.


49
Purification of T Cells from PBMC
Two methods were used to isolate purified T cell populations. One
method involved passing PBMC over T Cellect columns (Biotex, Edmunton,
Alberta, Canada). A second method involved a double rosetting technique
using neuraminidase-treated sheep red blood cells (NA-SRBC) (Spawski and
Lipsky, 1992). PBMC (5 ml at 107/ml) were mixed with 2.5 ml each of NA-
SRBC and FBS. PBMC were incubated for 10 min at 37C, centrifuged at 900
rpm to gently pellet the cells, and incubated for 90 min at 37C. Rosetted T cells
were separated from non-rosetted cells by ficoll hypaque centrifugation. NA-
SRBC were removed from the rosetted fraction by lysis with ammonium
chloride. The procedure was repeated on the rosetted fraction to insure purity.
Autologous rosetted T cells were plated at 2.8 x 10 cells/ml. Cells were plated
into wells of microtiter plates at 2.8 x 10 cells/ml. To insure that these cultures
were depleted of APC, T cells were stimulated with staphylococcal enterotoxins,
which stimulate T cells only in the presence of APC.
Autologous Antigen-Presenting Cells (APC1
For proliferation studies, APC consisted of PBMC that were inactivated
with 0.8% paraformaldehyde. For cytokine studies, APC consisted of cells that
did not form rosettes after two treatments with NA-SRBC. APC were plated at
2.8 x 106 cells/ml. To insure inactivation with paraformaldehyde was complete,
APC were tested for possible proliferation using anti-IgM and Con A.
Synthetic Nef Peptides and Antibodies to Nef Peptides
The sequences of the peptides used are listed in Table III. Peptides
were purified by reverse phase high performance liquid chromatography.
Polyclonal antibodies to Nef peptides were generated in rabbits. Anti-peptide


95
Terwillegar, E. F., J. G. Sodroski, and W. A. Haseltine. 1990.
Mechanisms of infectivity and replication of HIV-1 and implications for therapy.
Ann. Emer. Med. 19:233.
Terwillegar, E. F., J. G. Sodroski, C. A. Rosen, and W. A. Haseltine. 1986.
Effects of mutations within the 3' orf open reading frame region of human T-cell
lymphotropic virus type III on replication and cytopathogenicity. J. Virol. 60:754.
Torres, B. A., J. K. Yamamoto, and H. M. Johnson. 1982. Cellular
regulation of immune interferon (IFNy) production: Lyt phenotype of the
suppressor cell. Infect. Immun. 35:770.
Tsubura, A., M. Inabe, S. Imai, A. Murakami, N. Oyaizu, R. Yasumizu, Y.
Ohnishi, H. Tanaka, S. Morii, and S. Ikehara. 1988. Intervention of T-cells in
transportation of mouse mammary tumor virus (milk factor) to mammary gland
cells in vivo. Cancer Res. 48:6555.
Uchiyama, T., T. Miyoshi-Akiyama, H. Kato, W. Fujimaki, K. Imanishi, and
X. Yan. 1993. Superantigenic properties of a novel mitogenic substance
produced by Yersinia pseudotuberculosis isolated from patients manifesting
acute and systemic symptoms. J. Immunol. 151:4407.
Wain-Hobson, S., P. Sonigo, O. Danos, S. Cole, and M. Alizon. 1985.
Nucleotide sequence of the AIDS virus LAV. Cell 40:9.
Winslow, G. M., M. T. Scherer, J. Kappler, and P. Marrack. 1992
Detection and biochemical characterization of the mouse mammary tumor
virus-7 superantigen. Cell 71:719.
Woodland, D. L., M. P. Happ, K. J. Gollob, and E. Palmer. 1991. An
endogenous retrovirus mediating deletion of ap T cells? Nature 349:529.
Yoshida, M., I. Miyoshi, and Y. Hinuma. 1982. Isolation and
characterization of retrovirus from cell lines of human T-cell leukemia and its
implication in the disease. Proc. Natl. Acad. Sci. USA 79:2031.
Zack, J. A., S. J. Arrigo, S. R. Weitman, A. S.Go, A. Haislip, and I. S. Y.
Chen. 1990. HIV-1 entry into quiescent primary lymphocytes: molecular
analysis reveals a labile, latent viral structure. Cell 61:213.
Zinkernagel, R. and H. Hentgartner. 1994. T-cell mediated
immunopathology versus direct cytolysis by virus: implications for HIV and
AIDS. Immunol. Today 15:262.


% Control
38
Figure 7. Relative abilities of Nef(123-160), purified Nef protein, and SEA to
compete with 125I-SEA for binding to Raji cells. Experimental conditions were
the same as those described in Figure 1. Binding of 125I-SEA to Raji cells in
the absence of competitors was 30,406 2851 CPM. The data presented
represent the mean of three individual experiments, each performed in
duplicate. Each point represents the mean percent of SEA control binding in
the presence of competitors SD.


3
Table I.
List of bacterial superantigens implicated in disease.
Organism
Superantigenic
Protein
Disease/Effect3
Prototype:
Staphylococcal enterotoxins
Staphylococcus aureus
(?)
Enterotoxins
Food poisoning
Toxic shock
Kawasaki's
disease (?)b
Multiple sclerosis
Group A streptococci
Pyrogenic exotoxins
Psoriasis (?)
Rheumatic heart
disease (?)
Mycoplasma arthitidis
T-cell mitogen
Arthritis (?)
Mycobacterium tuberculosis
Not identified
Tuberculosis (?)
Yersinia pestis
Not identified
Reiter's syndrome
Reactive arthritis
Clostridium perfringens
Exotoxin
Sudden infant death
syndrome (?)
aJohnson et al., 1995.
bDenotes diseases in which superantigens have been implicated, but no direct
evidence is available.


34
Table IV.
Amino acid sequences of Nef peptides.
Net Peptide
Sequence
1 -38 MGGKWSKSSVVGWPTVRERMRRAEPAADGVGAASRDLE
31 -65 GAASRDLEKHGAITSSNTAATNAACAWLEAQEEEE
62-99 EEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEG
93-132 EKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPG
123-160 DWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGE
156-186 NKGENTSLLHPVSLHGMDDPEREVLEWRFD
182-206 EWRFDSRLAFHHVARELHPEYFKNC


RETROVIRAL SUPERANTIGENS
By
BARBARA AUREA TORRES
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995

ACKNOWLEDGMENTS
I would like to thank my mentor, Dr. Howard M. Johnson, for his
unflagging support, insight, and wisdom. All of these qualities have helped
guide me, not just a in scientific sense, but in finding my way through the
quagmire of academic science. On a scientific level, Howard has always
been unfailingly logical and knowledgeable, which makes him a wonderful
mentor.
I would also like to thank the other members of my committee. Dr.
Shanmugam and Dr. Hoffmann have been very supportive throughout this
venture. Dr. Yamamoto has given me excellent advice on steering my HIV
work towards clinical relevance. I give special thanks to Dr. Blalock, who
was a tough, but caring, committee member and confidante.
Many thanks must go to my fellow labmates who have always been
willing to lend a hand, ear, or a shoulder to cry on, whichever was needed at
the time. I thank Jeanne, Brian, Prem, and Taishi for generously sharing
their past experiences of graduate school. I also am grateful for my fellow
graduate school mates, Amy, George, and Mustafa, for their understanding
and help. And I thank Aaron, Laurie, and Tim for their warmth and humor.
Finally, I wish to thank my family for their unwavering support through
my graduate school tenure. My parents, Manuel and Antonia Torres, have
stood by with understanding and a warm hug whenever needed. Although
far away, my sister, Marisol Beaton, has cheered me on to complete my
doctoral work. I owe a great debt of gratitude to my wonderful family.
n

TABLE OF CONTENTS
ACKNOWLEDGMENTS ¡¡
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT viii
CHAPTERS
I INTRODUCTION 1
Overview 1
Bacterial Superantigens 6
Retroviruses 8
Mouse Mammary Tumor Virus Superantigen 10
Evidence for a Superantigen Associated with
Human Immunodeficiency Virus 12
II MMTV SUPERANTIGEN BINDING TO MHC CLASS II
ANTIGENS 16
Introduction 16
Materials and Methods 17
Synthetic Peptides 17
Cell Lines and Reagents 17
Radioiodinations 19
Binding Studies 19
Radioimmunoassay 20
Results 21
Competition of vSAg Peptides with Radiolabeled SEA for
MHC Binding 21
vSAg(76-119) Peptide Competition Is Dose-Dependent 21
Direct Binding of vSAg(76-119) Peptide to A20 Cells 24
Evidence that vSAg(76-119) Binds Specifically to MHC
Class II Antigens 26
Discussion 26
iii

III IDENTIFICATION OF AN HIV-1 NEF PEPTIDE THAT BINDS TO
MHC CLASS II ANTIGENS 32
Introduction 32
Materials and Methods 33
Synthetic Peptides 33
Cells and Reagents 33
Radioiodinations 35
Binding Studies 35
Results 35
Competition of Nef Peptides with Radiolabeled SEA for
MHC Binding 36
Nef(123-160) Peptide Competition Is Dose-Dependent 39
Ability of Nef Peptides to Compete with Ses for Binding to
Raji Cells 39
Direct Binding of Nef(123-160) Peptide to Raji Cells 39
MAbs to MHC Class II Antigens Block Nef(123-160) Peptide
Binding to Raji Cells 42
Discussion 42
IV ACTIVATION OF CD4 T CELLS BY THE NEF PROTEIN FROM
HIV-1
Introduction 45
Materials and Methods 45
Nef Protein 45
Reagents 48
Proliferation Assays 48
Purification of T Cells from PBMC 49
Autologous Antigen-Presenting Cells (APC) 49
Synthetic Peptides and Antibodies to Nef Peptides 49
Cytokine Production and Assays 50
Studies on Vp-Specific T Cell Expansion and Induction
of Anergy 51
Results 51
Nef Proliferative Response 51
Antigen-Presenting Cells (APC) Are Required for Nef Activity .53
Nef(123-160) Peptide Specifically Blocks Proliferation of
PBMC Induced by Nef and SEA 58
Induction of T Cell Cytokines by Nef 60
Discussion 64
V HIV ENCODES FOR ITS OWN CD4 T CELL MITOGEN 68
Introduction 68
IV

Materials and Methods 69
PBMC Cultures for HIV Infectivity Studies 69
PBMC Cultures for Proliferation Studies 69
Reverse Transcriptase (RT) Assay 70
ELISA for HIV p24 Antigen 70
Assay for Infectious Virus from PBMC Cultures 70
Fluorescent Antibody Cell Sorter (FACS) Analysis of
Nef-Activated Cells 71
Studies on Proliferation of PBMC Induced by Autlogous
HIV-Infected Cells 71
Anti-Nef Peptide Antibodies 71
Results 72
HIV Replication in Nef-Stimulated PBMC Cultures 72
Infectious Virus Is Produced by Nef-Stimulated Cultures 72
Nef-Stimulated PBMC Express T Cell Activation Markers 75
Expression of Nef by HIV-Infected Cells 75
Discussion 78
VI A MODEL FOR THE ROLE OF NEF IN HIV PATHOGENESIS 82
LIST OF REFERENCES 86
BIOGRAPHICAL SKETCH
100

LIST OF TABLES
Table Page
I. List of bacterial superantigens implicated in disease 3
II. List of viral superantigens implicated in disease 5
III. Amino acid sequences of vSAg peptides 18
IV. Amino acid sequences of Nef peptides 34
V. Ability of anti-peptide antibodies to block Nef-induced
proliferation 51
VI. IL 2 production induced by Nef 61
VII. IFNy is induced by Nef 63
VIII. Maximal proliferation, RT, and p24 values from stimulated
cultures 73
IX. Nef-induced activation of T cells as assessed by FACS 77
X. Ability of anti-Nef antibodies to block proliferation induced by
autologous HIV-infected cells 79
VI

LIST OF FIGURES
Figure Pa9e
1. Ability of vSAG peptides to compete with 125|-SEA for binding
to A20 cells 22
2. Ability of vSAG(76-119) to compete with 125|-SEA for binding
to A20 cells in dose response studeis 23
3. Ability of SEA, SEB, and TSST-1 to compete with i25|-vSAg(76-119)
for binding to A20 cells 25
4. Evidence that vSAg(76-119) peptide binds to MHC class II
antigens, and does not bind to class I antigens 27
5. Binding of i25|-vSAg(76-119) to MHC class II peptides 28
6. Blockage of 125|-SEA binding to Raji and DR 1-transfected
L cells by Nef peptides 37
7. Relative abilities of Nef(123-160), purified Nef protein, and SEA to
compete with 125|-SEA for binding to Raji cells 38
8. Blockage of 125|-SE binding to Raji cells by Nef peptides 40
9. Direct binding of i25|-Nef(123-160) to Raji cells 41
10. Blockage of i25|-Nef(123-160) binding to Raji cells by
antibodies to MHC class I and class II antigens 43
11. Comparison of the mitogenic activities of Nef protein
preparations 52
12. Lack of proliferation by PBMC in response to the Nef fusion
partner MBP 54
13. Nef-induced proliferative responses from a representative
sampling of donors 56
14. Nef-induced activation of T cells requires APC but does
not require prcessing of Nef 57
15. Nef(123-160) peptide specifically blocks proliferation
of PBMC induced by Nef and SEA 59
16. Kinetics of IFN production induced by Nef 62
17. Infectivity assay on supernatants from Nef-stimulated
HIV-infected cultures 76
18. Model for the role of Nef in the pathogenesis of HIV 85
vii

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
RETROVIRAL SUPERANTIGENS
By
Barbara Aurea Torres
December 1995
Chairman: Howard M. Johnson
Major Department: Microbiology and Cell Science
Superantigens (SAg) are potent inducers of T cell activation that cause
proliferation and massive cytokine release. The receptors for superantigens on
antigen-presenting cells (APC) are major histocompatibility (MHC) class II
molecules. Both Nef protein of human immunodeficiency virus (HIV) and
mouse mammary tumor virus (MMTV) SAgs are encoded in genes that overlap
the terminal repeat at the 3' end. Using synthetic peptides, a region was
identifed on the MMTV-1 SAg, corresponding to residues 76-119, that
specifically binds to mouse MHC class II antigens. MMTVSAg(76-119) also
bound to and competed with bacterial superantigens for binding to MHC class II
peptides, suggesting similar binding regions on class II. These studies are the
first demonstration that retroviral superantigens bind to MHC class II antigens.
viii

A peptide corresponding to an internal region of HIV Nef protein was
identified that specifically binds to to human class II antigens. Nef protein
induced proliferation of human peripheral blood mononuclear cells (PBMC) in
85-90% of the HIV-negative donors tested. Nef stimulation required APC, and
did not require processing. Interleukin 2 and gamma interferon were produced
in Nef-stimulated cultures. These results strongly suggest that Nef acts like a
retroviral SAg that stimulates CD4+ T cells.
Activated T cells are required for HIV replication. The role of Nef in HIV
pathogenesis was investigated by treating PBMC with Nef prior to in vitro
infection with HIV. Significant levels of infection were found in these cultures,
as compared to unstimulated controls. Nef-stimulated T cells were found to
express T cell activation markers. Inactivated HIV-infected cells were capable
of inducing proliferation in autologous fresh PBMC, and proliferation was
significantly reduced by anti-Nef antibodies. These results indicate that Nef is
expressed on the surface of infected T cells, possibly in the context of class II
antigens, and as such, Nef can activate a cellular reservoir in an paracrine
fashion for continual viral replication. Thus, Nef is an HIV-encoded SAg-like
mitogen that promotes HIV replication in T cells. As such, Nef is probabaly an
HIV virulence factor.
IX

CHAPTER I
INTRODUCTION
Overview
A novel class of antigens called superantigens has recently been
characterized. Unlike conventional antigens, which stimulate approximately 1
in 10,000 cells, superantigens can induce the proliferation and activation of as
many as 1 in 5 T cells (Johnson et al., 1991). The staphylococcal enterotoxins,
prototypic of this class of antigens, are among the most potent inducers of T cell
proliferation, and are effective at concentrations as low as 10'16 M (Langford et
al., 1978a).
Major differences exist between conventional antigens and
superantigens. Superantigens exert their effects as whole molecules and do
not require processing by antigen presenting cells for T cell recognition.
Although superantigens require MHC class II molecules, they bind at site(s)
outside the peptide antigen binding groove (Russell et al., 1990; Dellabonna et
al., 1990). Superantigens presented in the context of MHC class II molecules
are recognized by the variable region of the p chain (Vp) of the T cell receptor.
Only T cells that express T cell receptors with specific Vp regions will be
activated by a given superantigen. For example, toxic shock syndrome toxin-1
activates only Vp2-bearing human T cells (Abe et al., 1992) and staphylococcal
enterotoxin A activates several Vps on human T cells, including Vp5 and Vpl2,
(Kappler et al., 1989). Thus, T cell responses to superantigens are said to be
Vp-specific.
1

2
T cell activation by superantigens results in massive proliferation of Vp-
specific T cells. Concomitant with proliferation is prodigious production of
cytokines, in particular interleukin 2 and gamma interferon (IFNy). Such
responses can be detrimental to the host, as is the case with food poisoning
which occurs upon ingestion of staphylococcal enterotoxin-laden food
(Bergdoll, 1985). As an eventual consequence of superantigen activation, T
cells can become unresponsive to further stimulation (anergy) or undergo
programmed cell death (apoptosis) (Kawabe and Ochi, 1990, 1991; Rellahan et
al., 1990). Thus, the fate of T cells activated by superantigen differs from that of
cells activated by conventional antigens, with the possible consequence of loss
of immune function.
Superantigens are produced by a number of microorganisms, ranging from
bacteria to viruses. The prototypic bacterial superantigens are the enterotoxins
produced by Staphylococcus aureus (Table I). Staphylococcal enterotoxin A
(SEA) is the most common cause of food poisoning, and its superantigen
effects on immunocytes have been implicated in the syndrome of short-term
nausea, fever, and diarrhea that are symptomatic of food poisoning (Bergdoll,
1985; Johnson and Magazine, 1988). Another staphylococcal enterotoxin, toxic
shock syndrome toxin-1 (TSST-1), is produced during infection and its
superantigenic effects result in toxic shock syndrome (Bergdoll, 1985). Other
bacterial sources of superantigens are the group A streptococci, which produce
pyrogenic exotoxins that have been implicated in psoriasis (Kotzin et al., 1993)
and rheumatic heart disease (Lewis et al., 1993). Superantigens may also be
involved in tuberculosis (Ohmen et al., 1994) and Reiter's syndrome (Uchiyama
et al., 1993). One species of mycoplasma, M. arthiditis, has been shown to
produce a superantigen that may be involved in arthritis (Cole and Griffiths,
1993).

3
Table I.
List of bacterial superantigens implicated in disease.
Organism
Superantigenic
Protein
Disease/Effect3
Prototype:
Staphylococcal enterotoxins
Staphylococcus aureus
(?)
Enterotoxins
Food poisoning
Toxic shock
Kawasaki's
disease (?)b
Multiple sclerosis
Group A streptococci
Pyrogenic exotoxins
Psoriasis (?)
Rheumatic heart
disease (?)
Mycoplasma arthitidis
T-cell mitogen
Arthritis (?)
Mycobacterium tuberculosis
Not identified
Tuberculosis (?)
Yersinia pestis
Not identified
Reiter's syndrome
Reactive arthritis
Clostridium perfringens
Exotoxin
Sudden infant death
syndrome (?)
aJohnson et al., 1995.
bDenotes diseases in which superantigens have been implicated, but no direct
evidence is available.

4
Like their bacterial counterparts, viruses produce superantigens that
have been implicated in pathogenic processes (Table II). The prototypic viral
superantigen is that produced by the oncornavirus, mouse mammary tumor
virus (MMTV) (Marrack et al., 1991; Frankel et al., 1991; Woodland et al., 1991;
Dyson et al., 1991). The Gag protein of mouse leukemia virus, the causative
agent of mouse acquired immunodeficiency syndrome (MAIDS), has been
shown to possess superantigenic properties (Hugin et al., 1991). A
superantigen has been implicated in the pathogenesis of rabies virus (Lafon et
al., 1992) and recently the superantigenic viral component has been identified
as the nucleocapsid protein (Lafon et al., 1994). It has been suggested that
human immunodeficiency virus (HIV) encodes for a superantigen that may, at
least in part, be responsible for the immunopathogenesis associated with
infection (Laurence et al., 1992). Recent evidence points to a superantigen
encoded by human cytomegalovirus (CMV) that expands specific Vp-bearing T
cells, thereby enhancing HIV replication in CMV/HIV-infected individuals
(Dobrescu et al., 1995). Although several reports suggest that other viruses,
such as Epstein-Barr virus and human foamy virus, encode for superantigens,
to date no hard evidence has been obtained.
The possible harmful consequences of activation by superantigens make
this class of molecules relevant to human disease. The prodigious expansion
of T cells having diverse specificities may be important in the induction and
establishment of autoimmunity. Concomitant cytokine production may also
have deleterious effects. The potential for loss of immune function via
mechansisms such as anergy or apoptosis can also result in immunodeficiency
diseases. Superantigens have been shown to be encoded by retroviruses,
suggesting that they may play a role in the syndrome associated with human

5
Table II.
List of viral superantigens implicated in disease.
Virus
(Family or Subfamily)
Superantigenic
Protein
Disease/Effect3
Prototype: Superantigen from Mouse Mammary Tumor Virus
Mouse mammary tumor virus
vsag gene product
Mammary tumors
(oncornavirus)
Mouse leukemia virus
Gag protein
Murine AIDS
(oncornavirus)
Human immunodeficiency virus
Not identified
AIDS
(lentivirus)
Human foamy virus
bel3 gene product
Grave's disease
(?>b
(spumavirus)
Rabies
Nucleocapsid
Rabies
(rhabdovirus)
Epstein-Barr virus
Not identified
B cell lymphoma
(?)
(herpesvirus)
Chronic fatigue
Cytomegalovirus
Not identified
syndrome (?)
Enhanced HIV
(herpesvirus)
replication
aJohnson et al. 1995.
^Denotes diseases in which superantigens have been implicated, but no direct
evidence is available.

6
immunodeficiency virus. Thus, superantigens may be involved in disease
processes and, as such, can be classified as virulence factors.
Bacterial Superantiaens
In the 1970s, staphylococcal enterotoxins were recognized as inducers
of lymphocyte proliferation (Peavy et al., 1970). Responses to staphylococcal
enterotoxin A (SEA) stimulation were shown to be T cell specific (Johnson and
Bukovic, 1975), with high levels of gamma interferon (IFNy) produced by this
lymphocyte fraction (Langford et al., 1978a). SEA was shown to have
mitogenic effects on human lymphocytes at extremely low concentrations,
making it one of the most potent T cell activators known (Langford et al., 1978a).
The ability to produce large quantities of IFNy using SEA paved the way for the
characterization of the key immunomodulatory properties of this cytokine
(Johnson, 1985). Interestingly, intravenous administration of SEA to mice
resulted in the induction of suppressor cell activity that was present in spleen as
early as 24 hours after injection (Johnson, 1981; Torres et al., 1982). SEA-
induced suppressor cells were capable of blocking naive spleen cells from
responding to SEA stimulation. Thus, SEA was shown to stimulate T cells to
proliferate and produce cytokines, and to induce suppressor cell activity.
Further investigations on the interaction of bacterial enterotoxins with
human lymphocytes revealed that antigen-presenting cells are required for
proliferative effects and that binding to major histocompatibility (MHC) class II
antigens occurred (Carlsson et al., 1988; Fleischer and Schrezenmeier, 1988;
Mollick et al., 1989). Unlike most antigens which interact with MHC class II
molecules as peptides, the staphylococal enterotoxins were shown to bind in
an unprocessed form (Fleischer and Schrezenmeier, 1988). The site of

7
interaction of enterotoxins on the MHC class II molecule was shown to be
distinct from conventional antigens in that binding occurs distal to the peptide
antigen binding groove (Russell et al., 1990; Dellabonna et al., 1990). Thus,
this class of antigens differs significantly from classic antigens and were given
the designation of superantigens (Marrack and Kappler, 1990).
The study of staphylococcal enterotoxins has led to a wealth of
information on the interaction of superantigens with MHC class II molecules.
Through the use of synthetic peptides, regions of interaction of SEA and MHC
class II antigens have been identified. Regions on the SEA molecule that are
involved in binding to MHC class II molecules on antigen presenting cells
include the N-terminus and three internal sequences (Griggs et al., 1992). A
peptide corresponding to the N-terminus of SEA, SEA(1-45), blocked
proliferation of cells in response to whole native SEA (Pontzer et al., 1990).
Interestingly, a peptide corresponding to another of these MHC class ll-binding
regions, SEA(121-149), has agonist properties in that it induces cytokine
production (Pontzer et al., 1993) and proliferation (A.C. Hobeika, personal
communication). Conversely, sites on MHC class II molecules which bind
superantigens have been identified. Regions on mouse MHC class II
molecules that interact with SEA have been identified and include the a-helical
region of the p chain associated with the peptide binding groove encompassed
by amino acids 65-85 (Russell et al., 1991). A similar site has been found on
human MHC class II antigens (Herman et al., 1991). A superantigen-binding
region on the a helical region of the a chain associated with the peptide binding
groove of MHC class II has also been defined (Russell et al., 1991). Both of
these binding sites reside outside of the peptide binding groove and are
required for SEA-induced mitogenesis. Thus the two outside faces of the a
helices of the peptide binding groove are involved in binding of SEA.

8
Presentation of superantigen in the context of MHC class II molecules is
required for interaction with T cell receptor (TCR). For this reason superantigen
sites that interact with TCR have been more difficult to elucidate. However,
through the use of synthetic peptides, a TCR site that interacts with SEA has
been identified. A peptide corresponding to residues 57-77 of mouse Vp3
blocked SEA-induced proliferation and IFNy production (Pontzer et al., 1992).
Vp3-bearing mouse T cells are known to be stimulated by SEA. Future studies
may help determine the sequences involved in the formation of the ternary
complex of superantigen:MHC:TCR. Such studies are needed to determine
how superantigens activate T cells bearing specific Vps and induce anergy and
deletion of these Vp subsets.
Retroviruses
Retroviruses belong to a family of viruses that are characterized by the
use of a unique RNA-dependent DNA polymerase, reverse transcriptase.
Reverse transcriptase, in conjunction with another virally-encoded enzyme,
RNAse H, transcribes the single-stranded RNA genome into a double-stranded
linear DNA provirus. This DNA intermediate is then capable of integrating into
the host genome. Retroviruses were first described in studies in which cell-free
filtered extracts were shown to transmit sarcoma to chickens (Rous 1910, 1911).
Since that time, retroviruses have been found in several vertebrates including
mice, cats, and primates. The first human retrovirus to be discovered was
human foamy virus, which has been speculated to cause disease (Achong et
al., 1971), although definitive proof of pathogenicity is lacking. In 1980, the
causative agent of adult T-cell leukemia was discovered, human T-cell

9
leukemia virus (Poiesz et al., 1980; Yoshida et al 1982), and established this
family of viruses as human pathogens.
The retrovirus family consists of three subgroups based on similar
pathogenicities: oncornaviruses, lentiviruses, and spumaviruses.
Spumaviruses or foamy viruses are the least characterized of the three
subfamilies of retroviruses, and have been grouped according to their ability to
cause vacuolation of infected cells in vitro, which gives the cells a foamy"
appearance. Spumaviruses have been found in primates and humans, and are
generally considered benign, although data suggest these viruses may play a
role in human disease. Oncornaviruses are tumor-causing retroviruses, and
members of this subfamily include the avian leukosis-sarcoma viruses, mouse
mammary tumor virus, feline leukemia virus, and human T-cell leukemia virus.
As a result of proviral integration into the host genome, tumors arise by
upregulation of host genes that encode for growth factors or by retroviral-
encoded oncogenes.
Viruses belonging to the lentiviral subfamily include the "slow" viruses
maedi/visna and equine infectious anemia virus. More recently, the viruses that
cause human and feline acquired immundeofiency syndromes have been
classified as lentiviruses, based on several parameters including genomic
complexity and virion morphology. Unlike oncornaviruses, lentiviruses have
not been implicated directly in causing neoplastic disease. Members of the
lentiviral subfamily cause long-term disease characterized by autoimmunity,
encephalopathy, immunodeficiency, or a combination thereof. Lentiviruses are
considerably more complex than some of the other retroviral subfamilies, in that
the level of gene regulation is much greater.

10
Mouse Mammary Tumor Virus Superantiqen
The first viral superantigens to be described are the proteins encoded by
the open reading frame that overlaps the 3' long terminal repeat of the
oncornavirus, mouse mammary tumor virus (MMTV) (Marrack et al., 1991;
Frankel et al., 1991; Woodland et al., 1991; Dyson et al., 1991). Although
MMTV superantigens were recognized in the 1990s, they were originally
described by Festenstein in the early 1970s as minor lymphocyte stimulating
(mis) antigens (Festenstein, 1973). The ability of T cells from certain mouse
strains to be stimulated by lymphocytes from MHC-identical strains was
ascribed by Festenstein to the presence of mis antigens. More recently, mis
antigens were found to be endogenous superantigens from germline-encoded
MMTV provirus (Choi et al., 1991; Acha-Orbea et al., 1991). Like their bacterial
counterparts, MMTV superantigens are thought to be presented in the context of
MHC class II antigens. MMTV superantigens are known to stimulate T cells in a
Vp-specific fashion (Acha-Orbea and Palmer, 1991). MMTV superantigens
show MHC preference, with more efficient presentation occurring in the context
of l-E as opposed to l-A, although presentation by l-A does occur (MacDonald et
al., 1989). Interestingly, this MHC preference corresponds with the greater
infectivity of B cells bearing l-E (Held et al., 1994b).
MMTV is a type B retrovirus that was determined to be the causative
agent for the induction and transmission of mammary carcinomas in mice
(Heston et al., 1945). It has been known for several years that infectivity by
MMTV requires an intact immune system (Tsubura et al., 1988), and only
recently has this paradox been explained (Held et al., 1993, 1994a).
Transmission of MMTV to offspring can occur via infectious virions in mother's
milk or can occur vertically as endogenous provirus. Most mouse strains have

11
been shown to contain one or more copies of endogenous MMTV, and many
contain distinct MMTV strains (Salmons and Gunzburg, 1987). Upon passage
into the gut of the host, virus enters the gut-associated immune tissue and
infects B cells. B cells then express the MMTV-encoded superantigen,
presumably in the context of class II antigens, causing Vp-specific T cell
stimulation. Although investigators have been able to show that actual infection
of T cells occurs, it is thought that T cell stimulation and subsequent cytokine
production by these cells indirectly enhances the further infection of B cells
(Held et al., 1994a). Infected B cells migrate to the main site of viral infection,
the mammary gland. Epithelial cells then become infected and are the source
of infectious virions that are transmitted in milk (Ringold, 1983). Proviral
integration can occur during infection of mammary epithelial cells, resulting in
tumors.
Germline-encoded MMTV serves an important protective mechanism for
the host. It is known that bacterial superantigens can cause anergy and/or
deletion of Vp-specific T cells (Johnson et al., 1992). Similarly, expression of
MMTV superantigen early in the ontogeny of the immune system induces the
eventual deletion of T cells bearing Vps specific for that particular strain of
MMTV superantigen. In this manner, T cells that would otherwise be stimulated
are lost and the host is protected against subsequent infection by MMTV strains
that stimulate those Vp-specific T cell populations. This has been shown in
mice transgenic for a MMTV superantigen that stimulates Vpl4+ T cells
(Golovkina et al., 1992; Acha-Orbea, 1991a). Transgenic mice showed partial
to complete deletion of Vpi4+ T cells, depending on the level of superantigen
expression. Those mice in which Vpl4 + T cells were deleted were
subsequently protected from infection upon challenge with the same MMTV
strain.

12
Evidence for a Superantiaen Associated with Human Immundeficiencv Virus
In the early 1980s, unusual incidences of young homosexual males
stricken with Pneumocystis carinii pneumonia and Kaposi's sarcoma were
reported to the Centers foe Disease Control. These reports led to the first
description of acquired immunodeficiency syndrome (AIDS). Subsequently,
similar immunodeficiency-associated illnesses were reported among
hemophiliacs, recipients of blood and blood components, and intravenous drug
users and their heterosexual partners. The incidence of AIDS in these distinct
populations was suggestive of an infectious agent, and this was confirmed
when, in 1984, human immunodeficiency virus (HIV) was isolated from the
blood of an AIDS patient (Klatzmann et al., 1984; Gallo et al., 1984). With the
advent of diagnostic assays, the number of infected individuals was found to
exceed the number of patients afflicted with AIDS, indicative of a serious
epidemic. The number of deaths is expected to increase, as asymptomatic HIV-
infected individuals progress to AIDS.
HIV is a member of the lentivirus subfamily of retroviruses. Infection with
HIV is associated with a debilitating and eventually fatal immunodeficiency. In
addition to immunologic impairment, neurological dysfunctions can occur,
including encephalopathies, sleep disorders, and dementia (the latter is also
referred to as HIV-associated cognitive/motor complex).
The size of the HIV genome is approximately 9.8 kbases. The genome
encodes for a total of 16 proteins, some of which are post-translationally
cleaved by either cellular or viral proteases. The primary HIV transcript is gag-
pol mRNA, which is translated to yield Gag (group antigen) and Pol
(polymerase). Synthesis of Gag-Pol occurs at a ratio of 20:1 (Oroszlan and
Luftig, 1990). Gag is proteolytically cleaved by a viral-encoded protease,

13
yielding 5 distinct proteins. Cleavage of Pol results in 2 proteins, one of which is
reverse transcriptase. Two envelope (Env) proteins are the cleavage products
from an initial Env precursor. In this case, the precursor is cleaved by a cellular
protease. Two regulatory proteins, Tat (transactivator) and Rev (regulator for
viral expression) are translated from multiply-spliced mRNA transcripts.
One putative regulatory protein, Nef (negative factor) is encoded by a
gene that overlaps the terminal repeat at the 3' end of the HIV genome. It was
initially named based on its apparent negative effects on viral transcription and
replication in vitro (Terwillegar et al, 1986). However, subsequent reports have
been conflicting, and have suggested that Nef has no effect (Hammes et al.,
1989) or a positive effect (DeRonde et al., 1992) on HIV transcription. Such
widely disparate Nef effects may, in part, be explained by the fact that several
immortalized human T cell lines and primary human lymphocytes were used in
these studies. These data may reflect inherent differences in primary cells and
cell lines, such as activation signals. Data from studies on Nef-transfected T
cells suggest that Nef may play a role in down-regulation of the CD4 molecule
(Garcia and Miller, 1991; Mariani and Skowronski, 1993).
HIV-1 Nef is a 25-27K protein and is myristylated at the N-terminus.
Myristylation is thought to be a mechanism by which Nef associates with the
cytoplasmic membrane (Franchini et al., 1986; Guy et al., 1987). Nef primarily
localizes to the cytoplasm (Franchini et al., 1986) but has been shown to be
present on the surface of infected cells (Fujii et al., 1993). Antibodies to Nef
appear early in HIV-infected individuals (Allan et al., 1985) and cytotoxic T
lymphocyte activity has been detected against cells presenting Nef peptides
(Bahraoui et al., 1990). These data confirm that Nef is expressed during active
HIV infection.

14
A hallmark of infection with HIV is the alteration of CD4/CD8 T cell ratios
due to loss of CD4+ T helper cells. Such losses ultimately result in the inability
of an infected individual to mount effective immune responses against
opportunistic infections, thus resulting in death. Several possible mechanisms
by which CD4 loss occurs have been postulated, including direct cytolysis by
HIV (Lemaitre et al., 1990), HIV-induced syncitia formation (Lifson et al., 1986;
Sodroski et al., 1986) and/or cytolytic T cell activity against infected CD4 cells
(Zinkernagel and Hentgartner, 1994). In addition to skewing of the CD4/CD8
ratios, other immunologic alterations seen in HIV-infected individuals include
polyclonal activation of B cells with increased immunoglobulin production and
reduced antigen and mitogen responses (Edelman and Zolla-Pazner, 1989).
Initial increased natural killer cell activity is observed in asymptomatic HIV-
infected individuals, but these activities decrease during disease progression
(Edelman and Zolla-Pazner, 1989).
Because of the known induction of T cell anergy and/or deletion by
superantigens (Johnson et al., 1992), it has been speculated that the HIV
genome encodes for a superantigen that may cause some of the
immunopathologies observed with AIDS. Upon interaction with MHC class II
antigens and TCR, Vp-specific T cell populations would be activated and
expand, eventually leading to functional (anergy) or actual (deletion) loss of T
cells. Several points of evidence suggest a role for an HIV-derived
superantigen. Although initial studies suggested that AIDS patients had
deletions in Vp T cell populations (Imberti et al., 1991), such deletions could not
be found by others (Laurence et al., 1992). Vpl2+ T cells were shown to
support enhanced replication of HIV compared to other Vp subsets and
proliferated in response to cells from HIV+ patients, suggesting the presence of
a superantigen (Laurence et al., 1992). Asymptomatic patients exhibit altered

15
Vp profiles (Dalgleish et al., 1992) as well as skewed T cell Vp usage upon
stimulation with staphylococcal enterotoxins in vitro (Bisset et al., 1993).
Interestingly, one study found skewed Vp T cell profiles in monozygotic twins
that were discordant for HIV infection, with perturbations in several Vps (Rebai
et al., 1994). This last study is of particular importance in that the altered Vp
profiles in infected individuals cannot be ascribed to differences in MHC, since
only identical twins were used. Thus, these studies hint at the presence of a
superantigen encoded by HIV that may play a role in HIV pathogenesis by
augmenting a population of T cells, resulting in a reservoir for virus replication,
with eventual deletion of those T cells.

>
CHAPTER II
MMTV SUPERANTIGEN BINDING TO MHC CLASS II ANTIGENS
Introduction
SEA is one of the most potent T-cell mitogens known, and has been
classified as a bacterial superantigen based on its ability to stimulate Vp-
specific T-cell subsets (Johnson et al., 1991). Concurrent with studies on SEA,
minor lymphocyte stimulating (mis) antigens have recently been shown to be
products of MMTV (Choi et al., 1991; Acha-Orbea et al., 1991). Two exogenous
strains of MMTV encode for retroviral superantigens in genes that overlap the
terminal repeat at the 3' end of the viral genome (Pullen et al., 1992; Choi et al.,
1992). These genes have been designated vsag, denoting that they encode for
viral superantigens (Marrack and Kappler, 1990). No direct binding of the
putative MMTV superantigen to either MHC antigens or TCR has been shown.
To determine sites that interact with class II MHC antigens, overlapping
synthetic peptides were synthesized that encompass the putative extracellular
domain of MMTV-1 superantigen. The data presented here indicate that a site
that is encompassed by amino acid residues 76-119 of MMTV-1 superantigen
competes with SEA for binding to class II MHC antigens. Further, direct binding
studies show that this region of the MMTV superantigen binds directly to class II
MHC antigens. These data indicate that SEA and MMTV superantigen share at
least one common binding region on class II MHC molecules.
16

17
Materials and Methods
Synthetic Peptides
Overlapping peptides corresponding to the putative extracellular domain
of the MMTV-1 vSAg protein (Pullen et al., 1992) were synthesized with a
Biosearch 9500AT automated peptide synthesizer using N-(9-
flurenyl)methoxycarbonyl chemistry (Griggs et al., 1992). Peptides were
synthesized based on a surface profile which uses a composite of three
parameters: 1) HPLC hydophilicity; 2) accessibility; and 3) segmental mobility
(B value) (Parker et al., 1986). The sequences of the peptides are listed in
Table III. Peptides were cleaved from the resins using trifluoroacetic acid/
ethanedithiol/ thioanisole/ anisle at a ratio of 90:3:5:2. The cleaved peptides
were then extracted in ether and ethyl acetate and subsequently dissolved in
water and lyophilized. Peptides were extensively dialyzed against water to
remove the remaining cleavage products. Amino acid analysis of the peptides
showed that the amino acid contents and molecular weights corresponded
closely to theoretical values. Peptides were not purified further since reverse
phase HPLC analysis of crude peptides indicated one major peak in each
profile.
Cell Lines and Reagents
The A20 cell line (ATCC, Gaithersburg, MD) was used for MMTV
superantigen binding studies. A20 cells are a BALB/c B lymphoma line that
expresses la. Highly purified SEA and other staphylococcal enterotoxins were
obtained from Toxin Technology (Sarasota, FL). Several monoclonal
antibodies (mAb) were used in this study. MAbs were purchased from
Accurate Chemicals, Westbury, NY. MAb Kd (34-1-2S) is specific for m31
determinant of Kd (Ozato et al., 1982). MAb IAd (MK-D6) is specific for the p1

Table III.
Amino acid sequences of vSAg peptides
vSAg Peptide
Sequence
vSAg( 76-119)
DSFNNSSVQDYNLNDSENSTFLLGQGPQPTSSYKPHRLCPSEIE
vSAg(117-147)
EIEIRMLAKNYIFTNETNPIGRLLIMMLRNE
vSAg(142-172)
MMLRNESLSFSTIFTQIQRLEMGIENRKRRS
vSAg(166-195)
ENRKRRSTSVEEQVQGLRASGLEVKRGKRS
vSAg(190-222)
KRGKRSALVKIGDRWWQPGTYRGPYIYRPTDAP
vSAg(220-250)
DAPLPYTGRYDLNFDRWVTVNGYKVLYRSLP
vSAg(245-276)
LYRSLPFRERLARARPPWCVLSQEEKDDMKQQ
vSAg(274-313)
KQQVHDYIYLGTGMIHWKVFYNSREEAKRHIIEHIKALP
vSAg(76-119)
(scrambled)
PNSNEGLSQQSTDPSPHNFILSNENSYPCYSLLGDVQREDSTKF
The vSAg(76-119) scrambled peptide sequence was generated using the sequence edit
program (Devereax et al., 1984).

