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Subtype-A and Subtype-B HIV-1 P24 Based FIV Vaccines

Permanent Link: http://ufdc.ufl.edu/UFE0021406/00001

Material Information

Title: Subtype-A and Subtype-B HIV-1 P24 Based FIV Vaccines Efficacy and Mechanisms of Cross-Protection
Physical Description: 1 online resource (102 p.)
Language: english
Creator: Martin, Marcus
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: fiv, hiv, lentivirus, vaccine
Microbiology and Cell Science -- Dissertations, Academic -- UF
Genre: Microbiology and Cell Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Published data shows HIV-1UCD1 p24 based vaccine protects cats from FIV infection. Understanding the mechanism of vaccine protection could provide a blueprint to the development of an HIV-1 vaccine for humans. The studies were performed by evaluating the protective ability of the vaccine-induced antibody response in three separate passive-transfer studies. The first involving vaccine antiserum, the second involving ammonium sulfate purified antibodies, and the third using antibodies purified using ammonium sulfate precipitation and caprylic acid fractionation. The data suggested that the protective efficacy of HIV-1UCD1 p24 (subtype-B) based vaccine was not mediated by vaccine-induced antibody immunity but most likely mediated by vaccine-induced cellular immunity. In addition protective efficiency varied between cats vaccinated with subtype-B HIV-1UCD1 p24 (67% protection) and subtype-A HIV-1N20 p24 (25% protection). In addition, IFNy ELISpot analysis using separate peptide pools of overlapping 15mers of FIV and HIV-1 p24 showed cross reactivity between FIV peptides and HIV-1 p24-vaccinated cat sera and between HIV-1 peptides and sera from either FIV p24-vaccination a Fel-O-Vax FIV-vaccinated cats. Some vaccinated cats reacted to peptides corresponding to the KK10 HIV-1 epitiope in humans, which is associated with long term non-progressor (LTNP) HIV-1 infected individuals
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Marcus Martin.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Yamamoto, Janet K.
Local: Co-adviser: Johnson, Howard M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021406:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021406/00001

Material Information

Title: Subtype-A and Subtype-B HIV-1 P24 Based FIV Vaccines Efficacy and Mechanisms of Cross-Protection
Physical Description: 1 online resource (102 p.)
Language: english
Creator: Martin, Marcus
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: fiv, hiv, lentivirus, vaccine
Microbiology and Cell Science -- Dissertations, Academic -- UF
Genre: Microbiology and Cell Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Published data shows HIV-1UCD1 p24 based vaccine protects cats from FIV infection. Understanding the mechanism of vaccine protection could provide a blueprint to the development of an HIV-1 vaccine for humans. The studies were performed by evaluating the protective ability of the vaccine-induced antibody response in three separate passive-transfer studies. The first involving vaccine antiserum, the second involving ammonium sulfate purified antibodies, and the third using antibodies purified using ammonium sulfate precipitation and caprylic acid fractionation. The data suggested that the protective efficacy of HIV-1UCD1 p24 (subtype-B) based vaccine was not mediated by vaccine-induced antibody immunity but most likely mediated by vaccine-induced cellular immunity. In addition protective efficiency varied between cats vaccinated with subtype-B HIV-1UCD1 p24 (67% protection) and subtype-A HIV-1N20 p24 (25% protection). In addition, IFNy ELISpot analysis using separate peptide pools of overlapping 15mers of FIV and HIV-1 p24 showed cross reactivity between FIV peptides and HIV-1 p24-vaccinated cat sera and between HIV-1 peptides and sera from either FIV p24-vaccination a Fel-O-Vax FIV-vaccinated cats. Some vaccinated cats reacted to peptides corresponding to the KK10 HIV-1 epitiope in humans, which is associated with long term non-progressor (LTNP) HIV-1 infected individuals
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Marcus Martin.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Yamamoto, Janet K.
Local: Co-adviser: Johnson, Howard M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021406:00001


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SUBTYPE-A AND SUBTYPE-B HIV-1 P24 BASED FIV VACCINES: EFFICACY AND
MECHANISMS OF CROSS-PROTECTION






















By

MARCUS MAKES MARTIN


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

2007




























2007 Marcus Makesi Martin































To my loving family and dear friends; through their love and support, I was able to pursue my
dreams









ACKNOWLEDGMENTS

I would first like to thank the department of Microbiology and Cell Science for providing

me with the opportunity to fulfill my dream of becoming a research scientist. I am eternally

grateful to Dr. Yamamoto who invited me into her lab and provided me with exceptional

mentorship. It was wonderful to learn from someone who also sees no limitations. I would like to

specially thank Dr. Johnson, who provided continual support, and was instrumental in helping

me to achieve my research objectives; Dr. Maruniak who provided advice on several life issues

inside and outside of graduate instruction; Dr. Hoffmann who was very supportive throughout

my studies and Dr. Uhl whose interest in my overall wellbeing was a constant comfort. Also I

would like to specially thank Dr. Eiji Sato who advised me on the molecular biology aspect of

my work, and Dr. Pu who assisted me immeasurably with the animal handling during my studies.

I would like to especially acknowledge the essential support provided by my family,

relatives, my eternally supportive uncle Chick, and my grandparents Norman and Josephine who

recently passed on.

Peers help to provide support and Johnny Davis, Laurence Flowers, Karen Viera, Tiffany

Snipe, Carla Phillips and Gareth Jordan were there to share the journey. It was also my privilege

to have the pleasure of working with our lab team; James Coleman, Mayuko Omori,.Blerina

Hysi, Samantha Hass, Melissa Voltz, Taishi Tanabe, and Maki Tanabe who all provided research

camaraderie.









TABLE OF CONTENTS



A C K N O W L E D G M E N T S ..............................................................................................................4

L IST O F T A B L E S ......................................................................................................... ........ .. 7

LIST OF FIGURES ............................................. .. .......... ...........................8

L IST O F A B B R E V IA T IO N S ......................................................................... ...... ............... 10

A B S T R A C T .......................................................................................................... ..................... 12

CHAPTER

1 INTRODUCTION .................................. .. ........... ..................................... 13

B ack g rou n d ............... ......................................................................................... ........ .. 13
H um an Im m unodeficiency V irus .......................................... ......................... ................ 13
H IV -1 V iral Infectivity Factor (V if)........................................................... ............... 14
H IV -1 V iral Protein U (V pu) ................. ............................................................. 14
H IV -1 V iral P rotein R (V pr) ...................................................................... ............... 14
H IV -1 N negative Regulatory Factor (N ef)................................................... ................ 15
HIV-1 Trans-Activator of Transcription (Tat) ...........................................................15
H IV -1 R egulator of V iral Protein (R ev)..................................................... ................ 15
H IV -1 C classification ............. .. .................. .................. .......................... ............... 15
H IV 1 T reatm en t ............................................................................................................. 16
F eline Im m unodeficiency V irus ........................................................................ ................ 16
FIV Group Associated Antigen (Gag).................................................................... 17
F IV E n v elo p e (E n v ) ........................................................................................................ 17
FIV Polymerase (Pol) ...................... ............................ ........ .... ............... 18
F IV P protease (P R )............................................................................................................ 18
FIV Reverse Transcriptase (RT) ................. ......................................................... 18
FIV D eoxyuridine Triphosphate (D U ) ....................................................... ................ 18
F IV Integ rase (IN ) ........................................................................................................... 19
FIV V iral Infectivity Factor (V if)............................................................... ............... 19
FIV Open Reading Frame 2 (Orf2) ........................................................19
FIV R egulator of V iral Protein (R ev)......................................................... ............... 19
H IV /F IV V vaccine M odel ............................... ................................................................... 19
R ationale and G oals for the Proposed Studies................................................... ................ 21
Sp ecific A im s....................................................................................................... ....... .. 2 1

2 THE ROLE OF ANTIBODY IMMUNITY IN THE PROTECTION CONFERRED BY
HIV-1UCD1 P24 VACCINE ..................................................................30

In tro du ctio n ............................................................................................................. ........ .. 3 0
M e th o d s ....................................................................................................... ................... .. 3 1









A n im a ls ............... ... ... .. .. ..... .... ... ..................................................................... 3 1
Serum Collection for B-cell Epitope Mapping by FIV ELISA .................................31
Overlapping FIV P24 Peptides for ELISA.................................................... 32
Im m unoblot A analysis ................................................. ........................... .... ............. 33
Passive-transfer Studies with Antibodies from Vaccinated Cats ...............................33
A ntibody Preparation ................................................. .. ....................... .. .. ...............34
A m m onium sulfate precipitation......................................................... ................ 35
C aprylic acid purifi cation ......................................... ........................ ................ 35
V iru s iso latio n ..........................................................................................................3 6
V N A assay ................................................................................. ...................... 36
Results ............................................................. ... ..................... 37
B -cell E pitope A analysis ............... .. ............................... .. .... .... .... .. .... ..... ............ 37
Passive-transfer Studies: The Analysis of Purified Antibody Preparations for
T ra n sfe r ................. ... .. .......... ........... ... .. ................................................... 3 7
Passive-transfer Studies with Serum and Antibodies from Vaccinated Cats ...............39
D iscu ssio n .............................................................................................................. ........ .. 4 1

3 SUBTYPE A HIV-1 P24 AS AN FIV VACCINE IMMUNOGEN..................................61

In tro d u c tio n ............................................................................................................................. 6 1
M e th o d s ........................................................................................................ ..................... 6 3
V iru s S e le ctio n ................................................................................................................6 3
Co-culturing for V irus A m plification......................................................... ................ 63
Isolation of Proviral D N A ........................ ............................................................ 64
Sequencing and Expression of H IV P24 .................................................... ................ 64
HIV-1N20 primers........................ ....... ............... 65
Restriction enzym e digestion ........................................................ 66
Purification ................................................................................ ....................... 67
Statistical A analyses .................................................................................................... 70
R e su lts .......................................................... ................................................... .................. 7 1
Analysis of HIV-1N20 P24 for Vaccine.......................................................................71
HIV-1 and FIV Sequence Analyses .................... ........................................... 71
V vaccination Studies 1 and 2 ........................................ ................. ...... ... ................ 72
IFNy ELISpot Analysis of PBMC from Vaccination Study 2....................................73
Discussion ...................................................... .................. 74

4 F IN A L D ISC U S SIO N ....................................................... ............................................... 94

L IST O F R EFE R E N C E S ............................................................................................. 97

B IO G R A PH IC A L SK E T C H .................................................... ............................................. 102









6









LIST OF TABLES


Table page

1-1 Current drugs available to treat HIV infection a..................................................... 24

2-2 C current H IV vaccine trials .. ....................................................................... ................ 25

2-1 Passive-Transfer Study 1 with sera from vaccinated and non-vaccinated cats ..............44

2-3 Passive-Transfer Study 3 with purified antibodies from vaccinated and non-
v accin ated cat sera ............................................................................................................ 4 6

2-4 Summary of the results from Passive-Transfer Studies 1-3 .................... ..................... 47

2-5 VNA titers of highly purified antibodies and corresponding pooled serum ......................48

3-1 Vaccination Study 1 with HIV-lucD1 p24 vaccine and commercial Fel-O-Vax FIV
v accin e ............................................................................................................. ....... .. 8 0

3-2 Vaccination Study 2 with HIV-1 p24, FIV p24, and Fel-O-Vax FIV vaccines..............81









LIST OF FIGURES


Figure page

1-1 Proviral genomic organization of FIV, HIV-1, and HIV-2. FIV resembles HIV by
having LTR-gag-pol-env-LTR genomic organization, while missing few regulatory
genes (vpu, vpx, nef). The orf-2 gene was initially reported to express..........................26

1-2 Comparative gag phylogenetic tree of FIV, HIV-1, HIV-2, and SIV. An unrooted
phylogenetic tree was created using the BLOSUM matrix and neighbor-joining
algorithm based on the Kimura two-parameter correction[44], [45]. Support ...............27

1-3 FIV p24 cross-reactivity of the sera from HIV-1 p24-vaccinated cats. FIVBang p24
strip (200 ng/strip) was reacted with individual cat serum at 1:100 dilution. Tested
sera were pre- (pr) and post-3rd immunization(pre-challenge) sera of cats ...................29

2-1 Schedule of vaccination and challenge for passive-transfer studies. Nafve SPF cats
were transfused with antiserum or purified antibodies equivalent to 30% of
recipient's total blood volume (TBV). First transfusion was given on day -1 ...............49

2-2 Schematic of antibody preparation for passive-transfer studies. The figure illustrates
the procedure of three separate passive immunity-transfer studies. In the first study
four groups of two cats each were transfused with IWV ( inactivated...........................50

2-3 B-cell epitope analyses of sera and PBMC from HIV-1 p24-vaccinated cats. Sera
from HIV-1 p24-vaccinated cats (n = 15) and FIV p24-vaccinated cats (n= 14) at 3
weeks after the 3rd vaccination before challenge and from FIV-infected control ............51

2-4 Amino acid sequence alignment of HIV-1 and FIV strains, and B-cell peptide
sequence alignment. Sequence homology analysis between HIV-1uCD1 and FIVBang is
shown with (-) for amino acid homology and (:) for amino acid identity according to.....52

2-5 CBB-stained gel and immunoblot of purified antibody preparations for Passive-
Transfer Study 3. Purified antibodies from HIV-lucD1 p24 (panel A, lanes 1,2,5,6),
HIV-1N20 (panel A, lanes 3,4,8,9) Fel-O-Vax FIV (panel B, lanes 1,2,5,6), FIV p24.......54

2-6 Immunoblot of purified antibody preparations used in the six groups from Passive-
Transfer Study 3. Each lane contained 2 mg of purified preparation. The antibodies
were detected using goat anti-cat IgG heavy and light chain antibodies........................ 56

2-7 IgG concentrations of purified antibody preparations from Passive-Transfer Study 3.
The IgG concentrations of pre-purification pooled serum (Pre) and post-purification
antibody preparations (Post) were determined by commercial feline IgG ....................57

2-8 IgA concentrations of purified antibody preparations from Passive-Transfer Study 3.
The IgG concentrations of pre-purification pooled serum (Pre) and post-purification
antibody preparations (Post) were determined by commercial feline IgG..................... 58









2-9 IgM concentrations of purified antibody preparations from Passive-Transfer Study 3.
The IgG concentrations of pre-purification pooled serum (Pre) and post-purification
antibody preparations (Post) were determined by commercial feline IgG.....................59

2-10 HIV-1 and FIV p24 reactivity of purified antibody preparations from Passive-
Transfer Study 3. The p24 reactivity was based on immunoblot analysis using HIV-
IUCD1 p24 (A), HIV-1N20 p24 (B), FIVpet whole-virus (C), and uninfected FeT-J cell- ...60

3-1 Amino acid alignment of HIV-1N20 p24 (N20) with a subtype-A consensus sequence
(ConsA) and with HIV-1 p24. The stars under each amino acid pair indicate identical
b cases ........................................................................................................... 82

3-2 Schedule for vaccination and challenge for vaccination studies. Vaccinated cats
received three of vaccine at 3-week intervals and received FIV challenge 3-weeks
after the last boost. These animals were challenged IV with FIVFC1.............................. 83

3-3 The full length nucleotide sequence of HIV-1N20 gag. The sequence of HIV-1N2ogag
was amplified from proviral DNA and sequenced by direct sequencing methods.
The forward primer sequence used to amplify the p24 gene is the 18-base sequence ......84

3-4 Purity of PCR product and insert product of HIV-1N20 p24 gene. In panel A, PCR
product amplified from proviral DNA are shown at 0.04 (lane 2), 0.06 (lane 3), 0.08
(lane 4), and 0.1 (lane 5) |tg along with negative control (lane 6) and HIV-1-. .............85

3-5 Schematic ofp24 gene expression in E.coli M 15 cells. ................................ ............... 86

3-6 HIV-1N20 p24 sequence with adapter sequences. HIV-1N20 p24 sequence after PCR
amplification (sequence without bold) was identical to the p24 sequence of the gag
shown in Figure 3-3. The adaptors ligated to the gene create the restriction ...................87

3-7 Immunoblot and silver-stained gel of the HIV-1N20 p24 protein used for Vaccination
Study 2. The immunoblots were reacted with either sheep anti-HIV-1 p24 antibodies
(panel A) or normal sheep serum (panel B). The immunoblot....................................88

3-8 HIV-1 p24-specific IFNy responses of vaccinated cats from Vaccination Study 2.
IFNy responses to overlapping peptide pools of HIV-1 (Hpl-Hpl8) (A, B, C) were
determined by feline IFNy ELISpot assay. The IFNy responses of the PBMC ................90

3-9 FIV p24-specific IFNy responses of vaccinated cats from Vaccination Study 2. IFNy
responses to overlapping peptide pools of FIV p24 (Fpl-Fpl7) (A, B, C) were
determined by feline IFNy ELISpot assay ... ..................................... .................... 92









LIST OF ABBREVIATIONS


HIV:

HIV- UCD1:

HIV-1N20:

HIVlLAV:

FIV:

FIVBang:

FIVpet:

FIVFC1:

Fel-O-Vax:

IWV:

SPF:

LTNP:

KK10:

Gag:

Pol:

Env:

Vif:

Vpu:

Vpr:

Nef:

Tat:

Rev:

RT:

PR:


Human Immunodeficiency Virus

HIV subtype-B strain

HIV subtype-A strain

HIV subtype-B strain

Feline Immunodeficiency Virus

FIV-Bangston (subtype-A gag-pol-envv_-v2, B envy3-v9)

FIV-Petaluma (subtype-A)

FIV-FC1 (subtype-B)

Commercial FIV vaccine, Fort Dodge Animal Health, Fort Dodge, Iowa.

Inactivated whole virus

Specific pathogen free cats

Long term non-progressor

HIV epitope associated with LTNPs

Group specific antigen

Polymerase

Envelope

Viral Infectivity Factor

Viral Protein U

Viral Protein R

Negative Regulatory Factor

Trans-Activator of Transcription

Regulator of Viral Protein

FIV Reverse Transcriptase

Protease









PRIs: Protease inhibitors

IN: Integrase

DU: Deoxyuridine triphosphate

HAART: Highly active antiretroviral therapy

ART: Anti-retroviral therapy

MA: Matrix

CA: Capsid

NC: Nucleocapsid

HVTN: HIV Vaccine Trials Network

Orf2: Open Reading Frame 2

LANL: Los Alamos National Laboratories

NRTI: Nucleoside Reverse Transcriptase Inhibitor

NNRTI: Non-nucleoside Reverse Transcriptase Inhibitors

PI: Protease Inhibitor

FI: Fusion Inhibitors

VNA: Virus-neutralizing antibody

SC: Vaccinated subcutaneously

RID: Radial immunodiffusion

FeT-J: Feline T-cell line developed By J.K. Yamamoto

EU: Endotoxin

TBV: Total blood volume

SFU: Spot forming units

ELISpot: Enzyme-linked immunosorbent spot

pQE30: Expression vector is based on the T5 promoter transcription-translation
system









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

SUBTYPE-A AND SUBTYPE-B HIV-1 P24 BASED FIV VACCINES: EFFICACY AND
MECHANISMS OF CROSS-PROTECTION
By
Marcus Makesi Martin

August 2007

Chair: Janet K. Yamamoto
Major: Microbiology and Cell Science

Published data shows HIV-lucD1 p24 based vaccine protects cats from FIV infection.

Understanding the mechanism of vaccine protection could provide a blueprint to the

development of an HIV-1 vaccine for humans. The studies were performed by evaluating the

protective ability of the vaccine-induced antibody response in three separate passive-transfer

studies. The first involving vaccine antiserum, the second involving ammonium sulfate purified

antibodies, and the third using antibodies purified using ammonium sulfate precipitation and

caprylic acid fractionation. The data suggested that the protective efficacy of HIV-lucD1 p24

(subtype-B) based vaccine was not mediated by vaccine-induced antibody immunity but most

likely mediated by vaccine-induced cellular immunity. In addition protective efficiency varied

between cats vaccinated with subtype-B HIV-UCD1c p24 (67% protection) and subtype-A HIV-

IN20 p24 (25% protection). In addition, IFNy ELISpot analysis using separate peptide pools of

overlapping 15mers of FIV and HIV-1 p24 showed cross reactivity between FIV peptides and

HIV-1 p24-vaccinated cat sera and between HIV-1 peptides and sera from either FIV p24-

vaccination a Fel-O-Vax FIV-vaccinated cats. Some vaccinated cats reacted to peptides

corresponding to the KK10 HIV-1 epitiope in humans, which is associated with long term non-

progressor (LTNP) HIV-1 infected individuals.









CHAPTER 1
INTRODUCTION

Background

Human Immunodeficiency (HIV) and feline immunodeficiency virus (FIV) are members

of the lentiviral family Retroviradae [1],[2]. These viruses are characterized by their ability to

infect terminally differentiated, non-dividing cells such as macrophages; by their genomic

organization of group specific antigen (gag), polymerase (pol), and envelope (env) in addition to

regulatory genes (Figure 1-1); and by their utilization of the enzyme reverse transcriptase (RT) to

synthesize DNA using their RNA genome as a template [1]. The error-prone nature of RT

accounts for the high mutation rate observed among these viruses [2].

Human Immunodeficiency Virus

HIV-1 infection was first reported in 1983 as a retroviral infection of humans [3]. HIV-1 is

the etiological agent of acquired immune deficiency syndrome (AIDS) which causes a

progressive deterioration of the host immune system, characterized by the loss of CD4+ T-helper

lymphocytes. The CD4 receptor is the primary receptor of viral entry into the cell. Other major

receptors utilized by the virus include CCR5 and CXCR4 [4]. Its genome contains three major

genes, consisting of gag, pol, and env. The primary p160 mRNA transcript of HIV-1 is translated

into Gag and Pol proteins. Gag p55 is further cleaved into p24 core protein, p17 matrix protein,

p9 nucleocapsid, and proline-rich p6 protein involved in viral assembly. Pol precursor protein is

cleaved RT, protease, and integrase [5]. The HIV-1 genome also encodes two regulatory

proteins, Tat and Rev, which regulate transcription and viral RNA transport, respectively. The

virus also contains four accessory genes, vif vpu, vpr and nef, which are implicated in viral

pathogenesis [6]. The accessory and regulatory genes are described in detail below.









HIV-1 Viral Infectivity Factor (Vif)

Vif-like proteins are associated with all known lentiviruses with the exception of equine

infectious anemia virus (EIAV) [7]. Vif appears to be needed at the late stage of viral replication

and may suppress antiviral activity of T lymphocytes and macrophages, which are the main cells

infected in humans and cats [8,9]. Macrophages also serve as viral reservoirs of infection even

after the acute stage of infection. It is believed that Vif is important for the retention of viral

infectivity because it acts as an inhibitor to the antiviral pathway involving APOBEC3G, a

messenger RNA editing enzyme [10]. Vif may prevent the editing of the early single strand

product of reverse transcription. It is also believed that this activity results in the prevention of

many mutations, which could compromise the key structural proteins, regulatory proteins, and

enzymes and as a result, aids in retaining viral infectivity.

HIV-1 Viral Protein U (Vpu)

Vpu protein promotes the degredation of the CD4 antigen in the host cell, and this prevents

the binding of CD4 molecules to viral gp 120 in the infected cells [11]. Vpu also causes the

formation of ion channels in the surface membrane of the infected cells. The ability of Vpu to

form these channels seems to correlate with the cell's ability to release the virus [12].

HIV-1 Viral Protein R (Vpr)

Vpr protein appears to be involved in viral pathogenesis and is important in the infection of

macrophages [13]. Vpr is also involved, to a lesser extent in the infection of other cells. This

protein causes host cell division to stop at the G2 stage, and induces the apoptosis of infected

cells. Vpr acts as a shuttle protein from the cytoplasm to the nucleus by facilitating the transfer of

the pre-interaction complex through the nuclear pores.









HIV-1 Negative Regulatory Factor (Nef)

Nef protein is synthesized very early in HIV infection and promote viral fitness through

multiple mechanisms [14]. First, in order to prevent super-infection of the infected cells, Nef

causes the incorporation and destruction of surface CD4 in the lysosomes. Second, this protein

causes the down regulation of the major histocompatability complex-I (MHC-I) expression,

which affects antigen presentation, and thereby reduces the destruction of infected cells by

cytotoxic T lymphocytes. Third, Nef increases viral infectivity by inducing the infected

macrophages to secrete MIP-1 alpha and MIP-1 beta. These chemokines have a chemotaxic

effect on resting CD4+ T lymphocytes, causing the migration of the cells to the site of infection.

This process in turn increases the chance for these cells to become infected, since free virions do

not retain infectivity very long when circulating in the blood [7,15].

HIV-1 Trans-Activator of Transcription (Tat)

Tat protein is a spliced gene product of 14 kD in size [2].Tat protein interacts with tat

responsive element, an RNA loop structure on the 3' end of the viral long terminal repeat (LTR),

to upregulate HIV gene expression [16].

HIV-1 Regulator of Viral Protein (Rev)

Rev protein is 19 kD and is the product of doubly spliced mRNA. Rev protein is involved

in the regulation of viral RNA expression [2].

HIV-1 Classification

There are two types of HIV presently known to exist: HIV-1 and HIV-2 [Figures 1-1 and

1-2] [17]. Based on the genetic sequences, HIV-1 has been classified into three groups: the M

(major) group, 0 (outlier) group, and the N (non-outlier) group. HIV-1 M group is further

divided into nine subtypes (A-D, F-H, J, and K) [18]. The majority of HIV infections worldwide









are the result of M group viruses [18]. There is also an increasing amount of infection caused by

inter- and intra-subtype recombinants [19].

HIV-1 Treatment

When HIV-1 was first identified over two decades ago, there were no effective treatments

available. There were also few treatments for the secondary opportunistic infections, which are

associated with HIV-1 infection. Presently there are several drug options for HIV-1-positive

individuals. A number of drugs first approved for HIV-1 therapy were nucleoside analogs,

inhibitors of RT activity. Subsequent classes of drugs included protease inhibitors and non-

nucleoside RT inhibitors followed by more recent drugs shown in Table 1-1. The use of two or

more of these drugs together was initially called highly active antiretroviral therapy (HAART)

and more recently renamed as anti-retroviral therapy (ART) [20]. ART has been very effective in

prolonging the life expectancy of infected individuals and can reduce the amount of circulating

virus to nearly undetectable levels. During drug induced remission, the virus may still remain

dormant in viral reservoirs within the host, such as the testes, brain, and retina [21]. Financial

constraint has been the major limiting factor for these drugs to have a major global impact. With

most of the HIV-infected individuals residing in developing countries, a desirable strategy to

arrest the viral spread would be by the development of an effective prophylactic vaccine.

Feline Immunodeficiency Virus

FIV is a lentivirus of domestic cats, which causes an immunodeficiency syndrome

strikingly similar to HIV infection in humans [22]. This similarity between these two viruses

extends to the genome organization of 5'- LTR-gag-pol-env-LTR-3' and the presence of select

regulatory genes (Figure 1-1) [9].

The level of FIV infection of domestic cats worldwide has been estimated to be between

1% to 28% [23]. FIV variants have also been classified into five subtypes A-E [23]. FIV









infection usually results in CD4 T lymphocyte reduction [24]. Similar to HIV strains, the

virulence of FIV strains varies [23]. Like HIV-infected individuals, there is also the presence of

long term non-progressor (LTNP) subjects among FIV-infected cats [23]. The LTNP cats remain

disease free for a period of time, which is well over the mean disease onset of acute and

moderate progressors.

FIV has a broad tropism by infecting both CD4+ and CD8+ T lymphocytes, B cells,

circulating monocytes, and monocytes of the CNS [25]. The tropism of the virus is determined

by the viral envelope characteristics. FIV has the same genomic organization as most other

lentiviruses with coding sequences of the gag, pol, and env, in addition to accessory genes

flanked by open reading frames (ORFs) [7].

FIV Group Associated Antigen (Gag)

The Gag polyprotein is approximately 50 kD and is produced by translation of unspliced

viral genomic RNA transcript. This polyprotein is cleaved by viral protease, into three proteins

matrix (MA, 15kD), capsid (CA, 24 kD), and nucleocapsid (NC, 7 kD) [7]. The importance of

including Gag proteins into a potential HIV vaccine is now recognized throughout the HIV

vaccine community. The current trials in the HIV Vaccine Trials Network (HVTN) all

incorporate gag gene or Gag protein (Table 1-2). The presence of many conserved epitopes

coupled with the strong immune response stimulated by this protein support its use as a vaccine

immunogen [26]. Many T-cell epitopes are located within the Gag region. These epitopes would

be important to facilitate the removal of infected cells in order to stem the spread of the infection.

FIV Envelope (Env)

FIV Env is the product of a singly spliced 4.4 kD strand of mRNA [7], [27,28]. The Env

glycoprotein precursor is composed of SU (95 kD) and TM 39 (kD), and these two glycoproteins

are assembled on the viral membrane as non-covalently bonded surface glycoprotein. As is the









case with HIV, FIV SU is the most divergent of the viral proteins [9], and mutations of this

glycoprotein can affect the tropism of the virus.

FIV Polymerase (Pol)

Thepol gene region of the ORF encodes a polyprotein, which is cleaved by FIV protease

(PR) into four proteins: PR, RT, integrase (IN), and deoxyuridine triphosphate (DU) [7]. The

cleavage order of these proteins affect the number of infectious virions produced [29].

FIV Protease (PR)

Protease is a viral enzyme, which processes the Gag and Gag-Pol polyproteins to the final

viral components. Comparison between FIV and HIV-1 PRs have revealed common amino acid

(aa) recognition sites, facilitating the utilization of experimental protease inhibitors against both

lentiviruses [30]. However, the majority of commercial HIV-1 protease inhibitors (PRIs) do not

work against FIV PR.

FIV Reverse Transcriptase (RT)

FIV RT is composed of a 61kD polypeptide and is the product of cleavage of the Gag-Pol

polyprotein by FIV protease [7]. There is a significant sequence homology between HIV-1 RT

and FIV RT, and FIV RT is also sensitive to the nucleoside analogs utilized in the treatment of

HIV-1 [31]. FIV RT may also undergo mutations, which result in resistance to these drugs [31].

FIV Deoxyuridine Triphosphate (DU)

FIV DU is a protein trimer consisting of 14.3kD subunits [32]. DU exists in both

eukaryotic and prokaryotic cells, facilitating the hydrolysis of dUTP to dUMP to prevent the

miss-incorporation of deoxyuridine during the synthesis of DNA. Low levels of DU in the

infected non-dividing cells can reduce FIV replication. The virus overcomes this obstacle by

producing its own DU and thereby enhancing its fitness [7].









FIV Integrase (IN)

FIV IN is roughly 32kD and is derived from the C-terminus of the precursor Pol protein

[7]. The C-terminus of IN is important to the interaction of the enzyme with DNA. It is

responsible for site-specific cleavage of the ends of FIV DNA, and for the transfer and

disintegration of DNA strands [33].

FIV Viral Infectivity Factor (Vif)

Vifis produced from spliced RNA about 5.2 kB long [7]. Vif gene is found in all

lentiviruses except EIAV. The size and genome location are also well conserved. This gene

affects the infectivity of the virions by mechanisms thought to be similar to those of HIV-1 [34].

FIV Open Reading Frame 2 (Orf2)

FIV Orf2 is the transactivating factor of FIV [7] [35]. It is a 9.3 kD protein product of

multiply spliced strands of mRNA. Orf2 contains an N-terminus cystene-rich region but lacks the

basic domains which are essential for HIV-1 Tat activity. Recent studies suggest that Orf-2 may

also serve as Vpr [36].

FIV Regulator of Viral Protein (Rev)

Rev protein facilitates the triggering of late gene expression in the virus life cycle [37]. It

promotes nuclear export of mRNAs enabling their translation. Similar to HIV-1, FIV Rev is

mediated by the rev responsive element, which is located on the 3' end of target mRNAs. Rev is

involved in nuclear localization and the transfer of genetic material in and out of the nucleus.

HIV/FIV Vaccine Model

FIV is similar to HIV in morphology, genetic sequence, and pathogenesis [9]. In light of

this, vaccine approaches, which are effective against one of these viruses, are likely to provide

valuable insights into the other. Both HIV and FIV have common challenges of high mutation

rate due to the error prone nature of viral RT, inter- and intra-subtype recombinations, and the









need to generate broad vaccine protection against a range of different subtypes [38]. Cytotoxic T

lymphocytes (CTL) responses to HIV and FIV are mediated by CD8+ Tcells, which target

epitopes on the whole virus. Several of these epitopes target have been identified on Gag and

Env [23]. The period of time until the onset of clinical symptoms may vary between strains and

between subjects. There is however a significantly greater numbers of LTNP cats infected with

FIV. This may be due to the fact that HIV is believed to only have been discovered in the human

population for about two decades, while FIV has evolved with its host much longer in

evolutionary time [5,9]. HIV Gag protein appears to be less divergent phylogenetically than FIV

and simian immunodeficiency virus (SIV) (Figure 2). The epitopic characters of the attenuated

strains may well provide insights into the ideal targets for vaccine design. Currently the average

survival time for infected individuals has increased considerably causing the CDC to launch new

studies to recalculate this figure [39].

In 1993 at University of California at Davis, researchers in the School of Veterinary

Medicine were able to elicit a protective immune response in 96% of cats immunized with

whole-cell or cell-free FIV vaccines and subsequently challenged with heterologous FIV [40].

This was the first instance of an effective vaccine formulation against an AIDS lentivirus. The

exact nature of the protective response was not certain. The next step was to determine the

mechanism of the protective response. The protection observed initially appeared to correlate

with a strong humoral response, including in the generation of virus neutralizing

antibodies(VNAs) [41]. However, the VNA titer of the study animals did not correlate

completely with protection.

One of the first viral proteins to be recognized by the host immune system is the core p24.

There are also several CTL epitopes found on core p24 [42]. This protein is the structural









component of the viral capsid and is relatively conserved compared to the viral Env. HIV-1 and

FIV p24 have 31% aa identity and 63% aa homology based on length adjusted analysis. The

identity observed with p24 was much higher than those for SU, TM, and MA. Therefore, it was a

good target for a vaccine immunogen to explore the concept of evolutionarily conserved epitopes

among lentiviruses. The use of HIV-1 p24 as a vaccine immunogen facilitated the formation of

cross-reactive antibodies to FIV (Figure 1-3), despite having only 63% homology. Thus, the

possible utility of HIV-1 p24 protein as vaccine immunogen against FIV was noted, and this led

to the following evaluations.

Rationale and Goals for the Proposed Studies

Following immunization with a subtype-B HIV-IUCD-1 p24 vaccine, 82% of specific

pathogen free (SPF) cats were able to elicit a successful protective immune response to challenge

with FIV [38]. The 31% aa identity between HIV-1 and FIV p24 sequences in combination with

the cross-protective ability of the HIV-1 p24 vaccine suggested that there may be some

protective epitopes within the regions of homology, and these may be evolutionarily conserved

between the two lentiviruses. Based on these observations, our hypothesis is that some of these

epitopes conserved among the lentiviruses may serve as potential vaccine immunogens against

FIV. This hypothesis will be tested by performing the studies for the following specific aims: 1)

determine if humoral immunity is responsible for the protection conferred by HIV-lucD1 p24

vaccine against FIV and 2) determine if cross-protection against FIV is conserved among

different subtypes of HIV-1.

Specific Aims

Determine if humoral immunity is responsible for the protection conferred by HIV-

1UCD1 p24 vaccine against FIV: Cross-reactive antibodies to HIV-1 proteins were generated

in cats infected with FIV [38](Figure 3). This implied that there were some common epitopes









conserved between both viruses despite their evolution in different host species. The strongest

area of cross reactivity observed by HIV-1 whole-virus western blot was core p24 protein. Hence

the role that HIV-1 p24 antibodies play in cross-protection against FIV was evaluated. The

following two experimental approaches were undertaken to achieve this goal.

A. B-cell mapping of cross-reactive antibodies to FIV p24.

B. Protective efficacy of the antibodies from FIV p24-vaccinated cats was determined in vivo
by passive-transfer studies against FIV challenge.

In order to identify the cross-reactive B-cell epitopes, sera from HIV-1 p24-vaccinated cats

were analyzed by FIV p24 peptide-based ELISA. The substrates for the ELISA consisted of 28-

30mer peptides that spanned FIV p24 with 11-13 aa overlaps. The published B-cell and T-cell

epitopes available from Los Alamos National Laboratories (LANL) were compared to those on

the vaccine p24 antigen and the challenge virus. Since the p24 capsid of the virus is not

expressed on its surface such as its Env, it is unlikely that it would elicit a protective B-cell

response. However, this arm of the immune system could be used to identify T-cell epitopes,

such as T-helper epitopes for B-cell responses and possibly for CTL activity.

Determine if p24 from subtype-A HIV-1 with a different epitope repertoire can be

utilized as a vaccine antigen: The hypothesis proposed for this dissertation is that some of the

p24 epitopes conserved among HIV-1 subtypes may work together as a vaccine immunogen

against FIV. Whereas, the central hypothesis of our laboratory is that some of the cross-

protective HIV-1 epitopes, prevalent among viral structural and enzymatic proteins, against FIV

should be useful as vaccine epitopes against HIV-1 in humans. As of yet, there has been no

studies demonstrating the epitopes found on HIV-1 p24 proteins are all protective against FIV.

For this reason, p24 from subtype-A HIV-1 will be used to test our immediate hypothesis by

identifying the protective p24 epitopes, which are conserved among subtypes A and B of HIV-1.









Furthermore, the studies proposed for this dissertation are the first step towards answering the

central hypothesis of our laboratory. Hence, the effort to identify cross-protective HIV-1 epitopes

against FIV may provide a blueprint for the development of HIV-i/AIDS vaccines against HIV-

1 subtypes prevalent worldwide. Overall, our studies will not only benefit the development of

second-generation FIV vaccines for veterinary medicine but will also contribute to the

development of a HIV-1 vaccine for human medicine.