19
helix of l-Ad (Lee and Watts, 1990). All mAb were used at a final concentration
of 10 [ig/ml. MAbs used in this study had similar potencies as determined by
the Hybridoma Core Facility, Interdisciplinary Center for Biotechnology
Research, University of Florida. Polyclonal antibodies were used at a final
dilution of 1:100. Poly IAd (Cat.Y1-9-04-26-01) is specific for the m11
determinant of l-Ad and poly la.7 (Cat.YI-7-02-02-01) is specific for the m7
determinant of l-Ed. Both antibodies were obtained from NIAID, Rockville, MD.
Poly l-A^ was obtained from Accurate Chemicals, Westbury, NY.
Radioiodinations
SEA (2.5 jig) and vSAg peptide (10 pig) were radiolabeled using
chloramine T as described (Griggs et al., 1992). Briefly, ligands were labeled
with 500 pCi of Na125l (15 mCi/pg, Amersham Corp., Arlington Heights, IL) in
25 pi of 0.5 M potassium phosphate buffer, pH 7.4, and 10 pi of chloramine T
(5 mg/ml) for 2 min. After neutralization of the reaction with 10 pi each of
sodium bisulfite (10 mg/ml), potassium iodide (70 mg/ml), bovine serum
albumin (BSA; 20 mg/ml), and 15 pi of NaCI (4 M), the preparation was sieved
on a 5 ml Sepharose G-10 column. The two fractions with the highest
radioactivity in the first eluted peak were pooled and used in the radiolabeled
binding assays. The specific activities of the SEs and vSAg peptide ranged
from 130-150 pCi/pg and 30-60 pCi/pg.
Binding Studies
In binding studies using A20 cells, unlabeled competitors (SEs and
vSAg peptides) were added in 50 pi volumes (in PBS with 1% BSA) at
indicated final concentrations to 50 pi of cells in "Eppendorf" tubes.
Competitors were incubated with cells at room temperature for 45 minutes,

20
followed by the addition of radiolabeled SEs or vSAg peptide. After 45 minutes,
the cells were washed three times and the radioactivity associated with the cell
pellets was quantified using a gamma counter. L cells were used as an MHC
class ll-negative control cell line.
L cells were grown to confluence in the wells of microtiter plates and
were subjected to the same incubation times and volumes of competitors and
labeled ligands as used for A20 cells. After three washes, the cells were
solubilized in 1% SDS, the liquid was absorbed by cotton-tipped applicators,
and radioactivity was quantified using a gamma counter.
Radioimmunoassay
Two MHC peptides l-Apb(30-60) and l-Apb(60-90) were used to for
binding of SEA and ORF(76-119) to class II antigens. The l-A(3b(60-90) site was
previously shown to be involved in SEA binding to MHC class II antigens
(Russell et al., 1990). MHC peptides were dissolved in PBS at a concentration
of 25 pg/ml. Peptide solutions were pipetted into polystyrene plastic tubes and
tubes were placed at 10C for 4 hours to allow adherence of the peptides. The
tubes were washed three times with PBS. Nonspecific sites on the plastic were
blocked using 2 ml of PBS containing 1% BSA at 10C overnight. After the
tubes were washed three times of PBS containing 1% BSA, unlabeled
competitors (SEA and vSAg peptide) were added in 0.1 ml and allowed to bind
at room temperature for 3 hours. The tubes were washed three times with 2 ml
of PBS/1 % BSA prior to addition of 0.1 ml of either radiolabeled SEA or
radiolabeled vSAg(76-119)peptide, at final concentrations of 2 nm and 5 nm,
respectively. Radiolabeled ligands were allowed to bind to MHC peptides at
room temperature for 4 hours. After washing three times with 2 ml of PBS
containing 1% BSA, the tubes were placed in a gamma counter and bound

21
radioactivity was quantified. Experiments were performed at least four times
each using replicates of three.
Results
Competition of vSAg Peptides with Radiolabeled SEA for MHC Binding
Overlapping peptides (referred to as vSAg peptides) corresponding to
the predicted extracellular domain of the MMTV-1 superantigen were
synthesized and their sequences are listed in Table III. The vSAg peptides
were initially tested at a concentration of 200 pM for their relative abilities to
compete with 125I-SEA for binding to A20 cells, which express l-Ad and l-Ed
(which are MHC class II antigens of the d haplotype, A isotype and E isotype,
respectively). The vSAg(76-119) peptide reduced 125I-SEA binding to A20
cells by 63%, while the other peptides had no effect (Figure 1). Thus, only one
of the overlapping vSAg peptides, vSAg(76-119), significantly blocked the
binding of 125I-SEA binding to A20 cells.
vSAa(76-1191 Peptide Competition Is Dose-Dependent
Dose response studies were performed on A20 cells with several vSAg
peptides, including vSAg(76-119). The vSAg(76-119) peptide reduced ^SI
SEA binding by 50% at a concentration of 20 pM (Figure 2). Further, vSAg(76-
119) peptide consistently competed with 125I-SEA in a manner similar to
unlabeled SEA, although SEA was 20 times more effective. Unlabeled SEA at
a concentration of 1 pM reduced 125I-SEA binding to A20 cells by 50%. These
data are consistent with the reported Kd for SEA binding to l-Ed of
approximately 10'6 M (Lee and Watts, 1990). The peptide corresponding toa

22
160
140-
76-119 117-147 142-172 166-195 190-222 220-250 245-276 274-313
vSAg peptides
Figure 1. Ability of vSAg peptides to compete with 125I-SEA for binding to A20
cells. Binding of 125I-SEA in the absence of competitors was 6384+400 CPM.
The data presented represent the mean of three individual experiments each
performed in duplicate. Each bar represents the mean percent of SEA control
binding in the presence of vSAg peptides SD.

% Control
23
Competitor (|iM)
Figure 2. Ability of vSAg(76-119) to compete with 125I-SEA for binding to A20
cells in dose response studies. 125I-SEA was used at 2.5 nM. Binding of ^SI
SEA to A20 cells in the absence of competitors was 5184380 CPM. The data
presented represent the mean of three individual experiments, each performed
in duplicate. Each point represents the mean percent of SEA control binding in
the presence of competitors SD.

24
C-terminal region of the MMTV superantigen, vSAg(245-276), was not a
consistent competitor for SEA binding. The peptide corresponding to the C-
terminal tail, vSAg(274-313), did not compete. A peptide having the same
amino acid content as vSAg(76-119) but in a scrambled sequence (see Table
III for sequence) did not compete, thus indicating that vSAg(76-119) peptide
competition was sequence specific. No direct binding of 125l-vSAg(76-119) to
SEA or to related superantigens was detected (data not shown). Thus, the
vSAg(76-119) peptide binds specifically to A20 cells, and competes for SEA
binding in a dose-dependent manner.
Direct Binding of vSAa(76-119) Peptide to A20 Cells
In direct binding studies, 125l-vSAg(76-119) peptide bound to A20 cells
and was effectively inhibited by both unlabeled SEA and unlabeled vSAg(76-
119) peptide (Figure 3). Unlabeled SEA and unlabeled vSAg(76-119) peptide
competed with 125l-vSAg(76-119) in a similar manner, although SEA was a
more potent competitor. SEA reduced 125l-vSAg(76-119) binding by 50% at a
concentration of 1.8 pM as compared to 25 pM for unlabeled vSAg(76-119)
peptide. Neither vSAg(76-119) scrambled peptide nor the C-terminal vSAg
peptide competed, thereby showing that vSAg(76-119) binding was sequence
and region specific. TSST-1 did not compete with 125l-vSAg(76-119) peptide
for binding to A20 cells, while SEB competed less effectively than SEA, which is
consistent with their relative abilities to compete with SEA for MHC class II
binding (Fraser, 1989; Pontzer et al., 1991). Thus these results indicate that
binding of vSAg(76-119) peptide occurs to murine MHC class II antigens.
Binding of vSAg(76-119) may occur at a region(s) to which SEA also binds or to
a neighboring site which interferes with the binding of SEA.

25
Figure 3. Ability of SEA, SEB, and TSST-1 to compete with 125l-vSAg(76-119)
for binding to A20 cells. Experimental conditions were the same as those
described in Figure 1. 125l-vSAg(76-119) was used at a final concentration of
2.3 nM. Binding of 125l-vSAg(76-119) in the absence of competitors was
2278224 CPM. The data presented represent the mean of three individual
experiments, each performed in duplicate. Each point represents the mean
percent of vSAg control binding in the presence of competitors SD.

26
Evidence that vSAa(76-1191 Binds Specifically to MHC Class II Antigens
Two further pieces of evidence indicate that vSAg(76-119) binds to class II
MHC antigens. Binding of vSAg(76-119) to class II negative mouse L cells was
insignificant as compared to mouse A20 cells even at concentrations as high as
5 nM (Figure 4A). Monoclonal antibodies to class I MHC antigens had no
effect on 125l-vSAg(76-119) binding to A20 cells (Figure 4B). Polyclonal anti-l-
A^ and anti-l-Eb significantly blocked binding and, in combination, these
antibodies reduced binding to A20 cells by 73%. These data suggest that
vSAg(76-119), like SEA (Lee and Watts, 1990), binds to both l-A and l-E. An I-
Ab p1 helix-specific monoclonal antibody reduced binding by approximately
30%, suggesting that this is a region on l-A to which vSAg(76-119) binds.
Polyclonal antibodies to l-A^ had minor effects on vSAg(76-119) binding. Thus,
these data indicate that vSAg(76-119) binds to class II MHC antigens.
In order to directly determine if vSAg(76-119) binds to the pi helix of l-A,
a competitive radioimmunoassay was performed in which 125I-SEA and 125l-
vSAg(76-119) were tested for their relative abilities to bind to l-Apb(60-90)
peptide. SEA and vSAg(76-119), but not scrambled VSAG(76-119), competed
with both 125I-SEA and 125l-vSAg(76-119) for binding to l-Apb(60-90) in a
manner similar to the competition seen on whole cells (Figure 5A). As
previously shown for SEA (Russell et al., 1990, 1991), vSAg(76-119) did not
directly bind to l-Apb(30-60) (Figure 5B). Thus, SEA and vSAg(76-119) bind to
a similar region on the p chain of the class II MHC molecule.
Discussion
To date, no information has been available on the ability of MMTV vSAg
protein to bind to MHC antigens and, in fact, this ability has been questioned
(Marrack and Kappler, 1990; Acha-Orbea and Palmer, 1991; Acha-Orbea et al.,

27
6000
0 200
Concentration of ORF(76-119)
OiM)
Antisera
Figure 4. Evidence that vSAg(76-119) peptide binds to MHC class II antigens,
and does not bind to class I antigens.
Panel A: Binding of 125l-vSAg(76-119) to class ll-positive A20 cells and class
ll-negative L cells. 125l-vSAg(76-119) was used at a final concentration of 5
nM.
Panel B: Blockage of 125l-vSAg(76-119) binding to A20 cells by antibodies to
class I and class II MHC antigens. 125l-vSAg(76-119) was used at a final
concentration of 5 nM. MAbs were used at a final dilution of 1:30.

28
Figure 5. Binding of 125l-vSAg(76-119) to MHC class II peptides.
Panel A: Binding of 125I-SEA and 125l-vSAg(76-119) to l-Apb(60-90) and
inhibition by vSAg(76-119). Each point represents the mean percent reduction
of control binding in the presence of competitors SD. Binding of 125I-SEA
and 125l-vSAg(76-119) in the absence of competitor was 265692 CPM and
154733 CPM, respectively.
Panel B: Relative ability of vSAg(76-119) to bind l-Ab(60-90) and l-Apb(60-90)
in the presence and absence of vSAg(76-119). Unlabeled and 125l-vSAg(76-
119) were used at final concentrations of 5 nM and 300 pM, respectively.

29
1991). Such binding studies are hampered by the difficulties inherent in
expressing the MMTV superantigen. A model has recently been proposed that
suggests that MMTV superantigens may act in the same or similar fashion to
bacterial superantigens by bridging MHC antigens and TCR on the appropriate
cell types (Acha-Orbea and Palmer, 1991). MMTV superantigens, presented in
the context of MHC class II antigens, may act by stimulating and expanding Vp-
specific T cell subsets, thereby indirectly enhancing the further infection of B
cells (Held et al., 1994a). The results presented in this chapter suggest that
MMTV vSAg protein binds to MHC antigens, thereby strengthening the
argument that bacterial and retroviral superantigens act in a similar manner.
It has been reported that MMTV superantigen is a 45 kd Type II integral
membrane protein with an intracellular N-terminus, an essential hydrophobic
transmembrane region near the N-terminus (residues 45-64), and a
glycosylated extracellular C-terminus (Choi et al., 1992). Although no direct
evidence exists, the variability of the C-terminal residues of vSAg proteins of
various MMTV strains seems to correlate with their differences in Vp specificity,
lending support to the concept that this region binds TCR (Pullen et al., 1992).
Truncated versions of the vsag gene were transfected into MHC class ll-bearing
cells and tested for superantigen activity. Complete loss of superantigen
activity occurred when the MMTV vsag gene was N-terminally truncated to the
third methionine (residue 122) and beyond (Choi et al., 1992). The authors
concluded that a hydrophobic region, which was missing in this N-terminally
truncated version, acts as a transmembrane region and is essential for
superantigen activity. However, our binding data suggest that loss of
superantigen activity may have been due, at least in part, to the deletion of the
MHC-binding domain which is encompassed by residues 76-119. In fact, the

30
superantigen activity of MMTV vSAg protein was lost in truncations that did not
contain the region encompassed by residues 76-119.
Subsequent studies by the same group of investigators suggest that
MMTV-7 superantigen is synthesized as a precursor molecule, is proteolytically
cleaved at an internal site (- residue 164) and is expressed as an 18.5 kd
surface protein consisting of the C-terminal residues (Winslow et al., 1992).
Membrane association of this truncated form may involve "tethering" to MHC
class II antigens or to the N-terminal portion of the precursor molecule. As a
cautionary note, the transfected cell line used to characterize this truncated form
of the MMTV-7 superantigen was shown to have high expression of the protein
but low activity. Conversely, a transfected cell line with moderate superantigen
expression and high activity was not used for characterization.
Recently, studies were reported on the binding of truncated forms of
MMTV-7 superantigen to human MHC class II antigens. Binding studies were
performed with 28K and 18K versions of the superantigen and showed that
both molecules bind to human MHC class II antigens (Mottershead et al., 1995).
Thus, studies by our laboratory indicate one site for binding to mouse MHC
class II antigens, whereas studies performed by others suggest that two sites on
MMTV superantigen are involved in binding to human MHC class II antigens.
Despite the diverse origins of SEA and MMTV vSAg protein, it is likely
that these two proteins exert superantigen activity by a similar mechanism. Two
different regions of the MMTV superantigen are probably involved in MHC
binding and Vp specificity. The variability of the C-terminal 30 residues
between Vp-specific MMTV superantigens is indirect evidence that the C-
terminus is responsible for Vp specificity (Pullen et al., 1992). Data presented
here on competitive and direct binding of vSAg peptides to A20 cells suggest
that the region encompassed by residues 76-119 is involved in MHC binding.

31
This region would be part of the extracellular domain based on a proposed
Type II membrane protein model for MMTV vSAg protein (Choi et al., 1992).
Thus, both the C-terminal tail and an N-terminal region may be required for
ternary complex formation with MHC and TCR by MMTV superantigen.
Conversely, SEA contains several N-terminal domains that bind MHC (Griggs
et al., 1992) and an internal domain that may be involved in binding to TCR, so
that the sites of interaction of these superantigens with MHC and TCR may not
be completely analogous. Although little sequence homology exists between
MMTV vSAg protein and SEA, circular dichroism analysis indicates that both
SEA and vSAg(76-119) have significant p structure, suggesting that similar
structural motifs may be important for MHC binding by superantigens.
Finally, results presented herein indicate that a peptide corresponding to
residues 76-119 of the MMTV superantigen binds directly to MHC class II
antigens. Further, competition studies indicate that SEA and vSAg(76-119)
peptide bind to at least one common region on mouse MHC antigens. Future
studies using synthetic peptides and peptide analogues may help further
elucidate the site(s) on MHC for which SEA and MMTV superantigen compete.

CHAPTER III
IDENTIFICATION OF AN HIV-1 NEF PEPTIDE THAT BINDS TO
MHC CLASS II ANTIGENS
Introduction
In Chapter Two, a site was identified on the MMTV-1 superantigen that
binds to class II MHC antigens, suggesting that retroviral and bacterial
superantigens exert their effects similarly. MMTV superantigen is encoded in a
gene that overlaps the terminal repeat at the 3' end of the viral genome. The
genome of human spumavirus also contains a gene {bel3) that overlaps the
terminal repeat at the 3' end, the product of which may have superantigenic
properties (Fluegel, 1993). Recently, it has been suggested that human
immunodeficiency virus (HIV) may possess superantigen activity, although no
specific superantigenic protein has been identified (Imberti et al., 1991;
Laurence et al., 1992; Dalgleish et al., 1992; Bisset et al., 1993). The HIV
genome also contains a gene that overlaps the terminal repeat at the 3' end,
the product of which is called Nef. Although Nef is one of the early proteins
produced during the replication of primate lentiviruses, the role of Nef in HIV
pathogenesis has yet to be established.
To determine if Nef had binding characteristics similar to superantigens,
a study was undertaken to determine its ability to bind to MHC class II antigens,
at sites that are known to bind superantigens. Using overlapping peptides
corresponding to the entire length of Nef (HIVlav). we have identified a region
which binds to MHC class II antigens, and which competes for binding with
known bacterial superantigens.
32

33
Materials and Methods
Synthetic Peptides
Overlapping peptides corresponding to the entire length of HIVlav Net
(Wain-Hobson et al., 1985) were synthesized with a Biosearch 9500AT
automated peptide synthesizer using N-(9-flurenyl)methoxycarbonyl chemistry
as described in chapter II of this text. Peptides were synthesized based on a
surface profile as described. The amino acid sequences of the peptides are
presented in Table IV.
Cells and Reagents
Two cell lines were used for the binding studies. Raji cells are EBV-
transformed B cells that express DR3, Dw10, DQw1, and DQw2 (Merryman
et al., 1989). DR 1-transfected L cells were kindly provided by Dr. Eric O. Long
and are described elsewhere (Lechler et al., 1988). SEs were obtained from
Toxin Technology (Sarasota, FL). Several mAb were used in this study. Anti-
HLA-DR clone L243 reacts with a nonpolymorphic DR epitope and does not
cross-react with DP or DQ (Robbins et al., 1987). Anti-HLA-DP clone B7/21
reacts with a monomorphic epitope present on DP1, DP2, DP3, DP4, and DP5
(Robbins et al., 1987). Anti-HLA-DQ clone SK10 reacts with a common
polymorphic epitope present on cells expressing DQw1 and DQw3 (associated
with DR1. DR2, DR4, DR5, w8, w9, and w10) (Brodsky, 1984). Anti-HLA-DR
clone L227 reacts with a nonpolymorphic region of DR (Barnstable et al., 1978).
Clone W6/32 reacts with a monomorphic epitope on HLA-A, B, and C
(Barnstable et al., 1978). Clones L243, B7/21, and SK10 were obtained from
Becton-Dickinson (Mountain View, CA) and clones L227 and W6/32 were
kindly provided by Dr. Robert Rich. All mAbs were used at a final concentration

34
Table IV.
Amino acid sequences of Nef peptides.
Net Peptide
Sequence
1 -38 MGGKWSKSSVVGWPTVRERMRRAEPAADGVGAASRDLE
31 -65 GAASRDLEKHGAITSSNTAATNAACAWLEAQEEEE
62-99 EEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEG
93-132 EKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPG
123-160 DWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGE
156-186 NKGENTSLLHPVSLHGMDDPEREVLEWRFD
182-206 EWRFDSRLAFHHVARELHPEYFKNC

35
of 10 pg/ml. Purified recombinant Nef protein was purchased from Repligen,
Cambridge, MA.
Radioiodinations
SEs (2.5 pg) and Nef peptide (10 pg) were radioiodinated using
chloramine T as described in chapter II of this text. The specific activities of the
SEs and Nef peptide ranged from 70-120 pCi/pg and 30-40 pCi/pg,
respectively.
Binding Studies
For binding studies on Raji cells, unlabeled competitors (SEs and Nef
peptides) were added in 50 pi (in PBS with 1% BSA) to 50 pi of cells in 1.5 ml
"Eppendorf" tubes. Cells and competitors were incubated at room temperature
for 45 min., followed by the addition of radiolabeled SEs or Nef peptide. After
45 min., the cells were washed three times and the bottoms of the tubes were
cut off. Radioactivity was quantified using a gamma counter. Similarly, DR-1
transfected L cells, which were grown to confluence in the wells of microtiter
plates, were subjected to the same incubation times and volumes of
competitors and labeled ligands as used for the Raji cells. After three washes,
the cells were solubilized in 1% SDS, and the liquid was absorbed by cotton-
tipped applicators and radioactivity was quantified using a gamma counter.
Results
Competition of Nef Peptides with Radiolabeled SEA for MHC Binding
Overlapping peptides corresponding to the entire length of the Nef
sequence (HIVlav) were synthesized (Table IV). The Nef peptides were

36
initially tested at a concentration of 300 pM for their relative abilities to compete
with 125I-SEA for binding to Raji cells, which express HLA-DR3 and HLA-
DRw10, and DR1-transfected L cells (Figure 6). Nef(123-160) reduced 125l-
SEA binding to Raji cells by 41% which was significant at p<0.002. This
degree of inhibition was consistent in repeated experiments. The N-terminal
peptide, Nef(1-38), had a marginal but insignificant effect (21% reduction,
p>0.05) on 125I-SEA binding to Raji cells in the same experiment. This slight
inhibition by Nef(1-38) was not seen in repeated experiments. The other
peptides did not affect SEA binding to Raji cells. A similar pattern was seen in
competitive binding studies performed on DR1-transfected L cells. Nef(123-
160) blocked 125I-SEA binding to DR1 cells by 35% (p<0.008), whereas Nef(1-
38) had only a slight effect (p>0.8). Thus, only one of the overlapping Nef
peptides, Nef(123-160), significantly and consistently blocked 125I-SEA binding
to both Raji cells and DR 1-transfected L cells.
Nef(123-160) Peptide Competition Is Dose-Dependent
The relative abilities of Nef(123-160), purified recombinant Nef protein
(Repligen, Cambridge, MA), and SEA to block SEA binding to MHC class II
were tested in dose response studies (Figure 7). Nef(123-160) reduced 125l-
SEA binding to Raji cells by 40% at the highest concentration tested (300 pM),
and competed with SEA in a dose-dependent manner. Two other Nef peptides,
Nef (1-38) and Nef(31-65), did not block SEA in a dose-dependent manner.
Unlabeled SEA reduced 125I-SEA binding by 50% at a concentration of 0.2 pM,
which is consistent with the reported Kd of SEA for human MHC class II
antigens (Chintagumpala et al., 1991). Thus, Nef protein and the internal Nef
sequence, Nef(123-160), competed for SEA binding to Raji cells in a dose-
dependent manner.

37
140
1-38 31-65 62-99 93-132 123-160 156-186 182-206
NeF Peptide
Figure 6. Blockage of 125I-SEA binding to Raji and DR 1-transfected L cells by
Nef peptides. Nef peptides were used at a final concentration of 300 pM. 125l-
SEA was used at a final concentration of 2 nM. 105 Raji or DR 1-transfected L
cells were used per tube. Binding of 125I-SEA to Raji and DR1-transfected L
cells in the absence of competitors was 31,469 2292 and 5,708 41 CPM,
respectively. Data represent the mean percent of control of three individual
experiments, each performed in duplicate. Bars represent binding to Raji and
DR1-transfected L cells in the presence of Nef peptides SD.

% Control
38
Figure 7. Relative abilities of Nef(123-160), purified Nef protein, and SEA to
compete with 125I-SEA for binding to Raji cells. Experimental conditions were
the same as those described in Figure 1. Binding of 125I-SEA to Raji cells in
the absence of competitors was 30,406 2851 CPM. The data presented
represent the mean of three individual experiments, each performed in
duplicate. Each point represents the mean percent of SEA control binding in
the presence of competitors SD.

39
Ability of Nef Peptides to Compete with SEs for Binding to Raii Cells
Net peptides were also tested for their relative abilities to compete with
other staphylococcal enterotoxins (SEs) for binding to Raji cells. As shown in
Figure 8, Nef(123-160) significantly blocked binding of 125I-SEE by 42%
(p<0.02) and 125I-SEC1 by 26% (p<0.03), but only blocked binding of 125l-
SEB by 10% (p>0.1). Thus, Nef(123-160) significantly inhibited the binding of
two highly homologous SEs, SEA and SEE, while it was less effective against
SEB and SEC1.
Direct Binding of Nef(123-160) Peptide to Raii Cells
In direct binding studies, 125l-Nef(123-160) bound to Raji cells and was
effectively inhibited by unlabeled Nef(123-160) and SEs (Figure 9). Unlabeled
Nef(123-160) reduced 125l-Nef(123-160) binding by 50% at a concentration of
30 pM. SEE was a better competitor and reduced 125l-Nef(123-160) binding
by 50% at a concentration of 2 pM. Both SEE and unlabeled Nef(123-160)
competed in a similar manner, but SEE was a more potent competitor. SEA did
not inhibit 125l-Nef(123-160) binding as well as SEE, but it was more effective
than SECi or SEB. This pattern of inhibition is reflective of the ability of
Nef(123-160) to block SE binding to MHC class II antigens. Although SEs
compete for similar sites on MHC class II antigens, our results suggest that
Nef(123-160) competes for sites that are more closely associated with SEE
than the other SEs tested. Nef(123-160) binding was not blocked by other Nef
peptides, such as Nef(1-38) and Nef (31-65), at 300 pM. These results suggest
that Nef(123-160) binds directly to Raji cells and competes for a site on MHC
class II antigens to which SEs bind.

% Control
40
1-38 31-65 62-99 93-132 123-160 156-186 182-206
NeF Peptide
Figure 8. Blockage of 125I-SE binding to Raji cells by Nef peptides. Nef
peptides were used at a final concentration of 300 pM. 125l-SEs were used at 2
nM. All other experimental conditions were the same as those described in
Figure 7. Binding of 125I-SEE, 125I-SEB, and 125I-SEC1 in the absence of
competitor was 5,917612 CPM, 6,438134 CPM, and 4,493425 CPM,
respectively. Data represent the mean of three individual experiments, each
performed in duplicate. Each bar represents the mean percent of the
appropriate SE control binding to Raji cells in the presence of Nef peptides
SD.

% Control
41
Concentration (p.M)
Figure 9. Direct binding of Nef(123-160) peptide to Raji cells. 125l-Nef(123-
160) was used at a final concentration of 3 nM. All experimental conditions
were the same as those described in Figure 7. Binding of 125l-Nef(123-160) in
the absence of competitor was 1956 150 CPM. The data presented represent
the mean of three individual experiments, each performed in duplicate. Each
point represents the mean percent of 125l-Nef(123-160) binding relative to
control in the presence of competitors SD.

42
MAbs to MHO Class II Antigens Block Nef(123-1601 Peptide Binding to Raji
Cells
To ascertain the receptor to which ^25|-Nef( 123-160) bound on Raji
cells, binding was performed in the presence of monoclonal antibodies (mAb)
to class II and class I antigens (Figure 10). Clone L243, a mAb specific for HLA-
DR, reduced 125I-Nef(123-160) binding by 35%. Another mAb specific for
HLA-DR, clone L227, also reduced Nef(123-160) binding, but was not as
effective as clone L243. This is consistent with reports that L227 mAb is not
very effective at blocking SEA binding to Raji cells (Chintagumpala et al., 1991).
MAb to other class II antigens (HLA-DP and DQ) had no effect on 125|-Nef(123-
160) binding, as was also the case for a mAb to class I antigens (clone W6/32).
Thus, our studies indicate that Nef(123-160) binds to class II MHC antigens, the
known receptors for superantigens on APC.
Discussion
Data presented here show that binding of bacterial superantigens to
MHC class II antigens can be blocked by a peptide corresponding to an internal
Nef region. Anti-HLA-DR mAb blocked Nef(123-160) binding to Raji cells,
suggesting that binding to class II molecules occurred. Binding of Nef(123-160)
to HLA-DR probably occurs outside the antigen binding groove, since Nef(123-
160) was able to block binding of the superantigens SEE and SEA. A recent
study has shown that HLA-DR is present on HIV-1 and SIV, and that it is
selectively incorporated into the virus membrane over HLA-DP or HLA-DQ
(Arthur et al., 1992). Antibodies to HLA-DR, but not antibodies to HLA-DP or
HLA-DQ, effectively inhibited viral infection of cells in vitro and these antibodies
target the HLA-DR antigens present on the virus particles and not those present

43
B7/21
SK10
W6/32
L243
L227
(Anti-DP)
(Anti-DQ)
(Anti-class I)
(Anti-DR)
(Anti-DR)
Antibody
Figure 10. Blockage of 125l-Nef(123-160) binding to Raji cells by antibodies to
MHC class I and class II antigens. 125l-Nef(123-160) was used at a final
concentration of 3 nM. MAbs were used at a final concentration of 10 |ig/ml.

44
on the cells. The ability of these antibodies to neutralize HIV suggest that HLA-
DR may play a role in infectivity. Our results suggest that Net may bind to HLA-
DR as it is being expressed on the surface of an infected cell in a manner
analogous to the putative expression of MMTV superantigen (Choi et al., 1992).
Further, Nef has been shown to be present on the surface of infected cells, as
assessed using anti-Nef antibodies labeled with fluorescein (Fujii et al., 1993).
This is in contrast to the situation with MMTV superantigen in that no one has
been able to show that cells from MMTV-infected animals express
superantigen, although superantigen functional activity is observed (Choi et al.,
1991). Transgenic mice expressing MMTV superantigen delete V(i14+ T cells,
probably during the ontogeny of the immune system, and these mice are
immune to infection by exogenous MMTV infection (Acha-Orbea and Palmer,
1991). Thus it is possible that Nef interacts with class II MHC antigens in a
manner somewhat similar to MMTV superantigen, suggesting a possible
superantigen function for Nef in HIV pathogenesis.

CHAPTER IV
ACTIVATION OF CD4 T CELLS BY THE NEF PROTEIN FROM HIV-1
Introduction
In the previous chapter, data was presented which showed that Net
binds specifically to class ll-bearing Raji cells at a site(s) involved in
staphylococcal enterotoxin binding. This binding was similar to that shown for
MMTV superantigen. These data suggest that Nef may have characteristics of
superantigens. The data presented in this chapter show that Nef induces
proliferation of human peripheral blood mononuclear cells. Further, the
proliferative response is T cell specific. The data presented in this chapter
indicate that Nef activates T cells and induces T cell cytokine production in a
manner reminiscent of staphylococcal superantigens.
Materials and Methods
Nef Protein
Nef protein was expressed and purified using a fusion protein and
purification system. The HIV-1 nef gene (HIV-1 IIIb, R & D Systems, Cambridge,
MA) was amplified by polymerase chain reaction (PCR), using the following
primer set:
5' ATG GGT GGC AAG TGG TCA AAA AGT (+)
51 GCC AAG CTT GAT GTC AGC AGT TCT (-)
45

46
The extra A added to the 3' end of the amplified nef gene fragment as a result
of PCR was removed by treatment with the Klenow fragment of DNA
polymerase. This DNA was ligated into the Xmn\/Hind\\\ cloning site of the
prokaryotic expression vector pMAL-c2 (New England BioLabs, Beverley,
MA). The sequence of the fusion construct was verified by the DNA
Sequencing Laboratory, Interdisciplinary Center for Biotechnology Research,
University of Florida. The fusion construct was engineered such that the N
terminus of Nef was immediately downstream from the Factor Xa cleavage site
at the C terminus of maltose binding protein (MBP). Thus, cleavage with Factor
Xa was predicted not to add any vector-derived amino acids. E. coli strain TB1
was transformed with the vector containing mbp/nef. Cultured E. coli TB1 were
suspended in column buffer consisting of 20 mM Tris-HCI (pH 7.4), 200 mM
NaCI, and 1 mM ethylenediaminetetraacetic acid (EDTA). The cells were
sonicated and the supernatants were recovered after centrifugation. Protease
inhibitors (2 mM phenylmethylsulfonyl fluoride (PMSF), pepstatin A, leupeptin,
and aprotinin) were added to extracts. The protein concentration of the extracts
was adjusted to 2.5 mg protein/ml prior to loading onto an amylose affinity
column containing a bed volume of 50 ml. The column was washed with 350
ml of column buffer. The fusion protein was eluted with 200 ml of column buffer
containing 10 mM maltose. Fractions (7 ml) were collected. The fusion protein
generally eluted within the first 70 ml. After cleaving the fusion protein with
factor Xa (New England BioLabs, Beverley, MA), the cleavage mixture was
loaded onto a hydroxyapatite column containing a bed volume of 35 ml. After
extensive washing of the column with 180 ml of 10 mM potassium phosphate
(pH 7.2) containing 2 mM PMSF and 1 mM benzamidine, fractions were eluted
using a linear gradient from 10 mM to 400 mM potassium phosphate buffer (pH
7.2) containing 2 mM PMSF. Fractions (5 ml) were collected. Nef eluted from

47
the column between 100-150 mM potassium phosphate, and was generally
contained within 7 fractions. The fraction containing Nef protein was dialyzed
against affinity column buffer containing 150 mM NaCI. The dialyzed material
was loaded onto a second amylose affinity column for removal of MBP. The
flow-through containing Nef protein was collected and loaded onto an High-Q
ion-exchange cartridge (BioRad Laboratories, Richmond, CA). The High-Q
cartridge was washed with 100 ml of 20 mM Tris buffer (pH 8.0) containing 150
mM NaCI. Fractions were eluted with a linear gradient from 150-400 mM NaCI
in the same buffer. The fractions containing Nef protein, which eluted at 180
mM NaCI, were collected and dialyzed overnight against PBS and stored at
-70C. The purity of Nef protein was assessed by SDS-PAGE, followed by
staining with silver. Upon staining with silver, a single band was found, and a
corresponding band was detected by Western blotting with monoclonal anti-Nef
antibody (Repligen, Cambridge, MA). MBP purified by this method and used at
the same concentrations as that used for the purified Nef preparation, did not
have proliferative activity on human peripheral blood mononuclear cells.
Nef proliferative activity was confirmed using recombinant Nef proteins
obtained from two other sources and which were produced using different
expression systems. The following reagent was obtained through the AIDS
Research and Reference Reagent Program, AIDS Program, NIAID, NIH: HIV-1
LAV Nef from the Division of AIDS, NIAID. The HIV-1 nef gene (LAV) used to
produce this Nef preparation was isolated from pBENN 6 and cloned into the
bacterial expression vector pPD-YN-61. The protein was produced in E. coli
strain Sf930 and isolated as inclusion bodies. Nef protein was also obtained
from Repligen (Cambridge, MA). In this case, the HIV-1 nef gene (LAV) was
cloned into the bacterial vector pD10, which adds a hexahistidine tag for protein
purification using nickel chelate chromatography. The protein was produced in

48
E. coli strain MC10611 P0L.1. All Net preparations were negative for endotoxin
as assessed by the limulus amoebocyte lysate assay.
Reagents
Staphylococcal enterotoxin A (SEA) was purchased from Toxin
Technology (Sarasota, FL). Concanavalin A (Con A), anti-CD3, and anti-IgM
were purchased from Sigma Chemical Co. (St. Louis, MO).
Proliferation Assays
Proliferation studies on human peripheral blood mononuclear cells
(PBMC) were performed as described previously (Pontzer et al., 1992).
Peripheral blood from healthy adult blood bank donors was obtained from
Civitan Regional Blood Center (Gainesville, FL) for use in these studies. All
donors were negative for cytomegalovirus, hepatitis B virus, and HIV. PBMC
were isolated from peripheral blood using ficoll hypaque gradient
centrifugation. After extensive washing, PBMC were plated into wells of
microtiter plates at 2.8 x 106 cells/ml, followed by addition of activators. Final
volumes were adjusted to 150 pl/well with RPMI 1640 tissue culture medium
containing 5% fetal bovine serum (FBS) and penicillin/streptomycin. 3h-
thymidine (IpCi/well; Amersham Corporation, Indianapolis, IN) was added at
90 h after initiation of cultures and the cells were incubated for an additional 6 h
prior to harvest onto filter paper. Filter paper was placed in liquid scintillation
fluid and radioactivity was quantified using a p scintillation counter. Stimulation
index (S.l.) was determined by dividing the experimental CPM by the CPM
obtained from control (unstimulated) cultures.

49
Purification of T Cells from PBMC
Two methods were used to isolate purified T cell populations. One
method involved passing PBMC over T Cellect columns (Biotex, Edmunton,
Alberta, Canada). A second method involved a double rosetting technique
using neuraminidase-treated sheep red blood cells (NA-SRBC) (Spawski and
Lipsky, 1992). PBMC (5 ml at 107/ml) were mixed with 2.5 ml each of NA-
SRBC and FBS. PBMC were incubated for 10 min at 37C, centrifuged at 900
rpm to gently pellet the cells, and incubated for 90 min at 37C. Rosetted T cells
were separated from non-rosetted cells by ficoll hypaque centrifugation. NA-
SRBC were removed from the rosetted fraction by lysis with ammonium
chloride. The procedure was repeated on the rosetted fraction to insure purity.
Autologous rosetted T cells were plated at 2.8 x 10 cells/ml. Cells were plated
into wells of microtiter plates at 2.8 x 10 cells/ml. To insure that these cultures
were depleted of APC, T cells were stimulated with staphylococcal enterotoxins,
which stimulate T cells only in the presence of APC.
Autologous Antigen-Presenting Cells (APC1
For proliferation studies, APC consisted of PBMC that were inactivated
with 0.8% paraformaldehyde. For cytokine studies, APC consisted of cells that
did not form rosettes after two treatments with NA-SRBC. APC were plated at
2.8 x 106 cells/ml. To insure inactivation with paraformaldehyde was complete,
APC were tested for possible proliferation using anti-IgM and Con A.
Synthetic Nef Peptides and Antibodies to Nef Peptides
The sequences of the peptides used are listed in Table III. Peptides
were purified by reverse phase high performance liquid chromatography.
Polyclonal antibodies to Nef peptides were generated in rabbits. Anti-peptide

50
antibodies were tested for their relative abilities to neutralize Nef mitogenic
activity by incubating Nef with antibodies at 37C for 60 minutes prior to
addition of PBMC. Antibodies to both Nef(123-160) and Nef(182-206) showed
strong reactivity to whole recombinant Nef protein, and these combined
antisera had significant neutralizing activity against Nef. Nef was treated with
preimmune sera as control.
Cytokine Production and Assays
PBMC were plated at 106 cell/ml into wells of 24-well plates (Corning
Corp., Corning, NY). Activators were added and final volumes were adjusted to
300 pl/well. Cultures were incubated at 37C over a 96 h period and samples
(100 pi) were obtained at 24 h intervals. Cell cultures were replenished with
RPMI/5% FBS (100 pi) after obtaining samples. Culture supernatants were
tested for interleukin 2 (IL 2) using the IL 2-dependent HT-2 cell line (Ho et al.,
1987). The amounts of IL 2 present in culture supernatants were determined
using recombinant human IL 2 (Genzyme, Cambridge, MA) as a standard.
Samples were tested for interferon (IFN) activity on human WISH cells by a
microplaque reduction method (Langford, et al., 1978b), using approximately
40 plaque-forming units (PFU) of vesicular stomatitis virus (VSV) per well. In
our studies, 1 U/ml of IFN is defined as the concentration required to decrease
the number of PFU per well by 50%.
IFN activity was typed by neutralization reactions with specific antisera,
as described (Johnson et al., 1982). Briefly, samples were pretreated with 1000
neutralizing units of either anti-human IFNa (Lee Biomolecular, San Diego, CA)
or anti-human IFNy (Genzyme, Cambridge, MA). Controls were sham-treated
with EMEM/2% FBS. The neutralizing activity of anti-IFN antisera was
confirmed using HuIFNa (Lee Biomolecular, San Diego, CA) and HuIFNy

51
(Genzyme, Cambridge, MA) as positive controls. Samples were incubated at
37C for 1 h prior to transfer to confluent human WISH cells. Residual IFN
activity in sham- and antiserum-treated samples was measured as described
above.
Studies on Vfi-Specific T Cell Expansion and Induction of Anerqy
PBMC cultured in the presence of Nef, TSST-1, and ConA were tested
by flow cytometry for specific Vp expansion as previously described (Soos and
Johnson, 1994) using a panel of anti-Vp antibodies. Induction of anergy in vitro
by Nef, TSST-1, and ConA was also assessed with anti-Vp antibodies as
described (Schiffenbauer et al., 1993). Antibodies to Vp5a, Vp5b, Vp5c, Vp6a,
Vp8a, and Vp12 were obtained from T Cell Sciences, Inc., Cambridge, MA.
Antibodies to Vp2, Vp3, Vp13, Vpl7, Vp18, Vp21, and Vp22 were purchased from
Immunotech, Marseilles, France.
Results
Nef Proliferative Response
Recombinant Nef proteins from two sources were tested for proliferative
activity. One Nef preparation (referred to as Nef 1) was purified and kindly
provided by Dr. Taishi Tanabe in the laboratory of Dr. Howard M. Johnson. This
Nef preparation was compared to purified Nef from Repligen (Nef 2). Both Nef
protein preparations, which are derived from the HIVlav sequence, induced
similar levels of proliferation (Figure 11). Nef proteins from both sources were
pure and neither preparation contained detectable levels of endotoxin. Thus,
Nef proteins from both sources had similar proliferative activity, and the
proliferation observed was significant. Nef from these two sources were

52
x
a>
-a
s
"5
E
"M
CZ3
1000
Concentration (ng/ml)
Figure 11. Comparison of the mitogenic activities of Nef protein
preparations. Two Nef preparations,Nef 1 and Nef 2, were compared for
proliferative activities on human PBMC. Data are from a representative
experiment, performed in triplicate, and are expressed as mean stimulation
index S.D.. Comparison of the proliferation of PBMC cultured for 4 days in the
presence of either Nef 1 or Nef 2. The mean value for 3H-thymidine
incorporation by unstimulated cultures was 90067.

53
subsequently used in these studies. The possibility was raised that the
proliferative activity ascribed to Net was due to a contaminant in the
recombinant preparation. As a control, the proliferative activity of the Nef fusion
partner, MBP, was also assessed. No proliferation was observed with MBP
(Figure 12). Further, polyclonal antisera generated against Nef synthetic
peptides were tested for neutralizing activity. As shown in Table V, anti-Nef
antibodies significantly reduced Nef-induced proliferative responses. Antibody
neutralization of Nef activity occurred in a dose-dependent manner. These data
confirm that the proliferative activity seen in these studies is due to Nef protein,
and not to a vector-derived contaminant.
Nef was also tested for the ability to induce proliferation of PBMC from
fifteen donors. Results from a representative sample of ten donors are shown in
Figure 13. Nef induced significant proliferation in 90% of the donors tested.
Variation in the Nef response was seen, similar to the responses to the potent
staphylococcal enterotoxin, SEA. It is unlikely that these donors were
sensitized to Nef, since they tested negative for HIV-1. Similar results were
obtained using purified recombinant Nef protein preparations from two different
sources, and which used different expression systems (see Materials and
Methods in this chapter). The high number of donors that responded to Nef
indicates Nef protein has similar mitogenic activity to SEA for PBMC from a
wide sampling of donors.
Antigen-Presenting Cells (APC1 Are Required for Nef Activity
The question arose as to whether Nef, like SEA, requires APC in order to
stimulate lymphocyte proliferation. Nef induced significant proliferation in
PBMC (Figure 14). Purified T cell cultures did not proliferate in response to Nef
or to SEA. Purified APC, which contained monocytes and B cells, did not

Stimulation Index
54
15
10
5
0
PBMC stimulated with Nef or E. coli MBP:
Expt. 1
I I Expt. 2
Nef MBP
Figure 12. Lack of proliferation by PBMC in response to the Nef fusion partner
MBP. MBP and Nef were used at 3 pg/ml. Data are expressed as mean
stimulation index SD. The mean values for ^H-thymidine incorporation by
unstimulated cultures in eperiments 1 and 2 were 85239 CPM and 1093136
CPM, respectively.