Table 1-1. Current drugs available to treat HIV infection a


Drug Class
Nucleoside Reverse
Transcriptase
Inhibitor (NRTI)


Non-nucleoside
Reverse
Transcriptase
Inhibitors (NNRTI)

Protease Inhibitor
(PI)




Fusion Inhibitors
(FI)



CCR5 Antagonists


Drug Name
Lamivudine (3TC), Zidovudine (AZT),
Zalcitabine (ddC), Stavudine (d4T),
Abacavir (ABC), Tenofovir (TDF)
Didanosine (ddl)

Efavirenz (EFV), Nevirapine (NVP),
Delaviridine (DLV)



Nelfinavir (NFV), Amprenavir (APV),
Fosamprenavir (FPV), Saquinavir (SQV),
Lopinavir (LPV/r), Ritonavir
(RTV),Atazanavir (ATZ), Indinavir
(IDV)Tipranavir (TPV)

Enfuvirtide (EFV)




Maraviroc (FDA approved April 24, 2007)


Drug Mechanism
Nucleotide or nucleoside
analogues lacking a
hydroxyl group at the 3' end.


Binds to HIV-1 RT and
alters the active site to
reduce nucleotide binding.


Mimics Gag-Pol cleavage
site and competes with
protease, causing the
production of immature
non-infectious virions.

Binds to gp41 and blocks
the fusion of the virus and
cellular membrane.


Blocks binding to CCR5


a Adapted from Sierra S. et al.2005 and updated with newly approved anti-HIV drugs [43].











Table 2-2. Current HIV vaccine trials
Phase Producer Product Name Prime Formulation Constituents
/ Sub- Gag Pol Env Nef Tat Rev Vpr Vpu PR RT
type
I/B Sanofi ALVAC + + + + +
Pasteaur vCP1452


I/B Chiron




I/B Merck


I/B Therion


I/B Wyeth

I/B Wyeth


Gag and Env +
DNA/PLG
Microparticles


MRKAd5
HIV-1 Gag

TBC-M358
TBC-M335

GENEVAX
gag-2962
GENEVAX
gag-2962


+ + + + + +

+


I/B Pharmexa- EP-1043 +
Epimmune EP HIV-1090

I/B GeoVax HIVB DNA
pGA2/JS7 #2

I/Ba NIH VRC VRC-ADV-
014/ VRC-
ADV-009

I/C AlphaVax AVX-201


Pharmexa-
Epimmune


I/B UP C

I/B Merck


EP HIV-1233 +


PENNVAX-B +

MRKAd5 +


+ + +


+ +


+ +


+ +




+ + +

+ + +


+ + +


+ +


+ +


+


I/A NIH VRC VRC-
HIVDNA-044
II/Bb NIH VRC VRC-
HIVDNA-016


II/B Merck

II/B Merck


MRKAd5
Trivalent
MRKAd5
Trivalent


+ + +


+ +

+ +


a Also contains clade A,B,C env.
b Includes multiple specific epitopes.
University of Pennsylvania.











LTR

I I gag


if or2 rev RRE


elv RRE


HIV-1


at ne


vpr


HIV-2 ~


vpr vpx


Figure 1-1. Proviral genomic organization of FIV, HIV-1, and HIV-2. FIV resembles HIV by
having LTR-gag-pol-env-LTR genomic organization, while missing few regulatory
genes (vpu, vpx, nef). The orf-2 gene was initially reported to express 79 aa Tat-like
protein. However, recent studies suggest that this gene may also serve as vpr based on
its gene product promoting the transport of the DNA pre-integration complex into the
cell nuclei. Diagram adapted from Sauter, S.L, et al. 2001 [7].


LTR

ErIl









Figure 1-2. Comparative gag phylogenetic tree of FIV, HIV-1, HIV-2, and SIV. An unrooted
phylogenetic tree was created using the BLOSUM matrix and neighbor-joining
algorithm based on the Kimura two-parameter correction[44], [45]. Support at each
internal node was assessed using 1000 bootstrap samplings and each tree was
visualized using Tree View. Branch lengths, drawn to scale, are based on number of
synonymous substitutions per site. The gag tree is based on sequences from NCBI
GenBank (FIV accession numbers NC_001482, AY684181, M36968, D37820,
DQ365596, AY13911, AY139112, AY139110, D37819, D37821, D37823, D37824,
AF474246, AY679785, D37822, AB027302, AB027304, AB027303; SIV accession
numbers AF301156, L06042, AF468659, AF075269, AF131870, M27470,
AF328295, 349680, M30931, L40990, M66437, U58991, AF334679, AF077017,
M80194, U72748, U79412, M32741, M33262, M19499, M83293, AJ271369,
AF103818, U42720; HIV-1 accession numbers L39106, M62320, M38429, M93258,
M17449, L31963, AF004394, U43096, U23487, AY679786, AF321523, AB023804,
M22639, K03454, U88826, AF005496, AJ006022, L20587, AJ302647, L20571;
HIV-2 accession numbers M31113, AF082339, M30502, D00835, X52223, J03654,
M30895, Z48731, X05291, J04498, U22047, X62240, U27200, L07625, U75441,
AF208027). HIV-1 distribution is shown in major groups (Group 0 for outlier group,
Group M for main group; Group N for non-M group); while HIV-2 distribution is
shown in subtypes (subtypes C, D, and E missing). SIVcol, SIVSYK, SIVGSN, SIVHOEST,
SIVsuN, SIVMND, SIVRCM, SIVsM, and SIVAGM are SIV species isolated from Colobus,
Sykes, greater-spot-nosed, L'Hoest, sun-tailed, mandril, red-capped mangabeys,
sooty mangabeys, and African green monkeys, respectively. SIVmac belongs to SIV
SM group and are isolates from captive macaques [46]. The gag phylogenetic tree
shows close relationship between SIVsM/SIVmac and HIV-2 and between SIVcpz and
HIV-1, but distant relationship between FIV and primate AIDS lentiviruses. This
figure was adapted from Yamamoto, J.K, et al. 2005.














Post
a #- Vaccination
[ LIN 626 R9 J V1= 1st
+ V2 = 2nd
V Pr V1 V 2 IV 3 Pr I V1 V 2 V 3 Pr V3 Pr 'V3 IPr V3 V3 = 3rd

p24 -"*" .... H E = 11 1 1 11 l o,.w,.


Figure 1-3. FIV p24 cross-reactivity of the sera from HIV-1 p24-vaccinated cats. FIVBang p24
strip (200 ng/strip) was reacted with individual cat serum at 1:100 dilution. Tested
sera were pre- (pr) and post-3rd immunization(pre-challenge) sera of cats vaccinated
with either HIVILAV or HIVlucD1 p24 proteins. In addition, serum reactivities of
post-1st and 2nd vaccinations are shown for cats #ID4 and #626. Based on
immunoblot and ELISA analyses, approximately 80% of HIV-1 p24-vaccinated cats
developed cross-reactive antibodies to FIV p24.









CHAPTER 2
THE ROLE OF ANTIBODY IMMUNITY IN THE PROTECTION CONFERRED BY HIV-
1UCD1 P24 VACCINE

Introduction

Many of the challenges faced during the development of an FIV vaccine are common to

the development of an HIV-1 vaccine [23]. Therefore, the insights derived from the

development of an FIV vaccine could also lead to clues in developing an effective HIV-1

vaccine. Identifying the epitopes for vaccine protection and the immunity required for such

protection are the two major aims of the proposed project on subtype-A HIV-1 p24-based FIV

vaccine. This section initiates the examination of the role of vaccine-induced antibody immunity

in p24 vaccine protection. Antibodies generated by vaccination were examined for patterns of

FIV and HIV-1 p24 epitope recognition among protected/vaccinated cats, unprotected/vaccinated

cats, and infected control cats. The vaccine immunogens consisted of subtype-A HIV-1 p24,

subtype-B HIV-1 p24, FIV p24, and as a control, inactivated dual-subtype FIV whole-virus (Fel-

O-Vax FIV vaccine, Fort Dodge Animal Health, Fort Dodge, Iowa). The reactivity of these sera

was evaluated by western blot analysis for antigen specificity, ELISA for level of affinity to

native immunogen and peptides, VNA assay for biological activity, and B-cell epitope mapping

to identify vaccine epitopes of protection. Overall, the structural and functional analyses of the

B-cell epitopes on the HIV-1 p24 were performed to determine the significance of HIV-1 p24

vaccine-induced antibodies in cross-protection against FIV.

In addition to the in vitro functional analysis, the vaccine-induced antibodies were tested

for in vivo efficacy against FIV challenge using passive-transfer studies (Table 2-1, 2-2, and 2-

3). Passive-transfer studies consisted of intravenous transfusion with vaccine-induced antibodies

into naive SPF cats one-day prior to and one day after FIV challenge (Figure 2-1). These cats

were then monitored for the levels of infection and FIV-specific antibody development. Three









separate passive-transfer studies were performed. The first study was performed using pooled

serum from vaccinated donor cats as the transfer material. The second study involved the use of

donor antibodies, which were partially purified by ammonium sulfate precipitation method. The

third study was performed using material purified by both ammonium sulfate precipitation and

caprylic acid fractionation methods (Figure 2-2). The purity and activity of these antibodies were

tested prior to transfusion.

In order to identify the B-cell epitopes responsible for the cross-reactive antibodies, B-cell

epitope mapping was performed using sera derived from both protected and unprotected cats,

which were vaccinated with HIV-1 p24. The intent of these studies was to examine the FIV p24

epitope reactivity of these different groups of animals and to determine if there was a particular

profile, which was associated with infection or vaccine protection [38].

Methods

Animals

Specific pathogen free (SPF) cats were obtained from Liberty Research (Waverly, New

York) or Harlan Sprague Dawley, Inc. (Indianapolis, Indiana). All of these animals tested

negative for FIV, toxoplasma, and feline leukemia virus, and they were maintained at the

University of Florida experimental animal care facility under SPF conditions during vaccination

or prior to FIV infection. Animals for FIV-challenge studies were transferred to BSL-2 facilities

before FIV inoculation.

Serum Collection for B-cell Epitope Mapping by FIV ELISA

The animals were vaccinated subcutaneously (SC) at 3-week intervals with FIV-lUCD1 p24

or FIV-Petaluma (FIVpet) p24 vaccine. The vaccine consisted of the p24 protein (200-250

[tg/dose) in Ribi adjuvant supplemented with 5 [tg/dose of human IL-12. In addition, sera from

FIV-Bangston (FIVPBang)-infected cats at 18 weeks post-challenge (wpc) were used as positive









control sera, while negative control sera were from uninfected SPF cats. Whole blood was

centrifuged at 2000 rpm for 20 minutes, and the serum was collected and stored at -200C until

use.

Overlapping FIV P24 Peptides for ELISA

Overlapping 28-30mer peptides of FIV p24 with 11-13 aa overlaps were designed using

LANL PeptGen program and synthesized by SynPrep Corp. (Dublin, California) based on the

p24 aa sequence of FIVBang, with the exception of four peptides designated FBI, FB2, FC2, and

FB4, which were derived from FIVFC1 (Figure 2-3). These were pre-screened at the company by

HPLC for purity and were determined to be > 95% pure. The peptides were diluted in coating

buffer (0.35 M sodium bicarbonate solution, pH 9.4). ELISA was performed by standard

laboratory method [40]. ELISA plates were coated with 400 ng of the appropriate peptide in 50

[IL of coating buffer per well and incubated overnight at 4C. These wells were subsequently

blocked for 1 hour with 200 [L of blocking solution (5% milk protein in PBS), washed 3X with

wash buffer (150 mM NaCl and 0.05% Tween 20), and incubated with 1:200 dilution of cat

serum for 1 hour at 37C. The wells were then washed 3X with wash buffer and incubated with

100 [iL of biotinylated anti-cat IgG (Vector Laboratories, Burlingame, California), diluted

1:5000 in Buffer 3 (3% BSA/PBS with 0.05% Tween-20) with 5% milk for 1 hour at 37C. After

incubation the plates were washed 3X, in wash buffer and incubated for 1 hour at 37C with 100

[IL per well strepavidin-conjugated horseradish preoxidase (Vector Laboratories). The plates

were then washed 3X and 100 [L of substrate solution (0.005% tetramethylbenzidine and

0.015% H202 in 0.96% citric acid solution) was added to each well and incubated for 15

minutes. The reaction was stopped by the addition of 100 [L per well of 0.12 % hydrogen

fluoride solution.









Immunoblot Analysis

The sera from vaccinated and unvaccinated cats before and after FIV challenge were tested

for reactivity to immunoblot strips, each containing 200 ng of virus proteins. Immunoblot strips

were incubated individually with 1:50 dilution of cat serum in Buffer 3 with 5% milk. The

control sera consisted of serum from an SPF cat for negative control and serum from an FIV-

infected cat for positive control. The remaining immunoblot method was performed using

laboratory protocol [41].

Passive-transfer Studies with Antibodies from Vaccinated Cats

Three separate passive-transfer studies were undertaken. In Passive-Transfer Study 1,

four groups of four cats were IV transfused with saline or pooled sera from either HIV-lCD1c

p24-vaccinated cats, Fel-O-Vax FIV-vaccinated (Fel-O-Vax-vaccinated) cats, or non-vaccinated

cats. These sera were collected from the SC-vaccinated donor cats 2-3 weeks after the 3rd

vaccination and before FIV challenge (vaccinated donors in Chapter 3). All pooled sera were

inactivated by incubation at 560C for 40 minutes. In order to prevent serum reaction, the pooled

serum was cross-matched with each recipient cat's blood. Each cat received a cross-match-

negative serum equivalent to 30% of the recipient's total blood volume. The amount of volume,

including serum protein level, was too large to administer in a single transfusion. For this

reason, cats received 20% volume in the first transfusion and 10% volume in the second

transfusion. The first transfusion was given on day -1 with FIV challenge on day 0 and second

transfer on day 1 (Figure 2-1). These cats were challenged IV with FIVpet (10 mean cat

infectious dose, CID5o).

In Passive-Transfer Study 2, the antibodies were purified from pooled serum by

ammonium sulfate precipitation, which partially purifies for IgG. SPF cats were transfused with

saline or partially-purified antibodies of pooled serum from HIV-lucD1 p24-vaccinated cats,









FIVpet p24-vaccinated cats, Fel-O-Vax-vaccinated cats, or non-vaccinated cats. In Passive-

Transfer Study 3, the antibody purification was taken further using a two-step purification

method involving caprylic acid fractionation followed by ammonium sulfate precipitation. After

these procedures were completed, the antibodies were dialyzed and sterilized by 0.45 |tm

filtration. Tests for biological activity, purity, and toxicity were performed on the material prior

to passive transfer. The vaccine specificity of the antibodies in Study 3 was identical to those of

Study 2, except for the additional antibody group derived from uninfected FeT-J cell/lysate-

immunized (FeT-J cell-immunized) cats. FeT-J cells were the cells used to produce the viruses

for the Fel-O-Vax FIV vaccine. These cats received a combined cell and cell lysate

immunization using SC (2x106 cells/dose), intradermal (250 [tg/dose cell lysate), intranasal (100

[tg/dose cell lysate), and transcutaneous (100 [tg/dose cell lysate) routes (see Chapter 3 for

additional detail). All immunizations were administered at 3-week intervals. The passive-

transfer schedule and transfusion procedure for both Studies 2 and 3 were identical to the one in

Study 1. Cats in Studies 2 and 3 included groups challenged IV with FIVFcl (Study 2, 15 CID5o;

Study 3, 10 CID5o) and another set of groups challenged IV with FIVpet (25 CID5o).

Antibody Preparation

Blood was collected from HIV-lCD1c p24, HIV-1N20, FIV p24, and FeT-J cell lysate-

immunized donor cats and from SPF cats in either serum or heparinized collection tubes. The

whole blood was centrifuged at 2000 rpm for 20 minutes and the serum or plasma was collected.

The sera were pooled according to immunization group and purified for IgG by ammonium

sulfate precipitation alone (Study 2) or in combination with caprylic acid fractionation (Study 3)

(Figure 2-2). The pooled serum and purified antibody preparations were tested by immunoblot

and radial immunodiffusion (RID) assay to determine the reactivity to the target immunogens









and to determine the immunoglobulin levels (IgG, IgA, IgM), respectively. Commercial RID

assay for feline IgG, IgA, and IgM (Bethyl Laboratories, Montgomery, Texas) were performed

according to the manufacturer's protocol.

Ammonium sulfate precipitation

The supernatant from caprylic acid purification was cooled to 4C then placed into a

container and stirred slowly using a magnetic stirring bar. Ammonium sulfate was added to each

sample 0.65 g/mL (Fisher Scientific Pittsburg PA) to achieve 100% saturation. Solid ammonium

sulfate was slowly added to the antibodies and the mixture was stirred at 4 C overnight.

Following overnight incubation, the sample was centrifuged (20g) for 20 minutes at 4C. The

solid fraction was pooled and dissolved in an equal volume of H20. The fractions were then

dialyzed against PBS using 600-800 MW Spectra/ Por (Sprectra Laboratories, Inc., Rancho

Dominguez, California) [47].

Caprylic acid purification

Serum samples were pooled for each group, transferred to a clean container, and mixed

with 60 mM of sodium acetate (Sigma-Aldrich USA, St. Louis, Missouri) solution at pH 4. Each

pool of antibodies was mixed with 0.75 mL caprylic acid (Sigma Aldrich USA) per 10 mL of

original sample volume. This mixture was stirred for 30 minutes and then centrifuged at 5000g

for 10 minutes. The supernatant was decanted and subsequently dialyzed against PBS using 600-

800 MW Spectra/Por (Sprectra Laboratories. Inc.) [47]. The antibodies were then concentrated to

half the previous volume using a Bio-Rad Ultra concentrator. The final product was filter

purified using a 0.45 [tm filter. The product was tested by western blot, ELISA, and gel analysis

using Coomassie Brilliant Blue (CBB) stain and silver stain to determine purity, and by VNA

assay to determine retention of biological activity [48].









Virus isolation

Whole blood was collected from the cats in passive-transfer studies at 3-4 week intervals.

The peripheral blood mononuclear cells (PBMC) purified by ficoll-hypaque gradient technique

[8] were used to determine the FIV infection status. These cells (1.5x106-3x106 cells) were co-

cultured with indicator cells (3x106 cells) in 3 mL of culture media for approximately 3 weeks.

The cells were recultured every 3 days and the spent culture fluid from each passage day was

collected. The culture fluid was then tested for RT activity [7]. The indicator cells consisted of

PBMC from SPF cats, which were stimulated with 5 [tg/mL of concanavalin A (Con A) for 3

days and recultured every 3 days in fresh culture media for 6-12 days before use in the assay.