55
Table V.
Ability of anti-peptide antibodies to block Nef-induced proliferation
PBMC cultured
in the presence ofa:
Anti-Nef
antibodies'3
Stimulation Index0
(Mean SD)
p value
Nef
-
4.7 0.2
Nef
+
1.3 0.1
<0.001
SEA
-
59.7 2.6
SEA
+
62.0 0.6
>0.15
-
+
0.9 0.4
-

-
aNef (3 pg/ml) and SEA (0.3 pg/ml) were incubated with anti-Nef antibodies for
60 minutes prior to addition of PBMC.
^A mixture of antisera to Nef(123-160) peptide and Nef( 182-206) peptide were
used, each at a final dilution of 1:1000. Preimmune sera had no effect. Both
anti-Nef(123-160) and anti-Nef(182-206) had strong reactivity to Nef protein by
ELISA.
cData are from a representative experiment performed in triplicate.

56
1000
Figure 13. Nef-induced proliferative responses from a representative sampling
of donors. PBMC of blood bank donors were tested for proliferation induced by
SEA or Nef. SEA and Nef were used at 300 ng/ml. Mean values for 3H-
thymidine incorporation by unstimulated cultures ranged from 35286 CPM to
1983+274 CPM.

57
Unfractionated HPMC APC
T cells
APC + T cells
Cells
Figure 14. Nef-induced activation of T cells requires APC but does not require
processing of Nef. Unfractionated PBMC, purified APC alone, purified T cells
alone, and purified T cells in the presence of APC were tested for proliferation
in response to Nef, SEA, and Con A. Data are from a representative
experiment, performed in triplicate, and are expressed as mean stimulation
index S.D. Purified T cells were reconstituted with paraformaldehyde-
inactivated APC. Con A was used at 10 pg/ml. Nef and SEA were both used at
100 ng/ml.

58
respond to stimulation by either Net or SEA. Upon reconstitution with
paraformaldehyde-inactivated autologous APC, significant Nef-induced
proliferation of T cells occurred, with essentially complete reconstitution of the
response. Similar proliferative responses were obtained using cells from other
donors. Thus, T cells responded to Nef stimulation only in the presence of
autologous APC, and Nef does not require processing to be presented by APC.
These results indicate that Nef-induced proliferation required APC in a manner
reminiscent of SEA.
Nef(123-160) Peptide Specifically Blocks Proliferation of PBMC Induced by Nef
and SEA
As shown in Chapter 3, a synthetic peptide corresponding to an internal
Nef sequence, Nef(123-160), blocked binding of Nef and SEA to Raji cells. It
was important to determine if this peptide could also block proliferation induced
by Nef and SEA. The results of this study are presented in Figure 15. Nef(123-
160) blocked both Nef-induced and SEA induced proliferation, consistent with
its ability to block binding of Nef and SEA to Raji cells. Further, the blocking
was specific in that proliferation induced by the T cell mitogens Con A and anti-
CD3, or the B cell mitogen anti-IgM, were not blocked by Nef(123-160).
Nef(157-186), whose sequence slightly overlaps that of Nef(123-160), had no
effect on the proliferative effects of either Nef, SEA, or Con A. These results
confirm that the proliferative responses observed in PBMC cultures were
specific for Nef, and were not due to a contaminant. Thus, proliferative
responses to Nef and SEA were specifically blocked by a peptide
corresponding to the region on Nef that binds to class II antigens and which
blocks binding of Nef and the superantigen SEA.

59
x
TJ
C
s
.o

3
E
5Z5
No Peptide
SEA Nef Anti-IgM Anti-CD3
Mitogen
Figure 15. Nef(123-160) peptide specifically blocks proliferation of PBMC
induced by Nef and SEA. PBMC cultures were stimulated for 96 h with
mitogens in the absence of peptides or in the presence of either Nef(123-160)
peptide or Nef(157-186) peptide. Data are from a representative experiment,
performed in triplicate, and are expressed as mean stimulation index SD. Nef
and SEA were used at 300 ng/ml. Anti-IgM and anti-CD3 were used at 10
pg/ml. Peptides were used at a final concentration of 100 pM. The mean value
for ^H-thymidine incorporation by unstimulated cultures was 1005+37 CPM.

60
Induction of T Cell Cytokines by Nef
T cell activation by the staphylococcal superantigens results in the
prodigious production of T cell cytokines, such as IL 2 and IFNy. Because of the
significant T cell proliferation induced by Nef, it was determined if Nef induced
Thelper cell cytokines. The results of a representative experiment are
presented in Table VI. Consistent with proliferation data, Nef induced
significant levels of IL 2, although IL 2 levels were lower than those induced by
SEA. The question arose as to the ability of Nef to also induce another
important T helper cell cytokine, IFNy. Samples were removed from cultures of
cells over the course of 96 h and tested for IFN antiviral activity. Nef induced
high levels of IFN activity, with peak production occurring by 96 h (Figure 16,
Panel A). IFN levels were lower than those induced by SEA, but were similar
than those induced by Con A. Purified T cells did not produce IFN upon
stimulation with either Nef or SEA (Figure 16, Panel B). However, purified T cell
cultures were capable of producing IFN upon Con A stimulation. No IFN activity
was produced in cultures of APC (consisting of B cells) stimulated with either
Nef, SEA, or Con A. Purified T cell cultures reconstituted with purified B cells for
antigen presentation produced significant levels of IFN upon either Nef or SEA
stimulation (Figure 16, Panel C). These results are consistent with T cell
mitogen activity for both Nef and SEA.
The type of IFN activity induced by Nef was determined by neutralization
reactions with specific antisera. Treatment of Nef-induced IFN with anti-IFNy
resulted in a significant reduction of activity (Table VII). Antisera to IFNa did not
affect the IFN activity in these samples. Consistent with previous data (Langford
et al., 1978a; Johnson et al., 1982), the IFN activity induced by SEA was
IFNy. Similar to the results found with unfractionated PBMC, the IFN produced

61
Table VI.
IL 2 production induced by Nef.
IL 2 (U/ml) produced atb:
Mitogen3
48 h
96 h
Nef
14.7 0.1
16.9 1.1
SEA
17.2 0.7
24.7 1.8
None
<3
<3
aSamples were obtained from PBMC cultures which had been
stimulated with Nef and SEA at 300 ng/ml and 500 ng/ml,
respectively.
b|L 2 in culture samples were determined using IL 2-dependent
HT-2 cells. The data presented are from a representative
experiment performed in triplicate.

IFN (U/ml) IFN (u/m|) IFN (U/ml)
62

PBMC + SEA
PBMC + Nef
PBMC + Con A
PBMC + media
T cells + SEA
T cells + Nef
T cells + Con A
T cells + media
10000
1000
100
10
1
24 48 72 96
Hours
T+APC+SEA
T+APC+Nef
T+APC+ConA
T+APC+media
Figure 16. Kinetics of IFN production induced by Nef. Cultures of PBMC (Panel
A), purified T cells (Panel B), and reconstituted cultures of purified APC and
purified T cells (Panel C) were tested for IFN production at 24, 48, 72, and 96 h.
Cultures were stimulated with either Nef at 100 ng/ml, SEA at 100 ng/ml, or Con
A at 10 pg/ml, or were left unstimulated. Cultures of purified APC produced <10
U/ml IFN at all timepoints.

63
Table Vil.
IFNy is induced by Net.
Samples from PBMC IFN titer (U/ml) after treatment with:
cultures stimulated with: EMEM anti-IFNa anti-IFNy
Nef
300
300
20
SEA
300
300
30
Con A
300
300
100
IFNa Control
300
<3
300
IFNy Control
30
30
<3

64
by T cell cultures reconstituted with B cells in response to Net was IFNy. Thus,
Net induced high levels of IFNy in cultures of PBMC, indicating that Nef
activates T cells to produce the cytokines IL 2 and IFNy, lymphokines that are
products of activated T helper 1 cells.
PBMC cultured in the presence of Nef were tested for specific Vp
expansion and anergy in vitro. No expansion of specific Vp populations was
observed with the anti-Vp antibodies used (data not shown). Unlike
pretreatment with TSST-1, which anergized the response to anti-Vp2, Nef did
not significantly anergize the responses of T cells to the tested anti-Vp
antibodies (data not shown). Thus, no specific Vp expansion or anergy was
detected using the available human Vp reagents.
Discussion
Nef protein induced significant levels of proliferation in unfractionated
PBMC from a number of donors. The observed Nef responses were significant,
with proliferation induced in cells from a large majority (85-90%) of the donors
tested. It is unlikely that prior sensitization to Nef is responsible for the
proliferative response, since these donors were negative for HIV. The following
evidence points to Nef as the inducer of the observed proliferation: 1)
proliferation was observed using recombinant Nef from three different
expression systems; 2) antibodies to synthetic Nef peptides neutralized the
proliferative activity of Nef; 3) the Nef fusion partner, MBP, did not induce
proliferation; and 4) a synthetic peptide, Nef(123-160), previously shown to
block binding of superantigens to MHC class II, blocked the Nef-induced
proliferative responses. Nef induced proliferation was generally lower than that
induced by SEA. This is not unusual in that SEA is extremely potent, causing
proliferation at concentrations as low as 10'16 M (Langford et al., 1978a).

65
Proliferation induced by Nef occurred only in the presence of APC.
Cultures of purified APC or purified T cells did not respond to Nef. T cells
cultured in the presence of inactivated autologous APC proliferated to a similar
extent to that seen in unfractionated cultures. These data suggest that Nef is
presented to T cells by APC in an unprocessed form. The production of the T
helper 1 lymphokines IL 2 and IFNy from Nef-induced cultures is evidence that
Nef activates T helper 1 cells.
In Chapter III, data was presented showing that Nef binds to Raji cells at
a site(s) involved in SEA binding. A peptide corresponding to an internal
region of Nef, Nef(123-160), specifically bound to DR 1-transfected L cells and
blocked proliferation induced by Nef and SEA, evidence that Nef binds to MHC
class II antigens. Con A-induced proliferation was not affected by Nef(123-
160). This region of Nef was shown to be involved in binding of Nef to Raji
cells. These functional data suggest that binding to MHC class II antigens is
required for Nef proliferative activity. Binding of Nef to MHC class II occurs on
APC, since Nef proliferative activity also requires presentation, but not
processing, by APC. However, these data do not preclude Nef presentation by
T cells expressing HLA-DR, a T cell activation marker.
Superantigenic activity of Nef was not observed using the anti-Vp mAb
currently available. Neither Vp-specific expansion or anergy were observed.
Further, preliminary results using reverse transcriptase-polymerase chain
reaction (RT-PCR), which quantifies the amount of mRNA produced, did not
show a Vp preference in Nef-activated cells. These results raise a question on
the nature of Nef receptor on T cells, and it is tempting to speculate on possible
candidates such as TCR or CD4.
Nef induces human B cells to differentiate into immunoglobulin secreting
cells (Chirmule et al., 1994). HLA-DR and adhesion molecules seem to be

66
involved in Nef-induced B cell differentiation, since monoclonal antibodies to
these surface proteins abrogated Nef-induced responses. Consistent with the
findings on T cell activation by Nef, B cell differentiation induced by Net
required T cells. In addition to activating T cells, staphylococcal superantigens
have also been shown to induce B cell differentiation (Stohl et al., 1994). Thus,
additional parallels can be drawn between the functional activities of
staphylococcal superantigens and Nef.
It has been shown that HIV requires activated T cells in which to
replicate. Specific antiviral immune responses may not be sufficient to activate
large numbers of T cells. For this reason, the identification of an HIV protein
that induces T cell proliferation is of considerable interest and may, in part,
explain the role of Nef in HIV pathogenesis. Activation of T cells may result from
interaction with Nef, either in soluble form released from lysed infected cells or
as a cell-associated complex with HLA-DR on the surface of infected T cells. T
cell activation by Nef could result in a stable cellular reservoir for virus
production as a result of continuous stimulation. In fact, hyperimmunization
against Nef has been proposed as a means of reducing viral load, either
prophylactically or therapeutically (Montagnier, 1995).
The T cell expansion observed in response to Nef may not be the only
mechanism of polyclonal activation of CD4+ T cells for HIV replication. Recent
evidence points to a superantigen encoded by the human herpesvirus,
cytomegalovirus (CMV), that expands Vpl2-bearing T cells, thereby enhancing
HIV replication in CMV-infected individuals (Dobrescu et al., 1995). It is not
surprising that no expansion of Vpl 2-bearing T cells was observed in response
to Nef, since the donors used in the studies discussed in this chapter were
negative for CMV. This versatility in polyclonal CD4+ T cell expansion via the
endogenous mitogen Nef and exogenous superantigens such as that of CMV

67
probably play an important role in HIV pathogenesis. Clearly, the control of the
mitogenic activity of these substances should help reduce the viral load in HIV-
infected individuals.

CHAPTER V
HIV ENCODES FOR ITS OWN CD4 T CELL MITOGEN
Introduction
Net protein is encoded in the genomes of both HIV-1 and the related
primate virus simian immunodeficiency virus (SIV). The function that Nef plays
in the pathogenesis of these viruses is uncertain, although its importance is
reflected in studies in which a nef-deleted SIV mutant did not cause disease
and protected against infection with the pathogenic wildtype strain (Daniel et
al., 1992). Further, the viral load in SIV mutant-infected animals was
considerably lower than in animals infected with the wildtype strain. However,
challenge of neonatal macaques with the same attenuated SIV strain caused
disease and, in some cases, death (Baba et al., 1995). The different outcomes
of these two studies may involve differences in the immune system
development between neonates and adults.
In Chapter IV, data was presented on the ability of Nef protein to induce
proliferation of human peripheral blood mononuclear cells (PBMC) from a wide
sampling of HIV-negative donors. Proliferative responses were T cell specific
and were accompanied by production of cytokines such as interleukin 2 (IL 2)
and gamma interferon (IFNy), indicative of CD4 T cell activation. Nef-induced T
cell proliferation and activation required the presence of antigen-presenting
cells. These results are interesting in that T cell activation has been shown to
be required for active HIV replication. Further, although resting or quiescent T
cells can be infected, evidence suggests that HIV replication only occurs upon
68

69
subsequent cellular activation. Herein, work is described that indicates that Net
activation of T cells is sufficient for HIV replication. These results show that Net
is an HIV-encoded mitogen that helps, at least in part, in establishing a cellular
reservoir for virus replication.
Materials and Methods
PBMC Cultures for HIV Infectivitv Studies
PBMC were isolated from peripheral blood of healthy adult donors as
described in Chapter IV. PBMC were resuspended in RPMI 1640 medium
containing 5% FBS and cultured in 25 cm2 flasks (Sarstedt Inc., Newton, NC) at
a concentration of 2 x 10 cells/ml in a final volume of 4 ml (or 8 x 10
cells/flask). Mitogens were added at the initiation of cultures. Nef, SEA, and
PHA were used at 3 pg/ml, 0.1 pg/ml, and 10 pg/ml, respectively. After four
days, PBMC were washed extensively in PBS, resuspended in RPMI 1640
medium containing 10% FBS, 106 M 2-mercaptoethanol, and 10 U rHulL 2/ml.
IL 2 is rapidly depleted in these cultures, and thus rHulL 2 was added to
medium. Cells were counted and all cultures were adjusted to 8 x 106
cells/flask. Cultures were infected with HIV at a final reverse transcriptase titer
of 20,000 CPM/ml. Culture supernatants were harvested and cells were fed
with fresh medium every third day for 12 days.
PBMC Cultures for Proliferation Assays
Concomitant with infectivity studies, PBMC were cultured in microtiter
plates to monitor proliferation in response to Nef and mitogens. The culture
conditions were the same as those described in Chapter IV. Nef and mitogens
were used at the same concentrations as described above. Stimulation indices
were calculated as described in Chapter IV.

70
Reverse Transcriptase (RT1 Assay
Harvested culture supernatants (1 ml) were placed in 1.5 ml "Eppendorf"
tubes and centrifuged at 17,000 rpm for 70 minutes. A cocktail containing Mg++
as the divalent cation, poly(rA)oligo(dTi2-i8) as template primer, and 2.5 pCi of
(3H)TTP per sample was used to resupend the virus. Tubes were incubated at
37C for 60 minutes, after which time samples were blotted onto filter paper
discs. Discs were allowed to air-dry and were washed extensively in sequential
baths. Specifically, discs were washed twice in 10% trichloroacetic acid (TCA)
for 15 and 5 minutes, respectively. Discs were then placed in 5% TCA
containing 0.1% SDS for 5 minutes. Finally, the discs were washed in 95%
ethanol, after which they were allowed to air-dry. Discs were placed in vials
containing scintillation fluid and radioactivity was quantified on a (3-scintillation
counter.
ELISA for HIV p24 Antigen
Levels of p24 antigen in infected culture supernatants were assessed
using a sandwich ELISA (DuPont, Boston, MA). Levels of p24 (in ng/ml) were
quantified according to the Manufacturer's recommendations based on a
standard curve using purified p24 antigen (provided by Manufacturer).
Assay for Infectious Virus from PBMC Cultures
Supernatants from PBMC cultures infected for 6 days with HIV were used
to assess the titer of infectious virus produced in these cultures. Supernatants
were adjusted to achieve RT titers of 12,000 CPM/ml and added to SEA-
stimulated PBMC cultures. Culture conditions were the same as those
described above. RT activity at Day 9 of cultures was assessed as described
above.

71
Fluorescent Antibody Cell Sorter (FACS1 Analysis of Nef-Activated Cells
PBMC were stimulated for 4 days with either Nef (3|ig/ml) or SEA (0.1
|ig/ml). PBMC were washed and resuspended in FACS buffer containing 0.5%
BSA and 10 mM sodium azide. Cells were incubated with fluorescein-labeled
mAb to HLA-DR for 45 minutes and washed with FACS buffer. Cells were then
incubated with phycoerythrin-labeled mAbs to either CD4 or IL 2 receptor (IL
2R) for 45 minutes. Cells were washed and analyzed on a FACScan (Becton-
Dickinson, Mountain View, CA) at 10,000 events/sample. All mAbs used were
obtained from Becton-Dickinson, San Jose, CA.
Studies on Proliferation of PBMC Induced bv Autologous HIV-Infected Cells
PBMC cultures were stimulated as described above. PBMC were
infected with HIV for 6 days, at which time the cells were washed and
inactivated by overnight treatment with 2% paraformaldehyde. Inactivated HIV-
infected cells were washed extensively to remove excess paraformaldehyde.
Fresh autologous PBMC were cultured in microtiter plates in the presence of
inactivated HIV-infected cells at a ratio of 3:1. After 4 days, 3H-thymidine
incorporation was assessed as described above.
Anti-Nef Peptide Antibodies
A mixture of antibodies to Nef(123-160) peptide and Nef(182-206)
peptide were used to block proliferation in response to autologous HIV-infected
cells. Antibodies were each used at a final dilution of 1:1000, as described in
Chapter IV. Inactivated HIV-infected cells were treated with antibodies for 60
minutes prior to addition of fresh autologous PBMC. Preimmune sera were
used and had no effect on proliferation induced by HIV-infected cells.

72
Results
HIV Replication in Nef-Stimulated PBMC Cultures
Since T cell activation occurs with exogenous Net protein, the question
arose as to whether Net stimulation of PBMC was sufficient to induce HIV-1
replication. To this end, PBMC of several HIV-negative donors were tested for
proliferation induced by Nef and other T cell mitogens (Table VIII). Consistent
with previous data, PBMC from most of the donors proliferated significantly in
response to Nef protein, as well as to the T cell mitogens SEA and
phytohemagglutinin A (PHA).
Unstimulated and mitogen-stimulated PBMC were infected with HIV-1
and cultures were monitored over a 12-day period for signs of viral replication.
Supernatants from unstimulated PBMC showed only marginal reverse
transcriptase (RT) activity and p24 antigen production (Table VIII). RT and p24
antigen levels were high in cells stimulated with T cell mitogens, in particular
SEA. Interestingly, Nef-stimulated cells produced moderate to high levels of RT
and p24 antigen, indicating that these cells were capable of sustaining virus
replication. In an attempt to quantify the number of infected cells in these
cultures, PCR analysis of serially-diluted DNA was performed using nested
primers for the gag gene. Results indicated 10- to 30-fold increases in the
number of cells harboring virus in Nef-stimulated cultures, as compared to
unstimulated controls. These data indicate that Nef stimulation resulted in a
large number of infected cells and a concomitant increase in viral load.
Infectious Virus Is Produced bv Nef-Stimulated Cultures
As a means of determining whether the virus produced by Nef-stimulated
was infective, supernatants from HIV-infected cultures were tested for
reinfectivity. SEA-stimulated PBMC were infected with virus from 6-day

Table VIII
Maximal proliferation, RT, and p24 antigen values from stimulated cultures
Donor
Stimulation
Maximal Proliferation
Mean 1 SD Fold increase
Maximal RT value
Mean 1 SD Fold increase
Maximal p24 antigen value
Mean 1 SD Fold increase
204
Nef
8108 238
8.0
1901417141
10.9
256 1 24
10240
SEA
4368414316
43.3
29680 1 3509
17.1
10241402
40960
PHA
54874 4711
54.4
3581715233
20.6
1814180
72560
None
1009+199
-
1740141
-
0.025 1 0.01
-
205
Nef
123631864
10.1
2661413119
7.1
731 2
1197
SEA
732431 10333
60.6
125515116634
33.5
15231332
24967
PHA
57267 1 455
47.6
913131 11325
24.4
1668149
27344
None
12281583
-
3732 1 947
-
0.061 1 0.052
-
206
Nef
187531 1711
15.3
9823 1 3709
18.8
30.610.7
124
SEA
1247421 10411
96.0
218412149382
412.9
8191153
3316
PHA
N.D.
-
1200614132
22.7
38.1 6.7
154
None
13131356
-
529 1 35
-
0.24710.03


Table VII --continued
Donor
Stimulation
Maximal Proliferation
Mean SD Fold increase
Maximal RT value
Mean + SD Fold increase
Maximal p24 antigen value
Mean SD Fold increase
207
Net
14480 707
7.8
19229 + 3284
8.7
1408 38
14667
SEA
44040 2424
19.4
26700 3117
12.0
2541 178
26469
PHA
N.D.
-
N.D.
-
N.D.
-
None
2389 690
-
2118 162
-
0.096 0.01
-
208
Net
19473 3713
17.7
13133 2083
19.7
434172
4667
SEA
38033 7971
33.6
14424 318
21.7
520 61
5591
PHA
N.D.
-
N.D.
-
N.D.
-
None
1167 + 58
-
1009 249
-
0.093 0.01
-
209
Net
3314 + 830
4.3
1310 291
1.3
51 25
70
SEA
202441204
21.2
4558 360
4.5
226 81
310
PHA
N.D.
-
N.D.
-
N.D.
-
None
1004185
-
666130
-
0.73 0.1
-

75
supernatants of cultures stimulated with Nef, SEA, and PHA. All supernatants
were adjusted to achieve equivalent RT units per culture. The results of two
representative experiments are shown in Figure 17. Supernatants from Nef-
stimulated cultures had high titers of infectious virus similar to supernatants
from cultures stimulated with SEA and PHA. These results indicate that virus
replication in Nef-stimulated cultures, like the replication in SEA- and PHA-
stimulated cultures, yielded infectious virus.
Nef-Stimulated PBMC Express T Cell Activation Markers
It has been shown that HIV requires activated T cells for virus replication.
Cellular activation was investigated by double-staining FACS analysis of Nef-
stimulated cells using antibodies to CD4 and HLA-DR (Table IX). Increased
levels of dually-stained cells were observed in cultures of PBMC stimulated with
Nef protein, as compared to unstimulated cultures. HLA-DR expression on
CD4+ human T cells has been shown to correlate with an activated T cell state.
No increase was seen in cells that stained for CD8, either alone or in
conjunction with HLA-DR. These results are consistent with our previous
studies showing production of T helper cytokines upon stimulation with Nef.
Thus, stimulation of PBMC with Nef protein induces activation of T helper cells.
Expression of Nef bv HIV-Infected Cells
Culture supernatants of infected cells were tested for soluble Nef and none
could be detected, suggesting that release of soluble Nef may not be occurring
in these cultures. However, it has been shown that Nef is expressed on the
surface of infected cells (Fujii et al., 1993). This leads to the question of
whether Nef, in association with the extracellular membrane of infected cells,
plays a role in the activation event(s) required for continuous HIV replication.

RT (in CPM)
76
40000
30000 -
20000
10000 -
Expt 1
Expt 2
Figure 17. Infectivity assay on supernatants from Nef-stimulated HIV-infected
cultures.

77
Table IX
Nef-induced activation of T cells as assessed by FACS
Cultures from:
% Total of dually-stained populations
CD4 + HLA-DR CD4 + IL2R
Donor 206
Unstimulated
3.17
N.D.c
Nef-stimulated
7.39
N.D.
SEA-stimulated
29.46
N.D.
Donor 213
Unstimulated
6.65
N.D.
Nef-stimulated
16.74
N.D.
Donor 214
Unstimulated
3.45
N.D.
Nef-stimulated
11.15
N.D.
Donor 215
Unstimulated
N.D.
7.75
Nef-stimulated
N.D.
10.55
Donor 216
Unstimulated
1.52
4.66
Nef-stimulated
9.02
7.68

78
Thus, we investigated the possibility that HIV-infected cells could activate
autologous PBMC, similar to the activation seen with exogenously added Net.
To achieve maximal infection, PBMC were stimulated with mitogen prior to
infection with HIV. After 5 days, HIV-infected cells were washed and inactivated
with paraformaldehyde. Fresh autologous PBMC were cultured in the presence
of infected cells and monitored for proliferation. Proliferation was observed in
co-cultures of fresh and infected autologous cells (Table X). Uninfected
stimulated cells served as controls, and no proliferation was observed in these
co-cultures. Proliferative responses were significantly reduced by the addition
of polyclonal anti-Nef antibodies, while control anti-SEA antibodies were
without effect. Anti-Nef inhibition of proliferation was dose dependent, with
lower concentrations of antibodies having less inhibitory activity. Similar to the
results observed on HIV-infected cells, anti-Nef antibodies were capable of
reducing proliferation in response to Nef, but not to SEA. Thus, a significant
portion of the observed mitogenic activity of HIV-infected cells is due to Nef
protein.
Discussion
It is interesting that HIV-infected cells were more potent at inducing T cell
proliferation than was soluble Nef. Given that Nef interacts with MHC class II
antigens, it is conceivable that optimal conditions for Nef association with MHC
are achieved in HIV-infected cells, analogous to the expression of the mouse
mammary tumor virus superantigen in infected B cells. These data suggest that
proliferation occurred as a result of Nef expressed on the surface of HIV-
infected cells, thereby indicating that Nef is a virally-encoded mitogen. Thus,
the genome of HIV encodes for its own T cell mitogen, which would amplify the

79
Table X.
Ability of anti-Nef peptide antibodies to block proliferation induced by
autologous HIV-infected cells
Fresh PBMC cultured Anti-Nef Proliferation
in the presence of: antibodies CPM Fold increase p value
(Mean SD)
HIV-infected PBMC
-
137187 1923
162.2
HIV-infected PBMC
+
79740 8764
94.3
<0.003
_
+
1973 880
2.3
846 271

80
replication of virus in the host. As such, Nef could be classified as a virulence
factor for HIV.
It has been demonstrated that activated T cells are required for
replication of HIV-1. On the other hand, the ability of HIV-1 to infect quiescent or
resting cells is still questioned. In two studies using quiescent cells, HIV-1
replication in vitro was blocked at the level of virus entry (Gowda et al., 1989;
Tang and Levy, 1990). Others have shown that blockage of replication
occurred at an intracellular event, possibly at reverse transcription (Zack et al.,
1990) or proviral integration (Stevenson et al., 1990; Bukrinsky et al., 1991).
Upon entry into quiescent cells, replication could be induced by subsequent T
cell activation. Consistent with data from in vitro studies are reports that high
numbers of quiescent CD4+ cells from asymptomatic HIV-infected patients
harbor unintegrated viral DNA capable of integration subsequent to PHA
stimulation. Further, AIDS patients have increased numbers of cells containing
integrated virus, corresponding with greater production of virus at this stage of
the disease. These findings indicate that HIV-1 may infect quiescent cells, but
such infection is nonproductive until cell activation occurs.
Studies suggest that Nef plays an important role in HIV pathogenesis.
Infection of adult rhesus monkey with a neAdeleted mutant of SIVmac239
resulted in low viral burden and did not cause disease, although infected
animals were persistently seropositive (Daniel et al., 1992). Recently, genomic
analysis of an HIV strain from a longterm survivor revealed a 118 bp deletion in
the nef gene which resulted in an out-of-frame shift for downstream sequences
(Kirchhoff et al., 1995). These studies hint at the importance of Nef in HIV
pathogenesis in that repeated attempts to isolate virus from this patient with
nonprogressive disease were unsuccessful. Mutations in nef may result in
altered mitogenic activity of the protein, with concomitant reduction or loss of the

81
ability to activate T cells. Possible consequences of nef mutations, then, would
be a reduction both in the cellular reservoir for virus replication and in the viral
load in the infected host. Other investigators have not found alterations in the
nef gene in strains isolated from other longterm survivors (Cao et al., 1995). It
would be interesting to determine the T cell reactivity of these individuals to Nef,
as we have found that 10-15% of individuals that we tested did not respond to
Nef. It is possible that nonresponsiveness to Nef may play an important role in
longterm survival in some HIV-infected individuals. Thus, Nef may aid in the
establishment and maintenance of infection, suggesting its role as a virulence
factor for HIV.

CHAPTER VI
A MODEL FOR THE ROLE OF NEF IN THE PATHOGENESIS OF HIV
As shown in Chapters ll-V, Net binds to MHC class II antigens at a site(s)
involved in bacterial superantigen binding. Like MMTV superantigen, Net
protein is encoded in the 3' long terminal repeat of the genome of lentiviruses
such as HIV-1 and simian immunodeficiency virus (SIV). Nef induced
significant levels of proliferation in PBMC from a wide sampling of donors (85-
90%), although these responses are lower than those induced by SEA.
Further, Nef stimulation resulted in the production of the T helper cell cytokines,
IL 2 and interferon gamma (IFNy). Proliferation in response to Nef was
observed in reconstituted cultures consisting of T cells and inactivated APCs,
which is compelling evidence for Nef superantigen activity. Further, Nef did not
induce purified T cells to proliferate in the absence of APCs, which is consistent
with Nef superantigen activity. However, no apparent Vp expansions were
found, although such data do not preclude the Vp specificity of Nef activation.
Thus, Nef has superantigen-like characteristics in that it binds to class II
antigens, does not require processing by APC, and activates T cells to
proliferate and release cytokines such as IL 2 and IFNy.
The functional role that Nef plays in the lentiviral pathogenesis is
uncertain, although its importance is reflected in vaccine studies in which a nef-
deleted SIV mutant did not cause disease and protected animals upon
challenge with the pathogenic wildtype strain (Daniel et al. 1992). The viral
load in SIV mutant-infected animals was considerably lower than in animals
challenged with wildtype SIV. Within this context, the ability of Nef to activate T
82

83
cells is interesting in that active HIV replication requires T cell activation.
Further, although resting or quiescent T cells can be infected, evidence
suggests that HIV replication only occurs upon subsequent cellular activation.
Nef stimulation of PBMC was sufficient to induce HIV-1 replication. Virus
replication in Nef-stimulated cultures yielded infectious virus, rather than
defective particles. Flow cytometric analysis showed that increased
percentages of cells that stained for both CD4 and HLA-DR were present in
Nef-stimulated cultures. Further, increased percentages of cells that stained for
both CD4 and IL 2R were observed in Nef-stimulated cultures. These results
are consistent with the production of T helper cytokines upon stimulation with
Nef. Thus, stimulation of PBMC with Nef protein induces T cell activation and
such activation is sufficient for HIV replication.
Proliferation of fresh autologous PBMC cultured in the presence of
infected cells was observed. Further, proliferative responses were significantly
reduced by the addition of polyclonal anti-Nef antibodies. These data suggest
that proliferation occurred as a result of Nef present on the surface of HIV-
infected cells, thereby indicating that Nef may be a virally-encoded mitogen.
Nef has been shown to induce differentiation of human B cells to
immunoglobulin secreting cells (Chirmule et al., 1994). Monoclonal antibodies
to HLA-DR and adhesion molecules abrogated Nef-induced differentiation. B
cell stimulation required T cells and monocytes, the latter producing IL 6 upon
Nef stimulation. Interestingly, staphylococcal superantigens have been shown
to induce B cell differentiation analogous to that described for Nef (Stohl et al.,
1994). Thus, Nef can stimulate both T and B cells in a manner similar to the
staphylococcal enterotoxin superantigens.
This leads to the question of the activation event(s) that occurs during
HIV-1 exposure and that ultimately lead to the establishment of infection. Our

84
results suggest a model in which Net acts as a HIV-encoded T cell mitogen
(Figure 18). Net may interact with T cells either as an integral part of the
membranes of infected T cells or in a soluble form released as the result of lysis
of infected cells. Nef is present on the membranes of infected T cells (Fujii et
al., 1993), which is consistent with this model. Nef expression may occur in the
context of the T cell activation marker, HLA-DR, and the Nef/HLA-DR complex
would stimulate uninfected T cells, as well as B cells. Such stimulation would
allow for the expansion of a cellular reservoir for replication of the virus,
eventually leading to T cell anergy and/or apoptosis. B cell activation and
differentiation could ultimately lead to hypergammaglobulinemia. Thus, the HIV
genome may encode for its own T cell mitogen, which would induce the
amplification of virus replication in the host, with ultimate deleterious effects on
the immune system. As such, Nef could be classified as a virulence factor for
HIV.

85
l
Nef-induced
cellular
effects
Nef binding to HLA-DR
Presentation to uninfected T cells
Cellular proliferation
T cell activation
Cytokine production
B cell proliferation and differentiation
Potential
Nef-related
pathogenesis
I
Increased viral load
Immunosuppression via T cell
anergy/apoptosis
Autoimmunity via activation of B and T
cells
Hypergammaglobulinemia
Figure 18. Model for the role of Nef in the pathogenesis of HIV. Activation of
uninfected T cells occurs by Nef interaction in either of two forms: as soluble
Nef released from lysed cells, presented in the context of MHC class II antigens
on APC, or as an integral part of the membranes of infected T cells, complexed
to HLA-DR. Binding to uninfected T cells may occur via TCR or CD4. T cell
activation results in proliferation and release of cytokines such as IFNyand IL 2,
thereby creating a cellular reservoir for virus and increasing viral load in the
host. T cell activation results in depletion of T cells via virus production, anergy
and/or apoptosis. B cell differentiation, possibly mediated by T cell cytokine
release, results in hypergammaglobulinemia. Autoimmune-like sequelae may
result from B cell differentiation into lg-secreting cells, and activation of T cells.

LIST OF REFERENCES
Abe, J., B. L. Kotzin, K. Jujo, M. E. Melish, M. P. Glode, T. Kohsaka, and
D. Y. M. Leung. 1992. Selective expansion of T cells expressing T-cell
receptor variable region Vp2 and Vp8 in Kawasaki disease. Proc. Natl. Acad.
Sci. USA 89:4066.
Acha--Orbea, H. 1992. Retroviral superantigens. In B. Fleischer, Ed.
Ciological Significance of Superantigens. Basel: Karger, pp65-86.
Acha--Orbea, H. and E. Palmer. 1991. Mis a retrovirus exploits the
immune system. Immunol. Today 12:356.
Acha--Orbea, H., A. N. Shakhov, L. Scarpellino, E. Kolb, V. Muller, A.
Vessaz-Shaw, R. Fuchs, K. Blochinger, P. Rollini, J. Billotte, M. Sarafidou, H. R.
MacDonald, and H. Diggelmann. 1991. Clonal deletion of Vpl4-bearing T cells
in mice transgenic for mammary tumor viurs. Nature 350:207.
Achong, B. G., P. W. A. Mansell, M. A. Epstein, and P. Clifford. 1971. An
unusual virus in cultures from a human nasopharyngeal carcinoma. J. Nat.
Cancer Inst. 46:299.
Allan, J. S., J. E. Coligan, T. --H. Lee, M. F. McLane, P. J. Kanki, J. E.
Groopman, and M. Essex. 1985. A new HTLV-III/LAV encoded antigen
detected by antibodies from AIDS patients. Science 230:810.
Arthur, L. O., J. W. Bess, R. C. Sowder, R. E. Benveniste, D. L. Mann, J. C.
Chermann, and L. E. Henderson. 1992. Cellular proteins bound to
immunodeficiency viruses: implications for pathogenesis and vaccines.
Science 258:1935.
Baba, T. W., Y. S. Jeong, D. Penninck, R. Bronson, M. F. Greene, and R.
M. Ruprecht. 1995. Pathogenicity of live, attenuated SIV after mucosal
infection of neonatal macaques. Science 267:1820.
Bahraoui, E., M. Yagello, J. --N. Billaud, J. --M. Sabatier, B. Guy, E.
Muchmore, M. Girard, and J. -C. Gluckman. 1990. Immunogenicity of the
human immunodeficiency virus (HIV) recombinant nef gene product. Mapping
of T-cell and B-cell epitopes in immunized chimpanzees. AIDS Res. Hum.
Retroviruses 6:1087.
Barnstable, C. J., W. F. Bodmer, G. Brown, G. Galfre, C. Milstein, A. F.
Williams, and A. Ziegler. 1978. Production of monoclonal antibodies to group
A erythrocytes, HLA, and other human cell surface antigens new tools for
genetic analysis. Cell 14:9.
86

87
Bergdoll, M. S. 1985. The staphylococcal enterotoxins-an update. In J.
Jeljaszewics, Ed. The Staphylococci. New York: Gustav Fisher Verlag, pp247-
266.
Bisset, L. R., M. Opravil, E. Ludwig, and W. Fierz. 1993. T cell response
to staphylococcal superantigens by asymptomatic HIV-infected individuals
exhibits selective changes in T cell receptor V beta-chain usage. AIDS Res.
Hum. Retroviruses 9:241.
Brodsky, F.M. 1984. A matrix approach to human class II
histocompatibility antigens: reactions of four monoclonal antibodies with the
products of nine haplotypes. Immunogenetics 19:179.
Buhrinsky, M. I., T. L. Stanwick, M. P. Dempsey, and M. Stevenson.
1991. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1
infection. Science 254:423.
Cao, Y., L. Qin, L. Zhang, J. Safrit, and D. D. Ho. 1995. Virological and
immunological characterization of long-term survivors of huma
immunodeficiency virus type 1 infection. New Engl. J. Med. 332:233.
Carlsson, R., H. Fischer, and H. O. Sjogren. 1988. Binding of
staphylococcal enterotoxin A to accessory cells is a requirement for its ability to
activate human T cells. J. Immunol. 140:2484.
Chinatgumpala, M. M., J. A. Mollick, and R. R. Rich. 1991.
Staphylococcal toxins bind to different sites on HLA-DR. J. Immunol. 147:3876.
Chirmule, N., N. Oyaizu, C. Saxinger, and S. Pahwa. 1994. Nef protein
of HIV-1 has B-cell stimulatory activity. AIDS 8:733.
Choi, Y., J. Kappler, and P. Marrack. 1991. A superantigen encoded in
the open reading frame of the 3' long terminal repeat of mouse mammary tumor
virus. Nature 350:203.
Choi, Y., J. Kappler, and P. Marrack. 1992. Structural analysis of a
mouse mammary tumor virus superantigen. J. Exp. Med. 175:847.
Cole, B. C. and M. M. Griffiths. 1993. Triggering and exacerbation of
autoimmune arthritis by the Mycoplasma arthriditis superantigen MAM. Arth.
Rheum. 36:994.
Dalgleish, A. G., S. Wilson, M. Gompels, C. Ludlam, B. Gazzard, A. M.
Coates, and J. Habeshaw. 1992. T-cell receptor variable gene products and
early HIV-1 infection. Lancet 339:824.
Daniel, M. D., F. Kirchhoff, S. C. Czajak, K. Sehgal, and R. C. Desrosiers.
1992. Protective effects of a live attenuated SIV vaccine with a deletion in the
nef gene. Science 258:1938.

88
Dellabonna, P. J. Peccoud, J. Kappler, P. Marrack, C. Benoit, and D.
Mathis. 1990. Superantigens interact with MHC class II molecules outside of
the antigen binding groove. Cell 62:1115.
deRonde, A., B. Klaver, W. Keulen, L. Smit, and J. Goudsmit. 1992.
Natural HIV-1 Nef accelerates virus replication in primary human lymphocytes.
Virology 188:391.
Devereax, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set
of sequence analysis programs for the VAX. Nucl. Acid Res. 12:387.
Dobrescu, D., B. Ursea, M. Pope, A.S. Asch, and D.N. Posnett. 1995.
Enhanced HIV-1 replication in V(312 due to human cytomegalovirus in
monocytes: evidence for a putative herpesvirus superantigen. Cell 82:753.
Dyson, P. J., A. M. Knight, S. Fairchild, E. Simpson, and K. Tomonari.
1991. Genes encoding ligands for deletion of Vpl1 T cells cosegregate with
mammary tumor virus genomes. Nature 349:531.
Edelman, A. S. and S. Zolla-Pazner. 1989. AIDS: a syndrome of
immune dysregulation, dysfunction, and deficiency. FASEB J. 3:22.
Festenstein, H. 1973. Immunogenetic and biological aspects of in vitro
lymphocyte allotransformation (MLR) in the mouse. Transplant. Rev. 15:62.
Fischer, H., M. Dohlsten, M. Lindvall, H. O. Sjogren, and R. Carlsson.
1989. Binding of staphylococcal enterotoxin A to HLA-DR on B cell lines. J.
Immunol. 142:3151.
Fleischer, B. and H. Schrezenmeier. 1988. T cell stimulation by
staphylococcal enterotoxins: Clonally variable response and requirement for
major histocompatibility complex class II molecules on accessory or target cells.
J. Exp. Med. 167:1697.
Fluegel, R. M. 1993. The molecular biology of the human spumavirus.
In B. R. Cullen, Ed. Human Retroviruses. Oxford: IRL Press, pp193-214.
Franchini, G., M. Robert-Guroff, J. Ghrayeb, N. T. Chang, and F. Wong-
Staal. 1986. Cytoplasmic localization of the HTLV-III 3' orf protein in cultured T
cells. Virology 155:593.
Frankel, W. N., C. Rudy, J. M. Coffin, and B. T. Huber. 1991. Linkage of
Mis genes to endogenous mammary tumor virus. Nature 349:525.
Fraser, J.D. 1989. High-affinity binding of staphylococcal enterotoxins A
and B to HLA-DR. Nature 339:221.

89
Fujii, Y., Y. Nishino, T. Nakaya, K. Tokunaga, and K. Ikuta. 1993.
Expression of human immunodeficiency virus type 1 Nef antigen on the surface
of acutely and persistently infected human T cells. Vaccine 11:1240.
Gallo, R.C., Salahuddin, S.Z., M. Popovic, G. M. Shearer, M. Kaplan, B. F.
Haynes, T. J. Palker, R. Redfield, J. Oleske, and B. Safai. 1984. Frequent
detection and isolation of cytopathic retroviruses (HTLV-III) from patients with
AIDS and at risk for AIDS. Science 224:500.
Garcia, J. V. and A. D. Miller. 1991. Serine-phosphorylation-
independent downregulation of cell-surface CD4 by nef. Nature 350:508.
Golovkina, T. V., A. Chervonsky, J. P. Dudley, and S. R. Ross. 1992.
Transqenic mouse mammary tumor virus superantigen prevents viral infection.
Cell 69:637.
Gowda, S. D., B. S. Stein, N. Mohagheghpour, C. J. Benike, and E. G.
Engleman. 1989. Evidence that T cell activation is required for HIV-1 entry in
CD4+ lymphocytes. J. Immunol. 142:773.
Griggs, N. D., C. H. Pontzer, M. A. Jarpe, and H. M. Johnson. 1992,
Mapping of multiple binding domains of the superantigen staphylococcal
enterotoxin A for HLA. J. Immunol. 148:2516.
Guy, B., M. P. Kierny, Y. Riviere, C. Le Peuch, K. Dott, M. Girard, L.
Montagnier, and J. --P. Lecocq. 1987. HIV F/3' orf encodes a phosphorylated
GTP-binding protein resembling an oncogene product. Nature 330:266.
Hammes, S. R., E. P. Dixon, M. H. Malim, B. R. Cullen, and W. C. Greene.
1989. Nef protein of human immunodeficiency virus type 1: evidence against
its role as a transcriptional inhibitor. Proc. Natl. Acad. Sci. USA 86:9549.
Held, W., H. Acha--Orbea, H. R. MacDonald, and G. A. Waanders.
1994a. Superantigens and retroviral infections: insights from mouse mammary
tumorvirus. Immunol. Today 15:184.
Held, W., G. A. Waanders, H. R. MacDonald, and H. Acha-Orbea.
1994b. MHC class II hierarchy of superantigen presentation predicts efficiency
of infection with mouse mammary tumor virus. Int. Immunol. 6:1403.
Held, W., G. A. Waanders, A. N. Shakhov, L. Scarpellino, H. Acha-
Orbea, and H. R. MacDonald. 1993. Superantigen-induced immune
stimulation amplifies mouse mammary tumor virus infection and allows
transmission. Cell 74:529.
Herman, A., N. Labrecque, J. Thibodeau, P. Marrack, and R. R. Rich.
1991. Identification of the staphylococcal enterotoxin A superantigen binding
site in the beta 1 domain of the human histocompatibility antigen HLA-DR.
Proc. Natl. Acad. Sci. USA 88:9954.

90
Heston, W. E., M. K. Deringer, and H. B. Andervont. 1945. Gene-milk
agent relationship in mammary tumor development. J. Natl. Cancer Inst. 5:289.
Ho, S. N., R. T. Abraham, S. Gillis, and D. J. Mckean. 1987. Differential
bioassay of interleukin 2 and interleukin 4. J. Immunol. Methods. 98:99.
Hugin, A. W., M. S. Vacchio, and H. C. Morse III. 1991. A virus-encoded
"superantigen" in a retrovirus-induced immunodefieciency syndrome of mice.
Science 252:424.
Imberti, L, A. Sottini, A. Bettinardi, M. Puoti, and D. Primi. 1991.
Selective depletion in HIV infection of T cells that bear specific T cell receptor
Vp sequences. Science 254:860.
Johnson, H. M. 1981. Cellular regulation of immune interferon
production. Antiviral Res. 1:37.
Johnson, H. M. 1985 mechanism of lnterferon-y production and
assessment of immunoregulatory properties. In E. Pick and M. Landy, Eds.
Lymphokines Vol. 11. New York: Academic Press,pp 33-53.
Johnson, H. M. and J. A. Bukovic. 1975. Staphylococcal entertoxin A
inhibition of the primary in vitro antibody response to thymus-dependent
antigen. IRCS Med. Sci. 3:398.
Johnson, H. M., M. P. Langford, B. Lakhchaura, T. -S. Chan, and G. J.
Stanton. 1982. Neutralization of native human gamma interferon (HuIFNy) bt
antibodies to a synthetic peptide encoded by the 5' end of HuIFNy cDNA. J.
Immunol. 129:2357.
Johnson, H. M. and H. I. Magazine. 1988. Potent mitogenic activity of
staphylococcal enterotoxin A requires induction of interleukin 2. Int. Arch.
Allergy Appl. Immunol. 87:87.
Johnson, H. M., J. K. Russell, and C. H. Pontzer. 1991. Staphylococcal
enterotoxin superantigens. P.S.E.B.M. 198:765
Johnson, H. M., J. K. Russell, and C. H. Pontzer. 1992. Role of
superantigens in human disease. Sci. Am. 266(4):92.
Johnson, H. M., B. A. Torres, and J. M. Soos. 1995. Superantigens :
Structure and Relevance to Human Disease. Proc. Soc. Exp. Biol. Med. In
Press.
Kappler, J., B. Kotzin, L. Herron, E. W. Gelfand, R. D. Bigler, A. Boylston,
S. Carrel, D. N. Posnett, Y. Choi, and P. Marrack. 1989. Vp-specific stimulation
of human T cells by staphylococcal toxins. Science 244:811.

91
Kawabe.Y. and A. Ochi. 1990. Selective anergy of Vp8+ T cells in
staphylococcus enterotoxin B-primed mice. J. Exp. Med. 172:1065.
Kawabe.Y. and A. Ochi. 1991. Programmed cell death and extrathymic
reduction of Vp8+ CD4+ T cells in mice tolerant to Staphylococcus aureus
enterotoxin B. Nature 349:245.
Kirchhoff, F., T. C. Greenough, D. B. Brettler, J. L. Sullivan, and R. C.
Desrosiers. 1995. Absence of intact nef sequences in a long-term survivor with
nonprogressive HIV-1 infection. New Engl. J. Med. 332:228.
Klatzmann, D., F. Barre-Sinoussi, M. T. Nugeyre, C. Danquet, E. Vilmer,
C. Griscelli, F. Brun-Veziret, C. Rouzioux, J. C. Gluckman, J. C. Chermann, and
L. Montagnier. 1984. Selective tropism of lymphadenopathy associated virus
(LAV) for helper-inducer T lymphocytes. Science 225:59.
Kotzin, B. \J, D. Y. M. Leung, J. Kappler, and P. Marrack. 1993.
Superantigens and their potential role in human disease. Adv. Immunol, 54:99.
Lafon, M., M. Lafage, A. Martinez-Arends, R. Ramirez, F. Vuiller, D.
Charron, V. Lotteau, and D. Scott-Algara. 1992. Edidence for a viral
superantigen in humans. Nature 358:507.
Lafon, M., D. Scott-Algara, P. N. Marche, P. A Cazenave. and E. Jouvin-
Marche. 1994. Neonatal deletion and selctive expansion of mouse T cells by
exposure to rabies virus nuceocapsid superantigen. J. Exp. Med. 180:1207.
Langford, M. P., G. J. Stanton, and H. M. Johnson. 1978a. Biological
effects of staphylococcal enterotoxin A on human peripheral lymphocytes.
Infect. Immun. 22:62.
Langford, M. P., D. A. Weigent, G. J. Stanton, and S. Baron. 1978b.
Virus plaque-reduction assay for interferon. Microplaqye and regular
macroplaque reduction assays. Methods Enzymol. 78:339.
Laurence, J., A. S. Hodstev, and D. N. Posnett. 1992. Superantigen
implicated in dependence of HIV-1 replication in T cells on TCR Vp expression.
Nature 358:255.
Lechler, R. I., V. Bal, J. B. Rothbard, R. N. Germain, R. Sekaly, E. O. Long,
and J. Lamb. 1988. Structural and functional studies of HLA-DR restricted
antigen recognition by human helper T lymphocyte clones by using transfected
murine cell lines. J. Immuno. 141:3003.
Lee, J. M. and T. H. Watts. 1990. Binding of staphylococcal enterotoxin
A to purified murine MHC class II molecules in supported lipid bilayers. J.
Immunol. 145:3360.

92
Lemaitre, M., D. Guetard, Y. henin, L. Montagnier, and A. Zerial. 1990.
Protective activity of tetracycline analogs against the cytopathic effect of human
immunodeficiency viruses in CEM cells. Res. Virol. 141:5.
Lewis, H. M., B. S. Baker, S. Bokth, A. V. Powles, J. J. Garioch, H.
Valdimarsson, and L. Fry. 1993. Restricted T cell receptor V(3 gene usage in
the skin of patients with guttate and chronic plaque psoriasis. Brit. J. Dermatol.
129:514.
Lifson, J. D., M. B. Feinberg, G. R. Reyes, L. Rabin, B. Banapour, S.
Chakrabati, B. Moss, F. Wong-Staal, K. S. Steimer, and E. G. Engelman. 1986.
Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope
glycoprotein. Nature 323:725.
MacDonald, H. R., A. L. Glasebrook, R. Schneider, R. K. Lees, H. P.
Pircher, T. Pedrazzini, O. Kanagawa, J.-F. Nicholas, R. C. Howe, R. M.
Zinkernagel, and H. Hentgartner. 1989. T-cell reactivity and tolerance to Mlsa-
encoded antigens. Immunol. Rev. 107:89.
Mariani, R. and J. Skowronski. 1993. CD4 down-regulation by nef
alleles isolated from human immunodeficiency virus type 1-infected individuals.
Proc. Natl. Acad. Sci. USA 90:5549.
Marrack, P. and J. Kappler. 1990. The staphylococcal enterotoxins and
their relatives. Science 248:705.
Marrack, P., E. Kushnir, and J. Kappler. 1991. A maternally inherited
superantigen encoded by a mouse mammary tumor virus. Nature 349:524.
Merryman, P., J. Silver, P. K. Gregeersen, G. Solomon, and R.
Winchester. 1989. A novel association of DQ alpha and DQ beta genes in the
DRw10 haplotype. J. Immunol. 143:2068.
Montagnier, L. 1995 Nef vaccination against HIV disease. Lancet
346:1170.
Mollick, J. A., R. G. Cook, and R. R. Rich. 1989. Class II molecules are
specific receptors for staphylococcal enterotoxin A. Science 244:817.
Mottershead, D. G., P. -N. Hsu, R. G. Urban, J. L. Strominger, and B. T.
Huber. 1995. Direct binding of Mtv7 superantigen (Mls-1) to soluble MHC
class II molecules. Immunity 2:149.
Ohmen, J. D., P. F. Barnes, C. L. Grisso, B. R. Bloom, and R. L. Modlin.
1994. Evidence for a superantigen in human tuberculosis. Immunity 1:35.
Oroszlan, S. and R. B. Luftig. 1990. Retroviral proteinases. Curr. Top.
Microbiol. Immunol. 157:153.

93
Ozato, K, N. M. Mayer, and D. H. Sachs. 1982. Monoclonal antibodies to
mouse major histocompatibility complex antigens. Transplantation 34:113.
Parker, J. M. R., D. Guo, and R. S. Hodges. 1986. New hydrophilicity
scale derived from high-performance liquid chromatography peptide retention
data: correlation of predicted surface residues with antigenicity and x-ray-
derived accessible sites. Biochem. 25:5425.
Peavy, D. L, W. H. Adler, and R. T. Smith. 1970. The mitogenic effects of
endotoxin and staphylococcal enterotoxin B on mouse spleen cells and human
peripheral lymphocytes. J. Immunol. 105:1453.
Poiesz, B. J., F. W. Ruscetti, A. F. Gazdar, P. A. Bunn, J. D. Minna, and R.
C. Gallo. 1980. Detection and isolation of type C retrovirus particles from fresh
cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. natl.
Acad. Sci. USA 77:7415.
Pontzer, C. H., N. D. Griggs, and H. M. Johnson. 1993. Agonist
properties of a microbial superantigen peptide. J. Immunol. 193:191.
Pontzer, C. H., M. J. Irwin, N. R. J. Gascoigne, and H. M. Johnson. 1992.
T cell antigen receptor binding sites for the microbial superantigen
staphylococcal enterotoxin A. Proc Natl. Acad. Sci. USA 89:7727.
Pontzer C. H., J. K. Russell, and H. M. Johnson HM. 1991. Site of non-
restrictive binding of SEA to class II MHC antigens. Int. Arch. Allergy Appl.
Immunol. 93:107.
Pontzer, C. H., J. K. Russell, and H. M. Johnson. 1990. Site of
nonrestrictive binding of SEA to class II MHC antigens. Int. Arch. Allergy Appl.
Immunol. 93:107.
Pullen, A. M., Y. Choi, E. Kushnir, J. Kappler, and P. Marrack. 1992. The
open reading frames inthe 3' long terminal repeats of several mouse mammary
tumor virus integrants encode Vp3-specific superantigens. J. Exp. Med. 175:41.
Rebai, N, G. Pantaleo, J. F. Demarest, C. Ciurli, H. Soudeyns, J. W.
Adelsberger, M. Vaccarezza, R. E. Walker, R. P. Sekaly, and A. S. Fauci. 1994.
Analysis of the T-cell receptor beta-chain variable-region (Vp) repertoire in
monozygotic twins discordant for human immunodeficiency virus: evidence for
perturbations of specific V beta segments in CD4+ T cells of the virus-positive
twins. Proc. Natl. Acad. Sci. USA 91:1529.
Rellahan, B. L, L. A. Jones, A. M. Kruisbeek, A. M. Fry, and L. A. Mathis.
1990. In vivo induction of anergy in peripheral Vp8+ T cells by staphylococcal
enterotoxin B. J. Exp. Med. 172:1091.
Ringold, G.M. 1983. Regulation of mouse mammary tumor virus gene
expression by glucocorticoid hormones. Curr. Top. Microbiol. Immunol. 106:79.

94
Robbins, P. A., E. L. Evans, A. H. Ding, N. L. Warner, and F. M. Brodsky.
1987. Monoclonal antibodies that distinguish between class II antigens (HLA-
DP, DQ, and DR) in 14 haplotypes. Hum. Immunol. 18:301.
Rous, P. 1910. A transmissible avian neoplasm (sarcoma of common
fowl). J. Exp. Med. 12:696.
Rous, P. 1911. Transmission of a malignant new growth by means of a
cell-free filtrate. J. Amer. Med. Assoc. 56:198.
Russell, J. K., C. H. Pontzer, and H. M. Johnson. 1990. The l-Apb region
(65-85) is a binding site for the superantigen, staphylococcal entertoxin A.
Biochem. Biophys. Res. Comm. 168:696.
Russell, J. K., C. H. Pontzer, and H. M. Johnson. 1991. Both a-helices
along the major histocompatibility complex binding cleft are required for
staphylococcal enterotoxin A function. Proc. Natl. Acad. Sci. USA 88:7228.
Salmons, B. and W. H. Gunzburg. 1987. Current perspectives in the
biology of mouse mammary tumor virus. Virus Res. 8:81.
Schiffenbauer, J., H. M. Johnson, E. J. Butfilowski, L. Wegrzyn, and J. M.
Soos. 1993. Staphylococcal enterotoxins can reactivate experimental allergic
encephalomyelitis. Proc. Natl. Acad. Sci. USA 90:8543.
Sodrowski, J., W. C. Goh, C. Rosen, K. Campbell, and W. A. Haseltine.
1986. Role of the HTLV-III/LAV envelope in synctium formation and
cytopathicity. Nature 322:470.
Soos, J. M. and H. M. Johnson. 1994. Type I interferon inhibition of
superantigen stimulation: implications for treatment of superantigen-associated
disease. J. Interferon and Cytokine Res. 15:39.
Spawski, J. B. and P. E. Lipsky. 1992. Isolation of B cell populations. In
J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober,
Eds. Protocols in Immunology. New York: John Wiley and Sons, p7.5.1.
Stevenson, M., S. Haggerty, C. Lamonica, A. M. Mann, C. Meier, and A.
Wasiak. 1990. Cloning and characterization of human immunodeficiency virus
type 1 variants diminished in the ability to induce syncytium-independent
cytloysis. J. Virol. 64:3792.
Stohl, W., J. E. Elliott, and P. S. Linsley. 1994. Human T-cell dependent
B cell differentiation induced by staphylococcal superantigens. J. Immunol.
153:117.
Tang, S. and J. A. Levy. 1990. Parameters involved in the cell fusion
induced by HIV. AIDS 4:409.

95
Terwillegar, E. F., J. G. Sodroski, and W. A. Haseltine. 1990.
Mechanisms of infectivity and replication of HIV-1 and implications for therapy.
Ann. Emer. Med. 19:233.
Terwillegar, E. F., J. G. Sodroski, C. A. Rosen, and W. A. Haseltine. 1986.
Effects of mutations within the 3' orf open reading frame region of human T-cell
lymphotropic virus type III on replication and cytopathogenicity. J. Virol. 60:754.
Torres, B. A., J. K. Yamamoto, and H. M. Johnson. 1982. Cellular
regulation of immune interferon (IFNy) production: Lyt phenotype of the
suppressor cell. Infect. Immun. 35:770.
Tsubura, A., M. Inabe, S. Imai, A. Murakami, N. Oyaizu, R. Yasumizu, Y.
Ohnishi, H. Tanaka, S. Morii, and S. Ikehara. 1988. Intervention of T-cells in
transportation of mouse mammary tumor virus (milk factor) to mammary gland
cells in vivo. Cancer Res. 48:6555.
Uchiyama, T., T. Miyoshi-Akiyama, H. Kato, W. Fujimaki, K. Imanishi, and
X. Yan. 1993. Superantigenic properties of a novel mitogenic substance
produced by Yersinia pseudotuberculosis isolated from patients manifesting
acute and systemic symptoms. J. Immunol. 151:4407.
Wain-Hobson, S., P. Sonigo, O. Danos, S. Cole, and M. Alizon. 1985.
Nucleotide sequence of the AIDS virus LAV. Cell 40:9.
Winslow, G. M., M. T. Scherer, J. Kappler, and P. Marrack. 1992
Detection and biochemical characterization of the mouse mammary tumor
virus-7 superantigen. Cell 71:719.
Woodland, D. L., M. P. Happ, K. J. Gollob, and E. Palmer. 1991. An
endogenous retrovirus mediating deletion of ap T cells? Nature 349:529.
Yoshida, M., I. Miyoshi, and Y. Hinuma. 1982. Isolation and
characterization of retrovirus from cell lines of human T-cell leukemia and its
implication in the disease. Proc. Natl. Acad. Sci. USA 79:2031.
Zack, J. A., S. J. Arrigo, S. R. Weitman, A. S.Go, A. Haislip, and I. S. Y.
Chen. 1990. HIV-1 entry into quiescent primary lymphocytes: molecular
analysis reveals a labile, latent viral structure. Cell 61:213.
Zinkernagel, R. and H. Hentgartner. 1994. T-cell mediated
immunopathology versus direct cytolysis by virus: implications for HIV and
AIDS. Immunol. Today 15:262.

BIOGRAPHICAL SKETCH
Barbara Aurea Torres, a first-generation American, was born on June 4,
1956 at Hialeah Hospital in South Florida. Her parents, Manuel and Antonia
Torres, and sister, Marisol Torres Beaton, were born in Chile and emigrated to
the United States in 1950. She was blessed with parents who intentionally
gave her a first name that allowed her to "blend" into the American melting pot.
Her father's family in Chile has had a long-time connection to medicine and the
sciences. Barbara's grandfather was a physician in a rural community in Chile,
and her uncle is still a general practitioner in Ecuador. In 1989, Barbara's father
gave her a monograph on the atomic theory written by her great-grandfather,
who was an instructor at the Sorbonne University in Paris, France. This
monograph made a lasting impression on Barbara on the nature of scientific
inquiry. Barbara graduated in 1978 with a Bachelor of Arts degree from
Randolph-Macon Woman's College in Lynchburg, Virginia. In 1981, Barbara
did graduate work in the laboratory of Dr. Howard M. Johnson and received a
Master of Science degree from the Department of Microbiology at the University
of Texas Medical Branch in Galveston, Texas. Barbara continued working with
Dr. Johnson for several years, both in Galveston and at the University of Florida.
After a mundane two-year stint at Bio-Rad Laboratories in Richmond, California,
Barbara decided to return to Gainesville to work towards attaining her doctorate,
once again in the laboratory of Dr. Johnson. After graduation, Barbara plans on
continuing her research on HIV, as a postdoctoral fellow in the laboratory of Dr.
Johnson at the University of Florida.
96

I certify that I have read this study and that iri my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Howard M. Johns/n, Chair
Graduate Research Professor of
Microbiology and Cell Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
t L <-
Edward M. Hoffpiann
Professor of Microbiology and
Cell Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
K.T. Shanmugam
Professor of Microbiology and
Cell Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
>danetK. Y
Associate Professor of Pathology
and Laboratory Medicine

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Xdwin Blalock
Professor of Physiology and
Biophysics
University of Alabama
This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate School and was accepted as partial fulfillment
of the requirements for the degree of Doctor of Philosophy.
December, 1995
Dean, College of Agriculture
Dean, Graduate School



LIST OF FIGURES
Figure Pa9e
1. Ability of vSAG peptides to compete with 125|-SEA for binding
to A20 cells 22
2. Ability of vSAG(76-119) to compete with 125|-SEA for binding
to A20 cells in dose response studeis 23
3. Ability of SEA, SEB, and TSST-1 to compete with i25|-vSAg(76-119)
for binding to A20 cells 25
4. Evidence that vSAg(76-119) peptide binds to MHC class II
antigens, and does not bind to class I antigens 27
5. Binding of i25|-vSAg(76-119) to MHC class II peptides 28
6. Blockage of 125|-SEA binding to Raji and DR 1-transfected
L cells by Nef peptides 37
7. Relative abilities of Nef(123-160), purified Nef protein, and SEA to
compete with 125|-SEA for binding to Raji cells 38
8. Blockage of 125|-SE binding to Raji cells by Nef peptides 40
9. Direct binding of i25|-Nef(123-160) to Raji cells 41
10. Blockage of i25|-Nef(123-160) binding to Raji cells by
antibodies to MHC class I and class II antigens 43
11. Comparison of the mitogenic activities of Nef protein
preparations 52
12. Lack of proliferation by PBMC in response to the Nef fusion
partner MBP 54
13. Nef-induced proliferative responses from a representative
sampling of donors 56
14. Nef-induced activation of T cells requires APC but does
not require prcessing of Nef 57
15. Nef(123-160) peptide specifically blocks proliferation
of PBMC induced by Nef and SEA 59
16. Kinetics of IFN production induced by Nef 62
17. Infectivity assay on supernatants from Nef-stimulated
HIV-infected cultures 76
18. Model for the role of Nef in the pathogenesis of HIV 85
vii


81
ability to activate T cells. Possible consequences of nef mutations, then, would
be a reduction both in the cellular reservoir for virus replication and in the viral
load in the infected host. Other investigators have not found alterations in the
nef gene in strains isolated from other longterm survivors (Cao et al., 1995). It
would be interesting to determine the T cell reactivity of these individuals to Nef,
as we have found that 10-15% of individuals that we tested did not respond to
Nef. It is possible that nonresponsiveness to Nef may play an important role in
longterm survival in some HIV-infected individuals. Thus, Nef may aid in the
establishment and maintenance of infection, suggesting its role as a virulence
factor for HIV.


83
cells is interesting in that active HIV replication requires T cell activation.
Further, although resting or quiescent T cells can be infected, evidence
suggests that HIV replication only occurs upon subsequent cellular activation.
Nef stimulation of PBMC was sufficient to induce HIV-1 replication. Virus
replication in Nef-stimulated cultures yielded infectious virus, rather than
defective particles. Flow cytometric analysis showed that increased
percentages of cells that stained for both CD4 and HLA-DR were present in
Nef-stimulated cultures. Further, increased percentages of cells that stained for
both CD4 and IL 2R were observed in Nef-stimulated cultures. These results
are consistent with the production of T helper cytokines upon stimulation with
Nef. Thus, stimulation of PBMC with Nef protein induces T cell activation and
such activation is sufficient for HIV replication.
Proliferation of fresh autologous PBMC cultured in the presence of
infected cells was observed. Further, proliferative responses were significantly
reduced by the addition of polyclonal anti-Nef antibodies. These data suggest
that proliferation occurred as a result of Nef present on the surface of HIV-
infected cells, thereby indicating that Nef may be a virally-encoded mitogen.
Nef has been shown to induce differentiation of human B cells to
immunoglobulin secreting cells (Chirmule et al., 1994). Monoclonal antibodies
to HLA-DR and adhesion molecules abrogated Nef-induced differentiation. B
cell stimulation required T cells and monocytes, the latter producing IL 6 upon
Nef stimulation. Interestingly, staphylococcal superantigens have been shown
to induce B cell differentiation analogous to that described for Nef (Stohl et al.,
1994). Thus, Nef can stimulate both T and B cells in a manner similar to the
staphylococcal enterotoxin superantigens.
This leads to the question of the activation event(s) that occurs during
HIV-1 exposure and that ultimately lead to the establishment of infection. Our


III IDENTIFICATION OF AN HIV-1 NEF PEPTIDE THAT BINDS TO
MHC CLASS II ANTIGENS 32
Introduction 32
Materials and Methods 33
Synthetic Peptides 33
Cells and Reagents 33
Radioiodinations 35
Binding Studies 35
Results 35
Competition of Nef Peptides with Radiolabeled SEA for
MHC Binding 36
Nef(123-160) Peptide Competition Is Dose-Dependent 39
Ability of Nef Peptides to Compete with Ses for Binding to
Raji Cells 39
Direct Binding of Nef(123-160) Peptide to Raji Cells 39
MAbs to MHC Class II Antigens Block Nef(123-160) Peptide
Binding to Raji Cells 42
Discussion 42
IV ACTIVATION OF CD4 T CELLS BY THE NEF PROTEIN FROM
HIV-1
Introduction 45
Materials and Methods 45
Nef Protein 45
Reagents 48
Proliferation Assays 48
Purification of T Cells from PBMC 49
Autologous Antigen-Presenting Cells (APC) 49
Synthetic Peptides and Antibodies to Nef Peptides 49
Cytokine Production and Assays 50
Studies on Vp-Specific T Cell Expansion and Induction
of Anergy 51
Results 51
Nef Proliferative Response 51
Antigen-Presenting Cells (APC) Are Required for Nef Activity .53
Nef(123-160) Peptide Specifically Blocks Proliferation of
PBMC Induced by Nef and SEA 58
Induction of T Cell Cytokines by Nef 60
Discussion 64
V HIV ENCODES FOR ITS OWN CD4 T CELL MITOGEN 68
Introduction 68
IV


12
Evidence for a Superantiaen Associated with Human Immundeficiencv Virus
In the early 1980s, unusual incidences of young homosexual males
stricken with Pneumocystis carinii pneumonia and Kaposi's sarcoma were
reported to the Centers foe Disease Control. These reports led to the first
description of acquired immunodeficiency syndrome (AIDS). Subsequently,
similar immunodeficiency-associated illnesses were reported among
hemophiliacs, recipients of blood and blood components, and intravenous drug
users and their heterosexual partners. The incidence of AIDS in these distinct
populations was suggestive of an infectious agent, and this was confirmed
when, in 1984, human immunodeficiency virus (HIV) was isolated from the
blood of an AIDS patient (Klatzmann et al., 1984; Gallo et al., 1984). With the
advent of diagnostic assays, the number of infected individuals was found to
exceed the number of patients afflicted with AIDS, indicative of a serious
epidemic. The number of deaths is expected to increase, as asymptomatic HIV-
infected individuals progress to AIDS.
HIV is a member of the lentivirus subfamily of retroviruses. Infection with
HIV is associated with a debilitating and eventually fatal immunodeficiency. In
addition to immunologic impairment, neurological dysfunctions can occur,
including encephalopathies, sleep disorders, and dementia (the latter is also
referred to as HIV-associated cognitive/motor complex).
The size of the HIV genome is approximately 9.8 kbases. The genome
encodes for a total of 16 proteins, some of which are post-translationally
cleaved by either cellular or viral proteases. The primary HIV transcript is gag-
pol mRNA, which is translated to yield Gag (group antigen) and Pol
(polymerase). Synthesis of Gag-Pol occurs at a ratio of 20:1 (Oroszlan and
Luftig, 1990). Gag is proteolytically cleaved by a viral-encoded protease,


63
Table Vil.
IFNy is induced by Net.
Samples from PBMC IFN titer (U/ml) after treatment with:
cultures stimulated with: EMEM anti-IFNa anti-IFNy
Nef
300
300
20
SEA
300
300
30
Con A
300
300
100
IFNa Control
300
<3
300
IFNy Control
30
30
<3


4
Like their bacterial counterparts, viruses produce superantigens that
have been implicated in pathogenic processes (Table II). The prototypic viral
superantigen is that produced by the oncornavirus, mouse mammary tumor
virus (MMTV) (Marrack et al., 1991; Frankel et al., 1991; Woodland et al., 1991;
Dyson et al., 1991). The Gag protein of mouse leukemia virus, the causative
agent of mouse acquired immunodeficiency syndrome (MAIDS), has been
shown to possess superantigenic properties (Hugin et al., 1991). A
superantigen has been implicated in the pathogenesis of rabies virus (Lafon et
al., 1992) and recently the superantigenic viral component has been identified
as the nucleocapsid protein (Lafon et al., 1994). It has been suggested that
human immunodeficiency virus (HIV) encodes for a superantigen that may, at
least in part, be responsible for the immunopathogenesis associated with
infection (Laurence et al., 1992). Recent evidence points to a superantigen
encoded by human cytomegalovirus (CMV) that expands specific Vp-bearing T
cells, thereby enhancing HIV replication in CMV/HIV-infected individuals
(Dobrescu et al., 1995). Although several reports suggest that other viruses,
such as Epstein-Barr virus and human foamy virus, encode for superantigens,
to date no hard evidence has been obtained.
The possible harmful consequences of activation by superantigens make
this class of molecules relevant to human disease. The prodigious expansion
of T cells having diverse specificities may be important in the induction and
establishment of autoimmunity. Concomitant cytokine production may also
have deleterious effects. The potential for loss of immune function via
mechansisms such as anergy or apoptosis can also result in immunodeficiency
diseases. Superantigens have been shown to be encoded by retroviruses,
suggesting that they may play a role in the syndrome associated with human


85
l
Nef-induced
cellular
effects
Nef binding to HLA-DR
Presentation to uninfected T cells
Cellular proliferation
T cell activation
Cytokine production
B cell proliferation and differentiation
Potential
Nef-related
pathogenesis
I
Increased viral load
Immunosuppression via T cell
anergy/apoptosis
Autoimmunity via activation of B and T
cells
Hypergammaglobulinemia
Figure 18. Model for the role of Nef in the pathogenesis of HIV. Activation of
uninfected T cells occurs by Nef interaction in either of two forms: as soluble
Nef released from lysed cells, presented in the context of MHC class II antigens
on APC, or as an integral part of the membranes of infected T cells, complexed
to HLA-DR. Binding to uninfected T cells may occur via TCR or CD4. T cell
activation results in proliferation and release of cytokines such as IFNyand IL 2,
thereby creating a cellular reservoir for virus and increasing viral load in the
host. T cell activation results in depletion of T cells via virus production, anergy
and/or apoptosis. B cell differentiation, possibly mediated by T cell cytokine
release, results in hypergammaglobulinemia. Autoimmune-like sequelae may
result from B cell differentiation into lg-secreting cells, and activation of T cells.


29
1991). Such binding studies are hampered by the difficulties inherent in
expressing the MMTV superantigen. A model has recently been proposed that
suggests that MMTV superantigens may act in the same or similar fashion to
bacterial superantigens by bridging MHC antigens and TCR on the appropriate
cell types (Acha-Orbea and Palmer, 1991). MMTV superantigens, presented in
the context of MHC class II antigens, may act by stimulating and expanding Vp-
specific T cell subsets, thereby indirectly enhancing the further infection of B
cells (Held et al., 1994a). The results presented in this chapter suggest that
MMTV vSAg protein binds to MHC antigens, thereby strengthening the
argument that bacterial and retroviral superantigens act in a similar manner.
It has been reported that MMTV superantigen is a 45 kd Type II integral
membrane protein with an intracellular N-terminus, an essential hydrophobic
transmembrane region near the N-terminus (residues 45-64), and a
glycosylated extracellular C-terminus (Choi et al., 1992). Although no direct
evidence exists, the variability of the C-terminal residues of vSAg proteins of
various MMTV strains seems to correlate with their differences in Vp specificity,
lending support to the concept that this region binds TCR (Pullen et al., 1992).
Truncated versions of the vsag gene were transfected into MHC class ll-bearing
cells and tested for superantigen activity. Complete loss of superantigen
activity occurred when the MMTV vsag gene was N-terminally truncated to the
third methionine (residue 122) and beyond (Choi et al., 1992). The authors
concluded that a hydrophobic region, which was missing in this N-terminally
truncated version, acts as a transmembrane region and is essential for
superantigen activity. However, our binding data suggest that loss of
superantigen activity may have been due, at least in part, to the deletion of the
MHC-binding domain which is encompassed by residues 76-119. In fact, the


20
followed by the addition of radiolabeled SEs or vSAg peptide. After 45 minutes,
the cells were washed three times and the radioactivity associated with the cell
pellets was quantified using a gamma counter. L cells were used as an MHC
class ll-negative control cell line.
L cells were grown to confluence in the wells of microtiter plates and
were subjected to the same incubation times and volumes of competitors and
labeled ligands as used for A20 cells. After three washes, the cells were
solubilized in 1% SDS, the liquid was absorbed by cotton-tipped applicators,
and radioactivity was quantified using a gamma counter.
Radioimmunoassay
Two MHC peptides l-Apb(30-60) and l-Apb(60-90) were used to for
binding of SEA and ORF(76-119) to class II antigens. The l-A(3b(60-90) site was
previously shown to be involved in SEA binding to MHC class II antigens
(Russell et al., 1990). MHC peptides were dissolved in PBS at a concentration
of 25 pg/ml. Peptide solutions were pipetted into polystyrene plastic tubes and
tubes were placed at 10C for 4 hours to allow adherence of the peptides. The
tubes were washed three times with PBS. Nonspecific sites on the plastic were
blocked using 2 ml of PBS containing 1% BSA at 10C overnight. After the
tubes were washed three times of PBS containing 1% BSA, unlabeled
competitors (SEA and vSAg peptide) were added in 0.1 ml and allowed to bind
at room temperature for 3 hours. The tubes were washed three times with 2 ml
of PBS/1 % BSA prior to addition of 0.1 ml of either radiolabeled SEA or
radiolabeled vSAg(76-119)peptide, at final concentrations of 2 nm and 5 nm,
respectively. Radiolabeled ligands were allowed to bind to MHC peptides at
room temperature for 4 hours. After washing three times with 2 ml of PBS
containing 1% BSA, the tubes were placed in a gamma counter and bound



PAGE 1

RETROVIRAL SUPERANTIGENS By BARBARA AUREA TORRES A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1995

PAGE 2

ACKNOWLEDGMENTS I would like to thank my mentor, Dr. Howard M. Johnson, for his unflagging support, insight, and wisdom. All of these qualities have helped guide me, not just a in scientific sense, but in finding my way through the quagmire of academic science. On a scientific level, Howard has always been unfailingly logical and knowledgeable, which makes him a wonderful mentor. I would also like to thank the other members of my committee. Dr. Shanmugam and Dr. Hoffmann have been very supportive throughout this venture. Dr. Yamamoto has given me excellent advice on steering my HIV work towards clinical relevance. I give special thanks to Dr. Blalock, who was a tough, but caring, committee member and confidante. Many thanks must go to my fellow labmates who have always been willing to lend a hand, ear, or a shoulder to cry on, whichever was needed at the time. I thank Jeanne, Brian, Prem, and Taishi for generously sharing their past experiences of graduate school. I also am grateful for my fellow graduate school mates, Amy, George, and Mustafa, for their understanding and help. And I thank Aaron, Laurie, and Tim for their warmth and humor. Finally, I wish to thank my family for their unwavering support through my graduate school tenure. My parents, Manuel and Antonia Torres, have stood by with understanding and a warm hug whenever needed. Although far away, my sister, Marisol Beaton, has cheered me on to complete my doctoral work. I owe a great debt of gratitude to my wonderful family. ii

PAGE 3

TABLE OF CONTENTS ACKNOWLEDGMENTS ii LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT viii CHAPTERS I INTRODUCTION 1 Overview 1 Bacterial Superantigens 6 Retroviruses 8 Mouse Mammary Tumor Virus Superantigen 1 0 Evidence for a Superantigen Associated with Human Immunodeficiency Virus 12 II MMTV SUPERANTIGEN BINDING TO MHC CLASS II ANTIGENS 16 Introduction 16 Materials and Methods 1 7 Synthetic Peptides 17 Cell Lines and Reagents 17 Radioiodinations 19 Binding Studies 19 Radioimmunoassay 20 Results 21 Competition of vSAg Peptides with Radiolabeled SEA for MHC Binding 21 vSAg(76-119) Peptide Competition Is Dose-Dependent 21 Direct Binding of vSAg(76-1 19) Peptide to A20 Cells 24 Evidence that vSAg(76-1 19) Binds Specifically to MHC Class II Antigens 26 Discussion 26 iii

PAGE 4

III IDENTIFICATION OF AN HIV-1 NEF PEPTIDE THAT BINDS TO MHC CLASS II ANTIGENS 32 Introduction 32 Materials and Methods 33 Synthetic Peptides 33 Cells and Reagents 33 Radioiodinations 35 Binding Studies 35 Results 35 Competition of Nef Peptides with Radiolabeled SEA for MHC Binding 36 Nef(123-160) Peptide Competition Is Dose-Dependent 39 Ability of Nef Peptides to Compete with Ses for Binding to Raji Cells 39 Direct Binding of Nef(123-160) Peptide to Raji Cells 39 MAbs to MHC Class II Antigens Block Nef(123-160) Peptide Binding to Raji Cells 42 Discussion 42 IV ACTIVATION OF CD4 T CELLS BY THE NEF PROTEIN FROM HIV-1 Introduction 45 Materials and Methods 45 Nef Protein 45 Reagents 48 Proliferation Assays 48 Purification of T Cells from PBMC 49 Autologous Antigen-Presenting Cells (APC) 49 Synthetic Peptides and Antibodies to Nef Peptides 49 Cytokine Production and Assays 50 Studies on Vp-Specific T Cell Expansion and Induction of Anergy 51 Results 51 Nef Proliferative Response 51 Antigen-Presenting Cells (APC) Are Required for Nef Activity .53 Nef(123-160) Peptide Specifically Blocks Proliferation of PBMC Induced by Nef and SEA 58 Induction of T Cell Cytokines by Nef 60 Discussion 64 V HIV ENCODES FOR ITS OWN CD4 T CELL MITOGEN 68 Introduction 68 iv

PAGE 5

Materials and Methods 69 PBMC Cultures for HIV Infectivity Studies 69 PBMC Cultures for Proliferation Studies 69 Reverse Transcriptase (RT) Assay 70 ELISA for HIV p24 Antigen 70 Assay for Infectious Virus from PBMC Cultures 70 Fluorescent Antibody Cell Sorter (FACS) Analysis of Nef-Activated Cells 71 Studies on Proliferation of PBMC Induced by Autlogous HIV-infected Cells 71 Anti-Nef Peptide Antibodies 71 Results 72 HIV Replication in Nef-Stimulated PBMC Cultures 72 Infectious Virus Is Produced by Nef-Stimulated Cultures 72 Nef-Stimulated PBMC Express T Cell Activation Markers 75 Expression of Nef by HIV-infected Cells 75 Discussion 78 VI A MODEL FOR THE ROLE OF NEF IN HIV PATHOGENESIS 82 LIST OF REFERENCES 86 BIOGRAPHICAL SKETCH 100 v

PAGE 6

LIST OF TABLES Table page I. List of bacterial superantigens implicated in disease 3 II. List of viral superantigens implicated in disease 5 III. Amino acid sequences of vSAg peptides 1 8 IV. Amino acid sequences of Nef peptides 34 V. Ability of anti-peptide antibodies to block Nef-induced proliferation 51 VI. IL 2 production induced by Nef 61 VII. IFNy is induced by Nef 63 VIII. Maximal proliferation, RT, and p24 values from stimulated cultures 73 IX. Nef-induced activation of T cells as assessed by FACS 77 X. Ability of anti-Nef antibodies to block proliferation induced by autologous HIV-infected cells 79 vi

PAGE 7

LIST OF FIGURES Figure page 1 . Ability of vSAG peptides to compete with 125|-SEA for binding to A20 cells 22 2. Ability of vSAG(76-1 19) to compete with 125|-SEA for binding to A20 cells in dose response studeis 23 3. Ability of SEA, SEB, and TSST-1 to compete with i25|v SAg(76-1 19) for binding to A20 cells 25 4. Evidence that vSAg(76-1 19) peptide binds to MHC class II antigens, and does not bind to class I antigens 27 5. Binding of i25|v SAg(76-1 19) to MHC class II peptides 28 6. Blockage of 125|-SEA binding to Raji and DR1-transfected L cells by Nef peptides 37 7. Relative abilities of Nef(123-160), purified Nef protein, and SEA to compete with 125|-SEA for binding to Raji cells 38 8. Blockage of 125|-SE binding to Raji cells by Nef peptides 40 9. Direct binding of i25|-Nef(123-160) to Raji cells 41 10. Blockage of i25|-Nef(123-160) binding to Raji cells by antibodies to MHC class I and class II antigens 43 1 1 . Comparison of the mitogenic activities of Nef protein preparations 52 12. Lack of proliferation by PBMC in response to the Nef fusion partner MBP 54 13. Nef-induced proliferative responses from a representative sampling of donors 56 14. Nef-induced activation of T cells requires APC but does not require prcessing of Nef 57 15. Nef(123-160) peptide specifically blocks proliferation of PBMC induced by Nef and SEA 59 16. Kinetics of IFN production induced by Nef 62 17. Infectivity assay on supernatants from Nef-stimulated HIV-infected cultures 76 18. Model for the role of Nef in the pathogenesis of HIV 85 vii

PAGE 8

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RETROVIRAL SUPERANTIGENS By Barbara Aurea Torres December 1995 Chairman: Howard M. Johnson Major Department: Microbiology and Cell Science Superantigens (SAg) are potent inducers of T cell activation that cause proliferation and massive cytokine release. The receptors for superantigens on antigen-presenting cells (APC) are major histocompatibility (MHC) class II molecules. Both Nef protein of human immunodeficiency virus (HIV) and mouse mammary tumor virus (MMTV) SAgs are encoded in genes that overlap the terminal repeat at the 3' end. Using synthetic peptides, a region was identifed on the MMTV-1 SAg, corresponding to residues 76-119, that specifically binds to mouse MHC class II antigens. MMTVSAg(76-1 19) also bound to and competed with bacterial superantigens for binding to MHC class II peptides, suggesting similar binding regions on class II. These studies are the first demonstration that retroviral superantigens bind to MHC class II antigens. viii

PAGE 9

A peptide corresponding to an internal region of HIV Net protein was identified that specifically binds to to human class II antigens. Nef protein induced proliferation of human peripheral blood mononuclear cells (PBMC) in 85-90% of the HIV-negative donors tested. Nef stimulation required APC, and did not require processing. Interleukin 2 and gamma interferon were produced in Nef-stimulated cultures. These results strongly suggest that Nef acts like a retroviral SAg that stimulates CD4+ T cells. Activated T cells are required for HIV replication. The role of Nef in HIV pathogenesis was investigated by treating PBMC with Nef prior to in vitro infection with HIV. Significant levels of infection were found in these cultures, as compared to unstimulated controls. Nef-stimulated T cells were found to express T cell activation markers. Inactivated HIV-infected cells were capable of inducing proliferation in autologous fresh PBMC, and proliferation was significantly reduced by anti-Nef antibodies. These results indicate that Nef is expressed on the surface of infected T cells, possibly in the context of class II antigens, and as such, Nef can activate a cellular reservoir in an paracrine fashion for continual viral replication. Thus, Nef is an HIV-encoded SAg-like mitogen that promotes HIV replication in T cells. As such, Nef is probabaly an HIV virulence factor. ix

PAGE 10

CHAPTER I INTRODUCTION Overview A novel class of antigens called superantigens has recently been characterized. Unlike conventional antigens, which stimulate approximately 1 in 10,000 cells, superantigens can induce the proliferation and activation of as many as 1 in 5 T cells (Johnson et al., 1991). The staphylococcal enterotoxins, prototypic of this class of antigens, are among the most potent inducers of T cell proliferation, and are effective at concentrations as low as 10" 1 6 m (Langford et al., 1978a). Major differences exist between conventional antigens and superantigens. Superantigens exert their effects as whole molecules and do not require processing by antigen presenting cells for T cell recognition. Although superantigens require MHC class II molecules, they bind at site(s) outside the peptide antigen binding groove (Russell et al., 1990; Dellabonna et al., 1990). Superantigens presented in the context of MHC class II molecules are recognized by the variable region of the p chain (Vp) of the T cell receptor. Only T cells that express T cell receptors with specific Vp regions will be activated by a given superantigen. For example, toxic shock syndrome toxin-1 activates only Vp2-bearing human T cells (Abe et al., 1992) and staphylococcal enterotoxin A activates several Vps on human T cells, including Vp5 and Vpl2, (Kappler et al., 1989). Thus, T cell responses to superantigens are said to be Vp-specific. 1

PAGE 11

2 T cell activation by superantigens results in massive proliferation of Vpspecific T cells. Concomitant with proliferation is prodigious production of cytokines, in particular interleukin 2 and gamma interferon (IFNy). Such responses can be detrimental to the host, as is the case with food poisoning which occurs upon ingestion of staphylococcal enterotoxin-laden food (Bergdoll, 1985). As an eventual consequence of superantigen activation, T cells can become unresponsive to further stimulation (anergy) or undergo programmed cell death (apoptosis) (Kawabe and Ochi, 1990, 1991; Rellahan et al., 1990). Thus, the fate of T cells activated by superantigen differs from that of cells activated by conventional antigens, with the possible consequence of loss of immune function. Superantigens are produced by a number of microorganisms, ranging from bacteria to viruses. The prototypic bacterial superantigens are the enterotoxins produced by Staphylococcus aureus (Table I). Staphylococcal enterotoxin A (SEA) is the most common cause of food poisoning, and its superantigen effects on immunocytes have been implicated in the syndrome of short-term nausea, fever, and diarrhea that are symptomatic of food poisoning (Bergdoll, 1985; Johnson and Magazine, 1988). Another staphylococcal enterotoxin, toxic shock syndrome toxin-1 (TSST-1), is produced during infection and its superantigenic effects result in toxic shock syndrome (Bergdoll, 1985). Other bacterial sources of superantigens are the group A streptococci, which produce pyrogenic exotoxins that have been implicated in psoriasis (Kotzin et al., 1993) and rheumatic heart disease (Lewis et al., 1993). Superantigens may also be involved in tuberculosis (Ohmen et al., 1994) and Reiter's syndrome (Uchiyama et al., 1993). One species of mycoplasma, M. arthiditis, has been shown to produce a superantigen that may be involved in arthritis (Cole and Griffiths, 1993).