VNA assay

Blood from the cats in passive-transfer studies were collected pre- and post-challenge at 3-

4 week intervals. The serum from blood was separated by centrifugation at 2000 rpm for 20

minutes, and the serum was collected and frozen until the day of assay. VNA assay was

performed in accordance with established laboratory protocol [6]. On the day of assay, the

serum was incubated at 560C for 30 minutes to inactivate the complement. Serial dilutions of the

serum in culture media were incubated with 200 TCID50 of virus for one hour before addition of

0.25 mL mixture to 0.5x105 indicator cells in 0.25 mL/well. The final serum dilutions ranged

from 1:10 to 1:10,000, and the final virus titer was at 100 TCID5s. The cells were recultured

every 3 days in fresh culture media for 18-21 days, while the spent culture fluid was collected

and later tested for FIV levels by RT assay. The indicator cells were prepared the same way as

those used in the virus isolation assay.









Results


B-cell Epitope Analysis

ELISA analysis of the reactivity of HIV-1 p24-vaccinated cat serum to the overlapping 28-

30mer peptides of FIVBang p24 with 11 aa overlaps showed highest reactivity to peptide 71-100

(FB4) followed by peptide 197-223 (FBI 1). Less reactivity was also observed to peptide 53-81

(FB3), 161-188 (FB9), and 178-207 (FB10) (Figure 2-3). This indicates that at least 5 cross-

reactive B-cell epitopes are induced by vaccination of cats with HIV-1 p24. However, these

cross-reactive peptides did not correspond to the major homology regions (MHR) found among

FIV, SIV, and HIV [49-52] (Figure 2-4). As expected, FIV p24-vaccinated cats had the most

serum reactivity to FIV p24 peptides (11 of 12 peptides), while FIV-infected cats had a

moderate-to-high frequency of serum reactivity to three FIV p24 peptides (FBI, FB4, FB 11).

FB4 and FB 11 peptides, which reacted with the highest frequency to sera from infected cats, also

reacted with highest frequency to sera from HIV-1 p24-vaccinated cats. This observation

suggests that both vaccination with HIV-1 p24 and infection with FIV stimulate a similar B-cell

repertoire to FIV p24 epitopes. No VNA activity was observed with antibodies generated to

HIV-1 p24 and to FIV p24 (data not shown). In contrast, VNA activity was observed with sera

from dual-subtype FIV-vaccinated cats (both prototype and commercial dual-subtype FIV

vaccines tested, data not shown).

Passive-transfer Studies: The Analysis of Purified Antibody Preparations for Transfer

Greater than 90% of the contaminating serum proteins were removed with the use of

caprylic acid/ammonium sulfate precipitation procedure (Figure 2-5). This is evident from both

the CBB-gel and immunoblot analyses of the purified antibodies and the corresponding pre-

purification sera from each vaccination group. Albumin was used as a measure of contamination

in the purified antibody preparations. Albumin levels were determined by immunoblot analysis









using commercial polyclonal antibodies to feline albumin (Bethyl Laboratories). Purified

antibody preparations contained <95% albumin when compared to the amounts in corresponding

pre-purification sera (Figure 2-5). Thus, >90% of the contaminating proteins were removed

according to the minor albumin contamination remaining and few weak bands of other

contaminating proteins in the CBB-gels. Furthermore, the immunoblots show heavy and light

chains of the cat antibodies at the predicted molecular weight sizes of 50 kD and 25 kD,

respectively (Figure 2-6). The intensity of the bands for heavy and light chains suggests that

high levels of antibodies were retained after purification. Analysis for IgG levels using RID

assay indicates that most of the IgG in the purified antibody preparations (average recovery of

75%; recovery range of 67%-82%) were retained after the purification (Figure 2-6 and 2-7). The

purified antibody preparations also contained lower amounts of IgA (average recovery of 36%;

recovery range of 19-62%) and IgM (average recover of 53%; recovery range of 48-55%) than

those in the pre-purification sera (Figure 2-8 and 2-9). The loss of IgA and IgM was anticipated

since caprylic acid/ammonium sulfate precipitation procedure has been reported to be a

purification procedure for IgG [47].

The retention of biological activity was illustrated by the reactivity of the purified antibody

preparations to the vaccine immunogen used on the donors of their pre-purification pooled serum

(Figure 2-10). Furthermore, no significant loss in VNA titers to FIVpet were detected after

purification of pooled serum from Fel-O-Vax-vaccinated cats (Table 2-5; 1000 VNA titers of

pre- and post-purification). The VNA titers to FIVpet and FIVFC1 were not detected in pooled

serum from either HIV-1 p24- or FIV p24-vaccinated cats.

All purified antibodies were resuspended to the original volume to simulate the

concentrations equivalent to those in the pre-purification pooled sera. The same dilution of









1:200 was used for the purified preparation and pre-purification serum in the immunoblot

analysis. These adjustments allowed for the direct comparison between the pre- and post-

purification materials. Therefore, the intensity of the bands was used as a measure for

comparing specific-antibody titers in purified preparations to those in pre-purification sera.

Purified antibody preparations from pooled sera of HIV-lucD1- and HIV-1N20 p24-vaccinated

cats reacted strongly to HIV-lucD1- and HIV-1N20 p24 immunoblots (Figure 2-10A, 2-10B).

They reacted weakly to p24 of FIV whole-virus immunoblot (Figure 2-10C). However, their

weak p24 reactivity in the FIV whole-virus immunoblot was slightly stronger than their pre-

purification pooled serum (Figure 2-10C). The purified antibody preparation from pooled serum

of FIV p24-vaccinated cats reacted to HIV-1 p24 immunoblots at a similar weak intensity as that

of corresponding pre-purification serum (Figure 2-10A, 2-10B). As expected, purified antibody

preparations from sera of HIV-1 and FIV p24-vaccinated cats had no reaction to uninfected FeT-

J cell-lysate immunoblot (Figure 2-10D). Whereas, purified antibody preparation and pre-

purification pooled serum from Fel-O-Vax-vaccinated cats reacted weakly at similar bands (50

and 75kD on FeT-J immunoblots). These reactions to cellular components were anticipated

since Fel-O-Vax FIV vaccine, which is composed of inactivated infected cells and viruses,

should also induce anti-cellular antibodies.

Passive-transfer Studies with Serum and Antibodies from Vaccinated Cats

In Passive-Transfer Study 1, 3 of 4 (75%) cats transfused with HIV-ucmD1 p24-vaccinated

cat serum became infected with FIVpet (10 CID5o) (Table 2-1). Whereas, Fel-O-Vax-vaccinated

cat serum was able to protect 4 of 4 (100%) cats from this group. Furthermore, no protection

was observed in all four control cats, which received saline or pooled serum from non-vaccinated

cats.









In Passive-Transfer Study 2 with slightly higher challenge dose of 25 CID50, all three cats

transfused with partially-purified antibodies from pooled serum of HIV-lucDm and FIVpet p24-

vaccinated cats, were not protected against FIVpet (Table 2-2). Three of 4 cats, which received

similarly purified antibodies from Fel-O-Vax-vaccinated cat sera, were protected against FIVpet

but not against FIVFC1 even at a slightly lower challenge dose (15 CID5o). As expected, all five

recipients of saline or partially-purified antibodies from non-vaccinated cats were all infected

with either FIVpet or FIVFC1. These observations suggested that the antibodies induced by Fel-O-

Vax FIV vaccination can confer protection against homologous FIV challenge but not against

heterologous-subtype FIVFC1. Furthermore, antibodies induced by HIV-1 or FIV p24

vaccination conferred no protection against FIVpet.

In Passive-Transfer Study 3, 1 of 3 cats transfused with highly-purified antibodies from

HIV-1 UCD1 p24-vaccinated cat sera were protected against FIVFC1 (10 CID5o), while no

protection (0 of 4) was observed in recipients of purified antibodies from HIV-1N20 p24-

vaccinated cat sera (Table 2-3). Moreover, all four cats transfused with purified antibodies from

either FIV p24-vaccinated (n=2) or Fel-O-Vax-vaccinated (n=2) cat sera were not protected

against FIVFC1. Like the results from Study 2, all four cats transfused with purified antibodies

from Fel-O-Vax-vaccinated cat sera were protected against homologous FIVpet. VNA analysis of

antibodies removed from the transfused cats 6 days post transfusion showed the retention of

some VNA activity at an average dilution of 1:800 (800 VNA titer). All recipients of saline or

purified antibodies from FeT-J cell-immunized or non-vaccinated cat sera were infected with

either FIVpet or FIVFC1. Hence, the antibodies to HIV-1 p24 and FIV p24 do not mediate the

protection observed in HIV-1 p24-vaccinated cats and FIV p24-vaccinated cats.









Discussion

As summarized in Table 2-4, current studies demonstrated that only 1 of 8 cats transfused

with serum or antibodies to HIV-IucD1 p24, 0 of 3 cats transfused with antibodies to FIV p24,

and 11 of 12 cats transfused with antibodies to Fel-O-Vax antigen were protected against FIVpet

challenge. In contrast, 1 of 3 cats transfused with antibodies to HIVIucD1 p24 and none of the

cats transfused with antibodies to HIV-1N20 p24, FIV p24, or Fel-O-Vax immunogen were

protected against FIVFcl challenge. These challenge doses resulted in infection of all control

cats, which received saline, antibodies to FeT-J cells, or antibodies from SPF cats. Hence, no

significant levels of passive-transfer protection were observed in cats transfused with antibodies

to HIV-1 p24 or FIV p24. However, passive-transfer protection was conferred with antibodies

from Fel-O-Vax-vaccinated cats against homologous FIVpet challenge but not against FIVFC1

challenge. These results taken together suggested that cross-protection, previously observed in

HIV-1 p24-vaccinated cats against both FIVpet and FIVFC1 and in Fel-O-vax-vaccinated cats

against FIVFC1 was not mediated by vaccine-induced antibodies.

Antibodies to HIV-1 p24 and FIV 24 conferred no passive-transfer protection. This

finding was anticipated since VNAs to p24 have yet to be reported. Only antibodies to SU and

TM, which are exposed on the viral surface, have been reported [53]. The core p24 is masked by

the viral lipid envelope and blocked from interaction with the antibodies to p24. This also

explained why only antibodies induced by Fel-O-Vax FIV vaccination, which had high levels of

antibodies to FIV SU and TM, conferred passive-transfer protection against FIVpet. High VNA

titers to FIVpet, but not to FIVFC1, were observed only in the serum or purified antibodies from

Fel-O-Vax-vaccinated cats (Table 2-5). This observation suggested a correlation of passive-

transfer protection with high vaccine-induced VNAs to the challenge strain. Previous studies

using sera from inactivated FIVpet (single-strain)-vaccinated cats and inactivated sera from









FIVpet-infected cats demonstrated positive correlation between passive-transfer protection and

transfusion with sera containing high VNA titers to the challenge virus [54]. Similar observations

were made in passive-transfer studies of macaques with inactivated infect sera against the

homologous SIV strain [55] or with human monoclonal VNA to HIV-1 Env against a challenge

with SHIV, which had homologous HIV-1 Env [56]. Thus, vaccine-induced antibodies may play

a role in the protection of Fel-O-Vax-vaccinated cats against vaccine-induced VNA-sensitive,

challenge viruses.

The pattern of passive-protection observed in Studies 1 and 2 with pooled serum and

partially-purified antibodies was similar to the protection pattern observed in Study 3 with

highly-purified antibodies. The highly-purified antibody preparations were produced by a

combination of caprylic acid fractionation and ammonium sulfate precipitation. Such

purification procedure produced antibody preparations with high levels of IgG antibodies (IgG

levels by RID assay), biological activities (VNA activity and reactivity to HIV/FIV p24 on

immunoblot), and purity (>90% purity and <50 EU endotoxin activity). The removal of serum

albumin and other serum proteins were greatly reduced and had no affect on the results of the

passive-transfer studies. This observation indicated that the antibodies, and not other serum

proteins, were responsible for the passive-transfer protection observed against FIVpet challenge in

the recipients of Fel-O-Vax-vaccinated cat antibodies. These studies do not however determine

which anti-FIV immunoglobulin isotypes are more important for passive-transfer protection as

well as for protection observed after active vaccination. The high retention of IgG antibodies

and VNA titers in highly-purified preparations suggest that FIV-specific IgG antibodies play a

major role in both passive-transfer protection and vaccine protection against homologous FIVpet

challenge. This protection against IV homologous challenge may also extend towards









mucosal/vaginal challenge, since a recent vaccine study using a combination of Fel-O-Vax and

prototype vaccines demonstrated major protection against vaginal FIVpet challenge [57]. High

levels of IgG, but very low levels of IgA, were found in the vaginal vault of the vaccinated cats.

Furthermore, high levels of FIV-specific IgG antibodies, but only low levels of FIV-specific IgA

antibodies, were detected in the vaginal vault. Thus, the role of vaccine-induced IgG antibodies

appeared to be more important than IgA antibodies at least in the vaginal homologous challenge.

The lack of passive-transfer protection with antibodies to HIV-1 p24 and FIV p24

suggested that vaccine protection observed in previous studies with HIV-1 p24-vaccinated cats

and FIV p24-vaccinated cats [38] may be due to vaccine-induced cellular immunity. In previous

studies FIV 24-specific IFNy responses have been detected in PBMC from HIV-1 p24-

vaccinated cats [38]. Similarly, cellular immunity may have mediated the protection observed in

Fel-O-Vax-vaccinated cats against VNA-resistant FIVFC1. Strong FIV-specific cellular

immunity has been reported to be present in cats vaccinated with prototype dual-subtype FIV

vaccine (prototype to Fel-O-Vax vaccine) [48]. These observations further support the view that

HIV-1 p24 vaccination induces cross-protective cellular immunity against FIV challenge. The

studies in Chapter 3 were performed to test this concept.











Table 2-1. Passive-Transfer Study 1 with sera from vaccinated and non-vaccinated cats

FIV Challenge Weeks Post-Challenge (FIV Ab/VI) b Protection
Group Cat # Passive-Transfer Serum (CID5o) a 3 6 9 12 15 17 20 Rate (%)


A RT2 HIV1ucD1 p24-vaccinated cat serum Pet (10) -/- -/- -/- -/- -/- -/- -/- 1/4(25%)
RW1 HIVlucD1 p24-vaccinated cat serum Pet (10) -/+ +/+ +/+ +/+ +/+ + Eu
RX3 HIVlucD1 p24-vaccinated cat serum Pet (10) -/- +/+ +/+ +/+ +/+ +/+ Eu
RV1 HIVlucD1 p24-vaccinated cat serum Pet (10) -/- -/+ +/+ +/+ +/+ + Eu

B RT5 Fel-O-Vax FIV-vaccinated cat serum Pet (10) -/- -/- -/- -/- -/- -/- -/- 4/4(100%)
RW2 Fel-O-Vax FIV-vaccinated cat serum Pet (10)
EF3 Fel-O-Vax FIV-vaccinated cat serum Pet (10)
EH2 Fel-O-Vax FIV-vaccinated cat serum Pet (10)

C RT4 Non-vaccinated cat serum Pet (10) +/+ +/+ +/+ +/+ +/+ +/+ +/+ 0/4(0%)
RX1 Non-vaccinated cat serum Pet (10) -/- -/- -/+ -/+ -/+ +/+ +/+
RW4 Saline Pet (10) -/- +/- +/+ +/+ +/+ +/+ +
DQ1 Saline Pet (10) -/- -/- +/+ +/+ +/+ + Eu

a FIV challenge was subtype-A FIVpet (Pet) at 10 CID5o.

b FIV infection was based on the development of FIV antibodies (FIV Ab) determined by FIV-immunoblot
analysis and the presence of viruses determined by virus isolation (VI) using RTand PCR analyses. Results for the
samples collected at 3, 6, 9, 12, 15, 17, and 20 wpc are shown either as positive (+) or negative (-) for FIV
antibodies or viruses. Abbreviation is euthanized (Eu).











Table 2-2. Passive-Transfer Study 2 with partially-purified antibodies from vaccinated and non-
vaccinated cat sera

FIV Challenge Weeks Post-Challenge (FIV Ab/VI)b Protection
Group Cat # Passive-Transfer Antibody (CID5o) a 3 6 9 12 15 17 20 Rate (%)


A QC3 HIVlucD1 p24-vaccinated cat Ab Pet (25) -/+ +/+ +/+ +/+ +/+ +/+ + 0/4 (0%)
QD3 HIV1ucD1 p24-vaccinated cat Ab Pet (25) -/+ +/+ +/+ +/+ +/+ +/+ +
QE4 HIV1ucD1 p24-vaccinated cat Ab Pet (25) -/+ +/+ +/+ +/+ +/+ +/+ +
BDP HIVlucD1 p24-vaccinated cat Ab Pet (25) -/+ +/+ +/+ +/+ +/+ +/+ +

B QC1 FIVpet p24-vaccinated cat Ab Pet (25) -/+ -/+ -/+ -/+ ++ +/+ +/+ 0/3 (0%)
QD4 FIVpet p24-vaccinated cat Ab Pet (25) -/+ -/+ -/+ -/+ -/+ +/+ +/+
QE7 FIVpet p24-vaccinated cat Ab Pet (25) -/+ -/+ -/+ -/+ +/+ +/+ +/+

C QC2 Fel-O-Vax FIV-vaccinated cat Ab Pet (25) -/- -/- -/- -/- -/- -/- -/- 3/4(75%)
QD2 Fel-O-Vax FIV-vaccinated cat Ab Pet (25) -/+ -/+ -/+ -/+ -+ /+ +/+
QEl Fel-O-Vax FIV-vaccinated cat Ab Pet (25)
BDQ Fel-O-Vax FIV-vaccinated cat Ab Pet (25)

D QD5 Non-vaccinated cat Ab Pet (25) -/+ +/+ +/+ +/+ +/+ +/+ + 0/3 (0%)
BDS Non-vaccinated cat Ab Pet (25) -/- +/- +/+ +/+ +/+ +/+ +
QE2 Saline Pet (25) -/- -/- +/+ +/+ +/+ +/+ +

E QE2 Fel-O-Vax FIV-vaccinated cat Ab FC1 (15) -/- -/- -/+ -/+ -/- +/- +/+ 0/3 (0%)
PQ6 Fel-O-Vax FIV-vaccinated cat Ab FC1 (15) -/- +/+ +/+ +/+ +/+ +/+ +/+
75B Fel-O-Vax FIV-vaccinated cat Ab FC1 (15) -/- +/+ +/+ +/+ +/+ +/+ +/+

F QE6 Non-vaccinated cat Ab FC1 (15) -/- -/- +/+ +/+ +/+ +/+ +/+ 0/2(0%)
75C Saline FC1 (15) +/+ +/+ +/+ +/+ +/+ +/+ +/+
a FIV challenge was subtype-A FIVpet (Pet) at 25 CID50 and subtype-B FIVFcl (FC1) at 15 CID50

b FIV infection was based on the development of FIV antibodies (FIV Ab) determined by FIV-immunoblot
analysis and the presence of viruses determined by virus isolation (VI) using RTand PCR analyses. Results for the
samples collected at 3, 6, 9, 12, 15, 17, and 20 wpc are shown either as positive (+) or negative (-) for FIV
antibodies or viruses.












Table 2-3. Passive-Transfer Study 3 with purified antibodies from vaccinated and non-vaccinated
cat sera.