PAGE 12

3 Table I. List of bacterial superantigens implicated in disease. Organism Prototype: Staphylococcus aureus (?) Group A streptococci Mycoplasma arthitidis Mycobacterium tuberculosis Yersinia pestis Clostridium perfringens Superantigenic Protein Disease/Effect 3 Staphylococcal enterotoxins Enterotoxins Food poisoning Toxic shock Kawasaki's disease (?) D Multiple sclerosis Pyrogenic exotoxins T-cell mitogen Not identified Not identified Exotoxin Psoriasis (?) Rheumatic heart disease (?) Arthritis (?) Tuberculosis (?) Reiter's syndrome Reactive arthritis Sudden infant death syndrome (?) a Johnson et al., 1995. ^Denotes diseases in which superantigens have been implicated, but no direct evidence is available.

PAGE 13

4 Like their bacterial counterparts, viruses produce superantigens that have been implicated in pathogenic processes (Table II). The prototypic viral superantigen is that produced by the oncornavirus, mouse mammary tumor virus (MMTV) (Marrack et al., 1991; Frankel et al., 1991 ; Woodland et al., 1991; Dyson et al., 1991). The Gag protein of mouse leukemia virus, the causative agent of mouse acquired immunodeficiency syndrome (MAIDS), has been shown to possess superantigenic properties (Hugin et al., 1991). A superantigen has been implicated in the pathogenesis of rabies virus (Lafon et al., 1992) and recently the superantigenic viral component has been identified as the nucleocapsid protein (Lafon et al., 1994). It has been suggested that human immunodeficiency virus (HIV) encodes for a superantigen that may, at least in part, be responsible for the immunopathogenesis associated with infection (Laurence et al., 1992). Recent evidence points to a superantigen encoded by human cytomegalovirus (CMV) that expands specific Vp-bearing T cells, thereby enhancing HIV replication in CMV/HIV-infected individuals (Dobrescu et al., 1995). Although several reports suggest that other viruses, such as Epstein-Barr virus and human foamy virus, encode for superantigens, to date no hard evidence has been obtained. The possible harmful consequences of activation by superantigens make this class of molecules relevant to human disease. The prodigious expansion of T cells having diverse specificities may be important in the induction and establishment of autoimmunity. Concomitant cytokine production may also have deleterious effects. The potential for loss of immune function via mechansisms such as anergy or apoptosis can also result in immunodeficiency diseases. Superantigens have been shown to be encoded by retroviruses, suggesting that they may play a role in the syndrome associated with human

PAGE 14

5 Table II. List of viral superantigens implicated in disease. Virus (Family or Subfamily) Superantigenic Protein Disease/Effect 3 Prototype: Superantigen Mouse mammary tumor virus (oncornavirus) Mouse leukemia virus (oncornavirus) Human immunodeficiency virus (lentivirus) Human foamy virus (?)b (spumavirus) Rabies (rhabdovirus) Epstein-Barr virus (?) (herpesvirus) Cytomegalovirus (herpesvirus) from Mouse Mammary Tumor Virus vsag gene product Mammary tumors Gag protein Not identified Nucleocapsid Not identified Not identified Murine AIDS AIDS bel3 gene product Grave's disease Rabies B cell lymphoma Chronic fatigue syndrome (?) Enhanced HIV replication a Johnson et al. 1995. D Denotes diseases in which superantigens have been implicated, but no direct evidence is available.

PAGE 15

6 immunodeficiency virus. Thus, superantigens may be involved in disease processes and, as such, can be classified as virulence factors. Bacterial Superantigens In the 1970s, staphylococcal enterotoxins were recognized as inducers of lymphocyte proliferation (Peavy et al., 1970). Responses to staphylococcal enterotoxin A (SEA) stimulation were shown to be T cell specific (Johnson and Bukovic, 1975), with high levels of gamma interferon (IFNy) produced by this lymphocyte fraction (Langford et al., 1978a). SEA was shown to have mitogenic effects on human lymphocytes at extremely low concentrations, making it one of the most potent T cell activators known (Langford et al., 1978a). The ability to produce large quantities of IFNy using SEA paved the way for the characterization of the key immunomodulatory properties of this cytokine (Johnson, 1985). Interestingly, intravenous administration of SEA to mice resulted in the induction of suppressor cell activity that was present in spleen as early as 24 hours after injection (Johnson, 1981; Torres et al., 1982). SEAinduced suppressor cells were capable of blocking naive spleen cells from responding to SEA stimulation. Thus, SEA was shown to stimulate T cells to proliferate and produce cytokines, and to induce suppressor cell activity. Further investigations on the interaction of bacterial enterotoxins with human lymphocytes revealed that antigen-presenting cells are required for proliferative effects and that binding to major histocompatibility (MHC) class II antigens occurred (Carlsson et al., 1988; Fleischer and Schrezenmeier, 1988; Mollick et al., 1989). Unlike most antigens which interact with MHC class II molecules as peptides, the staphylococal enterotoxins were shown to bind in an unprocessed form (Fleischer and Schrezenmeier, 1988). The site of

PAGE 16

7 interaction of enterotoxins on the MHC class II molecule was shown to be distinct from conventional antigens in that binding occurs distal to the peptide antigen binding groove (Russell et al., 1990; Dellabonna et al., 1990). Thus, this class of antigens differs significantly from classic antigens and were given the designation of superantigens (Marrack and Kappler, 1990). The study of staphylococcal enterotoxins has led to a wealth of information on the interaction of superantigens with MHC class II molecules. Through the use of synthetic peptides, regions of interaction of SEA and MHC class II antigens have been identified. Regions on the SEA molecule that are involved in binding to MHC class II molecules on antigen presenting cells include the N-terminus and three internal sequences (Griggs et al., 1992). A peptide corresponding to the N-terminus of SEA, SEA(1-45), blocked proliferation of cells in response to whole native SEA (Pontzer et al., 1990). Interestingly, a peptide corresponding to another of these MHC class ll-binding regions, SEA(121-149), has agonist properties in that it induces cytokine production (Pontzer et al., 1993) and proliferation (A.C. Hobeika, personal communication). Conversely, sites on MHC class II molecules which bind superantigens have been identified. Regions on mouse MHC class II molecules that interact with SEA have been identified and include the a-helical region of the p chain associated with the peptide binding groove encompassed by amino acids 65-85 (Russell et al., 1991). A similar site has been found on human MHC class II antigens (Herman et al., 1991). A superantigen-binding region on the a helical region of the a chain associated with the peptide binding groove of MHC class II has also been defined (Russell et al., 1991). Both of these binding sites reside outside of the peptide binding groove and are required for SEA-induced mitogenesis. Thus the two outside faces of the a helices of the peptide binding groove are involved in binding of SEA.

PAGE 17

8 Presentation of superantigen in the context of MHC class II molecules is required for interaction with T cell receptor (TCR). For this reason superantigen sites that interact with TCR have been more difficult to elucidate. However, through the use of synthetic peptides, a TCR site that interacts with SEA has been identified. A peptide corresponding to residues 57-77 of mouse Vp3 blocked SEA-induced proliferation and IFNy production (Pontzer et al., 1992). Vp3-bearing mouse T cells are known to be stimulated by SEA. Future studies may help determine the sequences involved in the formation of the ternary complex of superantigen:MHC:TCR. Such studies are needed to determine how superantigens activate T cells bearing specific Vps and induce anergy and deletion of these Vp subsets. Retroviruses Retroviruses belong to a family of viruses that are characterized by the use of a unique RNA-dependent DNA polymerase, reverse transcriptase. Reverse transcriptase, in conjunction with another virally-encoded enzyme, RNAse H, transcribes the single-stranded RNA genome into a double-stranded linear DNA provirus. This DNA intermediate is then capable of integrating into the host genome. Retroviruses were first described in studies in which cell-free filtered extracts were shown to transmit sarcoma to chickens (Rous 1910, 191 1). Since that time, retroviruses have been found in several vertebrates including mice, cats, and primates. The first human retrovirus to be discovered was human foamy virus, which has been speculated to cause disease (Achong et al., 1971), although definitive proof of pathogenicity is lacking. In 1980, the causative agent of adult T-cell leukemia was discovered, human T-cell

PAGE 18

9 leukemia virus (Poiesz et al., 1980; Yoshida et al., 1982), and established this family of viruses as human pathogens. The retrovirus family consists of three subgroups based on similar pathogenicities: oncornaviruses, lentiviruses, and spumaviruses. Spumaviruses or foamy viruses are the least characterized of the three subfamilies of retroviruses, and have been grouped according to their ability to cause vacuolation of infected cells in vitro, which gives the cells a "foamy" appearance. Spumaviruses have been found in primates and humans, and are generally considered benign, although data suggest these viruses may play a role in human disease. Oncornaviruses are tumor-causing retroviruses, and members of this subfamily include the avian leukosis-sarcoma viruses, mouse mammary tumor virus, feline leukemia virus, and human T-cell leukemia virus. As a result of proviral integration into the host genome, tumors arise by upregulation of host genes that encode for growth factors or by retroviralencoded oncogenes. Viruses belonging to the lentiviral subfamily include the "slow" viruses maedi/visna and equine infectious anemia virus. More recently, the viruses that cause human and feline acquired immundeofiency syndromes have been classified as lentiviruses, based on several parameters including genomic complexity and virion morphology. Unlike oncornaviruses, lentiviruses have not been implicated directly in causing neoplastic disease. Members of the lentiviral subfamily cause long-term disease characterized by autoimmunity, encephalopathy, immunodeficiency, or a combination thereof. Lentiviruses are considerably more complex than some of the other retroviral subfamilies, in that the level of gene regulation is much greater.

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10 Mouse Mammary Tumor Virus Superantiaen The first viral superantigens to be described are the proteins encoded by the open reading frame that overlaps the 3' long terminal repeat of the oncornavirus, mouse mammary tumor virus (MMTV) (Marrack et al., 1991; Frankel et al., 1991; Woodland et al., 1991; Dyson et al., 1991). Although MMTV superantigens were recognized in the 1990s, they were originally described by Festenstein in the early 1970s as minor lymphocyte stimulating (mis) antigens (Festenstein, 1973). The ability of T cells from certain mouse strains to be stimulated by lymphocytes from MHC-identical strains was ascribed by Festenstein to the presence of mis antigens. More recently, mis antigens were found to be endogenous superantigens from germline-encoded MMTV provirus (Choi et al., 1991; Acha-Orbea et al., 1991). Like their bacterial counterparts, MMTV superantigens are thought to be presented in the context of MHC class II antigens. MMTV superantigens are known to stimulate T cells in a Vp-specific fashion (Acha-Orbea and Palmer, 1991). MMTV superantigens show MHC preference, with more efficient presentation occurring in the context of l-E as opposed to l-A, although presentation by l-A does occur (MacDonald et al., 1989). Interestingly, this MHC preference corresponds with the greater infectivity of B cells bearing l-E (Held et al., 1994b). MMTV is a type B retrovirus that was determined to be the causative agent for the induction and transmission of mammary carcinomas in mice (Heston et al., 1945). It has been known for several years that infectivity by MMTV requires an intact immune system (Tsubura et al., 1988), and only recently has this paradox been explained (Held et al., 1993, 1994a). Transmission of MMTV to offspring can occur via infectious virions in mother's milk or can occur vertically as endogenous provirus. Most mouse strains have

PAGE 20

11 been shown to contain one or more copies of endogenous MMTV, and many contain distinct MMTV strains (Salmons and Gunzburg, 1987). Upon passage into the gut of the host, virus enters the gut-associated immune tissue and infects B cells. B cells then express the MMTV-encoded superantigen, presumably in the context of class II antigens, causing Vp-specific T cell stimulation. Although investigators have been able to show that actual infection of T cells occurs, it is thought that T cell stimulation and subsequent cytokine production by these cells indirectly enhances the further infection of B cells (Held et al., 1994a). Infected B cells migrate to the main site of viral infection, the mammary gland. Epithelial cells then become infected and are the source of infectious virions that are transmitted in milk (Ringold, 1983). Proviral integration can occur during infection of mammary epithelial cells, resulting in tumors. Germline-encoded MMTV serves an important protective mechanism for the host. It is known that bacterial superantigens can cause anergy and/or deletion of Vp-specific T cells (Johnson et al., 1992). Similarly, expression of MMTV superantigen early in the ontogeny of the immune system induces the eventual deletion of T cells bearing Vps specific for that particular strain of MMTV superantigen. In this manner, T cells that would otherwise be stimulated are lost and the host is protected against subsequent infection by MMTV strains that stimulate those Vp-specific T cell populations. This has been shown in mice transgenic for a MMTV superantigen that stimulates Vp14 + T cells (Golovkina et al., 1992; Acha-Orbea, 1991a). Transgenic mice showed partial to complete deletion of Vpl4 + T cells, depending on the level of superantigen expression. Those mice in which Vpi4 + T cells were deleted were subsequently protected from infection upon challenge with the same MMTV strain.

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12 Evidence for a Superantiaen Associated with Human Immundeficiencv Virus In the early 1980s, unusual incidences of young homosexual males stricken with Pneumocystis carinii pneumonia and Kaposi's sarcoma were reported to the Centers foe Disease Control. These reports led to the first description of acquired immunodeficiency syndrome (AIDS). Subsequently, similar immunodeficiency-associated illnesses were reported among hemophiliacs, recipients of blood and blood components, and intravenous drug users and their heterosexual partners. The incidence of AIDS in these distinct populations was suggestive of an infectious agent, and this was confirmed when, in 1984, human immunodeficiency virus (HIV) was isolated from the blood of an AIDS patient (Klatzmann et al., 1984; Gallo et al., 1984). With the advent of diagnostic assays, the number of infected individuals was found to exceed the number of patients afflicted with AIDS, indicative of a serious epidemic. The number of deaths is expected to increase, as asymptomatic HIVinfected individuals progress to AIDS. HIV is a member of the lentivirus subfamily of retroviruses. Infection with HIV is associated with a debilitating and eventually fatal immunodeficiency. In addition to immunologic impairment, neurological dysfunctions can occur, including encephalopathies, sleep disorders, and dementia (the latter is also referred to as HIV-associated cognitive/motor complex). The size of the HIV genome is approximately 9.8 kbases. The genome encodes for a total of 16 proteins, some of which are post-translationally cleaved by either cellular or viral proteases. The primary HIV transcript is gagpol mRNA, which is translated to yield Gag (group antigen) and Pol (polymerase). Synthesis of Gag-Pol occurs at a ratio of 20:1 (Oroszlan and Luftig, 1990). Gag is proteolytically cleaved by a viral-encoded protease,

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13 yielding 5 distinct proteins. Cleavage of Pol results in 2 proteins, one of which is reverse transcriptase. Two envelope (Env) proteins are the cleavage products from an initial Env precursor. In this case, the precursor is cleaved by a cellular protease. Two regulatory proteins, Tat (transactivator) and Rev (regulator for viral expression) are translated from multiply-spliced mRNA transcripts. One putative regulatory protein, Nef (negative factor) is encoded by a gene that overlaps the terminal repeat at the 3' end of the HIV genome. It was initially named based on its apparent negative effects on viral transcription and replication in vitro (Terwillegar et al, 1986). However, subsequent reports have been conflicting, and have suggested that Nef has no effect (Hammes et al., 1989) or a positive effect (DeRonde et al., 1992) on HIV transcription. Such widely disparate Nef effects may, in part, be explained by the fact that several immortalized human T cell lines and primary human lymphocytes were used in these studies. These data may reflect inherent differences in primary cells and cell lines, such as activation signals. Data from studies on Nef-transfected T cells suggest that Nef may play a role in down-regulation of the CD4 molecule (Garcia and Miller, 1991; Mariani and Skowronski, 1993). HIV-1 Nef is a 25-27K protein and is myristylated at the N-terminus. Myristylation is thought to be a mechanism by which Nef associates with the cytoplasmic membrane (Franchini et al., 1986; Guy et al., 1987). Nef primarily localizes to the cytoplasm (Franchini et al., 1986) but has been shown to be present on the surface of infected cells (Fujii et al., 1993). Antibodies to Nef appear early in HIV-infected individuals (Allan et al., 1985) and cytotoxic T lymphocyte activity has been detected against cells presenting Nef peptides (Bahraoui et al., 1990). These data confirm that Nef is expressed during active HIV infection.

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14 A hallmark of infection with HIV is the alteration of CD4/CD8 T cell ratios due to loss of CD4+ T helper cells. Such losses ultimately result in the inability of an infected individual to mount effective immune responses against opportunistic infections, thus resulting in death. Several possible mechanisms by which CD4 loss occurs have been postulated, including direct cytolysis by HIV (Lemaitre et al., 1990), HIV-induced syncitia formation (Lifson et al., 1986; Sodroski et al., 1986) and/or cytolytic T cell activity against infected CD4 cells (Zinkernagel and Hentgartner, 1994). In addition to skewing of the CD4/CD8 ratios, other immunologic alterations seen in HIV-infected individuals include polyclonal activation of B cells with increased immunoglobulin production and reduced antigen and mitogen responses (Edelman and Zolla-Pazner, 1989). Initial increased natural killer cell activity is observed in asymptomatic HIVinfected individuals, but these activities decrease during disease progression (Edelman and Zolla-Pazner, 1989). Because of the known induction of T cell anergy and/or deletion by superantigens (Johnson et al., 1992), it has been speculated that the HIV genome encodes for a superantigen that may cause some of the immunopathologies observed with AIDS. Upon interaction with MHC class II antigens and TCR, Vp-specific T cell populations would be activated and expand, eventually leading to functional (anergy) or actual (deletion) loss of T cells. Several points of evidence suggest a role for an HIV-derived superantigen. Although initial studies suggested that AIDS patients had deletions in Vp T cell populations (Imberti et al., 1991), such deletions could not be found by others (Laurence et al., 1992). Vpl2+ T cells were shown to support enhanced replication of HIV compared to other Vp subsets and proliferated in response to cells from HIV+ patients, suggesting the presence of a superantigen (Laurence et al., 1992). Asymptomatic patients exhibit altered

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15 Vp profiles (Dalgleish et al., 1992) as well as skewed T cell Vp usage upon stimulation with staphylococcal enterotoxins in vitro (Bisset et al., 1993). Interestingly, one study found skewed Vp T cell profiles in monozygotic twins that were discordant for HIV infection, with perturbations in several Vps (Rebai et al., 1994). This last study is of particular importance in that the altered Vp profiles in infected individuals cannot be ascribed to differences in MHC, since only identical twins were used. Thus, these studies hint at the presence of a superantigen encoded by HIV that may play a role in HIV pathogenesis by augmenting a population of T cells, resulting in a reservoir for virus replication, with eventual deletion of those T cells.

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CHAPTER II MMTV SUPERANTIGEN BINDING TO MHC CLASS II ANTIGENS Introduction SEA is one of the most potent T-cell mitogens known, and has been classified as a bacterial superantigen based on its ability to stimulate V(3specific T-cell subsets (Johnson et al., 1991). Concurrent with studies on SEA, minor lymphocyte stimulating (mis) antigens have recently been shown to be products of MMTV (Choi et al., 1991; Acha-Orbea et al., 1991). Two exogenous strains of MMTV encode for retroviral superantigens in genes that overlap the terminal repeat at the 3' end of the viral genome (Pullen et al., 1992; Choi et al., 1992). These genes have been designated vsag, denoting that they encode for viral superantigens (Marrack and Kappler, 1990). No direct binding of the putative MMTV superantigen to either MHC antigens or TCR has been shown. To determine sites that interact with class II MHC antigens, overlapping synthetic peptides were synthesized that encompass the putative extracellular domain of MMTV-1 superantigen. The data presented here indicate that a site that is encompassed by amino acid residues 76-119 of MMTV-1 superantigen competes with SEA for binding to class II MHC antigens. Further, direct binding studies show that this region of the MMTV superantigen binds directly to class II MHC antigens. These data indicate that SEA and MMTV superantigen share at least one common binding region on class II MHC molecules. 16

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17 Materials and Methods Synthetic Peptides Overlapping peptides corresponding to the putative extracellular domain of the MMTV-1 vSAg protein (Pullen et ai., 1992) were synthesized with a Biosearch 9500AT automated peptide synthesizer using N-(9flurenyl)methoxycarbonyl chemistry (Griggs et al., 1992). Peptides were synthesized based on a surface profile which uses a composite of three parameters: 1) HPLC hydophilicity; 2) accessibility; and 3) segmental mobility (B value) (Parker et al., 1986). The sequences of the peptides are listed in Table III. Peptides were cleaved from the resins using trifluoroacetic acid/ ethanedithiol/ thioanisole/ anisole at a ratio of 90:3:5:2. The cleaved peptides were then extracted in ether and ethyl acetate and subsequently dissolved in water and lyophilized. Peptides were extensively dialyzed against water to remove the remaining cleavage products. Amino acid analysis of the peptides showed that the amino acid contents and molecular weights corresponded closely to theoretical values. Peptides were not purified further since reverse phase HPLC analysis of crude peptides indicated one major peak in each profile. Cell Lines and Reagents The A20 cell line (ATCC, Gaithersburg, MD) was used for MMTV superantigen binding studies. A20 cells are a BALB/c B lymphoma line that expresses la. Highly purified SEA and other staphylococcal enterotoxins were obtained from Toxin Technology (Sarasota, FL). Several monoclonal antibodies (mAb) were used in this study. MAbs were purchased from Accurate Chemicals, Westbury, NY. MAb K d (34-1 -2S) is specific for m31 determinant of K d (Ozato et al., 1982). MAb IA d (MK-D6) is specific for the p1

PAGE 27

18 w T3 Q. CD Q< CO > _ O — CO 0 0 O" CD to g o c E < CD o c CD CT CD CO LU LU CO Q_ O _l x X Q_ > co CO \CL O CL CD O CD CO z LU CO Q > Q O > CO CO CO Q LU DC Q_ z ILU z ILL X LU LU CO X CC * DC Z LU CD LU _1 DC g o i— LL Ico LL CO _l CO LU z CC LU CO DC > _l > > > <: CC Q Q > CC o h> D_l Q_ < Q O o Q Q * LU LU O CO > o D_ CC < DC < _l DC LU DC CO DC > < I LU I CC < LU LU DC CO z > u_ > o ICD _i > > Q X > O o LL hco Q LU X o > Q CD CO > O Q_ > CO z LU Z CO x Q_ CO CL Q hCO O O CO _i cd LU Z CO z CL 0 < Q. 0 Q_ < CO > CD r< CO > CVJ in a> cd CD O) O) O) < < < CO CO CO > > > CM CM OJ 6 a> < CO > o ID C\J I o CM CM < CO > CD CM ID CM^ < CO > CO ^ iCD CO iCM CD 0 .Q E Cfl O) O) CO < < w CO CO > >

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19 helix of l-A d (Lee and Watts, 1990). All mAb were used at a final concentration of 10 ug/ml. MAbs used in this study had similar potencies as determined by the Hybridoma Core Facility, Interdisciplinary Center for Biotechnology Research, University of Florida. Polyclonal antibodies were used at a final dilution of 1:100. Poly IA d (Cat.YI -9-04-26-01) is specific for the m11 determinant of l-A d and poly la.7 (Cat.YI -7-02-02-01) is specific for the m7 determinant of l-E d . Both antibodies were obtained from NIAID, Rockville, MD. Poly l-A k was obtained from Accurate Chemicals, Westbury, NY. Radioiodinations SEA (2.5 |ig) and vSAg peptide (10 |xg) were radiolabeled using chloramine T as described (Griggs et al., 1992). Briefly, ligands were labeled with 500 uCi of Na 125 l (15 mCi/u.g, Amersham Corp., Arlington Heights, IL) in 25 ul of 0.5 M potassium phosphate buffer, pH 7.4, and 10 uJ of chloramine T (5 mg/ml) for 2 min. After neutralization of the reaction with 10 (il each of sodium bisulfite (10 mg/ml), potassium iodide (70 mg/ml), bovine serum albumin (BSA; 20 mg/ml), and 15 u.l of NaCI (4 M), the preparation was sieved on a 5 ml Sepharose G-10 column. The two fractions with the highest radioactivity in the first eluted peak were pooled and used in the radiolabeled binding assays. The specific activities of the SEs and vSAg peptide ranged from 130-150 uCi/ug and 30-60 uCi/ug. Binding Studies In binding studies using A20 cells, unlabeled competitors (SEs and vSAg peptides) were added in 50 ui volumes (in PBS with 1% BSA) at indicated final concentrations to 50 ul of cells in "Eppendorf" tubes. Competitors were incubated with cells at room temperature for 45 minutes,

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20 followed by the addition of radiolabeled SEs or vSAg peptide. After 45 minutes, the cells were washed three times and the radioactivity associated with the cell pellets was quantified using a gamma counter. L cells were used as an MHC class ll-negative control cell line. L cells were grown to confluence in the wells of microtiter plates and were subjected to the same incubation times and volumes of competitors and labeled ligands as used for A20 cells. After three washes, the cells were solubilized in 1% SDS, the liquid was absorbed by cotton-tipped applicators, and radioactivity was quantified using a gamma counter. Radioimmunoassay Two MHC peptides l-Apb(30-60) and l-Apb(60-90) were used to for binding of SEA and ORF(76-1 19) to class II antigens. The l-Apb(60-90) site was previously shown to be involved in SEA binding to MHC class II antigens (Russell et al., 1990). MHC peptides were dissolved in PBS at a concentration of 25 u-g/ml. Peptide solutions were pipetted into polystyrene plastic tubes and tubes were placed at 10°C for 4 hours to allow adherence of the peptides. The tubes were washed three times with PBS. Nonspecific sites on the plastic were blocked using 2 ml of PBS containing 1% BSA at 10°C overnight. After the tubes were washed three times of PBS containing 1% BSA, unlabeled competitors (SEA and vSAg peptide) were added in 0.1 ml and allowed to bind at room temperature for 3 hours. The tubes were washed three times with 2 ml of PBS/1% BSA prior to addition of 0.1 ml of either radiolabeled SEA or radiolabeled vSAg(76-1 19)peptide, at final concentrations of 2 nm and 5 nm, respectively. Radiolabeled ligands were allowed to bind to MHC peptides at room temperature for 4 hours. After washing three times with 2 ml of PBS containing 1% BSA, the tubes were placed in a gamma counter and bound

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21 radioactivity was quantified. Experiments were performed at least four times each using replicates of three. Results Competition of vSAa Peptides with Radiolabeled SEA for MHC Binding Overlapping peptides (referred to as vSAg peptides) corresponding to the predicted extracellular domain of the MMTV-1 superantigen were synthesized and their sequences are listed in Table III. The vSAg peptides were initially tested at a concentration of 200 |iM for their relative abilities to compete with 125 I-SEA for binding to A20 cells, which express l-A d and l-E d (which are MHC class II antigens of the d haplotype, A isotype and E isotype, respectively). The vSAg(76-119) peptide reduced 125 I-SEA binding to A20 cells by 63%, while the other peptides had no effect (Figure 1). Thus, only one of the overlapping vSAg peptides, vSAg(76-1 19), significantly blocked the binding of 125 I-SEA binding to A20 cells. vSAa(76-119) Peptide Competition Is Dose-Dependent Dose response studies were performed on A20 cells with several vSAg peptides, including vSAg(76-1 19). The vSAg(76-119) peptide reduced 125 lSEA binding by 50% at a concentration of 20 uM (Figure 2). Further, vSAg(76119) peptide consistently competed with 125 I-SEA in a manner similar to unlabeled SEA, although SEA was 20 times more effective. Unlabeled SEA at a concentration of 1 \lM reduced 125 I-SEA binding to A20 cells by 50%. These data are consistent with the reported Kd for SEA binding to l-E d of approximately 10" 6 M (Lee and Watts, 1990). The peptide corresponding to a

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22 160 14076-119 117-147 142-172 166-195 190-222 220-250 245-276 274-313 vSAg peptides Figure 1. Ability of vSAg peptides to compete with 125 I-SEA for binding to A20 cells. Binding of 125 I-SEA in the absence of competitors was 6384±400 CPM. The data presented represent the mean of three individual experiments each performed in duplicate. Each bar represents the mean percent of SEA control binding in the presence of vSAg peptides ± SD.

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23 Figure 2. Ability of vSAg(76-119) to compete with 125 I-SEA for binding to A20 cells in dose response studies. 125 I-SEA was used at 2.5 nM. Binding of 125 lSEA to A20 cells in the absence of competitors was 5184±380 CPM. The data presented represent the mean of three individual experiments, each performed in duplicate. Each point represents the mean percent of SEA control binding in the presence of competitors ± SD.

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24 C-terminal region of the MMTV superantigen, vSAg(245-276), was not a consistent competitor for SEA binding. The peptide corresponding to the Cterminal tail, vSAg(274-313), did not compete. A peptide having the same amino acid content as vSAg(76-119) but in a scrambled sequence (see Table III for sequence) did not compete, thus indicating that vSAg(76-119) peptide competition was sequence specific. No direct binding of 125 l-vSAg(76-1 19) to SEA or to related superantigens was detected (data not shown). Thus, the vSAg(76-119) peptide binds specifically to A20 cells, and competes for SEA binding in a dose-dependent manner. Direct Binding of vSAa(76-1 19) Peptide to A20 Cells In direct binding studies, 125 l-vSAg(76-1 19) peptide bound to A20 cells and was effectively inhibited by both unlabeled SEA and unlabeled vSAg(76119) peptide (Figure 3). Unlabeled SEA and unlabeled vSAg(76-119) peptide competed with 125 l-vSAg(76-1 19) in a similar manner, although SEA was a more potent competitor. SEA reduced 125 l-vSAg(76-1 19) binding by 50% at a concentration of 1.8 uM as compared to 25 \iM for unlabeled vSAg(76-119) peptide. Neither vSAg(76-119) scrambled peptide nor the C-terminal vSAg peptide competed, thereby showing that vSAg(76-119) binding was sequence and region specific. TSST-1 did not compete with 125 l-vSAg(76-1 19) peptide for binding to A20 cells, while SEB competed less effectively than SEA, which is consistent with their relative abilities to compete with SEA for MHC class II binding (Fraser, 1989; Pontzer et al., 1991). Thus these results indicate that binding of vSAg(76-119) peptide occurs to murine MHC class II antigens. Binding of vSAg(76-1 19) may occur at a region(s) to which SEA also binds or to a neighboring site which interferes with the binding of SEA.

PAGE 34

25 1200.01 0.10 1.00 10.00 100.00 1000.00 Competitor (|iM) Figure 3. Ability of SEA, SEB, and TSST-1 to compete with 12 5|-vSAg(76-1 19) for binding to A20 cells. Experimental conditions were the same as those described in Figure 1. 125 l-vSAg(76-1 19) was used at a final concentration of 2.3 nM. Binding of 125 l-vSAg(76-1 19) in the absence of competitors was 2278±224 CPM. The data presented represent the mean of three individual experiments, each performed in duplicate. Each point represents the mean percent of vSAg control binding in the presence of competitors ± SD.

PAGE 35

26 Evidence that vSAa(76-119^ Binds Specifically to MHC Class II Antigens Two further pieces of evidence indicate that vSAg(76-119) binds to class II MHC antigens. Binding of vSAg(76-1 19) to class II negative mouse L cells was insignificant as compared to mouse A20 cells even at concentrations as high as 5 nM (Figure 4A). Monoclonal antibodies to class I MHC antigens had no effect on 125 l-vSAg(76-1 19) binding to A20 cells (Figure 4B). Polyclonal anti-lA d and anti-l-E d significantly blocked binding and, in combination, these antibodies reduced binding to A20 cells by 73%. These data suggest that vSAg(76-119), like SEA (Lee and Watts, 1990), binds to both l-A and l-E. An IA d p1 helix-specific monoclonal antibody reduced binding by approximately 30%, suggesting that this is a region on l-A to which vSAg(76-119) binds. Polyclonal antibodies to l-A k had minor effects on vSAg(76-1 19) binding. Thus, these data indicate that vSAg(76-1 19) binds to class II MHC antigens. In order to directly determine if vSAg(76-1 19) binds to the p1 helix of l-A, a competitive radioimmunoassay was performed in which 125 I-SEA and 125 lvSAg(76-119) were tested for their relative abilities to bind to l-Ap b (60-90) peptide. SEA and vSAg(76-1 19), but not scrambled VSAG(76-1 19), competed with both 125|.sea and 1 25|. v SAg(76-1 19) for binding to l-Ap b (60-90) in a manner similar to the competition seen on whole cells (Figure 5A). As previously shown for SEA (Russell et al., 1990, 1991), vSAg(76-119) did not directly bind to l-Ap b (30-60) (Figure 5B). Thus, SEA and vSAg(76-119) bind to a similar region on the p chain of the class II MHC molecule. Discussion To date, no information has been available on the ability of MMTV vSAg protein to bind to MHC antigens and, in fact, this ability has been questioned (Marrack and Kappler, 1990; Acha-Orbea and Palmer, 1991; Acha-Orbea et al.,

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27 c U A20 cells L cells 0 200 Concentration of ORF(76-119) (MM) 160 140 120 100 80' — c c U ^ 60 MAb Kd Poly IAd Combination flU MAb IAd (poly IAd + Ia.7) Ia.7 Poly IAk B Antisera Figure 4. Evidence that vSAg(76-119) peptide binds to MHC class II antigens, and does not bind to class I antigens. Panel A: Binding of 125 l-vSAg(76-1 19) to class ll-positive A20 cells and class ll-negative L cells. 125 l-vSAg(76-1 19) was used at a final concentration of 5 nM. Panel B: Blockage of 125 l-vSAg(76-1 19) binding to A20 cells by antibodies to class I and class II MHC antigens. 125 l-vSAg(76-1 19) was used at a final concentration of 5 nM. MAbs were used at a final dilution of 1 :30.

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28 o u e © 12010080604020SEA vs. radiolabeled SEA vSAg(76-119) vs. radiolabeled SEA SEA vs. radiolabeled vSAg(76-199) vSAg(76-119) vs. radiolabeled vSAg(76-119) 0 I i i i i i i i 1 1 0.1 1.0 I I I I I I 10.0 I I — I — I I I I I 1 I 1 I I I 100.0 1000.0 Concentration (|iM) 2000 _ 1500 H c 9 o * 1000eu U 500I I No competitor vSAg(76-119) 60-90 30-60 MHC Peptide B Figure 5. Binding of 125 l-vSAg(76-1 19) to MHC class II peptides. Panel A: Binding of 12 5|-SEA and 1 25|. v SAg(76-119) to l-A(Jb(60-90) and inhibition by vSAg(76-1 19). Each point represents the mean percent reduction of control binding in the presence of competitors ± SD. Binding of 125 I-SEA and 125 l-vSAg(76-119) in the absence of competitor was 2656+92 CPM and 1547133 CPM, respectively. Panel B: Relative ability of vSAg(76-119) to bind l-A b (60-90) and l-Ap b (60-90) in the presence and absence of vSAg(76-1 19). Unlabeled and 125 l-vSAg(76119) were used at final concentrations of 5 nM and 300 |iM, respectively.

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29 1991). Such binding studies are hampered by the difficulties inherent in expressing the MMTV superantigen. A model has recently been proposed that suggests that MMTV superantigens may act in the same or similar fashion to bacterial superantigens by bridging MHC antigens and TCR on the appropriate cell types (Acha-Orbea and Palmer, 1991). MMTV superantigens, presented in the context of MHC class II antigens, may act by stimulating and expanding Vpspecific T cell subsets, thereby indirectly enhancing the further infection of B cells (Held et al., 1994a). The results presented in this chapter suggest that MMTV vSAg protein binds to MHC antigens, thereby strengthening the argument that bacterial and retroviral superantigens act in a similar manner. It has been reported that MMTV superantigen is a 45 kd Type II integral membrane protein with an intracellular N-terminus, an essential hydrophobic transmembrane region near the N-terminus (residues 45-64), and a glycosylated extracellular C-terminus (Choi et al., 1992). Although no direct evidence exists, the variability of the C-terminal residues of vSAg proteins of various MMTV strains seems to correlate with their differences in Vp specificity, lending support to the concept that this region binds TCR (Pullen et al., 1992). Truncated versions of the vsag gene were transfected into MHC class ll-bearing cells and tested for superantigen activity. Complete loss of superantigen activity occurred when the MMTV vsag gene was N-terminally truncated to the third methionine (residue 122) and beyond (Choi et al., 1992). The authors concluded that a hydrophobic region, which was missing in this N-terminally truncated version, acts as a transmembrane region and is essential for superantigen activity. However, our binding data suggest that loss of superantigen activity may have been due, at least in part, to the deletion of the MHC-binding domain which is encompassed by residues 76-119. In fact, the

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30 superantigen activity of MMTV vSAg protein was lost in truncations that did not contain the region encompassed by residues 76-119. Subsequent studies by the same group of investigators suggest that MMTV-7 superantigen is synthesized as a precursor molecule, is proteolytically cleaved at an internal site (residue 164) and is expressed as an 18.5 kd surface protein consisting of the C-terminal residues (Winslow et al., 1992). Membrane association of this truncated form may involve "tethering" to MHC class II antigens or to the N-terminal portion of the precursor molecule. As a cautionary note, the transfected cell line used to characterize this truncated form of the MMTV-7 superantigen was shown to have high expression of the protein but low activity. Conversely, a transfected cell line with moderate superantigen expression and high activity was not used for characterization. Recently, studies were reported on the binding of truncated forms of MMTV-7 superantigen to human MHC class II antigens. Binding studies were performed with 28K and 18K versions of the superantigen and showed that both molecules bind to human MHC class II antigens (Mottershead et al., 1995). Thus, studies by our laboratory indicate one site for binding to mouse MHC class II antigens, whereas studies performed by others suggest that two sites on MMTV superantigen are involved in binding to human MHC class II antigens. Despite the diverse origins of SEA and MMTV vSAg protein, it is likely that these two proteins exert superantigen activity by a similar mechanism. Two different regions of the MMTV superantigen are probably involved in MHC binding and Vp specificity. The variability of the C-terminal 30 residues between Vp-specific MMTV superantigens is indirect evidence that the Cterminus is responsible for Vp specificity (Pullen et al., 1992). Data presented here on competitive and direct binding of vSAg peptides to A20 cells suggest that the region encompassed by residues 76-119 is involved in MHC binding.