FIV Challenge Weeks Post-Challenge (FIV Ab/VI)b Protection
Group Cat # Passive-Transfer Antibody (CIDso)a 3 6 9 12 15 17 20 Rate (%)

A LF3 HIV1ucD1 p24-vaccinated cat Ab FC1 (10) -/- -/- -/- -/- -/- -/- -/- 1/3 (33%)
KA4 HIV1ucD1 p24-vaccinated cat Ab FC1 (10) -/- +/- +/- +/+ +/+ +/+ +
KE2 HIV1ucD1 p24-vaccinated cat Ab FC1 (10) -/- -/- -/- -/- -/- +/+ +/+
B LK2 HIV1N20 p24-vaccinated cat Ab FC1 (10) -/- -/- -/- -/- -/+ +/+ + 0/4 (0%)
KA6 HIV1N20 p24-vaccinated cat Ab FC1 (10) -/- -/- +/- +/+ +/+ +/+ +/+
KE3 HIV1N20 p24-vaccinated cat Ab FC1 (10) -/- -/- -/- -/- +/- +/+ +/+
KB2 HIV1N20 p24-vaccinated cat Ab FC1 (10) -/- -/- -/- -/- -/- +/+ +/+
C KE4 FIVpet p24-vaccinated cat Ab FC1 (10) -/- +/- +/+ +/+ +/+ +/+ + 0/2 (0%)
LK4 FIVpet p24-vaccinated cat Ab FC1 (10) -/- -/- -/- -/- -/- +/+ +/+
D KE5 Fel-O-Vax FIV-vaccinated cat Ab FC1 (10) -/- -/- -/- -/+ -/+ +/+ + 0/2 (0%)
KA2 Fel-O-Vax FIV-vaccinated cat Ab FC1 (10) -/- -/- -/+ -+ /+ +/+ +/+
E KE6 FeT-J cell-immunized cat Ab FC1 (10) -/- -/- -- +/+ +/+ +/+ +/+ 0/3 (0%)
LF2 Non-vaccinated cat Ab FC1 (10) -/- -/- -/- +/+ +/+ +/+ +/+
LD5 Saline FC1 (10) -/- -/- +/+ +/+ +/+ +/+ +
F JX2 Fel-O-Vax FIV-vaccinated cat Ab Pet (25) -/- -/- -/- -/- -/- -/- -/- 4/4(100%)
LE5 Fel-O-Vax FIV-vaccinated cat Ab Pet (25)
KB4 Fel-O-Vax FIV-vaccinated cat Ab Pet (25)
JY1 Fel-O-Vax FIV-vaccinated cat Ab Pet (25)
G LE6 FeT-J cell-immunized cat Ab Pet (25) -/- +/- +/+ +/+ +/+ +/+ + 0/4 (0%)
KB3 FeT-J cell-immunized cat Ab Pet (25) -/- +/+ +/+ +/+ +/+ +/+ +/+
JX1 Non-vaccinated cat Ab Pet (25) -/- +/+ +/+ +/+ +/+ +/+ +/+
KB1 Saline Pet (25) -/- +/+ +/+ +/+ +/+ +/+ +/+


a FIV challenge was subtype-A FIVpet (Pet) at 25 CID50 and subtype-B FIVFcl (FC1) at 10 CID5o.

b FIV infection was based on the development of FIV antibodies (FIV Ab) determined by FIV-immunoblot analysis

and the presence of viruses determined by virus isolation (VI) using RT and PCR analyses. Results for the samples
collected at 3, 6, 9, 12, 15, 17, and 20 wpc are shown either as positive (+) or negative (-) for FIV antibodies or
viruses.













Table 2-4. Summary of the results from Passive-Transfer Studies 1-3.

Donor Challenge Virus

Antibodies
FIVptaluma FIVFcl


HIV-1ucD-1 p24 1/8 1/3

HIV-1N20 p24 ND1 0/4

FIVpet p24 0/3 0/2

Fel-O-Vax FIV 11/12 0/5

FeT-J cells 0/2 0/1

Non-vaccinated b 0/5 0/2

Salinec 0/4 0/2
a Donor antibodies represent a combination of partially purified, highly purified, and unpurified serum
antibodies.
b Donor antibodies were from non-vaccinated cats.

Recipient cats were infused with saline and did not receive donor antibodies.










Table 2-5. VNA titers of highly purified antibodies and corresponding pooled serum

VNA to VNA to
Pooled serum for passive transfer Purification a FIVpet FIVFcl


HIV1ucD1 p24-vaccinated cat serum Pre <10 <10
Post <10 <10

HIV1N20 p24-vaccinated cat serum Pre <10 <10
Post <10 <10

FIV p24-vaccinated cat serum Pre <10 <10
Post <10 <10

Fel-O-Vax FIV-vaccinated cat serum Pre 1000 10
Post 1000 Ub
a Pre-purification (Pre) samples represent pooled serum from SPF cats immunized with the

corresponding vaccine. Post-purification (Post) preparations represent highly purified
antibodies, which were purified by caprylic acid fractionation followed by ammonium sulfate
precipitation.


b Retesting or under investigation (U)












P;I-s\i c Trinlsfci of Se'i or Antiibodies
FI\ ChIlcin,'c

D;i\ -1 1 I

Week 0 1 2 3 4 5 6 7 8 9 10 11 12
i i i i i i i i i i i i i W 17



Figure 2-1. Schedule of vaccination and challenge for passive-transfer studies. Naive SPF cats
were transfused with antiserum or purified antibodies equivalent to 30% of recipient's
total blood volume (TBV). First transfusion was given on day -1 (equivalent to 20%
TBV) with FIV challenge on day 0 and a second transfer on day 1(equivalent to 10%
TBV).













Study II


Blood collection


Serum extraction


Pool serum


[eat inactivation


Filter sterilization


Blood collection


Serum extraction


Pool serum


Ammonium
sulfate
precipitation


Collection
and dialysis
of precipitate


Concentration
of antibodies


Filter sterilization







Pai% nc Trailnfuiion ol' Anilbodi'%


Blood collection


Serum extraction


Pool serum


Caprylic acid fractionation


Ammonium
sulfate
precipitation


Collection
and dialysis
of precipitate


Concentration
of antibodies


Filter sterilization


Figure 2-2. Schematic of antibody preparation for passive-transfer studies. The figure illustrates
the procedure of three separate passive immunity-transfer studies. In the first study
four groups of two cats each were transfused with IWV ( inactivated whole virus)
antiserum, Fel-O-Vax antiserum, SPF serum and saline solution. All antibodies were
collected prior to the donor cats being exposed to FIV. The donor and recipients were
cross-matched before use of the serum. The serum was inactivated by incubation at
560C for 40 minutes. In the second study, the antibodies were purified by ammonium
sulfate precipitation in order to isolate primarily IgG. Whilst in the third study, this
purification was taken further using a two-step purification method involving caprylic
acid separation followed by ammonium sulfate precipitation. After these procedures
were completed the antibodies were dialyzed and filtered using a 0.45pm filter. Tests
for biological activity, purity, and toxicity were performed on the material prior to
passive transfer.


Study I


Study III












100_
100 ----------- ------- m- --------- 11----- -i- --- -j-i----- -
90
80 -- HIV-1 p24(n=15)
70 0 FIV p24 (n=14)
70 D FIV infected (n=5)
S 60 -- --- -- -
: 50- ---- -- -- -
S340
230- -- --- -- -- --- --

20 --- -------- -- --- --


FB1 FB2 FC2 FB3 FB4 FB5 FB6 FB7 FB8 FB9 FB10 FB11 FIV HIV FIV
p24 p24 TM


Figure 2-3. B-cell epitope analyses of sera and PBMC from HIV-1 p24-vaccinated cats. Sera
from HIV-1 p24-vaccinated cats (n = 15) and FIV p24-vaccinated cats (n= 14) at 3
weeks after the 3rd vaccination before challenge and from FIV-infected control cats
(n = 5) at 18 wpc were tested for reactivity to 12 overlapping B-cell peptides by
ELISA. These overlapping peptides derived from FIV p24 sequence are shown with
their peptide designation. The reactivity of the sera is shown as percent positive (e.g.,
number of positive sera among total number of sera tested). The B-cell peptide codes
(without aa sequence designation) are shown below the corresponding bars the aa
sequence designations are shown in Figure 2-4. As positive ELISA substrate controls,
serum reactivity to FIVBang p24 protein, HIV-lucD1 p24 protein, and FIV
transmembrane peptide TM (695-705) were determined.









Figure 2-4. Amino acid sequence alignment of HIV-1 and FIV strains, and B-cell peptide
sequence alignment. Sequence homology analysis between HIV-IUCD1 and FIVBang is
shown with (-) for amino acid homology and (:) for amino acid identity according to
the GeneStream Align program (A). Only aa residues that are different from HIV-
IUCD1 and FIVBang are shown for HIV-ILAI/LAV (above HIV-IUCD1 sequence) and
FIVFC1 (below FIVBang sequence), respectively. The designations of FIV B-cell
peptides and HIV-1/FIV T-cell peptides (peptide code with aa position) are shown on
the top left (B). Each bar below the aa alignment represents an overlapping 28-30-
mer B-cell peptide, which was used in our ELISA analysis for cross-reactive B-cell
epitope mapping. The B-cell peptide overlaps by 11-13 aa, except for the four
peptides (FBI, FB2, FC2, FB4) on the amino-terminus. The 26-mer peptide FC2/34-
59 is based on FIVFC1 sequence and differs from FIVBang sequence by having Ser48
and Ser57 instead of Pro48 and Ala57. HIV-1 and FIV peptide sequences used for T-
cell epitopes are shown with red aa. All 18 T-cell peptides (9-11-mers) are boxed
with dotted line. T-cell peptide code is shown immediately above (HIV-1 p24
peptide) or below (FIV p24 peptide) the dotted box. Overlapping red bars at the
carboxyl-terminus represent two overlapping T-cell epitopes and their corresponding
sequences. HIV-1 T-cell peptides are identical to the HIV-IUCD1 peptide sequences.
FIV T-cell peptides, except for the four peptide sequences below the alignment
(F4/73-83, F4.5/122-131, F5/142-150, F7/183-191), are identical to the FIVBang
peptide sequences. FIV peptides F4/73-83 and F7/183-191 are identical to the
corresponding FIVFC1 peptide sequences and remaining two FIV peptides, F4.5/122-
131 and F5/142-150, are identical to the FIVshi peptide sequences. The published
major homology region (MHR) is boxed with a solid line. This figure is a
modification of our figure in Coleman, J., et al. 2005 [38].














FIV B-cell Peptides
FB1 / 1-30 FB6 / 107-136
FB2 / 10-37 FB7 / 126-153
FC2 / 34-59 FB8 / 143-171
FB3/53-81 FB9/161-188
FB4/71-100 FB10/178-207
FB5 / 90-119 FB11 /197-223


T-cell Peptides
HIV-1 FIV
H2 / 16-25 F2 / 15-25
H3 / 43-53 F3 / 42-51
H4 / 74-84 F4/73-83
H4.5 /130-140 F4.5 /122-131
H5/150-158 F5/142-150
H6 / 162-172 F6 / 155-164
H7/191-199 F7/183-191
H7.8 / 217-226 F7.8 / 210-219
H8 / 222-230 F8 / 214-223


LAI I I A LH.2L)_ s _H31 _(H4)_ V G T
UCD1 PVVQNLQGQMVHQP PRTLNAWVWVEEKAFSPEVIPMFTLSEGATPQDL TMLNTVGGHQAAMQMLKETJNEEAAENWDPLPVHAGPIAPDQMREPRGSDIAGITSTLQEQ
::I.:.. ::.. ..: ..::. ..... .. .......
BANG PI-QTVNGAPQYVAADPfMVSIFMEfKREGLGGEEVQLWFTAFSANLTPTDM4TLIMAAPGCAADKEILDESIKQLTAEYDRTHP ----PDGPRPLPYFTAAEIMGIGLT-QEQ
FC1 (F2) (F3) S S T M
10 20 30 40 50 60 70 80 I 90 100
__QMTAEYDRTH_
(F4)


FC2


FB2


FB4


FB3


FB5


(LH4.5L (H5) MH R (H6) E (H7) H7 (H8)
IGWMTNNPPIPVGEIrKRWIILGLNKr--VRMYSPTILDIIQGP PFI YVDRFYTLAEQSQDVNTETLQNANPDCI
. .. .. .. I ...
---QAEARFAPARMQORAWnYEALGK IKAKSP AV-QL QGAIEDYSSFIDPRLFQIDQEQNTAEVKLYLKQSLSIANANAECIMSHLKPESTLEE#KLcCQEGSPGYKMQLL
P I K I PD R (F7.8) 8)
EALSK IAVQMKQGVKI IAN PDCK 220
(F4.5) (F5) (F7)


FB9


FB7


FB11









Figure 2-5. CBB-stained gel and immunoblot of purified antibody preparations for Passive-
Transfer Study 3. Purified antibodies from HIV-lucD1 p24 (panel A, lanes 1,2,5,6),
HIV-1N20 (panel A, lanes 3,4,8,9) Fel-O-Vax FIV (panel B, lanes 1,2,5,6), FIV p24
(panel B, lanes 3,4,8,9), and FeT-J cell (panel C, lanes 3,4,7,8) vaccinated cat sera
and non-vaccinated cat sera (panel C, lanes 1,2,5,6) are shown. Pre-purification
pooled serum (Pre, 1,3,5,7) at 4 mg/lane and post-purification antibody preparation
(Post, lanes 2,4,6,8) at 2 mg/lane were used in both CBB gels and immunoblots. All
purified preparations in the CBB gel had strong heavy and light chain bands and weak
bands at 150 kD and 100 kD, which was most likely due to unreduced and partially-
reduced antibodies. The bands were also present in the immunoblot reacted to anti-
feline IgG heavy and light chain in Figure 2-6. The antibodies in the immunoblots
were detected using goat anti-cat albumin antibodies. Bovine serum albumin (BSA)
(500 .g/lane) was used as positive control for the immunoblots (panels A and B, lane
9). A weak band at 70 kD on lane 9 were those to BSA. Feline albumin
contamination was highest in the non-vaccinated cat antibody preparation. Arrows are
placed next to lanes 6 and 8 of immunoblots indicate the minimal amount of feline
albumin in the purified product.
















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HIV-1 ,, HI\-1, ... FIV-1, Fcl---\j\
P24 P24 P24 FI\ F.i-J Non- acc iilla.d
\ Vccinc \ CCI nc \I ccnc \ ,CCInc Im iii1u ti/cd SPF
Antibodics Ajuiibodics. Anibodics Anibodics Aniibodics Anubodic.



Hin Chain


L iti Cluh nt

Line 1 4




Figure 2-6. Immunoblot of purified antibody preparations used in the six groups from Passive-
Transfer Study 3. Each lane contained 2 mg of purified preparation. The antibodies
were detected using goat anti-cat IgG heavy and light chain antibodies (Kirkegaard &
Perry Laboratories, Gaithersburg, Maryland). Purified antibodies from HIV-1N20 p24
(lane 1), HIV-lucD1 p24 (lane 2), FIV p24 (lane 3), Fel-O-Vax FIV (lane 4), and FeT-
J cell (lane 5) vaccinated cat sera and from non-vaccinated SPF cat sera (lane 6) are
shown. The molecular weight marker indicated that the bands corresponded to the
predicted sizes for feline heavy and light chains. All purified antibody preparations
had weaker band at 100 kD, which may be the partially-reduced immunoglobulin
chain ( 2 heavy chains with disulphide bonds intact). The anti-cat IgG heavy/light
chain antibodies showed no cross-reactivity to sheep serum, mouse serum, or BSA
(data not shown).











200 -i


180- -
O3 Std

160-


140-


120-


= 100-


80-


60-- .


40-- -.


20-- "







Sam pie


Figure 2-7. IgG concentrations of purified antibody preparations from Passive-Transfer Study 3.
The IgG concentrations of pre-purification pooled serum (Pre) and post-purification
antibody preparations (Post) were determined by commercial feline IgG RID assay.
The IgG concentration for pooled pre-serum (grey bar) and purified antibody
preparations (dark grey bar) from Fel-O-Vax FIV (Sample A), HIV-lucD1 p24
(Sample B), HIV-1N20 p24 (Sample C), FIVpet p24 (Sample D), and FeT-J cell
(Sample E) vaccinated cats and non-vaccinated SPF cats (Sample SPF) are shown
with values from reference standards. Commercial reference standards corresponded
to 25 (Ref Std3), 100 (Ref Std2), and 200 (Ref Stdl) mg/dL. The % recovery for
each purified antibody preparations is shown above the corresponding bar. The
average IgG recovery was 75%, indicating an average loss of 25%.


I Pre
SPost


-v



























0 100- ------------
120






60- -


20 -- -- ----------- -- --








Sample


Figure 2-8. IgA concentrations of purified antibody preparations from Passive-Transfer Study 3.
The IgG concentrations ofpre-purification pooled serum (Pre) and post-purification
antibody preparations (Post) were determined by commercial feline IgG RID assay.
The IgG concentration for pooled pre-serum (grey bar) and purified antibody
preparations (dark grey bar) from Fel-O-Vax FIV (Sample A), HIV-lucmD1 p24
(Sample B), HIV-1N20 p24 (Sample C), FIVpet p24 (Sample D), and FeT-J cell
(Sample E) vaccinated cats and non-vaccinated SPF cats (Sample SPF) are shown
with values from reference standards. Commercial reference standards corresponded
to 25 (Ref Std3), 100 (Ref Std2), and 200 (Ref Stdl) mg/dL. The % recovery for
each purified antibody preparations is shown above the corresponding bar. The
average IgA recovery was 36%, indicating an average loss of 64%.
















200-

'Pre
180-- ost
ESid

160- -


140- -


I 120- -
0)
cE
.100- -


0 80-- -


60-


40-


20-







Sample



Figure 2-9. IgM concentrations of purified antibody preparations from Passive-Transfer Study 3.
The IgG concentrations of pre-purification pooled serum (Pre) and post-purification
antibody preparations (Post) were determined by commercial feline IgG RID assay.
The IgG concentration for pooled pre-serum (grey bar) and purified antibody
preparations (dark grey bar) from Fel-O-Vax FIV (Sample A), HIV-lucD1 p24
(Sample B), HIV-1N20 p24 (Sample C), FIVpet p24 (Sample D), and FeT-J cell
(Sample E) vaccinated cats and non-vaccinated SPF cats (Sample SPF) are shown
with values from reference standards. Commercial reference standards corresponded
to 25 (Ref Std3), 100 (Ref Std2), and 200 (Ref Stdl) mg/dL. The % recovery for
each purified antibody preparations is shown above the corresponding bar. The %
recovery for each purified antibody preparations is shown above the corresponding
bar. The average IgM recovery was 53%, indicating an average loss of 47%.














lI I II l II '


2 24 pi l


P k -P 0. I '., l .I1 ,, I ', ,1













I K


1 2 3 4


1 2 3 4i
2 cicin1 I V\t, 1,,p24 llp.




\ P0lI1' _nll l ll- ",III0
]1 ]l- Iltllll|i ln.'.lll>'n


I ii'
gp
I) S
11111


i ii I i ,, i .


I 1 I, p I P- P [ I- ,
12 3 4


2iii i'-' Il V 11\h,,h,.' 2_ ii 11n I 1 c'l- l ,.',l
\. ll'll- ,'lip 1\ ",Jli' lllp
Figure 2-10. HIV-1 and FIV p24 reactivity of purified antibody preparations from Passive-
Transfer Study 3. The p24 reactivity was based on immunoblot analysis using HIV-
IUCD1 p24 (A), HIV-1N20 p24 (B), FIVpet whole-virus (C), and uninfected FeT-J cell-
lysate (D) as substrates at 200 ng/strip. Pre-purification serum (pr) and post-
purification antibody preparation (po) from HIV-1N20 p24 (Pre/Post Strip Pair 1),
HIV-IUCD1 p24 (Pair 2), FIV p24 (Pair 3), and Fel-O-Vax FIV (FOV, Pair 4)
vaccinated cats sera were reacted at a dilution of 1:200.


Hl l I ll .
111 1 ,I
i':4 p2-I


II I''
i'24


i,_2 4
1j',2a


|i':'


Pk \ Cc )i L _\IIIII IIl01'1
]1 ."]i l', I' LI' IIIi l .'.IliI>'









CHAPTER 3
SUBTYPE A HIV-1 P24 AS AN FIV VACCINE IMMUNOGEN

Introduction

One of the major hurdles impeding the development of an effective HIV vaccine is the

necessity to elicit an immune response, which protects against different subtypes of the virus

[58]. Thus far, our work on the development of an improved FIV vaccine shows that this is an

achievable goal. With an aim to increase the efficacy of the FIV vaccine, a combination of two

virus strains isolated from LTNP cats was utilized [59,60]. The vaccine was able to induce from

moderate to significant levels of protection among SPF cats challenged with homologous and

heterologous FIV strains. The vaccine induced many antibodies, which were also reactive to

HIV-1 p24. The induction of these cross-reactive antibodies was expected, since several p24

antigens are conserved in the same viral subfamily. The principle of cross-protective epitopes as

the basis for vaccine design has been utilized previously in the development of smallpox and

canine distemper vaccines [61,62]. The studies described in this section will utilize this principle

to develop an FIV vaccine that is effective against strains from multiple FIV subtypes.

Although HIV-1 and FIV p24 proteins have only 31% aa identity, core p24 of these viruses

have a number of cross-reactive epitopes [38]. Previous studies from our laboratory tested the

use of subtype-B HIV-lucD1 p24 as an immunogen for FIV vaccines. This vaccine conferred

protection in 60%-100% of vaccinated cats against strains from multiple FIV subtypes [38].

Based on this observation, current studies were performed to test the following hypothesis. HIV-

1 p24 has epitopes that are highly immunogenic to cats and some of these immunogenic epitopes

that are common to HIV-1 and FIV can serve as conserved vaccine epitopes for protection of

domestic cats against FIV. In previous studies, the p24 proteins from two HIV-1 subtype-B

strains had cross-protective epitopes, but such epitopes have yet to be identified on p24 from









other HIV-1 subtypes. Therefore, current studies were performed to evaluate the prevalence of

cross-protective p24 epitopes on HIV-1 subtype-A. The studies included the sequencing,

production, and purification of HIV-1 subtype-A p24; T-cell epitope mapping; immunogenicity

analysis; and pilot vaccine-efficacy trials.

HIV-1 subtype-A p24 was sequenced from a subtype-A strain obtained from AIDS

Reagent Bank. Upon confirmation of its subtype-A origin, its sequence was compared to HIV-1

subtype-B p24 (Figure 3-1) and FIVFC1 p24 sequences to identify potential differences in

TH/CTL epitopes. Since there was no T-cell epitope database for FIV, LANL database for TH

and CTL epitopes on subtype-B HIV-1 p24 was used to identify aa differences in T-cell epitopes

between subtype-A and subtype-B HIV-1 p24 proteins. Vaccinated cats received 3 doses of

vaccine at 3-week intervals before FIV challenge at 3 weeks after the last vaccination (Figure 3-

2). These animals were challenged IV with FIVFC1 (Table 3-1 and 3-2).

SPF cats were immunized with either subtype-A or subtype-B HIV-1 p24 protein and their

PBMC were assessed for T-cell responses to FIV and HIV-1. The interferon-gamma (IFNy)

ELISpot analysis with overlapping FIV p24 peptides was used to identify the cross-reactive T-

cell epitopes. Results showing similar epitope recognition between the two vaccinated groups

will indicate strong potential for subtype-A p24 vaccine to work as effectively as subtype-B

HIV-1 p24 vaccine. However, results showing disparity in the T-cell epitope responses will

suggest potential difference in vaccine efficacy between these two vaccines. Thus, these analyses

were used to project the outcome of subsequent vaccine-efficacy trials against subtype-B

pathogenic FIVFC1.

Since the previous report had only one HIV-1 p24 vaccine study using FIVFC1 as challenge

virus, the goals of Vaccination Study 1 were to test the reproducibility of the original findings









and to determine the level of vaccine protection observed in the vaccinated donors for Passive-

Immunity Study 1 in Chapter 2. The vaccination groups in Vaccination Study 1 consisted of

subtype-B HIV-IUCD1 p24-vaccinated cats, Fel-O-Vax FIV-vaccinated cats, and age-matched

non-vaccinated cats. Upon conformation of protection with HIV-1 subtype-A p24 vaccine

against FIVFC1, Vaccination Study 2 was initiated with vaccinated donors from Passive-Immunity

Study 3 (Chapter 2). The vaccination groups consisted of subtype-A HIV-1N20 p24, subtype-B

HIV-IUCDl p24-vaccinated cats, FIVpet p24-vaccinated cats, Fel-O-Vax FIV-vaccinated cats, and

age-matched non-vaccinated cats.

Overall, these vaccination studies were performed to determine the prevalence of cross-

protective epitopes on HIV-1 subtype-A p24 and to test the hypothesis on cross-reactive epitopes

for vaccine prophylaxis. Furthermore, these studies were the first step towards our central goal to

provide insight into the development of an HIV-1 vaccine for humans.

Methods

Virus Selection

HIV-1 subtype-A isolates were obtained from AIDS Reagent Bank and stored in a secure

freezer at -80C. The isolates were amplified by cell culture and then sequenced to identify the

strain to be used as the p24 immunogen for subtype-A vaccine.

Co-culturing for Virus Amplification

Whole blood was collected from a healthy human donor, and PBMCs isolated using the

ficoll-hypaque gradient technique. These cells were resuspended in culture media (RPMI 1640,

FBS 10%, Gentomycin, and 2-mecaptoethanol) and stimulated with 0.1 [g/mL staphylococcal

enterotoxin A for 3 days. The cells were then ready to be co-cultured with HIV-1 infected cells.

Cells were cultured at 3 day intervals by removing two-third of the culture media and replacing it

with fresh culture media. The supernatant from each flask was stored for RT assay analysis. The









cells were monitored daily to observe changes in morphology or the formation of multinucleated

giant cells indicative of HIV-1 infection. When the cultures were observed to contain a

significant amount of these large multinucleated cells, they were terminated to harvest the

proviral DNA.

Isolation of Proviral DNA

The cell suspension from the HIV co-cultures were placed into 15-mL tubes and

centrifuged at 2000 rpm for 5 minutes. The supernatant was removed and the cells were washed

in PBS. The cell pellet was resuspended in 200 [tL lysis buffer (0.45%Tween 20, 0.45% NP40,

PCR Buffer, 200 tg/mL proteinase K) and incubated at 56C for 24 hours. After heat treatment,

an equal volume of phenol-chloroform-isomyl alcohol was added and the suspension gently

rocked for 4-18 hours. The sample was subsequently votexed followed by centrifugation at

13000 rpm for 5 minutes. The upper layer containing the proviral DNA was transferred to a new

microfuge tube and mixed with 50 [tl of 3M sodium acetate and 400 [tL of ethyl alcohol. The

supernatant was removed and the pellet was rinsed in 1 mL of 70% ethanol. The sample was then

centrifuged, and the supernatant removed to recover purified DNA. The DNA recovered was

resuspended in DNase free H20 and stored at -80C until its use in PCR amplification for

proviral p24 sequencing.

Sequencing and Expression of HIV P24

PCR primers were designed based on the consensus and published sequences and reacted

with the regions upstream and downstream of the p24 gene of HIV-1 (Figure 3-3). PCR

conditions were optimized, and the following condition was used: the initial denaturing step at

94C for 2 minutes followed by 30 cycles of 30 seconds at 94C, 1 minute at 43C (gag) or 52C

(p24), additional 1 minute at 72C, and the final denaturation step of 10 minutes at 72C. The

reaction was performed in several 25-itL PCR reactions. The products of these reactions were









pooled and run on an 0.8% tris acetate EDTA gel electrophoresis for 15 minutes to separate the

target band from primer dimers and other contaminating non-target nucleic acids (Figure 3-4).

This band was then carefully excised from the gel, and the product was purified with a Qiagen

gel purification kit. The purified product was measured (concentration of DNA/protein) and set

to 0.5 [g/4L, and then sent to ICBR Sequencing Laboratory with accompanying primers. This

procedure was performed 3X from start to finish ensuring the accuracy of the sequence result.

Both HIV-1 p24 and FIV p24 protein were expressed by E. coli M15 cells that were

transfected with recombinant construct of lentiviral p24 and pQE 30 expression vector (Figure 3-

5). This expression system produced p24 tagged with a 6X histidine residue chain. The tag was

utilized in the protein purification.

Altered primer pairs were designed with adaptors on both ends of the gene insert to

facilitate restriction enzyme digestion of the ends of the PCR product and to allow sticky-end

ligation of the target gene in the correct orientation to the promoter of the vector. The histidine

tag was placed on the N-terminus of the protein product. The endotoxin levels for each p24

protein preparation were measured using LAL E-toxate assay (Sigma Aldrich USA). All purified

proteins were below 50 endotoxin units/dose, which is the endotoxin dose previously tested to be

safe for use in cats.

HIV-1N20 primers

Two pairs of primers were used for sequencing of the whole gag and p24 regions of the

proviral genome. The whole gag sequence primer pair consisted of the forward primer HGagF

(5'- GGACTCGGCTTGCTGAAGCGCGC-3') and reverse primer HGagR (5'-

ATCATCTGCTCCTGTATCTAATAG-3') (Figure 2). The p24 primer pair consisted of forward

primer HG1 (5'-CAGCATTATCAGAAGGAGCCAC-3') and reverse primer HG2 (5'-

CACTCCCTGACATGCTGTCATCAT-3'). Using the PCR conditions mentioned earlier, the









Gag primer pair produced a fragment that was 1.685 kB long, while the p24 primer produced a

fragment 542 bp long, which was within the p24 gene. Alternate reactions with HGagF/HG2 and

HGl/HGagR produced fragments of 1.115 kB and 1050 bp, respectively.

The primers used for expression were designed to introduce restriction enzyme sites

(RES). The sequences for forward and reverse primers were

5'CGCG/GATCCGCGCCTATAGTGCAAAATGCA-3'and 5'-

CCCA/AGCTTGGGCTACAAAACCCTTGCTTTATG-3', respectively. The underlined bases

correspond to the cassette and RE integration sites in the primer (Figure 3-6).

Restriction enzyme digestion

The restriction enzyme reaction using Buffer E was designed to facilitate the digestion of

both the insert DNA and the plasmid DNA, and the simultaneous digestion of both enzymatic

sites with high fidelity. The digestion reaction of the pQE 30 vector contained 6 [L plasmid

DNA, 0.5 [L Hind III, 0.5 [L BamH1, 1 [L Buffer E, 1 [L RNase A, and 1 [L 10X BSA. The

digestion reaction of the HIVN20 p24 insert contained 3 [L plasmid DNA, 0.5 [L Hind III, 0.5

[L BamHl, 1 [L Buffer(E), 1 [L RNase A, and 1 [L lOx BSA. Both reactions were performed

at 37C for 3 hours.

Expression and purification of p24

The expression system utilized to produce recombinant HIV-1 p24 protein was the pQE-

30 expression vector transfected into chemically competent E. coli M15 cells (derived from E.

coli K12). This expression system produced p24 protein with a 6X histidine tag, which was used

later for purification. Altered primer pairs were designed to introduce adaptors on both ends for

facilitating restriction enzyme digestion of the ends of the PCR product and to allow sticky end

ligation of the target gene in the correct orientation to the promoter sequence. The histidine tag

was placed on the N-terminus of the protein product. The 6X histidine tag facilitated the binding









of the recombinantly expressed p24 protein to a Ni-NTA resin used in affinity purification to

remove contaminating E. coli proteins. M15 cells were transformed with vector plus insert under

standard conditions (Sambrook et al. 1989) and plated on selective media containing 25 [g/mL

of kanamycin and 100 [g/mL of ampicillin. After overnight incubation at 370C, the colonies

grown on the replica plate were screened by colony-direct PCR to ensure that they contained the

target insert. After screening, glycerol stock was prepared from confirmed colonies and stored at

-800C. Two flasks, both containing 20 mL Lauria Bertani (LB) broth with 100 [g/ml of

ampicillin and 25 [g/mL of kanamycin were inoculated with the selected Ml 5 colony and

cultured overnight at 370C. Culture flasks, each containing 500 mL LB broth (100 [g/mL of

ampicillin and 25 [g/mL of kanamycin), were inoculated with 1:50 dilution of the overnight

culture. The cells were allowed to grow for approximately 2.5 hours, until an optical density of

0.6 was reached. The culture was then induced using IPTG at a final concentration of ImM.

Throughout 4.5 hours of culturing, Iml of the sample was collected for analysis. The cultures

were then centrifuged at 4000g for 20 minutes, and the pelleted cells stored overnight at -200C.

Purification

E. coli pellets were thawed on ice, then re-suspend in Lysis Buffer (50 mM NaH2PO4, 300 mM

NaC1, 10 mM imidazole) (3 mL/g E. coli). The cells were then sonicated to disrupt cell walls,

which allowed complete lysis. Protease inhibitor was added to the lysate to inhibit the

degredation of the expressed protein, and the suspension was centrigufed at 100OOg for 30

minutes to remove the cellular debris. Some of supernatant was saved for analysis, and the

remaining fraction was purified under native conditions using Ni-NTA affinity chromotography

column according to Ni Super Flow specifications (Qiagen Inc., Valencia, California). The

purified products were tested by CBB and silver stain analyses, endotoxin test, Bradford protein









assay (BioRad Systems, Inc. Hercules, California) according to manufacturers protocol and

immunoblot analysis[40].

Vaccination Studies 1 and 2

SPF cats from Liberty Research (Vaccination Study 1) and Harlan Sprague Dawley, Inc.

(Vaccination Study 2) were immunized 3X at 3-week intervals with either HIV-1 p24, FIV p24,

or Fel-O-Vax FIV vaccine. Cats were also immunized with PBS or FeT-J cell/lysate

combination (as described in Chapter 2 and detailed below). The p24 vaccines were 200 |tg of

p24 formulated in Ribi (Study 1) or FD-1 (Study 2) adjuvant supplemented with recombinant

feline IL-12 (R&D Systems, Inc., Minnesota, MN). Fel-O-Vax FIV vaccine is a commercial

product formulated in FD-1 adjuvant. FD-1 adjuvant generally provided better efficacy results

than Ribi adjuvant. Study 1 consisted of HIV-lucD1 p24-vaccinated (n=4), Fel-O-Vax FIV-

vaccinated (n=4), and PBS-immunized (n=3) groups, and used an FIVFcl challenge of 15 CID50.

Study 2 consisted of HIV-lucD1 p24-vaccinated ( n=4), HIV-1N20 p24-vaccinated (n=4), FIVpet

p24-vaccinated (n=3), Fel-O-Vax FIV-vaccinated (n=2), and FeT-J cell-immunized (n=2)

groups, and used an FIVFC1 challenge of 10 CID50.

The FeT-J cell/lysate-immunized cats received a combined cell and cell-lysate

immunization using SC (2x106 cells/dose), intradermal (ID, 250 [tg/dose cell lysate), intranasal

(IN, 100 [tg/dose cell lysate), and transcutaneous (TC, 100 [tg/dose cell lysate) routes. The total

number of cells/dose for SC immunization was equivalent to the inactivated infected cells (2x106

cells/dose) and viral proteins (50 [tg/dose) in the Fel-O-Vax FIV vaccine. The total cell-lysate

dose for combined ID/IN/TC/SC immunization was 500 [tg/dose. This amount was equivalent to

the amount of inactivated dual-subtype whole-viruses in prototype vaccines, which also

contained a moderate level of cellular debris. Therefore, this cell-lysate dose can be used safely









in cats. This high dose was close to 2X the amount of p24 proteins and was 10X the whole-viral

proteins in Fel-O-Vax FIV vaccine. This high cell-lysate dose in combination with uninfected

cells was used to ensure that sufficient amount of MHC was in the preparation to serve as cell

control for Fel-O-Vax FIV vaccine. FIV-infected FeT-J cells have higher levels of MHC

expressed on the cells than uninfected FeT-J cells. Furthermore, the FIV virion, much like HIV-

1, has a concentrated amount of host's MHC on the viral membrane. Previous inactivated SIV

vaccine studies in macaques demonstrated protection against SIV with uninfected human cells

used to grow the vaccine virus. In this study, the xenogeneic MHC of the human cells present in

the inactivated SIV vaccine provided the protection against SIV challenge, which was also

grown in human cells.

Feline IFNy (Fe IFNy) ELISpot analysis

Commercial FeIFNy ELISpot (R&D Systems, Inc.) was used to determine HIV-1 p24

and FIV p24 peptide responses of the PBMC from the HIV-lucD1 p24-, HIV-1N20 p24-, FIV p24-,

and Fel-O-Vax FIV-vaccinated cats. Fresh cells (2x105 cells/well) suspended in feline assay

media were cultured with peptides at 1 |tg peptide/well in FeIFNy ELISpot plate for 18 hours.

FeIFNy ELISpot plates were processed according to manufacturer's methods (R&D Systems,

Inc.) with the exception of the assay media used, and analyzed with an ELISpot reader. Feline

assay media consisted of AIM-V media (Gibco-Invitrogen Co., Carlsbad, CA), 10% heat-

inactivated pooled SPF sera, and 25 [tg/ml gentamycin (Mediatech, Inc., Herndon, VA). All

assays included T-cell mitogen, concanavalin A (Con A) (Sigma-Aldrich USA), at 2-4 [tg/well

as the positive control; recombinant HIV-lucD1, HIV-1N20, and FIVpet p24 proteins and

inactivated FIVpet plus FIVshi whole-viruses at 2 [tg/well as virus-specific immunogens; and

FIVBang p24 peptide F8 at 5 [tg/well as the negative control. In addition, LPS at 5 and 50









EU/well was included as a control for recombinant products. The LPS used for control was from

E. coli M15, which was used to produce the recombinant p24 proteins. These products had 1.19

EU per ng, whereas the recombinant vaccine had <50 EU per vaccine dose of 200 |tg. All results

were in duplicates and adjusted to spot forming units (SFU) per lx106 cells, after subtracting the

background derived from non-specific peptide control or media control, whichever was higher in

value. The standard deviation of duplicate results for the feline IFNy ELISpot analysis was

<15% of the median SFU.