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31 This region would be part of the extracellular domain based on a proposed Type II membrane protein model for MMTV vSAg protein (Choi et al., 1992). Thus, both the C-terminal tail and an N-terminal region may be required for ternary complex formation with MHC and TCR by MMTV superantigen. Conversely, SEA contains several N-terminal domains that bind MHC (Griggs et al., 1992) and an internal domain that may be involved in binding to TCR, so that the sites of interaction of these superantigens with MHC and TCR may not be completely analogous. Although little sequence homology exists between MMTV vSAg protein and SEA, circular dichroism analysis indicates that both SEA and vSAg(76-119) have significant p structure, suggesting that similar structural motifs may be important for MHC binding by superantigens. Finally, results presented herein indicate that a peptide corresponding to residues 76-119 of the MMTV superantigen binds directly to MHC class II antigens. Further, competition studies indicate that SEA and vSAg(76-119) peptide bind to at least one common region on mouse MHC antigens. Future studies using synthetic peptides and peptide analogues may help further elucidate the site(s) on MHC for which SEA and MMTV superantigen compete.

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CHAPTER III IDENTIFICATION OF AN HIV-1 NEF PEPTIDE THAT BINDS TO MHC CLASS II ANTIGENS Introduction In Chapter Two, a site was identified on the MMTV-1 superantigen that binds to class II MHC antigens, suggesting that retroviral and bacterial superantigens exert their effects similarly. MMTV superantigen is encoded in a gene that overlaps the terminal repeat at the 3' end of the viral genome. The genome of human spumavirus also contains a gene (bel3) that overlaps the terminal repeat at the 3' end, the product of which may have superantigenic properties (Fluegel, 1993). Recently, it has been suggested that human immunodeficiency virus (HIV) may possess superantigen activity, although no specific superantigenic protein has been identified (Imberti et al., 1991; Laurence et al., 1992; Dalgleish et al., 1992; Bisset et al., 1993). The HIV genome also contains a gene that overlaps the terminal repeat at the 3' end, the product of which is called Nef. Although Nef is one of the early proteins produced during the replication of primate lentiviruses, the role of Nef in HIV pathogenesis has yet to be established. To determine if Nef had binding characteristics similar to superantigens, a study was undertaken to determine its ability to bind to MHC class II antigens, at sites that are known to bind superantigens. Using overlapping peptides corresponding to the entire length of Nef (HIVlav). we have identified a region which binds to MHC class II antigens, and which competes for binding with known bacterial superantigens. 32

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33 Materials and Methods Synthetic Peptides Overlapping peptides corresponding to the entire length of HIVlav Nef (Wain-Hobson et al., 1985) were synthesized with a Biosearch 9500AT automated peptide synthesizer using N-(9-flurenyl)methoxycarbonyl chemistry as described in chapter II of this text. Peptides were synthesized based on a surface profile as described. The amino acid sequences of the peptides are presented in Table IV. Cells and Reagents Two cell lines were used for the binding studies. Raji cells are EBVtransformed B cells that express DR3, Dw10, DQw1, and DQw2 (Merryman et al., 1989). DR1-transfected L cells were kindly provided by Dr. Eric O. Long and are described elsewhere (Lechler et al., 1988). SEs were obtained from Toxin Technology (Sarasota, FL). Several mAb were used in this study. AntiHLA-DR clone L243 reacts with a nonpolymorphic DR epitope and does not cross-react with DP or DQ (Robbins et al., 1987). Anti-HLA-DP clone B7/21 reacts with a monomorphic epitope present on DP1, DP2, DP3, DP4, and DP5 (Robbins et al., 1987). Anti-HLA-DQ clone SK10 reacts with a common polymorphic epitope present on cells expressing DQw1 and DQw3 (associated with DR1. DR2, DR4, DR5, w8, w9, and w10) (Brodsky, 1984). Anti-HLA-DR clone L227 reacts with a nonpolymorphic region of DR (Barnstable et al., 1978). Clone W6/32 reacts with a monomorphic epitope on HLA-A, B, and C (Barnstable et al., 1978). Clones L243, B7/21, and SK10 were obtained from Becton-Dickinson (Mountain View, CA) and clones L227 and W6/32 were kindly provided by Dr. Robert Rich. All mAbs were used at a final concentration

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34 Table IV. Amino acid sequences of Nef peptides. Nef Peptide Sequence 1 -38 MGGKWSKSSVVGWPTVRERMRRAEPAADGVGAASRDLE 31 -65 GAASRDLEKHGAITSSNTAATNAACAWLEAQEEEE 62-99 EEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEG 93-1 32 EKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPG 1 23-1 60 DWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGE 1 56-1 86 NKGENTSLLHPVSLHGMDDPEREVLEWRFD 182-206 EWRFDSRLAFHHVARELHPEYFKNC

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35 of 10 ng/ml. Purified recombinant Nef protein was purchased from Repligen, Cambridge, MA. Radioiodinations SEs (2.5 ug) and Nef peptide (10 ug) were radioiodinated using chloramine T as described in chapter II of this text. The specific activities of the SEs and Nef peptide ranged from 70-120 uCi/|ig and 30-40 uCi/ug, respectively. Binding Studies For binding studies on Raji cells, unlabeled competitors (SEs and Nef peptides) were added in 50 ul (in PBS with 1% BSA) to 50 ul of cells in 1.5 ml "Eppendorf" tubes. Cells and competitors were incubated at room temperature for 45 min., followed by the addition of radiolabeled SEs or Nef peptide. After 45 min., the cells were washed three times and the bottoms of the tubes were cut off. Radioactivity was quantified using a gamma counter. Similarly, DR-1 transfected L cells, which were grown to confluence in the wells of microtiter plates, were subjected to the same incubation times and volumes of competitors and labeled ligands as used for the Raji cells. After three washes, the cells were solubilized in 1% SDS, and the liquid was absorbed by cottontipped applicators and radioactivity was quantified using a gamma counter. Results Competition of Nef Peptides with Radiolabeled SEA for MHC Binding Overlapping peptides corresponding to the entire length of the Nef sequence (HIVlav) were synthesized (Table IV). The Nef peptides were

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36 initially tested at a concentration of 300 uM for their relative abilities to compete with 12 5|-SEA for binding to Raji cells, which express HLA-DR3 and HLADRw10, and DR1-transfected L cells (Figure 6). Nef(123-160) reduced 125 lSEA binding to Raji cells by 41% which was significant at p<0.002. This degree of inhibition was consistent in repeated experiments. The N-terminal peptide, Nef(1-38), had a marginal but insignificant effect (21% reduction, p>0.05) on 125 I-SEA binding to Raji cells in the same experiment. This slight inhibition by Nef(1-38) was not seen in repeated experiments. The other peptides did not affect SEA binding to Raji cells. A similar pattern was seen in competitive binding studies performed on DR1-transfected L cells. Nef(123160) blocked 12 5|-SEA binding to DR1 cells by 35% (p<0.008), whereas Nef(138) had only a slight effect (p>0.8). Thus, only one of the overlapping Nef peptides, Nef(123-160), significantly and consistently blocked 125 I-SEA binding to both Raji cells and DR1-transfected L cells. Nef(123-160) Peptide Competition Is Dose-Dependent The relative abilities of Nef(123-160), purified recombinant Nef protein (Repligen, Cambridge, MA), and SEA to block SEA binding to MHC class II were tested in dose response studies (Figure 7). Nef(123-160) reduced 125 lSEA binding to Raji cells by 40% at the highest concentration tested (300 (iM), and competed with SEA in a dose-dependent manner. Two other Nef peptides, Nef (1-38) and Nef(31-65), did not block SEA in a dose-dependent manner. Unlabeled SEA reduced 125 I-SEA binding by 50% at a concentration of 0.2 |iM, which is consistent with the reported Kd of SEA for human MHC class II antigens (Chintagumpala et al., 1991). Thus, Nef protein and the internal Nef sequence, Nef(123-160), competed for SEA binding to Raji cells in a dosedependent manner.

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37 140 1-38 31-65 62-99 93-132 123-160 156-186 182-206 NeF Peptide Figure 6. Blockage of 125 I-SEA binding to Raji and DR1-transfected L cells by Nef peptides. Net peptides were used at a final concentration of 300 [iM. 125 lSEA was used at a final concentration of 2 nM. 10 5 Raji or DR1-transfected L cells were used per tube. Binding of 125 I-SEA to Raji and DR1-transfected L cells in the absence of competitors was 31,469 + 2292 and 5,708 ± 41 CPM, respectively. Data represent the mean percent of control of three individual experiments, each performed in duplicate. Bars represent binding to Raji and DR1-transfected L cells in the presence of Nef peptides ± SD.

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38 125 2 — * s o U 100755025 NeF(123-160) NeF(31-65) -A Nef protein I I I I I I I I | 0.01 0.10 I 1 I I I I 1 1 1.00 I I I 1 1 1 10.00 rrrr| 100.00 I 1000.00 Concentration (|iM) Figure 7. Relative abilities of Nef(123-160), purified Nef protein, and SEA to compete with 125 I-SEA for binding to Raji cells. Experimental conditions were the same as those described in Figure 1. Binding of 125 I-SEA to Raji cells in the absence of competitors was 30,406 ± 2851 CPM. The data presented represent the mean of three individual experiments, each performed in duplicate. Each point represents the mean percent of SEA control binding in the presence of competitors ± SD.

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39 Ability of Nef Peptides to Compete with SEs for Binding to Raji Cells Nef peptides were also tested for their relative abilities to compete with other staphylococcal enterotoxins (SEs) for binding to Raji cells. As shown in Figure 8, Nef(123-160) significantly blocked binding of 125 I-SEE by 42% (p<0.02) and 125 I-SEC1 by 26% (p<0.03), but only blocked binding of 125 lSEB by 10% (p>0.1). Thus, Nef(123-160) significantly inhibited the binding of two highly homologous SEs, SEA and SEE, while it was less effective against SEB and SEC1. Direct Binding of Nef(123-160) Peptide to Raii Cells In direct binding studies, 125 l-Nef(123-160) bound to Raji cells and was effectively inhibited by unlabeled Nef(123-160) and SEs (Figure 9). Unlabeled Nef(123-160) reduced 125 l-Nef(123-160) binding by 50% at a concentration of 30 uM. SEE was a better competitor and reduced 125 l-Nef(123-160) binding by 50% at a concentration of 2 uA/l. Both SEE and unlabeled Nef(123-160) competed in a similar manner, but SEE was a more potent competitor. SEA did not inhibit 125 l-Nef(123-160) binding as well as SEE, but it was more effective than SECi or SEB. This pattern of inhibition is reflective of the ability of Nef(123-160) to block SE binding to MHC class II antigens. Although SEs compete for similar sites on MHC class II antigens, our results suggest that Nef(123-160) competes for sites that are more closely associated with SEE than the other SEs tested. Nef(123-160) binding was not blocked by other Nef peptides, such as Nef(1-38) and Nef (31-65), at 300 u.M. These results suggest that Nef(123-160) binds directly to Raji cells and competes for a site on MHC class II antigens to which SEs bind.

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40 1-38 31-65 62-99 93-132 123-160 156-186 182-206 NeF Peptide Figure 8. Blockage of 125 I-SE binding to Raji cells by Nef peptides. Nef peptides were used at a final concentration of 300 uM. 125 l-SEs were used at 2 nM. All other experimental conditions were the same as those described in Figure 7. Binding of 125 I-SEE, 12 5|-SEB, and 125 I-SEC1 in the absence of competitor was 5,917±612 CPM, 6,438±134 CPM, and 4,493±425 CPM, respectively. Data represent the mean of three individual experiments, each performed in duplicate. Each bar represents the mean percent of the appropriate SE control binding to Raji cells in the presence of Nef peptides ± SD.

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41 Figure 9. Direct binding of Nef(123-160) peptide to Raji cells. 125 l-Nef(123160) was used at a final concentration of 3 nM. All experimental conditions were the same as those described in Figure 7. Binding of 125 l-Nef(123-160) in the absence of competitor was 1956 ± 150 CPM. The data presented represent the mean of three individual experiments, each performed in duplicate. Each point represents the mean percent of 125 l-Nef(123-160) binding relative to control in the presence of competitors ± SD.

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42 MAbs to MHC Class II Antigens Block NefM 23-1 601 Peptide Binding to Raii Cells To ascertain the receptor to which 125 l-Nef(123-160) bound on Raji cells, binding was performed in the presence of monoclonal antibodies (mAb) to class II and class I antigens (Figure 10). Clone L243, a mAb specific for HLADR, reduced 125 l-Nef(123-160) binding by 35%. Another mAb specific for HLA-DR, clone L227, also reduced Nef(123-160) binding, but was not as effective as clone L243. This is consistent with reports that L227 mAb is not very effective at blocking SEA binding to Raji cells (Chintagumpala et al., 1991). MAb to other class II antigens (HLA-DP and DQ) had no effect on 1 25 I-Nef(1 23160) binding, as was also the case for a mAb to class I antigens (clone W6/32). Thus, our studies indicate that Nef(123-160) binds to class II MHC antigens, the known receptors for superantigens on APC. Discussion Data presented here show that binding of bacterial superantigens to MHC class II antigens can be blocked by a peptide corresponding to an internal Nef region. Anti-HLA-DR mAb blocked Nef(123-160) binding to Raji cells, suggesting that binding to class II molecules occurred. Binding of Nef(123-160) to HLA-DR probably occurs outside the antigen binding groove, since Nef(123160) was able to block binding of the superantigens SEE and SEA. A recent study has shown that HLA-DR is present on HIV-1 and SIV, and that it is selectively incorporated into the virus membrane over HLA-DP or HLA-DQ (Arthur et al., 1992). Antibodies to HLA-DR, but not antibodies to HLA-DP or HLA-DQ, effectively inhibited viral infection of cells in vitro and these antibodies target the HLA-DR antigens present on the virus particles and not those present

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43 Figure 10. Blockage of 125 l-Nef(123-160) binding to Raji cells by antibodies to MHC class I and class II antigens. 125 l-Nef(123-160) was used at a final concentration of 3 nM. MAbs were used at a final concentration of 10 ng/ml.

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44 on the cells. The ability of these antibodies to neutralize HIV suggest that HLADR may play a role in infectivity. Our results suggest that Net may bind to HLADR as it is being expressed on the surface of an infected cell in a manner analogous to the putative expression of MMTV superantigen (Choi et al., 1992). Further, Nef has been shown to be present on the surface of infected cells, as assessed using anti-Nef antibodies labeled with fluorescein (Fujii et al., 1993). This is in contrast to the situation with MMTV superantigen in that no one has been able to show that cells from MMTV-infected animals express superantigen, although superantigen functional activity is observed (Choi et al., 1991). Transgenic mice expressing MMTV superantigen delete Vp14+ T cells, probably during the ontogeny of the immune system, and these mice are immune to infection by exogenous MMTV infection (Acha-Orbea and Palmer, 1991). Thus it is possible that Nef interacts with class II MHC antigens in a manner somewhat similar to MMTV superantigen, suggesting a possible superantigen function for Nef in HIV pathogenesis.

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CHAPTER IV ACTIVATION OF CD4 T CELLS BY THE NEF PROTEIN FROM HIV-1 Introduction In the previous chapter, data was presented which showed that Net binds specifically to class ll-bearing Raji cells at a site(s) involved in staphylococcal enterotoxin binding. This binding was similar to that shown for MMTV superantigen. These data suggest that Nef may have characteristics of superantigens. The data presented in this chapter show that Nef induces proliferation of human peripheral blood mononuclear cells. Further, the proliferative response is T cell specific. The data presented in this chapter indicate that Nef activates T cells and induces T cell cytokine production in a manner reminiscent of staphylococcal superantigens. Materials and Methods Nef Protein Nef protein was expressed and purified using a fusion protein and purification system. The HIV-1 nef gene (HIV-1 1Mb, R&D Systems, Cambridge, MA) was amplified by polymerase chain reaction (PCR), using the following primer set : 5 ' ATG GGT GGC AAG TGG TCA AAA AGT (+) 5 ' GCC AAG CTT GAT GTC AGC AGT TCT (-) 45

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46 The extra A added to the 3' end of the amplified nef gene fragment as a result of PCR was removed by treatment with the Klenow fragment of DNA polymerase. This DNA was ligated into the Xmnl/H/'ndlll cloning site of the prokaryotic expression vector pMAL™-c2 (New England BioLabs, Beverley, MA). The sequence of the fusion construct was verified by the DNA Sequencing Laboratory, Interdisciplinary Center for Biotechnology Research, University of Florida. The fusion construct was engineered such that the N terminus of Nef was immediately downstream from the Factor Xa cleavage site at the C terminus of maltose binding protein (MBP). Thus, cleavage with Factor Xa was predicted not to add any vector-derived amino acids. E. coli strain TB1 was transformed with the vector containing mbp/nef. Cultured E. coli TB1 were suspended in column buffer consisting of 20 mM Tris-HCI (pH 7.4), 200 mM NaCI, and 1 mM ethylenediaminetetraacetic acid (EDTA). The cells were sonicated and the supernatants were recovered after centrifugation. Protease inhibitors (2 mM phenylmethylsulfonyl fluoride (PMSF), pepstatin A, leupeptin, and aprotinin) were added to extracts. The protein concentration of the extracts was adjusted to 2.5 mg protein/ml prior to loading onto an amylose affinity column containing a bed volume of 50 ml. The column was washed with 350 ml of column buffer. The fusion protein was eluted with 200 ml of column buffer containing 10 mM maltose. Fractions (7 ml) were collected. The fusion protein generally eluted within the first 70 ml. After cleaving the fusion protein with factor Xa (New England BioLabs, Beverley, MA), the cleavage mixture was loaded onto a hydroxyapatite column containing a bed volume of 35 ml. After extensive washing of the column with 180 ml of 10 mM potassium phosphate (pH 7.2) containing 2 mM PMSF and 1 mM benzamidine, fractions were eluted using a linear gradient from 10 mM to 400 mM potassium phosphate buffer (pH 7.2) containing 2 mM PMSF. Fractions (5 ml) were collected. Nef eluted from

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47 the column between 100-150 mM potassium phosphate, and was generally contained within 7 fractions. The fraction containing Nef protein was dialyzed against affinity column buffer containing 150 mM NaCI. The dialyzed material was loaded onto a second amylose affinity column for removal of MBP. The flow-through containing Nef protein was collected and loaded onto an High-Q ion-exchange cartridge (BioRad Laboratories, Richmond, CA). The High-Q cartridge was washed with 100 ml of 20 mM Tris buffer (pH 8.0) containing 150 mM NaCI. Fractions were eluted with a linear gradient from 150-400 mM NaCI in the same buffer. The fractions containing Nef protein, which eluted at 180 mM NaCI, were collected and dialyzed overnight against PBS and stored at -70°C. The purity of Nef protein was assessed by SDS-PAGE, followed by staining with silver. Upon staining with silver, a single band was found, and a corresponding band was detected by Western blotting with monoclonal anti-Nef antibody (Repligen, Cambridge, MA). MBP purified by this method and used at the same concentrations as that used for the purified Nef preparation, did not have proliferative activity on human peripheral blood mononuclear cells. Nef proliferative activity was confirmed using recombinant Nef proteins obtained from two other sources and which were produced using different expression systems. The following reagent was obtained through the AIDS Research and Reference Reagent Program, AIDS Program, NIAID, NIH: HIV-1 LAV Nef from the Division of AIDS, NIAID. The HIV-1 nef gene (LAV) used to produce this Nef preparation was isolated from pBENN 6 and cloned into the bacterial expression vector pPD-YN-61 . The protein was produced in E. coli strain Sf930 and isolated as inclusion bodies. Nef protein was also obtained from Repligen (Cambridge, MA). In this case, the HIV-1 nef gene (LAV) was cloned into the bacterial vector pD10, which adds a hexahistidine tag for protein purification using nickel chelate chromatography. The protein was produced in

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48 E. coli strain MC1 0611 POLL All Net preparations were negative for endotoxin as assessed by the limulus amoebocyte lysate assay. Reagents Staphylococcal enterotoxin A (SEA) was purchased from Toxin Technology (Sarasota, FL). Concanavalin A (Con A), anti-CD3, and anti-IgM were purchased from Sigma Chemical Co. (St. Louis, MO). Proliferation Assays Proliferation studies on human peripheral blood mononuclear cells (PBMC) were performed as described previously (Pontzer et al., 1992). Peripheral blood from healthy adult blood bank donors was obtained from Civitan Regional Blood Center (Gainesville, FL) for use in these studies. All donors were negative for cytomegalovirus, hepatitis B virus, and HIV. PBMC were isolated from peripheral blood using ficoll hypaque gradient centrifugation. After extensive washing, PBMC were plated into wells of microtiter plates at 2.8 x 10 6 cells/ml, followed by addition of activators. Final volumes were adjusted to 150 (xl/well with RPMI 1640 tissue culture medium containing 5% fetal bovine serum (FBS) and penicillin/streptomycin. 3 Hthymidine (1uCi/well; Amersham Corporation, Indianapolis, IN) was added at 90 h after initiation of cultures and the cells were incubated for an additional 6 h prior to harvest onto filter paper. Filter paper was placed in liquid scintillation fluid and radioactivity was quantified using a p scintillation counter. Stimulation index (S.I.) was determined by dividing the experimental CPM by the CPM obtained from control (unstimulated) cultures.

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49 Purification of T Cells from PBMC Two methods were used to isolate purified T cell populations. One method involved passing PBMC over T Cellect columns (Biotex, Edmunton, Alberta, Canada). A second method involved a double rosetting technique using neuraminidase-treated sheep red blood cells (NA-SRBC) (Spawski and Lipsky, 1992). PBMC (5 ml at 10 7 /ml) were mixed with 2.5 ml each of NASRBC and FBS. PBMC were incubated for 10 min at 37°C, centrifuged at 900 rpm to gently pellet the cells, and incubated for 90 min at 37°C. Rosetted T cells were separated from non-rosetted cells by ficoll hypaque centrifugation. NASRBC were removed from the rosetted fraction by lysis with ammonium chloride. The procedure was repeated on the rosetted fraction to insure purity. Autologous rosetted T cells were plated at 2.8 x 10 6 cells/ml. Cells were plated into wells of microtiter plates at 2.8 x 10 6 cells/ml. To insure that these cultures were depleted of APC, T cells were stimulated with staphylococcal enterotoxins, which stimulate T cells only in the presence of APC. Autologous Antigen-Presenting Cells (APC) For proliferation studies, APC consisted of PBMC that were inactivated with 0.8% paraformaldehyde. For cytokine studies, APC consisted of cells that did not form rosettes after two treatments with NA-SRBC. APC were plated at 2.8 x 106 cells/ml. To insure inactivation with paraformaldehyde was complete, APC were tested for possible proliferation using anti-IgM and Con A. Synthetic Nef Peptides and Antibodies to Nef Peptides The sequences of the peptides used are listed in Table III. Peptides were purified by reverse phase high performance liquid chromatography. Polyclonal antibodies to Nef peptides were generated in rabbits. Anti-peptide

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50 antibodies were tested for their relative abilities to neutralize Net mitogenic activity by incubating Nef with antibodies at 37°C for 60 minutes prior to addition of PBMC. Antibodies to both Nef(123-160) and Nef(1 82-206) showed strong reactivity to whole recombinant Nef protein, and these combined antisera had significant neutralizing activity against Nef. Nef was treated with preimmune sera as control. Cytokine Production and Assays PBMC were plated at 10^ cell/ml into wells of 24-well plates (Corning Corp., Corning, NY). Activators were added and final volumes were adjusted to 300 u.l/well. Cultures were incubated at 37°C over a 96 h period and samples (100 u.l) were obtained at 24 h intervals. Cell cultures were replenished with RPMI/5% FBS (100 u.l) after obtaining samples. Culture supernatants were tested for interleukin 2 (IL 2) using the IL 2-dependent HT-2 cell line (Ho et al., 1987). The amounts of IL 2 present in culture supernatants were determined using recombinant human IL 2 (Genzyme, Cambridge, MA) as a standard. Samples were tested for interferon (IFN) activity on human WISH cells by a microplaque reduction method (Langford, et al., 1978b), using approximately 40 plaque-forming units (PFU) of vesicular stomatitis virus (VSV) per well. In our studies, 1 U/ml of IFN is defined as the concentration required to decrease the number of PFU per well by 50%. IFN activity was typed by neutralization reactions with specific antisera, as described (Johnson et al., 1982). Briefly, samples were pretreated with 1000 neutralizing units of either anti-human IFNa (Lee Biomolecular, San Diego, CA) or anti-human IFNy (Genzyme, Cambridge, MA). Controls were sham-treated with EMEM/2% FBS. The neutralizing activity of anti-IFN antisera was confirmed using HulFNa (Lee Biomolecular, San Diego, CA) and HulFNy

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51 (Genzyme, Cambridge, MA) as positive controls. Samples were incubated at 37°C for 1 h prior to transfer to confluent human WISH cells. Residual IFN activity in shamand antiserum-treated samples was measured as described above. Studies on VB-Specific T Cell Expansion and Induction of Anerav PBMC cultured in the presence of Nef, TSST-1 , and ConA were tested by flow cytometry for specific Vp expansion as previously described (Soos and Johnson, 1994) using a panel of anti-Vp antibodies. Induction of anergy in vitro by Nef, TSST-1, and ConA was also assessed with anti-Vp antibodies as described (Schiffenbauer et al., 1993). Antibodies to Vp5a, Vp5b, Vp5c, Vp6a, Vp8a, and Vp12 were obtained from T Cell Sciences, Inc., Cambridge, MA. Antibodies to Vp2, Vp3, Vp13, Vp17, Vp18, Vp21 , and Vp22 were purchased from Immunotech, Marseilles, France. Results Nef Proliferative Response Recombinant Nef proteins from two sources were tested for proliferative activity. One Nef preparation (referred to as Nef 1) was purified and kindly provided by Dr. Taishi Tanabe in the laboratory of Dr. Howard M. Johnson. This Nef preparation was compared to purified Nef from Repligen (Nef 2). Both Nef protein preparations, which are derived from the HIVlav sequence, induced similar levels of proliferation (Figure 11). Nef proteins from both sources were pure and neither preparation contained detectable levels of endotoxin. Thus, Nef proteins from both sources had similar proliferative activity, and the proliferation observed was significant. Nef from these two sources were

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52 1000 100 10 1 Concentration (ng/ml) Figure 11. Comparison of the mitogenic activities of Nef protein preparations. Two Nef preparations, Nef 1 and Nef 2, were compared for proliferative activities on human PBMC. Data are from a representative experiment, performed in triplicate, and are expressed as mean stimulation index ± S.D.. Comparison of the proliferation of PBMC cultured for 4 days in the presence of either Nef 1 or Nef 2. The mean value for 3H-thymidine incorporation by unstimulated cultures was 900+67.

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53 subsequently used in these studies. The possibility was raised that the proliferative activity ascribed to Net was due to a contaminant in the recombinant preparation. As a control, the proliferative activity of the Nef fusion partner, MBP, was also assessed. No proliferation was observed with MBP (Figure 12). Further, polyclonal antisera generated against Nef synthetic peptides were tested for neutralizing activity. As shown in Table V, anti-Nef antibodies significantly reduced Nef-induced proliferative responses. Antibody neutralization of Nef activity occurred in a dose-dependent manner. These data confirm that the proliferative activity seen in these studies is due to Nef protein, and not to a vector-derived contaminant. Nef was also tested for the ability to induce proliferation of PBMC from fifteen donors. Results from a representative sample of ten donors are shown in Figure 13. Nef induced significant proliferation in 90% of the donors tested. Variation in the Nef response was seen, similar to the responses to the potent staphylococcal enterotoxin, SEA. It is unlikely that these donors were sensitized to Nef, since they tested negative for HIV-1. Similar results were obtained using purified recombinant Nef protein preparations from two different sources, and which used different expression systems (see Materials and Methods in this chapter). The high number of donors that responded to Nef indicates Nef protein has similar mitogenic activity to SEA for PBMC from a wide sampling of donors. Antigen-Presenting Cells (APC) Are Required for Nef Activity The question arose as to whether Nef, like SEA, requires APC in order to stimulate lymphocyte proliferation. Nef induced significant proliferation in PBMC (Figure 14). Purified T cell cultures did not proliferate in response to Nef or to SEA. Purified APC, which contained monocytes and B cells, did not

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54 Nef MBP PBMC stimulated with Nef or E. coli MBP: Figure 12. Lack of proliferation by PBMC in response to the Nef fusion partner MBP. MBP and Nef were used at 3 ng/ml. Data are expressed as mean stimulation index ± SD. The mean values for ^H-thymidine incorporation by unstimulated cultures in eperiments 1 and 2 were 852±39 CPM and 1093±136 CPM, respectively.

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55 Table V. Ability of anti-peptide antibodies to block Nef-induced proliferation PBMC cultured Anti-Nef Stimulation Index 0 in the presence of a : antibodies 0 (Mean ± SD) p value Nef Nef + SEA SEA + 4.7 ± 0.2 1.3 ±0.1 <0.001 59.7 ±2.6 62.0 ±0.6 >0.15 0.9 ± 0.4 a Nef (3 ^ig/ml) and SEA (0.3 u.g/ml) were incubated with anti-Nef antibodies for 60 minutes prior to addition of PBMC. b A mixture of antisera to Nef(123-160) peptide and Nef(1 82-206) peptide were used, each at a final dilution of 1:1000. Preimmune sera had no effect. Both anti-Nef(123-160) and anti-Nef( 182-206) had strong reactivity to Nef protein by ELISA. c Data are from a representative experiment performed in triplicate.

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56 Figure 13. Nef-induced proliferative responses from a representative sampling of donors. PBMC of blood bank donors were tested for proliferation induced by SEA or Nef. SEA and Nef were used at 300 ng/ml. Mean values for 3 Hthymidine incorporation by unstimulated cultures ranged from 352±86 CPM to 1983±274 CPM.

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57 25 Unfractionated HPMC APC T cells APC + T cells Cells Figure 14. Nef-induced activation of T cells requires APC but does not require processing of Nef. Unfractionated PBMC, purified APC alone, purified T cells alone, and purified T cells in the presence of APC were tested for proliferation in response to Nef, SEA, and Con A. Data are from a representative experiment, performed in triplicate, and are expressed as mean stimulation index ± S.D. Purified T cells were reconstituted with paraformaldehydeinactivated APC. Con A was used at 10 ug/ml. Nef and SEA were both used at 100 ng/ml.

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58 respond to stimulation by either Nef or SEA. Upon reconstitution with paraformaldehyde-inactivated autologous APC, significant Nef-induced proliferation of T cells occurred, with essentially complete reconstitution of the response. Similar proliferative responses were obtained using cells from other donors. Thus, T cells responded to Nef stimulation only in the presence of autologous APC, and Nef does not require processing to be presented by APC. These results indicate that Nef-induced proliferation required APC in a manner reminiscent of SEA. NefM23-160^ Peptide Specifically Blocks Proliferation of PBMC Induced bv Nef and SEA As shown in Chapter 3, a synthetic peptide corresponding to an internal Nef sequence, Nef(123-160), blocked binding of Nef and SEA to Raji cells. It was important to determine if this peptide could also block proliferation induced by Nef and SEA. The results of this study are presented in Figure 15. Nef(123160) blocked both Nef-induced and SEA induced proliferation, consistent with its ability to block binding of Nef and SEA to Raji cells. Further, the blocking was specific in that proliferation induced by the T cell mitogens Con A and antiCD3, or the B cell mitogen anti-IgM, were not blocked by Nef(123-160). Nef(157-186), whose sequence slightly overlaps that of Nef(123-160), had no effect on the proliferative effects of either Nef, SEA, or Con A. These results confirm that the proliferative responses observed in PBMC cultures were specific for Nef, and were not due to a contaminant. Thus, proliferative responses to Nef and SEA were specifically blocked by a peptide corresponding to the region on Nef that binds to class II antigens and which blocks binding of Nef and the superantigen SEA.

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59 No Peptide SEA Nef Anti-IgM Anti-CD3 Mitogen Figure 15. Nef(123-160) peptide specifically blocks proliferation of PBMC induced by Nef and SEA. PBMC cultures were stimulated for 96 h with mitogens in the absence of peptides or in the presence of either Nef(123-160) peptide or Nef(157-186) peptide. Data are from a representative experiment, performed in triplicate, and are expressed as mean stimulation index ± SD. Nef and SEA were used at 300 ng/ml. Anti-IgM and anti-CD3 were used at 10 |ig/ml. Peptides were used at a final concentration of 100 jiM. The mean value for ^H-thymidine incorporation by unstimulated cultures was 1005±37 CPM.

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60 Induction of T Cell Cytokines bv Nef T cell activation by the staphylococcal superantigens results in the prodigious production of T cell cytokines, such as IL 2 and IFNy. Because of the significant T cell proliferation induced by Nef, it was determined if Nef induced Thelper cell cytokines. The results of a representative experiment are presented in Table VI. Consistent with proliferation data, Nef induced significant levels of IL 2, although IL 2 levels were lower than those induced by SEA. The question arose as to the ability of Nef to also induce another important T helper cell cytokine, IFNy. Samples were removed from cultures of cells over the course of 96 h and tested for IFN antiviral activity. Nef induced high levels of IFN activity, with peak production occurring by 96 h (Figure 16, Panel A). IFN levels were lower than those induced by SEA, but were similar than those induced by Con A. Purified T cells did not produce IFN upon stimulation with either Nef or SEA (Figure 16, Panel B). However, purified T cell cultures were capable of producing IFN upon Con A stimulation. No IFN activity was produced in cultures of APC (consisting of B cells) stimulated with either Nef, SEA, or Con A. Purified T cell cultures reconstituted with purified B cells for antigen presentation produced significant levels of IFN upon either Nef or SEA stimulation (Figure 16, Panel C). These results are consistent with T cell mitogen activity for both Nef and SEA. The type of IFN activity induced by Nef was determined by neutralization reactions with specific antisera. Treatment of Nef-induced IFN with anti-IFNy resulted in a significant reduction of activity (Table VII). Antisera to IFNa did not affect the IFN activity in these samples. Consistent with previous data (Langford et al., 1978a; Johnson et al., 1982), the IFN activity induced by SEA was IFNy. Similar to the results found with unfractionated PBMC, the IFN produced

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61 Table VI. IL 2 production induced by Nef. IL 2 (U/ml) produced at b : Mitogen 3 48 h 96 h Nef 14.7 ±0.1 16.9 ±1.1 SEA 17.2 ±0.7 24.7 ±1.8 None <3 <3 a Samples were obtained from PBMC cultures which had been stimulated with Nef and SEA at 300 ng/ml and 500 ng/ml, respectively. b IL 2 in culture samples were determined using IL 2-dependent HT-2 cells. The data presented are from a representative experiment performed in triplicate.

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62 PBMC + SEA PBMC + Nef PBMC + Con A PBMC + media 10000 T cells + SEA T cells + Nef T cells + Con A -• T cells + media 10000 10H T+APC+SEA T+APC+Nef T+APC+ConA T+APC+media Hours Figure 16. Kinetics of IFN production induced by Nef. Cultures of PBMC (Panel A), purified T cells (Panel B), and reconstituted cultures of purified APC and purified T cells (Panel C) were tested for IFN production at 24, 48, 72, and 96 h. Cultures were stimulated with either Nef at 100 ng/ml, SEA at 100 ng/ml, or Con A at 10 M-g/ml, or were left unstimulated. Cultures of purified APC produced <10 U/ml IFN at all timepoints.

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63 Table VII. IFNy is induced by Net. Samples from PBMC IFN titer (U/ml) after treatment with: cultures stimulated with: EMEM anti-IFNa anti-IFNy Nef 300 300 20 SEA 300 300 30 Con A 300 300 100 IFNa Control 300 <3 300 IFNy Control 30 30 <3

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64 by T cell cultures reconstituted with B cells in response to Nef was IFNy. Thus, Nef induced high levels of IFNy in cultures of PBMC, indicating that Nef activates T cells to produce the cytokines IL 2 and IFNy, lymphokines that are products of activated T helper 1 cells. PBMC cultured in the presence of Nef were tested for specific Vp expansion and anergy in vitro. No expansion of specific Vp populations was observed with the anti-Vp antibodies used (data not shown). Unlike pretreatment with TSST-1 , which anergized the response to anti-Vp2, Nef did not significantly anergize the responses of T cells to the tested anti-Vp antibodies (data not shown). Thus, no specific Vp expansion or anergy was detected using the available human Vp reagents. Discussion Nef protein induced significant levels of proliferation in unfractionated PBMC from a number of donors. The observed Nef responses were significant, with proliferation induced in cells from a large majority (85-90%) of the donors tested. It is unlikely that prior sensitization to Nef is responsible for the proliferative response, since these donors were negative for HIV. The following evidence points to Nef as the inducer of the observed proliferation: 1) proliferation was observed using recombinant Nef from three different expression systems; 2) antibodies to synthetic Nef peptides neutralized the proliferative activity of Nef; 3) the Nef fusion partner, MBP, did not induce proliferation; and 4) a synthetic peptide, Nef(123-160), previously shown to block binding of superantigens to MHC class II, blocked the Nef-induced proliferative responses. Nef induced proliferation was generally lower than that induced by SEA. This is not unusual in that SEA is extremely potent, causing proliferation at concentrations as low as 10" 16 M (Langford et al., 1978a).

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65 Proliferation induced by Nef occurred only in the presence of APC. Cultures of purified APC or purified T cells did not respond to Nef. T cells cultured in the presence of inactivated autologous APC proliferated to a similar extent to that seen in unfractionated cultures. These data suggest that Nef is presented to T cells by APC in an unprocessed form. The production of the T helper 1 lymphokines IL 2 and IFNy from Nef-induced cultures is evidence that Nef activates T helper 1 cells. In Chapter III, data was presented showing that Nef binds to Raji cells at a site(s) involved in SEA binding. A peptide corresponding to an internal region of Nef, Nef(123-160), specifically bound to DR 1 -transfected L cells and blocked proliferation induced by Nef and SEA, evidence that Nef binds to MHC class II antigens. Con A-induced proliferation was not affected by Nef(123160). This region of Nef was shown to be involved in binding of Nef to Raji cells. These functional data suggest that binding to MHC class II antigens is required for Nef proliferative activity. Binding of Nef to MHC class II occurs on APC, since Nef proliferative activity also requires presentation, but not processing, by APC. However, these data do not preclude Nef presentation by T cells expressing HLA-DR, a T cell activation marker. Superantigenic activity of Nef was not observed using the anti-Vp mAb currently available. Neither Vp-specific expansion or anergy were observed. Further, preliminary results using reverse transcriptase-polymerase chain reaction (RT-PCR), which quantifies the amount of mRNA produced, did not show a Vp preference in Nef-activated cells. These results raise a question on the nature of Nef receptor on T cells, and it is tempting to speculate on possible candidates such as TCR or CD4. Nef induces human B cells to differentiate into immunoglobulin secreting cells (Chirmule et al., 1994). HLA-DR and adhesion molecules seem to be

PAGE 75

66 involved in Nef-induced B cell differentiation, since monoclonal antibodies to these surface proteins abrogated Nef-induced responses. Consistent with the findings on T cell activation by Nef, B cell differentiation induced by Nef required T cells. In addition to activating T cells, staphylococcal superantigens have also been shown to induce B cell differentiation (Stohl et al., 1994). Thus, additional parallels can be drawn between the functional activities of staphylococcal superantigens and Nef. It has been shown that HIV requires activated T cells in which to replicate. Specific antiviral immune responses may not be sufficient to activate large numbers of T cells. For this reason, the identification of an HIV protein that induces T cell proliferation is of considerable interest and may, in part, explain the role of Nef in HIV pathogenesis. Activation of T cells may result from interaction with Nef, either in soluble form released from lysed infected cells or as a cell-associated complex with HLA-DR on the surface of infected T cells. T cell activation by Nef could result in a stable cellular reservoir for virus production as a result of continuous stimulation. In fact, hyperimmunization against Nef has been proposed as a means of reducing viral load, either prophylactically or therapeutically (Montagnier, 1995). The T cell expansion observed in response to Nef may not be the only mechanism of polyclonal activation of CD4+ T cells for HIV replication. Recent evidence points to a superantigen encoded by the human herpesvirus, cytomegalovirus (CMV), that expands Vpl2-bearing T cells, thereby enhancing HIV replication in CMV-infected individuals (Dobrescu et al., 1995). It is not surprising that no expansion of Vpl2-bearing T cells was observed in response to Nef, since the donors used in the studies discussed in this chapter were negative for CMV. This versatility in polyclonal CD4+ T cell expansion via the endogenous mitogen Nef and exogenous superantigens such as that of CMV

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67 probably play an important role in HIV pathogenesis. Clearly, the control of the mitogenic activity of these substances should help reduce the viral load in HIVinfected individuals.

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CHAPTER V HIV ENCODES FOR ITS OWN CD4 T CELL MITOGEN Introduction Nef protein is encoded in the genomes of both HIV-1 and the related primate virus simian immunodeficiency virus (SIV). The function that Nef plays in the pathogenesis of these viruses is uncertain, although its importance is reflected in studies in which a nef-deleted SIV mutant did not cause disease and protected against infection with the pathogenic wildtype strain (Daniel et al., 1992). Further, the viral load in SIV mutant-infected animals was considerably lower than in animals infected with the wildtype strain. However, challenge of neonatal macaques with the same attenuated SIV strain caused disease and, in some cases, death (Baba et al., 1995). The different outcomes of these two studies may involve differences in the immune system development between neonates and adults. In Chapter IV, data was presented on the ability of Nef protein to induce proliferation of human peripheral blood mononuclear cells (PBMC) from a wide sampling of HIV-negative donors. Proliferative responses were T cell specific and were accompanied by production of cytokines such as interleukin 2 (IL 2) and gamma interferon (IFNy), indicative of CD4 T cell activation. Nef-induced T cell proliferation and activation required the presence of antigen-presenting cells. These results are interesting in that T cell activation has been shown to be required for active HIV replication. Further, although resting or quiescent T cells can be infected, evidence suggests that HIV replication only occurs upon 68

PAGE 78

69 subsequent cellular activation. Herein, work is described that indicates that Net activation of T cells is sufficient for HIV replication. These results show that Net is an HIV-encoded mitogen that helps, at least in part, in establishing a cellular reservoir for virus replication. Materials and Methods PBMC Cultures for HIV Infectivitv Studies PBMC were isolated from peripheral blood of healthy adult donors as described in Chapter IV. PBMC were resuspended in RPMI 1640 medium containing 5% FBS and cultured in 25 cm 2 flasks (Sarstedt Inc., Newton, NC) at a concentration of 2 x 10 6 cells/ml in a final volume of 4 ml (or 8 x 10 6 cells/flask). Mitogens were added at the initiation of cultures. Nef, SEA, and PHA were used at 3 ng/ml, 0.1 |xg/ml, and 10 ug/ml, respectively. After four days, PBMC were washed extensively in PBS, resuspended in RPMI 1640 medium containing 10% FBS, 10' 6 M 2-mercaptoethanol, and 10 U rHulL 2/ml. IL 2 is rapidly depleted in these cultures, and thus rHulL 2 was added to medium. Cells were counted and all cultures were adjusted to 8 x 10 6 cells/flask. Cultures were infected with HIV at a final reverse transcriptase titer of 20,000 CPM/ml. Culture supernatants were harvested and cells were fed with fresh medium every third day for 12 days. PBMC Cultures for Proliferation Assays Concomitant with infectivity studies, PBMC were cultured in microtiter plates to monitor proliferation in response to Nef and mitogens. The culture conditions were the same as those described in Chapter IV. Nef and mitogens were used at the same concentrations as described above. Stimulation indices were calculated as described in Chapter IV.

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70 Reverse Transcriptase (RT) Assay Harvested culture supernatants (1 ml) were placed in 1.5 ml "Eppendorf" tubes and centrifuged at 17,000 rpm for 70 minutes. A cocktail containing Mg ++ as the divalent cation, poly(rA)oligo(dTi2-i8) as template primer, and 2.5 uCi of ( 3 H)TTP per sample was used to resupend the virus. Tubes were incubated at 37°C for 60 minutes, after which time samples were blotted onto filter paper discs. Discs were allowed to air-dry and were washed extensively in sequential baths. Specifically, discs were washed twice in 1 0% trichloroacetic acid (TCA) for 15 and 5 minutes, respectively. Discs were then placed in 5% TCA containing 0.1% SDS for 5 minutes. Finally, the discs were washed in 95% ethanol, after which they were allowed to air-dry. Discs were placed in vials containing scintillation fluid and radioactivity was quantified on a (3-scintillation counter. ELISA for HIV p24 Antigen Levels of p24 antigen in infected culture supernatants were assessed using a sandwich ELISA (DuPont, Boston, MA). Levels of p24 (in ng/ml) were quantified according to the Manufacturer's recommendations based on a standard curve using purified p24 antigen (provided by Manufacturer). Assay for Infectious Virus from PBMC Cultures Supernatants from PBMC cultures infected for 6 days with HIV were used to assess the titer of infectious virus produced in these cultures. Supernatants were adjusted to achieve RT titers of 12,000 CPM/ml and added to SEAstimulated PBMC cultures. Culture conditions were the same as those described above. RT activity at Day 9 of cultures was assessed as described above.

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71 Fluorescent Antibody Cell Sorter (FACS) Analysis of Nef-Activated Cells PBMC were stimulated for 4 days with either Nef (3ng/ml) or SEA (0.1 fxg/ml). PBMC were washed and resuspended in FACS buffer containing 0.5% BSA and 10 mM sodium azide. Cells were incubated with fluorescein-labeled mAb to HLA-DR for 45 minutes and washed with FACS buffer. Cells were then incubated with phycoerythrin-labeled mAbs to either CD4 or IL 2 receptor (IL 2R) for 45 minutes. Cells were washed and analyzed on a FACScan (BectonDickinson, Mountain View, CA) at 10,000 events/sample. All mAbs used were obtained from Becton-Dickinson, San Jose, CA. Studies on Proliferation of PBMC Induced by Autologous HIV-infected Cells PBMC cultures were stimulated as described above. PBMC were infected with HIV for 6 days, at which time the cells were washed and inactivated by overnight treatment with 2% paraformaldehyde. Inactivated HIVinfected cells were washed extensively to remove excess paraformaldehyde. Fresh autologous PBMC were cultured in microtiter plates in the presence of inactivated HIV-infected cells at a ratio of 3:1. After 4 days, 3 H-thymidine incorporation was assessed as described above. Anti-Nef Peptide Antibodies A mixture of antibodies to Nef(123-160) peptide and Nef(1 82-206) peptide were used to block proliferation in response to autologous HIV-infected cells. Antibodies were each used at a final dilution of 1:1000, as described in Chapter IV. Inactivated HIV-infected cells were treated with antibodies for 60 minutes prior to addition of fresh autologous PBMC. Preimmune sera were used and had no effect on proliferation induced by HIV-infected cells.

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72 Results HIV Replication in Nef-Stimulated PBMC Cultures Since T cell activation occurs with exogenous Net protein, the question arose as to whether Net stimulation of PBMC was sufficient to induce HIV-1 replication. To this end, PBMC of several HIV-negative donors were tested for proliferation induced by Nef and other T cell mitogens (Table VIII). Consistent with previous data, PBMC from most of the donors proliferated significantly in response to Nef protein, as well as to the T cell mitogens SEA and phytohemagglutinin A (PHA). Unstimulated and mitogen-stimulated PBMC were infected with HIV-1 and cultures were monitored over a 12-day period for signs of viral replication. Supernatants from unstimulated PBMC showed only marginal reverse transcriptase (RT) activity and p24 antigen production (Table VIII). RT and p24 antigen levels were high in cells stimulated with T cell mitogens, in particular SEA. Interestingly, Nef-stimulated cells produced moderate to high levels of RT and p24 antigen, indicating that these cells were capable of sustaining virus replication. In an attempt to quantify the number of infected cells in these cultures, PCR analysis of serially-diluted DNA was performed using nested primers for the gag gene. Results indicated 10to 30-fold increases in the number of cells harboring virus in Nef-stimulated cultures, as compared to unstimulated controls. These data indicate that Nef stimulation resulted in a large number of infected cells and a concomitant increase in viral load. Infectious Virus Is Produced by Nef-Stimulated Cultures As a means of determining whether the virus produced by Nef-stimulated was infective, supernatants from HIV-infected cultures were tested for reinfectivity. SEA-stimulated PBMC were infected with virus from 6-day

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74 CD _D 03 > C CD 03 C 03 DC E X 03 CD co 03 O c T3 o LL Q CO +1 c CO CD 3 00 CM CO +1 ct CM CM co +1 o o hco CM CM CO i — +1 CO CT i1CM CO co o CM +1 CO CO CO CO +1 ^3CM CO LO T1 CM CO ^ ct o CT CD CM CM CO +1 +1 +1 CT O co O LO o CO LO o CO +1 CD CD CD C o 03 o "o 1— 0. 03 E x CO CD CO CO CD 1— o c o Q CO +1 c CO CD CO o +1 o co ^3" d CM CM +1 o <^ o "3" Q Z CD co ico co o CT CT CD CO N +1 +1 +1 CT CO CO CO CO CO o CM CT oo i — CO Q Z co LO +1 CD co CM CM o o CO CM co +1 +1 <* CO CM CO o CM LO oo T — +1 o o c o 3 E co CD < < w X CO Q_ CD ^ C CD O -7 < LU CO < a. CD „_ O "7 < < co a. CD C o o c o Q O CM 00 o CM CT O CM

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75 supernatants of cultures stimulated with Nef, SEA, and PHA. All supernatants were adjusted to achieve equivalent RT units per culture. The results of two representative experiments are shown in Figure 17. Supernatants from Nefstimulated cultures had high titers of infectious virus similar to supernatants from cultures stimulated with SEA and PHA. These results indicate that virus replication in Nef-stimulated cultures, like the replication in SEAand PHAstimulated cultures, yielded infectious virus. Nef-Stimulated PBMC Express T Cell Activation Markers It has been shown that HIV requires activated T cells for virus replication. Cellular activation was investigated by double-staining FACS analysis of Nefstimulated cells using antibodies to CD4 and HLA-DR (Table IX). Increased levels of dually-stained cells were observed in cultures of PBMC stimulated with Nef protein, as compared to unstimulated cultures. HLA-DR expression on CD4+ human T cells has been shown to correlate with an activated T cell state. No increase was seen in cells that stained for CD8, either alone or in conjunction with HLA-DR. These results are consistent with our previous studies showing production of T helper cytokines upon stimulation with Nef. Thus, stimulation of PBMC with Nef protein induces activation of T helper cells. Expression of Nef by HIV-infected Cells Culture supernatants of infected cells were tested for soluble Nef and none could be detected, suggesting that release of soluble Nef may not be occurring in these cultures. However, it has been shown that Nef is expressed on the surface of infected cells (Fujii et al., 1993). This leads to the question of whether Nef, in association with the extracellular membrane of infected cells, plays a role in the activation event(s) required for continuous HIV replication.

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76 40000 30000 a. U 2000010000Exptl Expt2 Figure 17. Infectivity assay on supernatants from Nef-stimulated HIV-infected cultures.

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77 Table IX Nef-induced activation of T cells as assessed by FACS % Total of dually-stained populations Cultures from: CD4 + HLA-DR CD4 + IL 2 R Donor 206 Unstimulated 3.17 N.D. C Nef-stimulated 7.39 N.D. SEA-stimulated 29.46 N.D. Donor 213 Unstimulated 6.65 N.D. Nef-stimulated 16.74 N.D. Donor 214 Unstimulated 3.45 N.D. Nef-stimulated 11.15 N.D. Donor 215 Unstimulated N.D. 7.75 Nef-stimulated N.D. 10.55 Donor 216 Unstimulated Nef-stimulated 1.52 9.02 4.66 7.68

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78 Thus, we investigated the possibility that HIV-infected cells could activate autologous PBMC, similar to the activation seen with exogenously added Net. To achieve maximal infection, PBMC were stimulated with mitogen prior to infection with HIV. After 5 days, HIV-infected cells were washed and inactivated with paraformaldehyde. Fresh autologous PBMC were cultured in the presence of infected cells and monitored for proliferation. Proliferation was observed in co-cultures of fresh and infected autologous cells (Table X). Uninfected stimulated cells served as controls, and no proliferation was observed in these co-cultures. Proliferative responses were significantly reduced by the addition of polyclonal anti-Nef antibodies, while control anti-SEA antibodies were without effect. Anti-Nef inhibition of proliferation was dose dependent, with lower concentrations of antibodies having less inhibitory activity. Similar to the results observed on HIV-infected cells, anti-Nef antibodies were capable of reducing proliferation in response to Nef, but not to SEA. Thus, a significant portion of the observed mitogenic activity of HIV-infected cells is due to Nef protein. Discussion It is interesting that HIV-infected cells were more potent at inducing T cell proliferation than was soluble Nef. Given that Nef interacts with MHC class II antigens, it is conceivable that optimal conditions for Nef association with MHC are achieved in HIV-infected cells, analogous to the expression of the mouse mammary tumor virus superantigen in infected B cells. These data suggest that proliferation occurred as a result of Nef expressed on the surface of HIVinfected cells, thereby indicating that Nef is a virally-encoded mitogen. Thus, the genome of HIV encodes for its own T cell mitogen, which would amplify the

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79 Table X. Ability of anti-Net peptide antibodies to block proliferation induced by autologous HIV-infected cells Fresh PBMC cultured Anti-Nef Proliferation in the presence of: antibodies CPM Fold increase p value (Mean ± SD) HIV-infected PBMC 137187+ 1923 162.2 HIV-infected PBMC + 79740 ± 8764 94.3 <0.003 + 1973± 880 2.3 846 ± 271

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80 replication of virus in the host. As such, Net could be classified as a virulence factor for HIV. It has been demonstrated that activated T cells are required for replication of HIV-1 . On the other hand, the ability of HIV-1 to infect quiescent or resting cells is still questioned. In two studies using quiescent cells, HIV-1 replication in vitro was blocked at the level of virus entry (Gowda et al., 1989; Tang and Levy, 1990). Others have shown that blockage of replication occurred at an intracellular event, possibly at reverse transcription (Zack et al., 1990) or proviral integration (Stevenson et al., 1990; Bukrinsky et al., 1991). Upon entry into quiescent cells, replication could be induced by subsequent T cell activation. Consistent with data from in vitro studies are reports that high numbers of quiescent CD4+ cells from asymptomatic HIV-infected patients harbor unintegrated viral DNA capable of integration subsequent to PHA stimulation. Further, AIDS patients have increased numbers of cells containing integrated virus, corresponding with greater production of virus at this stage of the disease. These findings indicate that HIV-1 may infect quiescent cells, but such infection is nonproductive until cell activation occurs. Studies suggest that Nef plays an important role in HIV pathogenesis. Infection of adult rhesus monkey with a nef-deleted mutant of SIVmac239 resulted in low viral burden and did not cause disease, although infected animals were persistently seropositive (Daniel et al., 1992). Recently, genomic analysis of an HIV strain from a longterm survivor revealed a 1 18 bp deletion in the nef gene which resulted in an out-of-frame shift for downstream sequences (Kirchhoff et al., 1995). These studies hint at the importance of Nef in HIV pathogenesis in that repeated attempts to isolate virus from this patient with nonprogressive disease were unsuccessful. Mutations in nef may result in altered mitogenic activity of the protein, with concomitant reduction or loss of the

PAGE 90

81 ability to activate T cells. Possible consequences of net mutations, then, would be a reduction both in the cellular reservoir for virus replication and in the viral load in the infected host. Other investigators have not found alterations in the nef gene in strains isolated from other longterm survivors (Cao et al., 1 ?95). It would be interesting to determine the T cell reactivity of these individuals to Nef, as we have found that 10-15% of individuals that we tested did not respond to Nef. It is possible that nonresponsiveness to Nef may play an important role in longterm survival in some HIV-infected individuals. Thus, Nef may aid in the establishment and maintenance of infection, suggesting its role as a virulence factor for HIV.

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CHAPTER VI A MODEL FOR THE ROLE OF NEF IN THE PATHOGENESIS OF HIV As shown in Chapters ll-V, Nef binds to MHC class II antigens at a site(s) involved in bacterial superantigen binding. Like MMTV superantigen, Nef protein is encoded in the 3' long terminal repeat of the genome of lentiviruses such as HIV-1 and simian immunodeficiency virus (SIV). Nef induced significant levels of proliferation in PBMC from a wide sampling of donors (8590%), although these responses are lower than those induced by SEA. Further, Nef stimulation resulted in the production of the T helper cell cytokines, IL 2 and interferon gamma (IFNy). Proliferation in response to Nef was observed in reconstituted cultures consisting of T cells and inactivated APCs, which is compelling evidence for Nef superantigen activity. Further, Nef did not induce purified T cells to proliferate in the absence of APCs, which is consistent with Nef superantigen activity. However, no apparent Vp expansions were found, although such data do not preclude the Vp specificity of Nef activation. Thus, Nef has superantigen-like characteristics in that it binds to class II antigens, does not require processing by APC, and activates T cells to proliferate and release cytokines such as IL 2 and IFNy. The functional role that Nef plays in the lentiviral pathogenesis is uncertain, although its importance is reflected in vaccine studies in which a nefdeleted SIV mutant did not cause disease and protected animals upon challenge with the pathogenic wildtype strain (Daniel et al. 1992). The viral load in SIV mutant-infected animals was considerably lower than in animals challenged with wildtype SIV. Within this context, the ability of Nef to activate T 82

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83 cells is interesting in that active HIV replication requires T cell activation. Further, although resting or quiescent T cells can be infected, evidence suggests that HIV replication only occurs upon subsequent cellular activation. Net stimulation of PBMC was sufficient to induce HIV-1 replication. Virus replication in Nef-stimulated cultures yielded infectious virus, rather than defective particles. Flow cytometric analysis showed that increased percentages of cells that stained for both CD4 and HLA-DR were present in Nef-stimulated cultures. Further, increased percentages of cells that stained for both CD4 and IL 2R were observed in Nef-stimulated cultures. These results are consistent with the production of T helper cytokines upon stimulation with Nef. Thus, stimulation of PBMC with Nef protein induces T cell activation and such activation is sufficient for HIV replication. Proliferation of fresh autologous PBMC cultured in the presence of infected cells was observed. Further, proliferative responses were significantly reduced by the addition of polyclonal anti-Nef antibodies. These data suggest that proliferation occurred as a result of Nef present on the surface of HIVinfected cells, thereby indicating that Nef may be a virally-encoded mitogen. Nef has been shown to induce differentiation of human B cells to immunoglobulin secreting cells (Chirmule et al., 1994). Monoclonal antibodies to HLA-DR and adhesion molecules abrogated Nef-induced differentiation. B cell stimulation required T cells and monocytes, the latter producing IL 6 upon Nef stimulation. Interestingly, staphylococcal superantigens have been shown to induce B cell differentiation analogous to that described for Nef (Stohl et al., 1994). Thus, Nef can stimulate both T and B cells in a manner similar to the staphylococcal enterotoxin superantigens. This leads to the question of the activation event(s) that occurs during HIV-1 exposure and that ultimately lead to the establishment of infection. Our

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84 results suggest a model in which Nef acts as a HIV-encoded T cell mitogen (Figure 18). Nef may interact with T cells either as an integral part of the membranes of infected T cells or in a soluble form released as the result of lysis of infected cells. Nef is present on the membranes of infected T cells (Fujii et al., 1993), which is consistent with this model. Nef expression may occur in the context of the T cell activation marker, HLA-DR, and the Nef/HLA-DR complex would stimulate uninfected T cells, as well as B cells. Such stimulation would allow for the expansion of a cellular reservoir for replication of the virus, eventually leading to T cell anergy and/or apoptosis. B cell activation and differentiation could ultimately lead to hypergammaglobulinemia. Thus, the HIV genome may encode for its own T cell mitogen, which would induce the amplification of virus replication in the host, with ultimate deleterious effects on the immune system. As such, Nef could be classified as a virulence factor for HIV.

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85 l Nef binding to HLA-DR Presentation to uninfected T cells Cellular proliferation T cell activation Cytokine production B cell proliferation and differentiation I Increased viral load Immunosuppression via T cell anergy/apoptosis Autoimmunity via activation of B and T cells Hypergammaglobulinemia Figure 18. Model for the role of Nef in the pathogenesis of HIV. Activation of uninfected T cells occurs by Nef interaction in either of two forms: as soluble Nef released from lysed cells, presented in the context of MHC class II antigens on APC, or as an integral part of the membranes of infected T cells, complexed to HLA-DR. Binding to uninfected T cells may occur via TCR or CD4. T cell activation results in proliferation and release of cytokines such as IFNy and IL 2, thereby creating a cellular reservoir for virus and increasing viral load in the host. T cell activation results in depletion of T cells via virus production, anergy and/or apoptosis. B cell differentiation, possibly mediated by T cell cytokine release, results in hypergammaglobulinemia. Autoimmune-like sequelae may result from B cell differentiation into Ig-secreting cells, and activation of T cells. Nef-induced cellular effects Potential Nef-related pathogenesis

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LIST OF REFERENCES Abe, J., B. L. Kotzin, K. Jujo, M. E. Melish, M. P. Glode, T. Kohsaka, and D. Y. M. Leung. 1992. Selective expansion of T cells expressing T-cell receptor variable region Vp2 and V(38 in Kawasaki disease. Proc. Natl. Acad. Sci. USA 89:4066. Acha-Orbea, H. 1992. Retroviral superantigens. In B. Fleischer, Ed. Ciological Significance of Superantigens. Basel: Karger, pp65-86. Acha-Orbea, H. and E. Palmer. 1991. Mis a retrovirus exploits the immune system. Immunol. Today 12:356. Acha-Orbea, H., A. N. Shakhov, L. Scarpellino, E. Kolb, V. Muller, A. Vessaz-Shaw, R. Fuchs, K. Blochinger, P. Rollini, J. Billotte, M. Sarafidou, H. R. MacDonald, and H. Diggelmann. 1991. Clonal deletion of Vp14-bearing T cells in mice transgenic for mammary tumor viurs. Nature 350:207. Achong, B. G., P. W. A. Mansell, M. A. Epstein, and P. Clifford. 1971. An unusual virus in cultures from a human nasopharyngeal carcinoma. J. Nat. Cancer Inst. 46:299. Allan, J. S., J. E. Coligan, T. -H. Lee, M. F. McLane, P. J. Kanki, J. E. Groopman, and M. Essex. 1985. A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients. Science 230:810. Arthur, L. O., J. W. Bess, R. C. Sowder, R. E. Benveniste, D. L. Mann, J. C. Chermann, and L. E. Henderson. 1992. Cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines. Science 258:1935. Baba, T. W., Y. S. Jeong, D. Penninck, R. Bronson, M. F. Greene, and R. M. Ruprecht. 1995. Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science 267:1820. Bahraoui, E., M. Yagello, J. -N. Billaud, J. -M. Sabatier, B. Guy, E. Muchmore, M. Girard, and J. -C. Gluckman. 1990. Immunogenicity of the human immunodeficiency virus (HIV) recombinant nef gene product. Mapping of T-cell and B-cell epitopes in immunized chimpanzees. AIDS Res. Hum. Retroviruses 6:1087. Barnstable, C. J., W. F. Bodmer, G. Brown, G. Galfre, C. Milstein, A. F. Williams, and A. Ziegler. 1978. Production of monoclonal antibodies to group A erythrocytes, HLA, and other human cell surface antigens new tools for genetic analysis. Cell 14:9. 86

PAGE 96

87 Bergdoll, M. S. 1985. The staphylococcal enterotoxins-an update. In J. Jeljaszewics, Ed. The Staphylococci. New York: Gustav Fisher Verlag, pp247266. Bisset, L. R., M. Opravil, E. Ludwig, and W. Fierz. 1993. T cell response to staphylococcal superantigens by asymptomatic HIV-infected individuals exhibits selective changes in T cell receptor V beta-chain usage. AIDS Res. Hum. Retroviruses 9:241 . Brodsky, F.M. 1984. A matrix approach to human class II histocompatibility antigens: reactions of four monoclonal antibodies with the products of nine haplotypes. Immunogenetics 19:179. Buhrinsky, M. I., T. L. Stanwick, M. P. Dempsey, and M. Stevenson. 1991. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection. Science 254:423. Cao, Y., L. Qin, L. Zhang, J. Safrit, and D. D. Ho. 1995. Virological and immunological characterization of long-term survivors of huma immunodeficiency virus type 1 infection. New Engl. J. Med. 332:233. Carlsson, R., H. Fischer, and H. O. Sjogren. 1988. Binding of staphylococcal enterotoxin A to accessory cells is a requirement for its ability to activate human T cells. J. Immunol. 140:2484. Chinatgumpala, M. M., J. A. Mollick, and R. R. Rich. 1991. Staphylococcal toxins bind to different sites on HLA-DR. J. Immunol. 147:3876. Chirmule, N., N. Oyaizu, C. Saxinger, and S. Pahwa. 1994. Nef protein of HIV-1 has B-cell stimulatory activity. AIDS 8:733. Choi, Y., J. Kappler, and P. Marrack. 1991. A superantigen encoded in the open reading frame of the 3' long terminal repeat of mouse mammary tumor virus. Nature 350:203. Choi, Y., J. Kappler, and P. Marrack. 1992. Structural analysis of a mouse mammary tumor virus superantigen. J. Exp. Med. 175:847. Cole, B. C. and M. M. Griffiths. 1993. Triggering and exacerbation of autoimmune arthritis by the Mycoplasma arthriditis superantigen MAM. Arth. Rheum. 36:994. Dalgleish, A. G., S. Wilson, M. Gompels, C. Ludlam, B. Gazzard, A. M. Coates, and J. Habeshaw. 1992. T-cell receptor variable gene products and early HIV-1 infection. Lancet 339:824. Daniel, M. D., F. Kirchhoff, S. C. Czajak, K. Sehgal, and R. C. Desrosiers. 1992. Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science 258:1938.

PAGE 97

88 Dellabonna, P. J. Peccoud, J. Kappler, P. Marrack, C. Benoit, and D. Mathis. 1990. Superantigens interact with MHC class II molecules outside of the antigen binding groove. Cell 62:1 115. deRonde, A., B. Klaver, W. Keulen, L. Smit, and J. Goudsmit. 1992. Natural HIV-1 Net accelerates virus replication in primary human lymphocytes. Virology 188:391. Devereax, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucl. Acid Res. 12:387. Dobrescu, D., B. Ursea, M. Pope, A.S. Asch, and D.N. Posnett. 1995. Enhanced HIV-1 replication in Vp12 due to human cytomegalovirus in monocytes: evidence for a putative herpesvirus superantigen. Cell 82:753. Dyson, P. J., A. M. Knight, S. Fairchild, E. Simpson, and K. Tomonari. 1991. Genes encoding ligands for deletion of Vp11 T cells cosegregate with mammary tumor virus genomes. Nature 349:531. Edelman, A. S. and S. Zolla-Pazner. 1989. AIDS: a syndrome of immune dysregulation, dysfunction, and deficiency. FASEB J. 3:22. Festenstein, H. 1973. Immunogenetic and biological aspects of in vitro lymphocyte allotransformation (MLR) in the mouse. Transplant. Rev. 1 5:62. Fischer, H., M. Dohlsten, M. Lindvall, H. 0. Sjogren, and R. Carlsson. 1989. Binding of staphylococcal enterotoxin A to HLA-DR on B cell lines. J. Immunol. 142:3151. Fleischer, B. and H. Schrezenmeier. 1988. T cell stimulation by staphylococcal enterotoxins: Clonally variable response and requirement for major histocompatibility complex class II molecules on accessory or target cells. J. Exp. Med. 167:1697. Fluegel, R. M. 1993. The molecular biology of the human spumavirus. In B. R. Cullen, Ed. Human Retroviruses. Oxford: IRL Press, pp193-214. Franchini, G., M. Robert-Guroff, J. Ghrayeb, N. T. Chang, and F. WongStaal. 1986. Cytoplasmic localization of the HTLV-III 3' orf protein in cultured T cells. Virology 155:593. Frankel, W. N., C. Rudy, J. M. Coffin, and B. T. Huber. 1991. Linkage of Mis genes to endogenous mammary tumor virus. Nature 349:525. Fraser, J.D. 1989. High-affinity binding of staphylococcal enterotoxins A and B to HLA-DR. Nature 339:221 .

PAGE 98

89 Fujii, Y., Y. Nishino, T. Nakaya, K. Tokunaga, and K. Ikuta. 1993. Expression of human immunodeficiency virus type 1 Nef antigen on the surface of acutely and persistently infected human T cells. Vaccine 1 1 :1240. Gallo, R.C., Salahuddin, S.Z., M. Popovic, G. M. Shearer, M. Kaplan, B. F. Haynes, T. J. Palker, R. Redfield, J. Oleske, and B. Safai. 1984. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224:500. Garcia, J. V. and A. D. Miller. 1991. Serine-phosphorylationindependent downregulation of cell-surface CD4 by nef. Nature 350:508. Golovkina, T. V., A. Chervonsky, J. P. Dudley, and S. R. Ross. 1992. Transgenic mouse mammary tumor virus superantigen prevents viral infection. Cell 69:637. Gowda, S. D., B. S. Stein, N. Mohagheghpour, C. J. Benike, and E. G. Engleman. 1989. Evidence that T cell activation is required for HIV-1 entry in CD4+ lymphocytes. J. Immunol. 142:773. Griggs, N. D., C. H. Pontzer, M. A. Jarpe, and H. M. Johnson. 1992, Mapping of multiple binding domains of the superantigen staphylococcal enterotoxin A for HLA. J. Immunol. 148:2516. Guy, B., M. P. Kierny, Y. Riviere, C. Le Peuch, K. Dott, M. Girard, L. Montagnier, and J. --P. Lecocq. 1987. HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature 330:266. Hammes, S. R., E. P. Dixon, M. H. Malim, B. R. Cullen, and W. C. Greene. 1989. Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor. Proc. Natl. Acad. Sci. USA 86:9549. Held, W., H. Acha-Orbea, H. R. MacDonald, and G. A. Waanders. 1994a. Superantigens and retroviral infections: insights from mouse mammary tumor virus. Immunol. Today 15:184. Held, W„ G. A. Waanders, H. R. MacDonald, and H. Acha-Orbea. 1994b. MHC class II hierarchy of superantigen presentation predicts efficiency of infection with mouse mammary tumor virus. Int. Immunol. 6:1403. Held, W., G. A. Waanders, A. N. Shakhov, L. Scarpellino, H. AchaOrbea, and H. R. MacDonald. 1993. Superantigen-induced immune stimulation amplifies mouse mammary tumor virus infection and allows transmission. Cell 74:529. Herman, A., N. Labrecque, J. Thibodeau, P. Marrack, and R. R. Rich. 1991. Identification of the staphylococcal enterotoxin A superantigen binding site in the beta 1 domain of the human histocompatibility antigen HLA-DR. Proc. Natl. Acad. Sci. USA 88:9954.

PAGE 99

90 Heston, W. E., M. K. Deringer, and H. B. Andervont. 1945. Gene-milk agent relationship in mammary tumor development. J. Natl. Cancer Inst. 5:289. Ho, S. N., R. T. Abraham, S. Gillis, and D. J. Mckean. 1987. Differential bioassay of interleukin 2 and interleukin 4. J. Immunol. Methods. 98:99. Hugin, A. W., M. S. Vacchio, and H. C. Morse III. 1991. A virus-encoded "superantigen" in a retrovirus-induced immunodefieciency syndrome of mice. Science 252:424. Imberti, L, A. Sottini, A. Bettinardi, M. Puoti, and D. Primi. 1991. Selective depletion in HIV infection of T cells that bear specific T cell receptor Vp sequences. Science 254:860. Johnson, H. M. 1981. Cellular regulation of immune interferon production. Antiviral Res. 1 :37. Johnson, H. M. 1985 mechanism of Interferon-y production and assessment of immunoregulatory properties. In E. Pick and M. Landy, Eds. Lymphokines Vol. 1 1 . New York: Academic Press, pp 33-53. Johnson, H. M. and J. A. Bukovic. 1975. Staphylococcal entertoxin A inhibition of the primary in vitro antibody response to thymus-dependent antigen. IRCS Med. Sci. 3:398. Johnson, H. M., M. P. Langford, B. Lakhchaura, T. -S. Chan, and G. J. Stanton. 1982. Neutralization of native human gamma interferon (HulFNy) bt antibodies to a synthetic peptide encoded by the 5' end of HulFNy cDNA. J. Immunol. 129:2357. Johnson, H. M. and H. I. Magazine. 1988. Potent mitogenic activity of staphylococcal enterotoxin A requires induction of interleukin 2. Int. Arch. Allergy Appl. Immunol. 87:87. Johnson, H. M., J. K. Russell, and C. H. Pontzer. 1991. Staphylococcal enterotoxin superantigens. P.S.E.B.M. 198:765 Johnson, H. M., J. K. Russell, and C. H. Pontzer. 1992. Role of superantigens in human disease. Sci. Am. 266(4):92. Johnson, H. M., B. A. Torres, and J. M. Soos. 1995. Superantigens : Structure and Relevance to Human Disease. Proc. Soc. Exp. Biol. Med. In Press. Kappler, J., B. Kotzin, L. Herron, E. W. Gelfand, R. D. Bigler, A. Boylston, S. Carrel, D. N. Posnett, Y. Choi, and P. Marrack. 1989. Vp-specific stimulation of human T cells by staphylococcal toxins. Science 244:81 1 .

PAGE 100

91 Kawabe.Y. and A. Ochi. 1990. Selective anergy of Vp8+ T cells in staphylococcus enterotoxin B-primed mice. J. Exp. Med. 172:1065. Kawabe.Y. and A. Ochi. 1991. Programmed cell death and extrathymic reduction of Vp8+ CD4+ T cells in mice tolerant to Staphylococcus aureus enterotoxin B. Nature 349:245. Kirchhoff, F., T. C. Greenough, D. B. Brettler, J. L. Sullivan, and R. C. Desrosiers. 1995. Absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. New Engl. J. Med. 332:228. Klatzmann, D., F. Barre-Sinoussi, M. T. Nugeyre, C. Danquet, E. Vilmer, C. Griscelli, F. Brun-Veziret, C. Rouzioux, J. C. Gluckman, J. C. Chermann, and L. Montagnier. 1984. Selective tropism of lymphadenopathy associated virus (LAV) for helper-inducer T lymphocytes. Science 225:59. Kotzin, B. U, D. Y. M. Leung, J. Kappler, and P. Marrack. 1993. Superantigens and their potential role in human disease. Adv. Immunol, 54:99. Lafon, M., M. Lafage, A. Martinez-Arends, R. Ramirez, F. Vuiller, D. Charron, V. Lotteau, and D. Scott-Algara. 1992. Edidence for a viral superantigen in humans. Nature 358:507. Lafon, M., D. Scott-Algara, P. N. Marche, P. A Cazenave. and E. JouvinMarche. 1994. Neonatal deletion and selctive expansion of mouse T cells by exposure to rabies virus nuceocapsid superantigen. J. Exp. Med. 180:1207. Langford, M. P., G. J. Stanton, and H. M. Johnson. 1978a. Biological effects of staphylococcal enterotoxin A on human peripheral lymphocytes. Infect. Immun. 22:62. Langford, M. P., D. A. Weigent, G. J. Stanton, and S. Baron. 1978b. Virus plaque-reduction assay for interferon. Microplaqye and regular macroplaque reduction assays. Methods Enzymol. 78:339. Laurence, J., A. S. Hodstev, and D. N. Posnett. 1992. Superantigen implicated in dependence of HIV-1 replication in T cells on TCR Vp expression. Nature 358:255. Lechler, R. I., V. Bal, J. B. Rothbard, R. N. Germain, R. Sekaly, E. O. Long, and J. Lamb. 1988. Structural and functional studies of HLA-DR restricted antigen recognition by human helper T lymphocyte clones by using transfected murine cell lines. J. Immuno. 141:3003. Lee, J. M. and T. H. Watts. 1990. Binding of staphylococcal enterotoxin A to purified murine MHC class II molecules in supported lipid bilayers. J. Immunol. 145:3360.

PAGE 101

92 Lemaitre, M., D. Guetard, Y. henin, L. Montagnier, and A. Zerial. 1990. Protective activity of tetracycline analogs against the cytopathic effect of human immunodeficiency viruses in CEM cells. Res. Virol. 141:5. Lewis, H. M., B. S. Baker, S. Bokth, A. V. Powles, J. J. Garioch, H. Valdimarsson, and L. Fry. 1993. Restricted T cell receptor Vp gene usage in the skin of patients with guttate and chronic plaque psoriasis. Brit. J. Dermatol. 129:514. Lifson, J. D., M. B. Feinberg, G. R. Reyes, L. Rabin, B. Banapour, S. Chakrabati, B. Moss, F. Wong-Staal, K. S. Steimer, and E. G. Engelman. 1986. Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope glycoprotein. Nature 323:725. MacDonald, H. R., A. L. Glasebrook, R. Schneider, R. K. Lees, H. P. Pircher, T. Pedrazzini, O. Kanagawa, J.-F. Nicholas, R. C. Howe, R. M. Zinkernagel, and H. Hentgartner. 1989. T-cell reactivity and tolerance to Mls a encoded antigens. Immunol. Rev. 107:89. Mariani, R. and J. Skowronski. 1993. CD4 down-regulation by nef alleles isolated from human immunodeficiency virus type 1 -infected individuals. Proc. Natl. Acad. Sci. USA 90:5549. Marrack, P. and J. Kappler. 1990. The staphylococcal enterotoxins and their relatives. Science 248:705. Marrack, P., E. Kushnir, and J. Kappler. 1991. A maternally inherited superantigen encoded by a mouse mammary tumor virus. Nature 349:524. Merryman, P., J. Silver, P. K. Gregeersen, G. Solomon, and R. Winchester. 1989. A novel association of DQ alpha and DQ beta genes in the DRw10 haplotype. J. Immunol. 143:2068. Montagnier, L. 1995 Nef vaccination against HIV disease. Lancet 346:1170. Mollick, J. A., R. G. Cook, and R. R. Rich. 1989. Class II molecules are specific receptors for staphylococcal enterotoxin A. Science 244:817. Mottershead, D. G., P. -N. Hsu, R. G. Urban, J. L. Strominger, and B. T. Huber. 1995. Direct binding of Mtv7 superantigen (Mls-1) to soluble MHC class II molecules. Immunity 2:149. Ohmen, J. D., P. F. Barnes, C. L. Grisso, B. R. Bloom, and R. L. Modlin. 1994. Evidence for a superantigen in human tuberculosis. Immunity 1:35. Oroszlan, S. and R. B. Luftig. 1990. Retroviral proteinases. Curr. Top. Microbiol. Immunol. 157:153.

PAGE 102

93 Ozato, K, N. M. Mayer, and D. H. Sachs. 1982. Monoclonal antibodies to mouse major histocompatibility complex antigens. Transplantation 34:1 13. Parker, J. M. R., D. Guo, and R. S. Hodges. 1986. New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-rayderived accessible sites. Biochem. 25:5425. Peavy, D. L, W. H. Adler, and R. T. Smith. 1970. The mitogenic effects of endotoxin and staphylococcal enterotoxin B on mouse spleen cells and human peripheral lymphocytes. J. Immunol. 105:1453. Poiesz, B. J., F. W. Ruscetti, A. F. Gazdar, P. A. Bunn, J. D. Minna, and R. C. Gallo. 1980. Detection and isolation of type C retrovirus particles from fresh cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. natl. Acad. Sci. USA 77:7415. Pontzer, C. H., N. D. Griggs, and H. M. Johnson. 1993. Agonist properties of a microbial superantigen peptide. J. Immunol. 193:191. Pontzer, C. H., M. J. Irwin, N. R. J. Gascoigne, and H. M. Johnson. 1992. T cell antigen receptor binding sites for the microbial superantigen staphylococcal enterotoxin A. Proc Natl. Acad. Sci. USA 89:7727. Pontzer C. H., J. K. Russell, and H. M. Johnson HM. 1991. Site of nonrestrictive binding of SEA to class II MHC antigens. Int. Arch. Allergy Appl. Immunol. 93:107. Pontzer, C. H., J. K. Russell, and H. M. Johnson. 1990. Site of nonrestrictive binding of SEA to class II MHC antigens. Int. Arch. Allergy Appl. Immunol. 93:107. Pullen, A. M., Y. Choi, E. Kushnir, J. Kappler, and P. Marrack. 1992. The open reading frames inthe 3' long terminal repeats of several mouse mammary tumor virus integrants encode Vp3-specific superantigens. J. Exp. Med. 175:41. Rebai, N, G. Pantaleo, J. F. Demarest, C. Ciurli, H. Soudeyns, J. W. Adelsberger, M. Vaccarezza, R. E. Walker, R. P. Sekaly, and A. S. Fauci. 1994. Analysis of the T-cell receptor beta-chain variable-region (Vp) repertoire in monozygotic twins discordant for human immunodeficiency virus: evidence for perturbations of specific V beta segments in CD4+ T cells of the virus-positive twins. Proc. Natl. Acad. Sci. USA 91:1529. Rellahan, B. L, L. A. Jones, A. M. Kruisbeek, A. M. Fry, and L. A. Mathis. 1990. In vivo induction of anergy in peripheral Vp8+ T cells by staphylococcal enterotoxin B. J. Exp. Med. 172:1091. Ringold, G.M. 1983. Regulation of mouse mammary tumor virus gene expression by glucocorticoid hormones. Curr. Top. Microbiol. Immunol. 106:79.

PAGE 103

94 Robbins, P. A., E. L. Evans, A. H. Ding, N. L. Warner, and F. M. Brodsky. 1987. Monoclonal antibodies that distinguish between class II antigens (HLADP, DQ, and DR) in 14 haplotypes. Hum. Immunol. 18:301. Rous, P. 1910. A transmissible avian neoplasm (sarcoma of common fowl). J. Exp. Med. 12:696. Rous, P. 191 1 . Transmission of a malignant new growth by means of a cell-free filtrate. J. Amer. Med. Assoc. 56:198. Russell, J. K., C. H. Pontzer, and H. M. Johnson. 1990. The l-Ap b region (65-85) is a binding site for the superantigen, staphylococcal entertoxin A. Biochem. Biophys. Res. Comm. 168:696. Russell, J. K., C. H. Pontzer, and H. M. Johnson. 1991. Botha-helices along the major histocompatibility complex binding cleft are required for staphylococcal enterotoxin A function. Proc. Natl. Acad. Sci. USA 88:7228. Salmons, B. and W. H. Gunzburg. 1987. Current perspectives in the biology of mouse mammary tumor virus. Virus Res. 8:81 . Schiffenbauer, J., H. M. Johnson, E. J. Butfilowski, L. Wegrzyn, and J. M. Soos. 1993. Staphylococcal enterotoxins can reactivate experimental allergic encephalomyelitis. Proc. Natl. Acad. Sci. USA 90:8543. Sodrowski, J., W. C. Goh, C. Rosen, K. Campbell, and W. A. Haseltine. 1986. Role of the HTLV-III/LAV envelope in synctium formation and cytopathicity. Nature 322:470. Soos, J. M. and H. M. Johnson. 1994. Type I interferon inhibition of superantigen stimulation: implications for treatment of superantigen-associated disease. J. Interferon and Cytokine Res. 15:39. Spawski, J. B. and P. E. Lipsky. 1992. Isolation of B cell populations. In J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, Eds. Protocols in Immunology. New York: John Wiley and Sons, p7.5.1. Stevenson, M., S. Haggerty, C. Lamonica, A. M. Mann, C. Meier, and A. Wasiak. 1990. Cloning and characterization of human immunodeficiency virus type 1 variants diminished in the ability to induce syncytium-independent cytloysis. J. Virol. 64:3792. Stohl, W., J. E. Elliott, and P. S. Linsley. 1994. Human T-cell dependent B cell differentiation induced by staphylococcal superantigens. J. Immunol. 153:117. Tang, S. and J. A. Levy. 1990. Parameters involved in the cell fusion induced by HIV. AIDS 4:409.

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95 Terwillegar, E. F., J. G. Sodroski, and W. A. Haseltine. 1990. Mechanisms of infectivity and replication of HIV-1 and implications for therapy. Ann. Emer. Med. 19:233. Terwillegar, E. F., J. G. Sodroski, C. A. Rosen, and W. A. Haseltine. 1986. Effects of mutations within the 3' orf open reading frame region of human T-cell lymphotropic virus type III on replication and cytopathogenicity. J. Virol. 60:754. Torres, B. A., J. K. Yamamoto, and H. M. Johnson. 1982. Cellular regulation of immune interferon (IFNy) production: Lyt phenotype of the suppressor cell. Infect. Immun. 35:770. Tsubura, A., M. Inabe, S. Imai, A. Murakami, N. Oyaizu, R. Yasumizu, Y. Ohnishi, H. Tanaka, S. Morii, and S. Ikehara. 1988. Intervention of T-cells in transportation of mouse mammary tumor virus (milk factor) to mammary gland cells In vivo. Cancer Res. 48:6555. Uchiyama, T., T. Miyoshi-Akiyama, H. Kato, W. Fujimaki, K. Imanishi, and X. Yan. 1993. Superantigenic properties of a novel mitogenic substance produced by Yersinia pseudotuberculosis isolated from patients manifesting acute and systemic symptoms. J. Immunol. 151:4407. Wain-Hobson, S., P. Sonigo, O. Danos, S. Cole, and M. Alizon. 1985. Nucleotide sequence of the AIDS virus LAV. Cell 40:9. Winslow, G. M., M. T. Scherer, J. Kappler, and P. Marrack. 1992 Detection and biochemical characterization of the mouse mammary tumor virus-7 superantigen. Cell 71:719. Woodland, D. L, M. P. Happ, K. J. Gollob, and E. Palmer. 1991. An endogenous retrovirus mediating deletion of ap T cells? Nature 349:529. Yoshida, M., I. Miyoshi, and Y. Hinuma. 1982. Isolation and characterization of retrovirus from cell lines of human T-cell leukemia and its implication in the disease. Proc. Natl. Acad. Sci. USA 79:2031 . Zack, J. A., S. J. Arrigo, S. R. Weitman, A. S.Go, A. Haislip, and I. S. Y. Chen. 1990. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 61 :213. Zinkernagel, R. and H. Hentgartner. 1994. T-cell mediated immunopathology versus direct cytolysis by virus: implications for HIV and AIDS. Immunol. Today 15:262.