Overlapping 15mer peptides for HIV-1LAI/LAV (identical to NIH reference subtype-B virus

HIV-1HXB2) with 11 aa overlap were produced by Synpep Corporation, USA). Each peptide pool

was designated as HIV-1 peptide pool number 1-18 (Hpl-Hpl8). Peptide-pool Hpl consisted of

the first four overlapping peptides from the amino-terminal, and each peptide pool Hp2 through

Hpl8 contained three consecutive overlapping peptides starting from the amino-terminal. The

FIV peptides consisted of overlapping 15mer peptides for FIVBang (FIV subtype-A p24 sequence)

with 11 aa overlap and also produced by (Synpep Corporation, USA). Each FIV peptide pools

consisted of 3-4 consecutive peptides and were designated as FIV peptide pool number 1-17

(Fp l-Fp 1l7).

Statistical Analyses

Individual immunization groups, including protected versus infected cats in each study,

were analyzed for statistical significant difference by Mann-Whitney Rank Sum Test using

Sigma Stat (Windows Version 3.11). The difference was considered statistically significant

when P<0.05.









Results


Analysis of HIV-1N20 P24 for Vaccine

Silver-stained PAGE gel (Figure 3-7) and CBB-stained gel (data not shown) analyses showed

minimal contamination and a purity of 95% using BSA standard to extrapolate the p24

concentration. The silver-stained gel showed three bands at 24 kD (62.5-500 ng lanes), 20 kD

(250-500 ng lanes), and 48 kD (500 ng lane) (panel C). The HIV-1 p24 specificity of the 24 kD

product and truncated products was detected by immunoblot analysis using sheep anti-HIV-1

p24 antibodies (Cliniqa Corporation, Fallbrook, California) (panel A). The HIV-1 p24 bands at

24 kD were detectable from 31.25-500 ng, and a higher band at 48 kD at 500 ng appeared to be

p24 dimer. The two lower bands in the immunoblot at 15 kD and 20 kD were observed at 62.5-

500 ng and 125-500 ng, respectively, and appeared to be truncated p24 products (panel A). The

level of endotoxin in the HIV-1N20 p24 preparation used for vaccine was 1.19 EU/ng (2.38

EU/dose of vaccine), which was safe level for use as vaccine (LPS safety level previously tested

as <50 EU/dose).

HIV-1 and FIV Sequence Analyses

Sequences comparison of FIVFC1 p24 Challenge virus) with HIV-1N20 and HIV-1UCD1 p24

revealed the challenge virus to possess 52.1% homology and 31.3% identity with HIV-lucD1 p24

compared to 50.8% homology and 30.5% identity with HIV-1N20 p24. The HIV-1 p24 sequence

is 231 aa long compared to 223 of FIV p24. This resulted in alignment gap generation of 6.0%

and 7.6% gaps for HIV-lucD1 and HIV-1N20 p24, respectively. HIV-1N20 p24 contained aa

substitutions compared to HIV-lucD1 p24 within the 3 of the 4 published p24 CTL epitopes

shown on Figure 3-1, with a P S and I M substitution in the first epitope shown, a K to R

substitution in the third and a S T-- and D --E substitution in the fourth. The second epitope

was identical between the two sequences.









Vaccination Studies 1 and 2

In Vaccination Study 1 (Table 3-1), 3 of 4 HIVlucD1 p24-vaccinated cats (Group lA) and

4 of 4 Fel-O-Vax FIV-vaccinated cats (Group 1B) were protected against FIVFC1 challenge (15

CID5o), which infected all three PBS-immunized control cats (Group IC). The

unprotected/vaccinated cat (#AA1) and infected control cats had a major decrease in CD4+-cell

(CD4) counts and CD4+-cell/CD8+-cell (CD4/CD8) ratio when compared to the values from

protected/vaccinated cats. A comparison between infected cats and protected/vaccinated cats at

20 wpi showed only statistically significant difference for CD4/CD8 ratio (CD4 count, p=0.14;

CD4/CD8 ratio, p=0.001). However, statistical difference was observed between the mean

CD4/CD8 ratios of Fel-O-Vax FIV-vaccinated group and PBS-immunized control group at 20

wpi. These results demonstrate immune protection in the protected/vaccinated cats and further

support the results from FIV antibody and virus isolation analyses that these cats were clearly not

infected.

In Vaccination Study 2 (Table 3-2), 2 of 4 HIVlucD1 p24-vaccinated cats (Group 2A), 1 of

4 HIV1N20 p24-vaccinated cats (Group 2B), and 0 of 3 FIV p24-vaccinated cats (Group 2C) were

protected against FIVFC1 challenge (10 CID5o). All Fel-O-Vax FIV-vaccinated cats (Group 2D,

n=2) were also protected, while all control cats immunized with FeT-J cell/lysate (Group 2E,

n=2) were infected. Overall, the protected/vaccinated cats had higher CD4 counts and CD4/CD8

ratios than unprotected/vaccinated cats and infected control cats. In fact, statistical differences in

both the mean CD4 counts (p=0.019) and the mean CD4/CD8 ratios (p<0.001) were observed

between protected/vaccinated cats and infected cats. These statistical differences were due to the

infection and were not due to the difference in the mean CD4 counts of the pre-challenge groups

(Table 3-2; Groups A vs. B vs. C vs. D vs. E: CD4 count, p>0.10; CD4/CD8 ratio, p>0.2). This

pilot study confirms the results from Vaccination Study 1 that HIV1ucD1 p24 vaccine and Fel-O-









Vax FIV vaccine are effective against FIVFC1. In contrast, minimal protection was achieved by

HIVIN20 p24 vaccination. The level of protection (2 of 4) achieved with HIVuCD1 vaccine in

Vaccination Study 1 was slightly lower than Vaccination Study 2 (3 of 4) even though the

challenge dose in Study 1 was slightly higher than Study 2.

IFNy ELISpot Analysis of PBMC from Vaccination Study 2

As a measure for vaccine-induced cellular immunity, FIV-specific IFNy responses of

PBMC from vaccinated cats were compared. PBMC from HIV-lucD1 p24-vaccinated cats had

more cats responding to overlapping HIV-1LAI/LAV p24 and FIV p24 peptides (Hp3, Hpl 1, Hpl2;

Fp9) than PBMC from HIV-1N20 p24-vaccinated cats (Figures 3-8, and 3-9, Panels A and B).

However, the PBMC from both HIV-lucD1 p24- and HIV-1N20 p24-vaccinated cats (2 of 4 cats

from each group) responded to dual-subtype FIV whole-virus (IWV) immunogen. These results

clearly indicated that the PBMC from HIV-1 p24-vaccinated cats recognized cross-reactive

epitopes on FIV p24. Two HIV-1 p24-vaccinated cats also recognized Fp5 although one cat had

response slightly below the threshold value (figure 3-9A). The PBMC of all protected cats from

the HIV-1 p24-vaccinated groups (#185, #205, #207) responded to Fp9 peptide pool but the

response from cat #207 was way below the threshold level of significance. Moreover, the

PBMC from Fel-O-Vax FIV-vaccinated cat #171 and FIV p24-vaccinated cat #163 had a

significant cross-reactive response to Hpl 1 (Figure 3-8C). Hence, two-way cross-reactivity was

observed, whereby HIV-1 p24-vaccinated cats recognized Fp9 epitope, while Fel-O-Vax FIV-

and FIV p24-vaccinated cats recognized Hp 11.

The PBMC from the two Fel-O-Vax FIV-vaccinated cats had robust IFNy response (Figure

3-9C). One cat (#171) had significant responses (>50 SFU threshold) to a number of FIV p24

peptide pools, including Fp9, but the other protected cat (#183) had no response to Fp9 (Figure









3-9A, C). Both cats had a significant response to Fp3, suggesting the potential of multiple

epitopes for protection. Thus, 4 of 5 protected cats vaccinated with HIV-1 p24 or Fel-O-Vax

FIV vaccine reacted to Fp9, of which 3 responses were above the threshold response, while none

of the unprotected/vaccinated cats had significant response to Fp9 (Figure 3-9A, B and C). In

any event, the numbers of cats are much too limited in the current study to determine the cross-

protective epitope(s) solely based on one parameter (i.e., IFNy response) of cellular immune

response.

Discussion

The efficacy of Vaccination Studies 1 and 2 are summarized in Table 3-2 along with

results from other published and unpublished studies of our laboratory. The combined results

from current two studies show subtype-B HIV-lucD1 p24 vaccine conferring the highest

protection rate of 62% (5 of 8 cats) among the two HIV-1 p24 vaccines tested against FIVFC1

strain. Only minimal efficacy (1 of 4) was observed with subtype-A HIV-1N20 p24 vaccine in

Vaccination Study 2. This observation was somewhat in conflict with the sequence results since

considerable sequence identity and homology existed between HIV-1N20 and HIV-lucD1 p24

sequences. However, the numbers of animals used in each group were much too small to make

major conclusion. In fact, the efficacy of HIV-lucD1 p24 in Study 2 had the lowest rate, along

with another study, among the seven vaccination studies performed so far (Table 3-2). Since the

efficacy was also low for HIV-lucD1 p24 vaccine in Study 2, more studies will be needed to

determine if significant difference in efficacy exists between subtype-B HIV-lucD1 p24 vaccine

and subtype-A HIV-1N20 p24 vaccine.

One potential reason for lower efficacy in Study 2 than Study 1 may be due to the SPF cats

used. SPF cats from Liberty Research and Harlan Sprague Dawley, Inc. were used in Studies 1









and 2, respectively. In general, cats from Harlan Sprague Dawley. were more susceptible to FIV

infection and more difficult to confer protection with HIV-1 p24 and Fel-O-Vax FIV vaccines

than cats from Liberty Research (R. Pu and J.K. Yamamoto, personal communique). Based on

MHC-I sequence analysis of one cat from Harlan Sprague Dawley and our semi-inbred cats

derived from backcross inbreeding with tom from Liberty Research, cats from Harlan Sprague

Dawley had MHC-I sequence lineage different from the predominant lineages of the cats from

Liberty Research (E. Sato and J.K. Yamamoto, personal communique). Furthermore, according

to our findings from Chapter 2, both HIV-1 p24 and Fel-O-Vax FIV vaccines conferred

protection against FIVFC1 by cellular immunity. Recent adoptive-transfer studies by our

laboratory demonstrated protection with prototype dual-subtype FIV vaccine was mediated by

CD4+ CTL and CD8+ CTL [57]. Hence, MHC-restricted T-cell immunity may play a central role

in protection observed with HIV-1 p24 vaccine.

The importance of cellular immunity was determined in current studies by monitoring the

FIV-specific IFNy responses of the vaccinated cats. HIV-lucD1 p24 vaccine was more

immunogenic than HIV-1N20 p24 vaccine, but not as immunogenic as Fel-O-Vax FIV vaccine.

In current study, only 1 of 3 cats vaccinated with FIVpet p24 had robust IFNy responses (Fp7,

Fp8, Fpl4, Fpl5), while another cat (#195) from the same group had a single significant

response to Fp3. The PBMC from both Fel-O-Vax FIV-vaccinated cats had significant

responses to two or more FIV peptide pools. Thus, Fel-O-Vax FIV vaccine appeared to be more

immunogenic than FIV p24 vaccine. This observation supported the previous study of our

laboratory, which showed only 2 of 7 FIV p24-vaccinated cats with low but above the threshold

responses to FIV p24 peptide pools (Fp3, Fp7, and Fpl0O). Fp3 and Fp7 were the common FIV









peptide pools recognized by the cats from both studies. Furthermore, all cats, which responded

to these peptide epitopes, were from the same vendor (Harlan Sprague Dawley).

The PBMC from HIV-lucD1 p24-vaccinated cats had IFNy responses to Hp3 (2 of 4),

Hpl 1 (3 of 4), and Hpl2 (4 of 4), while the PBMC from Fel-O-Vax FIV-vaccinated cats and FIV

p24-vaccinated cats had a significant response to Hpl 1 (2 of 5). In the previous unpublished

studies of our laboratory, the PBMC from HIV-IUCD1 and HIV-ILAI/LAV p24-vaccinated cats had

significant responses to Hp3 (1 of 3), Hp6 (4 of 8), Hpll 1 (6 of 8), Hpl2 (2 of 8), Hpl4 (3 of 8),

and Hp 17 (2 of 8). Current study had lower numbers of peptide epitopes recognized by the HIV-

1 p24-vaccinated cats than the previous studies. This may be due to the more diverse MHC

patterns present in the cats from previous studies, since previous studies used three sources of cat

vendors. Only 2 of the 8 cats were from the same vendor as current study. Moreover, the one

cat that responded significantly to all Hp3, Hpl 1, and Hpl2 in the previous study was from the

same vendor as the current study. Whereas, the other cat from the same vendor in previous study

responded slightly below the threshold value to Hpl 1 and Hpl2, but had no responses to other

peptide epitopes. In current study, the PBMC from the two Fel-O-Vax FIV-vaccinated cats had

a significant response to Fp3. In the previous study, the PBMC from 8 of 8 cats vaccinated with

prototype vaccine of Fel-O-Vax FIV vaccine, had significant responses to Fp3. Thus, Fp3 may

be the immunodominant epitope for both prototype and commercial vaccines. Other major

epitopes recognized by prototype vaccine were Fp7, Fpl0, and Fpl3 (5 of 8 each) followed by

Fpl (3 of 8), Fp2 (4 of 8), Fp8 (3 of 8) Fp9 (3 of 8), Fp 11 (3 of 8), and Fpl5 (4 of 8). Thus, both

prototype and commercial vaccines induced strong IFNy responses to multiple epitopes.

Findings from recent adoptive-transfer studies from our laboratory [7] further support the

importance of MHC-restricted cellular immunity in prototype vaccine of the commercial









vaccine. These findings taken together with results from HIV-1 p24 vaccine studies and passive-

transfer studies in Chapter 2 suggest the importance of MHC-restricted cellular immunity in

protection conferred by subtype-B HIV-1 p24 vaccines.

FIVpet 24 vaccine conferred no protection (0 of 3) in Vaccination Study 2. This result

taken together with other FIV p24 studies demonstrated a very minimal protection with FIV p24

vaccine (Table 3-2, 3 of 21, 14%). Moreover, subtype-B HIV-1 p24 vaccines were far more

effective against FIV challenge than FIV p24 vaccine (64-67% versus 14%, Table 3-3). A

comparison of HIV-lucD1 and FIVFC1 p24 sequences demonstrated only 31.3% identity and

52.1% homology between them. Two potential explanations can be provided. FIV p24 may

possess immunodominant epitopes, which restrict the development of immune responses to

protective epitopes. Protective epitopes need to stimulate functional activity essential for

vaccine protection such as CTL activity to viral antigen. Many CTL epitopes have been

identified on HIV-1 p24, including those that are immunodominant [LANL]. However, these

CTL epitopes were identified by analyzing the CTL responses to HIV-1 p24 peptides made by

the PBMC from HIV-1-positive human subjects. Since cats may not recognize the same

epitopes as human, our IFNy ELISpot analysis with overlapping FIV and HIV-1 p24 peptides

were the first step towards identifying cross-protective CTL and TH epitopes induced by

effecticacious vaccines (HIV-lucD1 p24 and Fel-O-Vax FIV vaccines). The other possibility is

that cellular response elicited by HIV-1 p24 is not reacting to the cross-reactive epitopes on the

FIV p24 but instead reacting to the mimotopes elsewhere on the FIV virus. One way to examine

this possibility was by performing NCBI Blast search on both homologous and divergent

segments of the HIV-lucD1 p24 sequence against other structural and enzymatic proteins (Env,









Pol, MA) of FIV. Initial analysis suggests no significant mimicry of p24 aa sequences elsewhere

on the virus.

This study is the first to simultaneously examine the cross-protective epitopes of FIV/HIV

by IFNy ELISpot. The results seem to indicate that there is cross-recognition of epitopes with

HIV-1 p24-vaccinated cats responding to FIV IWV immunogen and FIV p24 epitope Fp9.

Furthermore, both Fel-O-Vax FIV-vaccinated cat #171 and FIV p24-vaccinated cat #163

responded to HIV-IUCD1 p24 protein and HIV-1 p24 epitope Hpl 1. Moreover, 3 of 4 HIV-

IUCD1 p24-vaccinated cats responded to Hp 11, with the response of the fourth cat near the

threshold. Overall, these results demonstrated two-way cross-reactivity and also confirmed the

existence of conserved epitopes between subtype-B HIV-1 p24 and FIV p24.

Though HIV-1N20 and HIV-IUCD1 p24 sequences are 90.5% identical and 95.2%

homologous, there was a considerable difference in the protection rate of the two groups with

62% protection for HIV-IUCD1 p24 vaccinated cats and only 25% efficacy for cats vaccinated

with HIV-1N20 p24. Both vaccinated groups generated antibodies to HIV-1 p24, but the ELISpot

result showed less overall reactivity of HIV-1N20 p24-vaccinated cats compared to HIV-IUCD1

p24-vaccinated cats to HIV-1 p24 peptides. Two reasons for this disparity in IFNy responses can

be speculated. This could also be the result of aa substitutions within the corresponding regions

of HIV-1N20 p24. It is also possible that the difference in reactivity could be based on the fact that

the peptide pool was designed based on HIV-1HXB2 (identical to HIV-1LAV/LAI), which is more

similar to HIV-IUCD1 p24 than to HIV-1N20 p24. One reasons for selecting this sequence was

because of its sequence variation within the CTL epitopes shown in Figure 3-1, which are

recognized by both cats and humans. Though the substitutions were similar in charge this change









or the steric alteration of cleavage patterns by flanking non-homologous substitutions may be

responsible for the disparity in the immunogenicity and the protection rate of these vaccines.

In current studies, Fel-O-Vax FIV vaccine conferred 100% protection (6 of 6). The high

protection rate against heterologous-subtype FIVFC1 was in agreement with previous published

and unpublished results (Table 3-2). These data taken together clearly demonstrate that the

commercial Fel-O-Vax FIV vaccine provides considerable protection (Table 3-2, 17 of 18)

against heterologous-subtype-B virus. Furthermore, long-duration vaccine analyses of Fel-O-

Vax FIV vaccine showed 50-56% protection in cats challenged with FIVBang and FIVFC1 (both 25

CID5o), respectively, after 1-year boost. Since natural transmission doses are reported to be far

lower than the lowest dose (10 CID5o) used in current studies [23,57,63], commercial Fel-O-Vax

FIV vaccine is likely to be more effective against natural transmission. A pilot study performed

by our laboratory also showed long-term efficacy (67%, 2 of 3) with HIV-lucD1 p24 vaccine

against subtype-A/B recombinant FIVBang challenge administered after 1-year boost (Table 3-2).