PAGE 105

BIOGRAPHICAL SKETCH Barbara Aurea Torres, a first-generation American, was born on June 4, 1956 at Hialeah Hospital in South Florida. Her parents, Manuel and Antonia Torres, and sister, Marisol Torres Beaton, were born in Chile and emigrated to the United States in 1950. She was blessed with parents who intentionally gave her a first name that allowed her to "blend" into the American melting pot. Her father's family in Chile has had a long-time connection to medicine and the sciences. Barbara's grandfather was a physician in a rural community in Chile, and her uncle is still a general practitioner in Ecuador. In 1989, Barbara's father gave her a monograph on the atomic theory written by her great-grandfather, who was an instructor at the Sorbonne University in Paris, France. This monograph made a lasting impression on Barbara on the nature of scientific inquiry. Barbara graduated in 1978 with a Bachelor of Arts degree from Randolph-Macon Woman's College in Lynchburg, Virginia. In 1981, Barbara did graduate work in the laboratory of Dr. Howard M. Johnson and received a Master of Science degree from the Department of Microbiology at the University of Texas Medical Branch in Galveston, Texas. Barbara continued working with Dr. Johnson for several years, both in Galveston and at the University of Florida. After a mundane two-year stint at Bio-Rad Laboratories in Richmond, California, Barbara decided to return to Gainesville to work towards attaining her doctorate, once again in the laboratory of Dr. Johnson. After graduation, Barbara plans on continuing her research on HIV, as a postdoctoral fellow in the laboratory of Dr. Johnson at the University of Florida. 96

PAGE 106

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Howard M. Johnspn, Chair Graduate Research Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Edward M. Hoffmann Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. K.T. Shanmugam Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. ^danet K. Yamajgsmo Associate Professor of Pathology and Laboratory Medicine

PAGE 107

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. J^Edwin Blalock Professor of Physiology and Biophysics University of Alabama This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1995 dC. Dean, College of Agriculture Dean, Graduate School


39
Ability of Nef Peptides to Compete with SEs for Binding to Raii Cells
Net peptides were also tested for their relative abilities to compete with
other staphylococcal enterotoxins (SEs) for binding to Raji cells. As shown in
Figure 8, Nef(123-160) significantly blocked binding of 125I-SEE by 42%
(p<0.02) and 125I-SEC1 by 26% (p<0.03), but only blocked binding of 125l-
SEB by 10% (p>0.1). Thus, Nef(123-160) significantly inhibited the binding of
two highly homologous SEs, SEA and SEE, while it was less effective against
SEB and SEC1.
Direct Binding of Nef(123-160) Peptide to Raii Cells
In direct binding studies, 125l-Nef(123-160) bound to Raji cells and was
effectively inhibited by unlabeled Nef(123-160) and SEs (Figure 9). Unlabeled
Nef(123-160) reduced 125l-Nef(123-160) binding by 50% at a concentration of
30 pM. SEE was a better competitor and reduced 125l-Nef(123-160) binding
by 50% at a concentration of 2 pM. Both SEE and unlabeled Nef(123-160)
competed in a similar manner, but SEE was a more potent competitor. SEA did
not inhibit 125l-Nef(123-160) binding as well as SEE, but it was more effective
than SECi or SEB. This pattern of inhibition is reflective of the ability of
Nef(123-160) to block SE binding to MHC class II antigens. Although SEs
compete for similar sites on MHC class II antigens, our results suggest that
Nef(123-160) competes for sites that are more closely associated with SEE
than the other SEs tested. Nef(123-160) binding was not blocked by other Nef
peptides, such as Nef(1-38) and Nef (31-65), at 300 pM. These results suggest
that Nef(123-160) binds directly to Raji cells and competes for a site on MHC
class II antigens to which SEs bind.


67
probably play an important role in HIV pathogenesis. Clearly, the control of the
mitogenic activity of these substances should help reduce the viral load in HIV-
infected individuals.


TABLE OF CONTENTS
ACKNOWLEDGMENTS ¡¡
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT viii
CHAPTERS
I INTRODUCTION 1
Overview 1
Bacterial Superantigens 6
Retroviruses 8
Mouse Mammary Tumor Virus Superantigen 10
Evidence for a Superantigen Associated with
Human Immunodeficiency Virus 12
II MMTV SUPERANTIGEN BINDING TO MHC CLASS II
ANTIGENS 16
Introduction 16
Materials and Methods 17
Synthetic Peptides 17
Cell Lines and Reagents 17
Radioiodinations 19
Binding Studies 19
Radioimmunoassay 20
Results 21
Competition of vSAg Peptides with Radiolabeled SEA for
MHC Binding 21
vSAg(76-119) Peptide Competition Is Dose-Dependent 21
Direct Binding of vSAg(76-119) Peptide to A20 Cells 24
Evidence that vSAg(76-119) Binds Specifically to MHC
Class II Antigens 26
Discussion 26
iii


21
radioactivity was quantified. Experiments were performed at least four times
each using replicates of three.
Results
Competition of vSAg Peptides with Radiolabeled SEA for MHC Binding
Overlapping peptides (referred to as vSAg peptides) corresponding to
the predicted extracellular domain of the MMTV-1 superantigen were
synthesized and their sequences are listed in Table III. The vSAg peptides
were initially tested at a concentration of 200 pM for their relative abilities to
compete with 125I-SEA for binding to A20 cells, which express l-Ad and l-Ed
(which are MHC class II antigens of the d haplotype, A isotype and E isotype,
respectively). The vSAg(76-119) peptide reduced 125I-SEA binding to A20
cells by 63%, while the other peptides had no effect (Figure 1). Thus, only one
of the overlapping vSAg peptides, vSAg(76-119), significantly blocked the
binding of 125I-SEA binding to A20 cells.
vSAa(76-1191 Peptide Competition Is Dose-Dependent
Dose response studies were performed on A20 cells with several vSAg
peptides, including vSAg(76-119). The vSAg(76-119) peptide reduced ^SI
SEA binding by 50% at a concentration of 20 pM (Figure 2). Further, vSAg(76-
119) peptide consistently competed with 125I-SEA in a manner similar to
unlabeled SEA, although SEA was 20 times more effective. Unlabeled SEA at
a concentration of 1 pM reduced 125I-SEA binding to A20 cells by 50%. These
data are consistent with the reported Kd for SEA binding to l-Ed of
approximately 10'6 M (Lee and Watts, 1990). The peptide corresponding toa


79
Table X.
Ability of anti-Nef peptide antibodies to block proliferation induced by
autologous HIV-infected cells
Fresh PBMC cultured Anti-Nef Proliferation
in the presence of: antibodies CPM Fold increase p value
(Mean SD)
HIV-infected PBMC
-
137187 1923
162.2
HIV-infected PBMC
+
79740 8764
94.3
<0.003
_
+
1973 880
2.3
846 271


77
Table IX
Nef-induced activation of T cells as assessed by FACS
Cultures from:
% Total of dually-stained populations
CD4 + HLA-DR CD4 + IL2R
Donor 206
Unstimulated
3.17
N.D.c
Nef-stimulated
7.39
N.D.
SEA-stimulated
29.46
N.D.
Donor 213
Unstimulated
6.65
N.D.
Nef-stimulated
16.74
N.D.
Donor 214
Unstimulated
3.45
N.D.
Nef-stimulated
11.15
N.D.
Donor 215
Unstimulated
N.D.
7.75
Nef-stimulated
N.D.
10.55
Donor 216
Unstimulated
1.52
4.66
Nef-stimulated
9.02
7.68


84
results suggest a model in which Net acts as a HIV-encoded T cell mitogen
(Figure 18). Net may interact with T cells either as an integral part of the
membranes of infected T cells or in a soluble form released as the result of lysis
of infected cells. Nef is present on the membranes of infected T cells (Fujii et
al., 1993), which is consistent with this model. Nef expression may occur in the
context of the T cell activation marker, HLA-DR, and the Nef/HLA-DR complex
would stimulate uninfected T cells, as well as B cells. Such stimulation would
allow for the expansion of a cellular reservoir for replication of the virus,
eventually leading to T cell anergy and/or apoptosis. B cell activation and
differentiation could ultimately lead to hypergammaglobulinemia. Thus, the HIV
genome may encode for its own T cell mitogen, which would induce the
amplification of virus replication in the host, with ultimate deleterious effects on
the immune system. As such, Nef could be classified as a virulence factor for
HIV.


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31
This region would be part of the extracellular domain based on a proposed
Type II membrane protein model for MMTV vSAg protein (Choi et al., 1992).
Thus, both the C-terminal tail and an N-terminal region may be required for
ternary complex formation with MHC and TCR by MMTV superantigen.
Conversely, SEA contains several N-terminal domains that bind MHC (Griggs
et al., 1992) and an internal domain that may be involved in binding to TCR, so
that the sites of interaction of these superantigens with MHC and TCR may not
be completely analogous. Although little sequence homology exists between
MMTV vSAg protein and SEA, circular dichroism analysis indicates that both
SEA and vSAg(76-119) have significant p structure, suggesting that similar
structural motifs may be important for MHC binding by superantigens.
Finally, results presented herein indicate that a peptide corresponding to
residues 76-119 of the MMTV superantigen binds directly to MHC class II
antigens. Further, competition studies indicate that SEA and vSAg(76-119)
peptide bind to at least one common region on mouse MHC antigens. Future
studies using synthetic peptides and peptide analogues may help further
elucidate the site(s) on MHC for which SEA and MMTV superantigen compete.


72
Results
HIV Replication in Nef-Stimulated PBMC Cultures
Since T cell activation occurs with exogenous Net protein, the question
arose as to whether Net stimulation of PBMC was sufficient to induce HIV-1
replication. To this end, PBMC of several HIV-negative donors were tested for
proliferation induced by Nef and other T cell mitogens (Table VIII). Consistent
with previous data, PBMC from most of the donors proliferated significantly in
response to Nef protein, as well as to the T cell mitogens SEA and
phytohemagglutinin A (PHA).
Unstimulated and mitogen-stimulated PBMC were infected with HIV-1
and cultures were monitored over a 12-day period for signs of viral replication.
Supernatants from unstimulated PBMC showed only marginal reverse
transcriptase (RT) activity and p24 antigen production (Table VIII). RT and p24
antigen levels were high in cells stimulated with T cell mitogens, in particular
SEA. Interestingly, Nef-stimulated cells produced moderate to high levels of RT
and p24 antigen, indicating that these cells were capable of sustaining virus
replication. In an attempt to quantify the number of infected cells in these
cultures, PCR analysis of serially-diluted DNA was performed using nested
primers for the gag gene. Results indicated 10- to 30-fold increases in the
number of cells harboring virus in Nef-stimulated cultures, as compared to
unstimulated controls. These data indicate that Nef stimulation resulted in a
large number of infected cells and a concomitant increase in viral load.
Infectious Virus Is Produced bv Nef-Stimulated Cultures
As a means of determining whether the virus produced by Nef-stimulated
was infective, supernatants from HIV-infected cultures were tested for
reinfectivity. SEA-stimulated PBMC were infected with virus from 6-day


57
Unfractionated HPMC APC
T cells
APC + T cells
Cells
Figure 14. Nef-induced activation of T cells requires APC but does not require
processing of Nef. Unfractionated PBMC, purified APC alone, purified T cells
alone, and purified T cells in the presence of APC were tested for proliferation
in response to Nef, SEA, and Con A. Data are from a representative
experiment, performed in triplicate, and are expressed as mean stimulation
index S.D. Purified T cells were reconstituted with paraformaldehyde-
inactivated APC. Con A was used at 10 pg/ml. Nef and SEA were both used at
100 ng/ml.


60
Induction of T Cell Cytokines by Nef
T cell activation by the staphylococcal superantigens results in the
prodigious production of T cell cytokines, such as IL 2 and IFNy. Because of the
significant T cell proliferation induced by Nef, it was determined if Nef induced
Thelper cell cytokines. The results of a representative experiment are
presented in Table VI. Consistent with proliferation data, Nef induced
significant levels of IL 2, although IL 2 levels were lower than those induced by
SEA. The question arose as to the ability of Nef to also induce another
important T helper cell cytokine, IFNy. Samples were removed from cultures of
cells over the course of 96 h and tested for IFN antiviral activity. Nef induced
high levels of IFN activity, with peak production occurring by 96 h (Figure 16,
Panel A). IFN levels were lower than those induced by SEA, but were similar
than those induced by Con A. Purified T cells did not produce IFN upon
stimulation with either Nef or SEA (Figure 16, Panel B). However, purified T cell
cultures were capable of producing IFN upon Con A stimulation. No IFN activity
was produced in cultures of APC (consisting of B cells) stimulated with either
Nef, SEA, or Con A. Purified T cell cultures reconstituted with purified B cells for
antigen presentation produced significant levels of IFN upon either Nef or SEA
stimulation (Figure 16, Panel C). These results are consistent with T cell
mitogen activity for both Nef and SEA.
The type of IFN activity induced by Nef was determined by neutralization
reactions with specific antisera. Treatment of Nef-induced IFN with anti-IFNy
resulted in a significant reduction of activity (Table VII). Antisera to IFNa did not
affect the IFN activity in these samples. Consistent with previous data (Langford
et al., 1978a; Johnson et al., 1982), the IFN activity induced by SEA was
IFNy. Similar to the results found with unfractionated PBMC, the IFN produced


89
Fujii, Y., Y. Nishino, T. Nakaya, K. Tokunaga, and K. Ikuta. 1993.
Expression of human immunodeficiency virus type 1 Nef antigen on the surface
of acutely and persistently infected human T cells. Vaccine 11:1240.
Gallo, R.C., Salahuddin, S.Z., M. Popovic, G. M. Shearer, M. Kaplan, B. F.
Haynes, T. J. Palker, R. Redfield, J. Oleske, and B. Safai. 1984. Frequent
detection and isolation of cytopathic retroviruses (HTLV-III) from patients with
AIDS and at risk for AIDS. Science 224:500.
Garcia, J. V. and A. D. Miller. 1991. Serine-phosphorylation-
independent downregulation of cell-surface CD4 by nef. Nature 350:508.
Golovkina, T. V., A. Chervonsky, J. P. Dudley, and S. R. Ross. 1992.
Transqenic mouse mammary tumor virus superantigen prevents viral infection.
Cell 69:637.
Gowda, S. D., B. S. Stein, N. Mohagheghpour, C. J. Benike, and E. G.
Engleman. 1989. Evidence that T cell activation is required for HIV-1 entry in
CD4+ lymphocytes. J. Immunol. 142:773.
Griggs, N. D., C. H. Pontzer, M. A. Jarpe, and H. M. Johnson. 1992,
Mapping of multiple binding domains of the superantigen staphylococcal
enterotoxin A for HLA. J. Immunol. 148:2516.
Guy, B., M. P. Kierny, Y. Riviere, C. Le Peuch, K. Dott, M. Girard, L.
Montagnier, and J. --P. Lecocq. 1987. HIV F/3' orf encodes a phosphorylated
GTP-binding protein resembling an oncogene product. Nature 330:266.
Hammes, S. R., E. P. Dixon, M. H. Malim, B. R. Cullen, and W. C. Greene.
1989. Nef protein of human immunodeficiency virus type 1: evidence against
its role as a transcriptional inhibitor. Proc. Natl. Acad. Sci. USA 86:9549.
Held, W., H. Acha--Orbea, H. R. MacDonald, and G. A. Waanders.
1994a. Superantigens and retroviral infections: insights from mouse mammary
tumorvirus. Immunol. Today 15:184.
Held, W., G. A. Waanders, H. R. MacDonald, and H. Acha-Orbea.
1994b. MHC class II hierarchy of superantigen presentation predicts efficiency
of infection with mouse mammary tumor virus. Int. Immunol. 6:1403.
Held, W., G. A. Waanders, A. N. Shakhov, L. Scarpellino, H. Acha-
Orbea, and H. R. MacDonald. 1993. Superantigen-induced immune
stimulation amplifies mouse mammary tumor virus infection and allows
transmission. Cell 74:529.
Herman, A., N. Labrecque, J. Thibodeau, P. Marrack, and R. R. Rich.
1991. Identification of the staphylococcal enterotoxin A superantigen binding
site in the beta 1 domain of the human histocompatibility antigen HLA-DR.
Proc. Natl. Acad. Sci. USA 88:9954.


22
160
140-
76-119 117-147 142-172 166-195 190-222 220-250 245-276 274-313
vSAg peptides
Figure 1. Ability of vSAg peptides to compete with 125I-SEA for binding to A20
cells. Binding of 125I-SEA in the absence of competitors was 6384+400 CPM.
The data presented represent the mean of three individual experiments each
performed in duplicate. Each bar represents the mean percent of SEA control
binding in the presence of vSAg peptides SD.


87
Bergdoll, M. S. 1985. The staphylococcal enterotoxins-an update. In J.
Jeljaszewics, Ed. The Staphylococci. New York: Gustav Fisher Verlag, pp247-
266.
Bisset, L. R., M. Opravil, E. Ludwig, and W. Fierz. 1993. T cell response
to staphylococcal superantigens by asymptomatic HIV-infected individuals
exhibits selective changes in T cell receptor V beta-chain usage. AIDS Res.
Hum. Retroviruses 9:241.
Brodsky, F.M. 1984. A matrix approach to human class II
histocompatibility antigens: reactions of four monoclonal antibodies with the
products of nine haplotypes. Immunogenetics 19:179.
Buhrinsky, M. I., T. L. Stanwick, M. P. Dempsey, and M. Stevenson.
1991. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1
infection. Science 254:423.
Cao, Y., L. Qin, L. Zhang, J. Safrit, and D. D. Ho. 1995. Virological and
immunological characterization of long-term survivors of huma
immunodeficiency virus type 1 infection. New Engl. J. Med. 332:233.
Carlsson, R., H. Fischer, and H. O. Sjogren. 1988. Binding of
staphylococcal enterotoxin A to accessory cells is a requirement for its ability to
activate human T cells. J. Immunol. 140:2484.
Chinatgumpala, M. M., J. A. Mollick, and R. R. Rich. 1991.
Staphylococcal toxins bind to different sites on HLA-DR. J. Immunol. 147:3876.
Chirmule, N., N. Oyaizu, C. Saxinger, and S. Pahwa. 1994. Nef protein
of HIV-1 has B-cell stimulatory activity. AIDS 8:733.
Choi, Y., J. Kappler, and P. Marrack. 1991. A superantigen encoded in
the open reading frame of the 3' long terminal repeat of mouse mammary tumor
virus. Nature 350:203.
Choi, Y., J. Kappler, and P. Marrack. 1992. Structural analysis of a
mouse mammary tumor virus superantigen. J. Exp. Med. 175:847.
Cole, B. C. and M. M. Griffiths. 1993. Triggering and exacerbation of
autoimmune arthritis by the Mycoplasma arthriditis superantigen MAM. Arth.
Rheum. 36:994.
Dalgleish, A. G., S. Wilson, M. Gompels, C. Ludlam, B. Gazzard, A. M.
Coates, and J. Habeshaw. 1992. T-cell receptor variable gene products and
early HIV-1 infection. Lancet 339:824.
Daniel, M. D., F. Kirchhoff, S. C. Czajak, K. Sehgal, and R. C. Desrosiers.
1992. Protective effects of a live attenuated SIV vaccine with a deletion in the
nef gene. Science 258:1938.


52
x
a>
-a
s
"5
E
"M
CZ3
1000
Concentration (ng/ml)
Figure 11. Comparison of the mitogenic activities of Nef protein
preparations. Two Nef preparations,Nef 1 and Nef 2, were compared for
proliferative activities on human PBMC. Data are from a representative
experiment, performed in triplicate, and are expressed as mean stimulation
index S.D.. Comparison of the proliferation of PBMC cultured for 4 days in the
presence of either Nef 1 or Nef 2. The mean value for 3H-thymidine
incorporation by unstimulated cultures was 90067.


64
by T cell cultures reconstituted with B cells in response to Net was IFNy. Thus,
Net induced high levels of IFNy in cultures of PBMC, indicating that Nef
activates T cells to produce the cytokines IL 2 and IFNy, lymphokines that are
products of activated T helper 1 cells.
PBMC cultured in the presence of Nef were tested for specific Vp
expansion and anergy in vitro. No expansion of specific Vp populations was
observed with the anti-Vp antibodies used (data not shown). Unlike
pretreatment with TSST-1, which anergized the response to anti-Vp2, Nef did
not significantly anergize the responses of T cells to the tested anti-Vp
antibodies (data not shown). Thus, no specific Vp expansion or anergy was
detected using the available human Vp reagents.
Discussion
Nef protein induced significant levels of proliferation in unfractionated
PBMC from a number of donors. The observed Nef responses were significant,
with proliferation induced in cells from a large majority (85-90%) of the donors
tested. It is unlikely that prior sensitization to Nef is responsible for the
proliferative response, since these donors were negative for HIV. The following
evidence points to Nef as the inducer of the observed proliferation: 1)
proliferation was observed using recombinant Nef from three different
expression systems; 2) antibodies to synthetic Nef peptides neutralized the
proliferative activity of Nef; 3) the Nef fusion partner, MBP, did not induce
proliferation; and 4) a synthetic peptide, Nef(123-160), previously shown to
block binding of superantigens to MHC class II, blocked the Nef-induced
proliferative responses. Nef induced proliferation was generally lower than that
induced by SEA. This is not unusual in that SEA is extremely potent, causing
proliferation at concentrations as low as 10'16 M (Langford et al., 1978a).


Stimulation Index
54
15
10
5
0
PBMC stimulated with Nef or E. coli MBP:
Expt. 1
I I Expt. 2
Nef MBP
Figure 12. Lack of proliferation by PBMC in response to the Nef fusion partner
MBP. MBP and Nef were used at 3 pg/ml. Data are expressed as mean
stimulation index SD. The mean values for ^H-thymidine incorporation by
unstimulated cultures in eperiments 1 and 2 were 85239 CPM and 1093136
CPM, respectively.


11
been shown to contain one or more copies of endogenous MMTV, and many
contain distinct MMTV strains (Salmons and Gunzburg, 1987). Upon passage
into the gut of the host, virus enters the gut-associated immune tissue and
infects B cells. B cells then express the MMTV-encoded superantigen,
presumably in the context of class II antigens, causing Vp-specific T cell
stimulation. Although investigators have been able to show that actual infection
of T cells occurs, it is thought that T cell stimulation and subsequent cytokine
production by these cells indirectly enhances the further infection of B cells
(Held et al., 1994a). Infected B cells migrate to the main site of viral infection,
the mammary gland. Epithelial cells then become infected and are the source
of infectious virions that are transmitted in milk (Ringold, 1983). Proviral
integration can occur during infection of mammary epithelial cells, resulting in
tumors.
Germline-encoded MMTV serves an important protective mechanism for
the host. It is known that bacterial superantigens can cause anergy and/or
deletion of Vp-specific T cells (Johnson et al., 1992). Similarly, expression of
MMTV superantigen early in the ontogeny of the immune system induces the
eventual deletion of T cells bearing Vps specific for that particular strain of
MMTV superantigen. In this manner, T cells that would otherwise be stimulated
are lost and the host is protected against subsequent infection by MMTV strains
that stimulate those Vp-specific T cell populations. This has been shown in
mice transgenic for a MMTV superantigen that stimulates Vpl4+ T cells
(Golovkina et al., 1992; Acha-Orbea, 1991a). Transgenic mice showed partial
to complete deletion of Vpi4+ T cells, depending on the level of superantigen
expression. Those mice in which Vpl4 + T cells were deleted were
subsequently protected from infection upon challenge with the same MMTV
strain.


44
on the cells. The ability of these antibodies to neutralize HIV suggest that HLA-
DR may play a role in infectivity. Our results suggest that Net may bind to HLA-
DR as it is being expressed on the surface of an infected cell in a manner
analogous to the putative expression of MMTV superantigen (Choi et al., 1992).
Further, Nef has been shown to be present on the surface of infected cells, as
assessed using anti-Nef antibodies labeled with fluorescein (Fujii et al., 1993).
This is in contrast to the situation with MMTV superantigen in that no one has
been able to show that cells from MMTV-infected animals express
superantigen, although superantigen functional activity is observed (Choi et al.,
1991). Transgenic mice expressing MMTV superantigen delete V(i14+ T cells,
probably during the ontogeny of the immune system, and these mice are
immune to infection by exogenous MMTV infection (Acha-Orbea and Palmer,
1991). Thus it is possible that Nef interacts with class II MHC antigens in a
manner somewhat similar to MMTV superantigen, suggesting a possible
superantigen function for Nef in HIV pathogenesis.


25
Figure 3. Ability of SEA, SEB, and TSST-1 to compete with 125l-vSAg(76-119)
for binding to A20 cells. Experimental conditions were the same as those
described in Figure 1. 125l-vSAg(76-119) was used at a final concentration of
2.3 nM. Binding of 125l-vSAg(76-119) in the absence of competitors was
2278224 CPM. The data presented represent the mean of three individual
experiments, each performed in duplicate. Each point represents the mean
percent of vSAg control binding in the presence of competitors SD.


I certify that I have read this study and that iri my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Howard M. Johns/n, Chair
Graduate Research Professor of
Microbiology and Cell Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
t L <-
Edward M. Hoffpiann
Professor of Microbiology and
Cell Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
K.T. Shanmugam
Professor of Microbiology and
Cell Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
>danetK. Y
Associate Professor of Pathology
and Laboratory Medicine


8
Presentation of superantigen in the context of MHC class II molecules is
required for interaction with T cell receptor (TCR). For this reason superantigen
sites that interact with TCR have been more difficult to elucidate. However,
through the use of synthetic peptides, a TCR site that interacts with SEA has
been identified. A peptide corresponding to residues 57-77 of mouse Vp3
blocked SEA-induced proliferation and IFNy production (Pontzer et al., 1992).
Vp3-bearing mouse T cells are known to be stimulated by SEA. Future studies
may help determine the sequences involved in the formation of the ternary
complex of superantigen:MHC:TCR. Such studies are needed to determine
how superantigens activate T cells bearing specific Vps and induce anergy and
deletion of these Vp subsets.
Retroviruses
Retroviruses belong to a family of viruses that are characterized by the
use of a unique RNA-dependent DNA polymerase, reverse transcriptase.
Reverse transcriptase, in conjunction with another virally-encoded enzyme,
RNAse H, transcribes the single-stranded RNA genome into a double-stranded
linear DNA provirus. This DNA intermediate is then capable of integrating into
the host genome. Retroviruses were first described in studies in which cell-free
filtered extracts were shown to transmit sarcoma to chickens (Rous 1910, 1911).
Since that time, retroviruses have been found in several vertebrates including
mice, cats, and primates. The first human retrovirus to be discovered was
human foamy virus, which has been speculated to cause disease (Achong et
al., 1971), although definitive proof of pathogenicity is lacking. In 1980, the
causative agent of adult T-cell leukemia was discovered, human T-cell


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
RETROVIRAL SUPERANTIGENS
By
Barbara Aurea Torres
December 1995
Chairman: Howard M. Johnson
Major Department: Microbiology and Cell Science
Superantigens (SAg) are potent inducers of T cell activation that cause
proliferation and massive cytokine release. The receptors for superantigens on
antigen-presenting cells (APC) are major histocompatibility (MHC) class II
molecules. Both Nef protein of human immunodeficiency virus (HIV) and
mouse mammary tumor virus (MMTV) SAgs are encoded in genes that overlap
the terminal repeat at the 3' end. Using synthetic peptides, a region was
identifed on the MMTV-1 SAg, corresponding to residues 76-119, that
specifically binds to mouse MHC class II antigens. MMTVSAg(76-119) also
bound to and competed with bacterial superantigens for binding to MHC class II
peptides, suggesting similar binding regions on class II. These studies are the
first demonstration that retroviral superantigens bind to MHC class II antigens.
viii


36
initially tested at a concentration of 300 pM for their relative abilities to compete
with 125I-SEA for binding to Raji cells, which express HLA-DR3 and HLA-
DRw10, and DR1-transfected L cells (Figure 6). Nef(123-160) reduced 125l-
SEA binding to Raji cells by 41% which was significant at p<0.002. This
degree of inhibition was consistent in repeated experiments. The N-terminal
peptide, Nef(1-38), had a marginal but insignificant effect (21% reduction,
p>0.05) on 125I-SEA binding to Raji cells in the same experiment. This slight
inhibition by Nef(1-38) was not seen in repeated experiments. The other
peptides did not affect SEA binding to Raji cells. A similar pattern was seen in
competitive binding studies performed on DR1-transfected L cells. Nef(123-
160) blocked 125I-SEA binding to DR1 cells by 35% (p<0.008), whereas Nef(1-
38) had only a slight effect (p>0.8). Thus, only one of the overlapping Nef
peptides, Nef(123-160), significantly and consistently blocked 125I-SEA binding
to both Raji cells and DR 1-transfected L cells.
Nef(123-160) Peptide Competition Is Dose-Dependent
The relative abilities of Nef(123-160), purified recombinant Nef protein
(Repligen, Cambridge, MA), and SEA to block SEA binding to MHC class II
were tested in dose response studies (Figure 7). Nef(123-160) reduced 125l-
SEA binding to Raji cells by 40% at the highest concentration tested (300 pM),
and competed with SEA in a dose-dependent manner. Two other Nef peptides,
Nef (1-38) and Nef(31-65), did not block SEA in a dose-dependent manner.
Unlabeled SEA reduced 125I-SEA binding by 50% at a concentration of 0.2 pM,
which is consistent with the reported Kd of SEA for human MHC class II
antigens (Chintagumpala et al., 1991). Thus, Nef protein and the internal Nef
sequence, Nef(123-160), competed for SEA binding to Raji cells in a dose-
dependent manner.


42
MAbs to MHO Class II Antigens Block Nef(123-1601 Peptide Binding to Raji
Cells
To ascertain the receptor to which ^25|-Nef( 123-160) bound on Raji
cells, binding was performed in the presence of monoclonal antibodies (mAb)
to class II and class I antigens (Figure 10). Clone L243, a mAb specific for HLA-
DR, reduced 125I-Nef(123-160) binding by 35%. Another mAb specific for
HLA-DR, clone L227, also reduced Nef(123-160) binding, but was not as
effective as clone L243. This is consistent with reports that L227 mAb is not
very effective at blocking SEA binding to Raji cells (Chintagumpala et al., 1991).
MAb to other class II antigens (HLA-DP and DQ) had no effect on 125|-Nef(123-
160) binding, as was also the case for a mAb to class I antigens (clone W6/32).
Thus, our studies indicate that Nef(123-160) binds to class II MHC antigens, the
known receptors for superantigens on APC.
Discussion
Data presented here show that binding of bacterial superantigens to
MHC class II antigens can be blocked by a peptide corresponding to an internal
Nef region. Anti-HLA-DR mAb blocked Nef(123-160) binding to Raji cells,
suggesting that binding to class II molecules occurred. Binding of Nef(123-160)
to HLA-DR probably occurs outside the antigen binding groove, since Nef(123-
160) was able to block binding of the superantigens SEE and SEA. A recent
study has shown that HLA-DR is present on HIV-1 and SIV, and that it is
selectively incorporated into the virus membrane over HLA-DP or HLA-DQ
(Arthur et al., 1992). Antibodies to HLA-DR, but not antibodies to HLA-DP or
HLA-DQ, effectively inhibited viral infection of cells in vitro and these antibodies
target the HLA-DR antigens present on the virus particles and not those present


92
Lemaitre, M., D. Guetard, Y. henin, L. Montagnier, and A. Zerial. 1990.
Protective activity of tetracycline analogs against the cytopathic effect of human
immunodeficiency viruses in CEM cells. Res. Virol. 141:5.
Lewis, H. M., B. S. Baker, S. Bokth, A. V. Powles, J. J. Garioch, H.
Valdimarsson, and L. Fry. 1993. Restricted T cell receptor V(3 gene usage in
the skin of patients with guttate and chronic plaque psoriasis. Brit. J. Dermatol.
129:514.
Lifson, J. D., M. B. Feinberg, G. R. Reyes, L. Rabin, B. Banapour, S.
Chakrabati, B. Moss, F. Wong-Staal, K. S. Steimer, and E. G. Engelman. 1986.
Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope
glycoprotein. Nature 323:725.
MacDonald, H. R., A. L. Glasebrook, R. Schneider, R. K. Lees, H. P.
Pircher, T. Pedrazzini, O. Kanagawa, J.-F. Nicholas, R. C. Howe, R. M.
Zinkernagel, and H. Hentgartner. 1989. T-cell reactivity and tolerance to Mlsa-
encoded antigens. Immunol. Rev. 107:89.
Mariani, R. and J. Skowronski. 1993. CD4 down-regulation by nef
alleles isolated from human immunodeficiency virus type 1-infected individuals.
Proc. Natl. Acad. Sci. USA 90:5549.
Marrack, P. and J. Kappler. 1990. The staphylococcal enterotoxins and
their relatives. Science 248:705.
Marrack, P., E. Kushnir, and J. Kappler. 1991. A maternally inherited
superantigen encoded by a mouse mammary tumor virus. Nature 349:524.
Merryman, P., J. Silver, P. K. Gregeersen, G. Solomon, and R.
Winchester. 1989. A novel association of DQ alpha and DQ beta genes in the
DRw10 haplotype. J. Immunol. 143:2068.
Montagnier, L. 1995 Nef vaccination against HIV disease. Lancet
346:1170.
Mollick, J. A., R. G. Cook, and R. R. Rich. 1989. Class II molecules are
specific receptors for staphylococcal enterotoxin A. Science 244:817.
Mottershead, D. G., P. -N. Hsu, R. G. Urban, J. L. Strominger, and B. T.
Huber. 1995. Direct binding of Mtv7 superantigen (Mls-1) to soluble MHC
class II molecules. Immunity 2:149.
Ohmen, J. D., P. F. Barnes, C. L. Grisso, B. R. Bloom, and R. L. Modlin.
1994. Evidence for a superantigen in human tuberculosis. Immunity 1:35.
Oroszlan, S. and R. B. Luftig. 1990. Retroviral proteinases. Curr. Top.
Microbiol. Immunol. 157:153.


51
(Genzyme, Cambridge, MA) as positive controls. Samples were incubated at
37C for 1 h prior to transfer to confluent human WISH cells. Residual IFN
activity in sham- and antiserum-treated samples was measured as described
above.
Studies on Vfi-Specific T Cell Expansion and Induction of Anerqy
PBMC cultured in the presence of Nef, TSST-1, and ConA were tested
by flow cytometry for specific Vp expansion as previously described (Soos and
Johnson, 1994) using a panel of anti-Vp antibodies. Induction of anergy in vitro
by Nef, TSST-1, and ConA was also assessed with anti-Vp antibodies as
described (Schiffenbauer et al., 1993). Antibodies to Vp5a, Vp5b, Vp5c, Vp6a,
Vp8a, and Vp12 were obtained from T Cell Sciences, Inc., Cambridge, MA.
Antibodies to Vp2, Vp3, Vp13, Vpl7, Vp18, Vp21, and Vp22 were purchased from
Immunotech, Marseilles, France.
Results
Nef Proliferative Response
Recombinant Nef proteins from two sources were tested for proliferative
activity. One Nef preparation (referred to as Nef 1) was purified and kindly
provided by Dr. Taishi Tanabe in the laboratory of Dr. Howard M. Johnson. This
Nef preparation was compared to purified Nef from Repligen (Nef 2). Both Nef
protein preparations, which are derived from the HIVlav sequence, induced
similar levels of proliferation (Figure 11). Nef proteins from both sources were
pure and neither preparation contained detectable levels of endotoxin. Thus,
Nef proteins from both sources had similar proliferative activity, and the
proliferation observed was significant. Nef from these two sources were


17
Materials and Methods
Synthetic Peptides
Overlapping peptides corresponding to the putative extracellular domain
of the MMTV-1 vSAg protein (Pullen et al., 1992) were synthesized with a
Biosearch 9500AT automated peptide synthesizer using N-(9-
flurenyl)methoxycarbonyl chemistry (Griggs et al., 1992). Peptides were
synthesized based on a surface profile which uses a composite of three
parameters: 1) HPLC hydophilicity; 2) accessibility; and 3) segmental mobility
(B value) (Parker et al., 1986). The sequences of the peptides are listed in
Table III. Peptides were cleaved from the resins using trifluoroacetic acid/
ethanedithiol/ thioanisole/ anisle at a ratio of 90:3:5:2. The cleaved peptides
were then extracted in ether and ethyl acetate and subsequently dissolved in
water and lyophilized. Peptides were extensively dialyzed against water to
remove the remaining cleavage products. Amino acid analysis of the peptides
showed that the amino acid contents and molecular weights corresponded
closely to theoretical values. Peptides were not purified further since reverse
phase HPLC analysis of crude peptides indicated one major peak in each
profile.
Cell Lines and Reagents
The A20 cell line (ATCC, Gaithersburg, MD) was used for MMTV
superantigen binding studies. A20 cells are a BALB/c B lymphoma line that
expresses la. Highly purified SEA and other staphylococcal enterotoxins were
obtained from Toxin Technology (Sarasota, FL). Several monoclonal
antibodies (mAb) were used in this study. MAbs were purchased from
Accurate Chemicals, Westbury, NY. MAb Kd (34-1-2S) is specific for m31
determinant of Kd (Ozato et al., 1982). MAb IAd (MK-D6) is specific for the p1


Materials and Methods 69
PBMC Cultures for HIV Infectivity Studies 69
PBMC Cultures for Proliferation Studies 69
Reverse Transcriptase (RT) Assay 70
ELISA for HIV p24 Antigen 70
Assay for Infectious Virus from PBMC Cultures 70
Fluorescent Antibody Cell Sorter (FACS) Analysis of
Nef-Activated Cells 71
Studies on Proliferation of PBMC Induced by Autlogous
HIV-Infected Cells 71
Anti-Nef Peptide Antibodies 71
Results 72
HIV Replication in Nef-Stimulated PBMC Cultures 72
Infectious Virus Is Produced by Nef-Stimulated Cultures 72
Nef-Stimulated PBMC Express T Cell Activation Markers 75
Expression of Nef by HIV-Infected Cells 75
Discussion 78
VI A MODEL FOR THE ROLE OF NEF IN HIV PATHOGENESIS 82
LIST OF REFERENCES 86
BIOGRAPHICAL SKETCH
100


LIST OF TABLES
Table Page
I. List of bacterial superantigens implicated in disease 3
II. List of viral superantigens implicated in disease 5
III. Amino acid sequences of vSAg peptides 18
IV. Amino acid sequences of Nef peptides 34
V. Ability of anti-peptide antibodies to block Nef-induced
proliferation 51
VI. IL 2 production induced by Nef 61
VII. IFNy is induced by Nef 63
VIII. Maximal proliferation, RT, and p24 values from stimulated
cultures 73
IX. Nef-induced activation of T cells as assessed by FACS 77
X. Ability of anti-Nef antibodies to block proliferation induced by
autologous HIV-infected cells 79
VI


7
interaction of enterotoxins on the MHC class II molecule was shown to be
distinct from conventional antigens in that binding occurs distal to the peptide
antigen binding groove (Russell et al., 1990; Dellabonna et al., 1990). Thus,
this class of antigens differs significantly from classic antigens and were given
the designation of superantigens (Marrack and Kappler, 1990).
The study of staphylococcal enterotoxins has led to a wealth of
information on the interaction of superantigens with MHC class II molecules.
Through the use of synthetic peptides, regions of interaction of SEA and MHC
class II antigens have been identified. Regions on the SEA molecule that are
involved in binding to MHC class II molecules on antigen presenting cells
include the N-terminus and three internal sequences (Griggs et al., 1992). A
peptide corresponding to the N-terminus of SEA, SEA(1-45), blocked
proliferation of cells in response to whole native SEA (Pontzer et al., 1990).
Interestingly, a peptide corresponding to another of these MHC class ll-binding
regions, SEA(121-149), has agonist properties in that it induces cytokine
production (Pontzer et al., 1993) and proliferation (A.C. Hobeika, personal
communication). Conversely, sites on MHC class II molecules which bind
superantigens have been identified. Regions on mouse MHC class II
molecules that interact with SEA have been identified and include the a-helical
region of the p chain associated with the peptide binding groove encompassed
by amino acids 65-85 (Russell et al., 1991). A similar site has been found on
human MHC class II antigens (Herman et al., 1991). A superantigen-binding
region on the a helical region of the a chain associated with the peptide binding
groove of MHC class II has also been defined (Russell et al., 1991). Both of
these binding sites reside outside of the peptide binding groove and are
required for SEA-induced mitogenesis. Thus the two outside faces of the a
helices of the peptide binding groove are involved in binding of SEA.


43
B7/21
SK10
W6/32
L243
L227
(Anti-DP)
(Anti-DQ)
(Anti-class I)
(Anti-DR)
(Anti-DR)
Antibody
Figure 10. Blockage of 125l-Nef(123-160) binding to Raji cells by antibodies to
MHC class I and class II antigens. 125l-Nef(123-160) was used at a final
concentration of 3 nM. MAbs were used at a final concentration of 10 |ig/ml.


33
Materials and Methods
Synthetic Peptides
Overlapping peptides corresponding to the entire length of HIVlav Net
(Wain-Hobson et al., 1985) were synthesized with a Biosearch 9500AT
automated peptide synthesizer using N-(9-flurenyl)methoxycarbonyl chemistry
as described in chapter II of this text. Peptides were synthesized based on a
surface profile as described. The amino acid sequences of the peptides are
presented in Table IV.
Cells and Reagents
Two cell lines were used for the binding studies. Raji cells are EBV-
transformed B cells that express DR3, Dw10, DQw1, and DQw2 (Merryman
et al., 1989). DR 1-transfected L cells were kindly provided by Dr. Eric O. Long
and are described elsewhere (Lechler et al., 1988). SEs were obtained from
Toxin Technology (Sarasota, FL). Several mAb were used in this study. Anti-
HLA-DR clone L243 reacts with a nonpolymorphic DR epitope and does not
cross-react with DP or DQ (Robbins et al., 1987). Anti-HLA-DP clone B7/21
reacts with a monomorphic epitope present on DP1, DP2, DP3, DP4, and DP5
(Robbins et al., 1987). Anti-HLA-DQ clone SK10 reacts with a common
polymorphic epitope present on cells expressing DQw1 and DQw3 (associated
with DR1. DR2, DR4, DR5, w8, w9, and w10) (Brodsky, 1984). Anti-HLA-DR
clone L227 reacts with a nonpolymorphic region of DR (Barnstable et al., 1978).
Clone W6/32 reacts with a monomorphic epitope on HLA-A, B, and C
(Barnstable et al., 1978). Clones L243, B7/21, and SK10 were obtained from
Becton-Dickinson (Mountain View, CA) and clones L227 and W6/32 were
kindly provided by Dr. Robert Rich. All mAbs were used at a final concentration