Since single boost 1-year later was sufficient enough to provide 67% protection, the immunity

elicited during the first three vaccinations must have sustained, in order for a single 1-year boost

to confer such level of protection.










Table 3-1. Vaccination Study 1 with HIV-lucD1 p24 vaccine and commercial Fel-O-Vax FIV
vaccine
FIV Chal. CD4# (CD4/CD8) b Weeks Post-Challenge (FIV Ab/VI) Protection
Group Cat # Vaccination (CIDs0)a Pre Post 3 6 9 12 15 17 20 P/L/B c Rate (%)


A AA1 HIV1ucDI p24 FC1 (15) 3.47(3.6) 2.44(1.4) +/+ +/+ +/+ +/+ +/+ ++ ++ ND 3/4(75%)
MD1 HIVlucDI p24 FC1 (15) 3.47(4.9) 3.88(2.5)
MG1 HIV1ucDI p24 FC1 (15) 3.53(2.7) 2.90(1.8)
MF3 HIVlucDlp24 FC1 (15) 1.83(3.4) 1.71(1.7)
3.07(3.6) 2.73(1.8)
B
AA2 Fel-O-Vax FIV FC1 (15) 3.58(3.4) 3.56(2.7) -/- -/- -/- -/- -/- -/- -/- -/-/- 4/4(100%)
MD2 Fel-O-Vax FIV FC1 (15) 3.41 (3.8) 2.83 (2.6)
MG2 Fel-O-Vax FIV FC1 (15) 3.61(2.8) 2.92(2.2)
MF4 Fel-O-Vax FIV FC1 (15) 1.44(2.7) 2.13 (2.1)
3.01 (3.2) 2.86(2.4)

C MD3 PBS FC1 (15) 3.23(2.5) 2.89(1.4) -/- +/- +/+ +/+ +/+ +/+ +/+ +/+/- 0/3(0%)
MG5 PBS FC1 (15) 1.99(2.6) 1.50(0.8) -/- -/- +/+ +/+ +/+ +/+ +/+ +/-/+
MK4 PBS FC1 (15) 3.03(4.3) 1.62(1.1) -/- -/- +/+ +/+ +/+ +/+ +/+ +/-/+
2.75(3.1) 2.00(1.1)


a FIV challenge (FIV chal.) was subtype-B FIVFC1 (FC1) at 15 CID50.

b The CD4 counts (CD4#; xlOOO/[tL) and CD4/CD8 ratios (CD4/CD8) determined at -2 (Pre) and
20 (Post) weeks. The average count and ratio are shown in bold italics for each group.

c FIV infection was based on the development of FIV antibodies (FIV Ab) determined by FIV-
immunoblot analysis and the presence of viruses determined by virus isolation (VI) using RT and
PCR analyses. Results for the samples collected at 3, 6, 9, 12, 15, 17, and 20 wpc are shown either as
positive (+) or negative (-) for FIV antibodies or viruses. Virus isolation was also performed on
PBMC (P), lymph node (L), and bone marrow (B) cells at 34-46 wpc. Abbreviation is not done
(ND).









Table 3-2. Vaccination Study 2 with HIV-1 p24, FIV p24, and Fel-O-Vax FIV vaccines


Vaccination FIV Challenge CD4# (CD4/CD8) b Weeks Post-Challenge (FIV Abs/VI) c Protection
Group Cat # Status (CIDso) a Pre Post 3 6 9 12 15 17 20 Rate (%)

2 A 147 HIV1uCDI p24 FC1 (10) 3.85(5.59) 0.68(1.98) -/- -/- +/- +/+ +/+ +/+ + 2/4(50%)
185 HIV1uCDI p24 FC1 (10) 2.39 (3.43) 1.80(2.42) -/- -/- -/- -/- -/- -/- -
199 HIV1uCDI p24 FC1 (10) 1.19(2.27) 1.06(1.55) -/- -/- /- +/+ +/+ +/+ +
205 HIV1uCDI p24 FC1 (10) 2.31 (3.66) 2.98 (2.25) -/- -/- -/- -/- -/- -/- -/-
2.44(3.74) 1.63(2.05)

2B 175 HIVIN20 p24 FC1 (10) 0.87(2.09) 0.40(1.71) -/- +- +/- +/- +/- +/+ 1/4 (2"..'
187 HIVIN20 p24 FC1 (10) 3.05(4.54) 1.40(0.88) -- +/- +/- +/+ +/+ +/+ +
201 HIVIN20p24 FC1 (10) 0.89(3.22) 1.55(1.20) -/- -/- +/- +/+ +/+ +/+ +
207 HIVIN20 p24 FC1 (10) 1.90 (2.72) 2.90(2.62) -/- -- -/- -/- -/- -/- -/-
1.68(3.14) 1.56(1.60)

2C 163 FIVpet p24 FC1 (10) 1.56(3.20) 0.56(1.07) -/- +/- +/- +/+ +/+ +/+ +/+ 0/3 ..
173 FIVpet p24 FC1 (10) 1.03(2.78) 0.76(0.76) -/- +/- +/- +/+ +/+ +/+ +
195 FIVpetp24 FC1 (10) 1.72(2.54) 1.76(1.58) -/- -/- -/- +/- +/+ +/+ +/+
1.44 (2.84) 1.03 (1.14)

2D 171 Fel-O-Vax FIV FC1 (10) 1.22(2.49) 1.09(2.44) -/- -/- -/- -/- -/- -/- -/- 2/2(1""..
183 Fel-O-Vax FIV FC1 (10) 1.86 (2.78) 2.51 (2.25) -/- -/- -/- -/- -/- -/- -/-
1.54(2.64) 1.80(2.35)

2 E 189 FeT-J cell lysate FC1 (10) 1.98(3.22) 1.31(1.15) -/- -/ +/- +/+ +/+ +/+ +/+ 0/2 ..
203 FeT-J cell lysate FC1 (10) 2.57(3.20) 2.61(1.60) -/- -/- -/- -/+ /+ +/+ +/+
2.28(3.21) 1.96(1.38)

a FIV challenge was subtype-B FIVFC1 (FC1) at 10CID50.

b The CD4 counts (CD4#; xlOOO/[tL) and CD4/CD8 ratios (CD4/CD8) determined at -2 (Pre)
and 20 (Post) weeks. The average count and ratio are shown in bold italics for each group.

FIV infection was based on the development of FIV antibodies (FIV Ab) determined by FIV-
immunoblot analysis and the presence of viruses determined by virus isolation (VI) using RT and
PCR analyses. Results for the samples collected at 3, 6, 9, 12, 15, 17, and 20 wpc are shown
either as positive (+) or negative (-) for FIV antibodies or viruses. Virus isolation was also
performed on PBMC (P), lymph node (L), and bone marrow (B) cells at 34-46 wpc.










N20 PIVQNAQGQMVHQSMSPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDLNMMLNIVG
ConsA PIVQNAQGQMVHQSLSPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDLNMMLNIVG


N20 GHQAAMQMLKDTINEEAAEWDRVHPVHAGPIPPGQMREPRGSDIAGTTSTLQEQIGWMTS
ConsA GHQAAMQMLKDTINEEAAEWDRLHPVHAGPIPPGQMREPRGSDIAGTTSTPQEQIGWMTG


N20 NPPIPVGEIYRRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQATQE
ConsA NPPIPVGDIYKRWIILGLNKIVRMYSPVSILDIKQGPKEPFRDYVDRFFKTLRAEQATQE


N20 VKNWMTETLLVQNANPDCKAILKSLGPGATLEEMMTACQGVGGPSHKARVL
ConsA VKNWMTETLLVQNANPDCKSILRALGPGATLEEMMTACQGVGGPGHKARVL



N20 PIVQNAQGQM\HQSMSPRTLNAVKVIEEKAFSPEVIPM]SALSEGATPQDLNMMLNIVG
UCD PVVQNLQGQM\HQPISPRTLNA-TVKVVEEKAFSPEVIPM]TALSEGATPQDLNTMLNTVG
N20 GHQAAMQMLKDTINEEAAEWDRVHPVHAGPIPPGQMREPRGSDIAGTTSTLQEQIGWMTS

N2UCD GHQAAMQMLKDTINEEAAEWDRVHPVHAGPIPPGQMREPRGSDIAGTTSTLQEQIGWMTS
UCD GHQAAMQMLKETINEEAAEWDRLHPVHAGPIAPDQMREPRGSDIAGITSTLQEQIGWMTN


N20 NPPIPVGEITRRWIILGLNKIVtMYSPVSILDIRQGPKEPFRDYVDRFYKT
UCD NPPIPVGEI"KRWIILGLNKIVFMYSPTSILDIRQGPKEPFRDYVDRFYKT


N20 IRAEQATQEVENWMTETLLVQNANPDCKAILKSLGPGATLEEMMTACQGVGGPSHKARVL
UCD IRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVL





Figure 3-1. Amino acid alignment of HIV-1N20 p24 (N20) with a subtype-A consensus sequence
(ConsA) and with HIV-1 p24. The stars under each amino acid pair indicate identical
bases. The number of dots listed below the non-identical pairs indicates the charge
similarity of the amino acids with 2 dots indicating a greater similarity in charge
signature than 1 dot, with no dots being indicative of least charge similarity. The
alignment comparison between HIV-1N20 and consensus sequences indicates that
HIV-1N20 was a good representative sequence of the subtype-A, and is not an outlier.
Therefore it should contain epitopes typical of this subtype. The boxes highlight
established CTL epitopes in the Los Alamos Database.

















Vaccination & Challenge


Vaccinalion FIV Challenge

Week -9 -6 -3 0 3
.= .=


9 12 15 18


21 24 27
..IL flC


Figure 3-2. Schedule for vaccination and challenge for vaccination studies. Vaccinated cats
received three of vaccine at 3-week intervals and received FIV challenge 3-weeks
after the last boost. These animals were challenged IV with FIVFC1.


.Je..









TTCGCGCTTAACCCTAGCCTTTTAGAAACAGCGGAAGGATGTCAGCAACTAATGGAACAGTT
ACAATCAGCTCTCAAGACAGGGTCAGAAGAACTTAAATCATTGTTTAACACCATAGCAACCC
TTTGGTGCGTGCATCAAAGGATAGATGTAAAAGACACCAAGGAAGCCTTAGATAAAGTAGA
GGAAGTACAGAACAAGAGCAAACAAAAGACACAGCAGGCAGCAGCTGCCACAGGAAGCGG
CAGCCAAAATTACCCTATAGTGCAAAATGCACAAGGGCAAATGGTACATCAGTCCATGTCA
CCTAGGACTTTGAATGCATGGGTGAAGGTAATAGAAGAAAAGGCTTTCAGTCCAGAAGTAA
TACCCATGTTTTCAGCATTATCAGAGGGAGCCACCCCACAAGATTTGAATATGATGCTAAAC
ATAGTGGGGGGACACCAGGCAGCAATGCAGATGCTAAAAGATACCATCAATGAGGAAGCTG
CAGAATGGGATAGGGTACACCCAGTACATGCAGGGCCTATTCCACCAGGCCAGATGAGGGA
ACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAAGAACAAATAGGATGGATG
ACCAGCAATCCACCTATCCCAGTGGGAGAAATCTATAGAAGATGGATAATTCTGGGATTAA
ATAAAATAGTAAGAATGTATAGCCCTGTCAGCATTTTGGACATAAGACAAGGGCCAAAAGA
ACCCTTTAGAGATTATGTAGATCGGTTCTATAAAACTTTGAGAGCTGAACAAGCTACACAGG
AAGTAAAAAATTGGATGACAGAAACCTTGCTGGTCCAAAATGCGAATCCAGACTGTAAGGC
CATTTTAAAATCATTAGGACCAGGGGGCTACATTAGAAGAGATGATGACAGCATGTCAGGG
AGTAGGGGGACCTAGCCATAAAGCAAGGGTTTTGGCTGAGGCAATGAGTCAAGCACAACA
GGCCAACATAATGATGCAGAGGGGCAATTTTAGGGGCCAGAGAACAATAAAATGCTTCAAC
TGTGGCAAAGAAGGACATCTAGCCAGAAATTGCAAGGCCCCTAGAAAAAAGGGCTGTTGGA
AATGTGGGAAGGAGGGACACCAAATGAAGGACTGTACTGAGAGACAGGCTAATTTTTTAGG
GAAAATCTGGCCTTCCAGCAAAGGGAGGCCAGGAAATTTTCCTCAGAGCAGACCGGAAACC
AGACCGGAACCAACAG


Figure 3-3. The full length nucleotide sequence of HIV-lN20 gag. The sequence of HIV-lN2ogag
was amplified from proviral DNA and sequenced by direct sequencing methods. The
forward primer sequence used to amplify the p24 gene is the 18-base sequence in
bold/underline, and the reverse primer sequence is the 18-base sequence in bold (no
underline).













111I I llll 11 l1i II .. II I


1)24








I 2 3 -I 5 6


I'QI 11,



p-1 4


I .. 1 2 3 4


Figure 3-4. Purity of PCR product and insert product of HIV-1N20 p24 gene. In panel A, PCR
product amplified from proviral DNA are shown at 0.04 (lane 2), 0.06 (lane 3), 0.08
(lane 4), and 0.1 (lane 5) |tg along with negative control (lane 6) and HIV-1-positive
control (lane 7) samples at 1 |tg. The molecular weight marker (MM) is shown on
lane 1. In panel B, the p24 insert (lane 3) and pQE30 vector (lane 4) after restriction
enzyme digestion are shown, along with low (lane 1, 1-MM) and high (lane 2, h-MM)
molecular weight markers. All samples were run on 0.8% tris-acetate EDTA gel for
20 minutes.


I \1\1 h l\l Iln~ll %lI..I
















BamHl Forward primer


HindIII
Reverse primer



PCR




Products


BamHl


Cut by BamH1, HindIII


BamHl Hindlil


Gel extraction


Gel extraction


Ligation (T4 DNA Ligase)


Transformation into E. coh M15 chemically competent cells


Sequence colonies selected to confirm insert and start large scale culture for induction of p24
expression

Figure 3-5. Schematic of p24 gene expression in E.coli M15 cells.


HindIII










BamH1
CGCG/GATCCGCGCCTATAGTGCAAAATGCACAAGGGCAAATGGTACATCAGTCC
ATGTCACCTAGGACTTTGAATGCATGGGTGAAGGTAATAGAAGAAAAGGCTTTCAGTCCA
GAAGTAATACCCATGTTTTCAGCATTATCAGAGGGAGCCACCCCACAAGATTTGAATATG
ATGCTAAACATAGTGGGGGGACACCAGGCAGCAATGCAGATGCTAAAAGATACCATCAAT
GAGGAAGCTGCAGAATGGGATAGGGTACACCCAGTACATGCAGGGCCTATTCCACCAGGC
CAGATGAGGGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAAGAACAA
ATAGGATGGATGACCAGCAATCCACCTATCCCAGTGGGAGAAATCTATAGAAGATGGATA
ATTCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTGTCAGCATTTTGGACATAAGA
CAAGGGCCAAAAGAACCCTTTAGAGATTATGTAGATCGGTTCTATAAAACTTTGAGAGCT
GAACAAGCTACACAGGAAGTAAAAAATTGGATGACAGAAACCTTGCTGGTCCAAAATGCG
AATCCAGACTGTAAGGCCATTTTAAAATCATTAGGACCAGGGGCTACATTAGAAGAGATG
ATGACAGCATGTCAGGGAGTAGGGGGACCTAGCCATAAAGCAAGGGTTTTGTAGCCC
A/AGCTTGGG
Lambda Hind III



Figure 3-6. HIV-1N20 p24 sequence with adapter sequences. HIV-1N20 p24 sequence after PCR
amplification (sequence without bold) was identical to the p24 sequence of the gag
shown in Figure 3-3. The adaptors ligated to the gene create the restriction enzyme
sites and are shown in bold on either ends of the gene sequence.









Figure 3-7. Immunoblot and silver-stained gel of the HIV-1N20 p24 protein used for Vaccination
Study 2. The immunoblots were reacted with either sheep anti-HIV-1 p24 antibodies
(panel A) or normal sheep serum (panel B). The immunoblot consisted of HIV-lucD1
p24 (UCD1) at 500 ng (lane 2) and HIV-1N20 p24 (N20) at 500 (lane 3), 250 (lane 4),
125 (lane 5), 62.5 (lane 6), 31.25 (lane 7), and 15.62 (lane 8) ng. No non-specific
reactivity was observed with normal sheep serum (panel B). The purity of the HIV-
IN20 p24 preparation was determined by silver-stain analysis of the p24 preparation
on 12% SDS-PAGE gel (panel C). The gel consisted of HIV-lucD1 p24 (UCD1) at
500 ng (lane 2) and HIV-1N20 p24 (N20) at 500 (lane 3), 250 (lane 4), 125 (lane 5),
and 62.5 (lane 6) ng. The gel also contained BSA standards at 25 (lane 7), 12.5 (lane
8), 6.25 (lane 9), and 3.12 (lane 10) ng, which was used to derive the concentration
curve. The molecular weight marker (MM) is shown on lane 1 for all panels.













HI\, ,, p24 Iini Lunoblot


SIhee) Anti-HIV p2-I4 Antibodies
HI\ ,, p114


Normal Sheep Scrum
HI\ -,, p24


7-----
.2'


4 I,


I.,, I


HI'-1 .,, p2-4 SiIer-Stain Gel


HI\, ,, p2-4 BSA standards


C

II)


5.4



















89


, ,,, I


L-









Figure 3-8. HIV-1 p24-specific IFNy responses of vaccinated cats from Vaccination Study 2.
IFNy responses to overlapping peptide pools of HIV-1 (Hpl-Hpl8) (A, B, C) were
determined by feline IFNy ELISpot assay. The IFNy responses of the PBMC from
HIV-IUCD1 p24 (panel A), HIV-1N20 p24 (panel B), FIVpet p24 (panel C, cats #163,
#173, #195), and Fel-O-Vax FIV (panel C, cats #171, #183) vaccinated cats are
shown. The virus-specific positive controls included IFNy responses to recombinant
HIV-lUCD1 p24 (UCD1 p24), HIV-1N20 p24 (N20 p24), and FIVpet p24 (Pet p24)
proteins, and inactivated dual-subtype FIV whole-viruses (IWV). The PBMC from
all vaccinated cats had moderate-to-high levels of responses to T-cell mitogen
stimulation with ConA (data not shown). ELISpot assays to both HIV-1 and FIV
peptides were performed at the same time. As a result, the positive control values for
set 1 and set 2 of each vaccination group are identical. No IFNy responses to LPS
and FeT-J cell lysate were detected in the PBMC from vaccinated cats, with the
exception of those from Fel-O-Vax FIV-vaccinated cats, which responded to cell
lysate. The IWV-stimulation results of PBMC from Fel-O-Vax FIV-vaccinated cats
are shown as SFU values after subtracting out the SFU values for FeT-J cell lysate
stimulation. SFU values (bars) above or at the dashed red line at 50 SFU (stringent
threshold) were considered significant.















- HIV-UCD p24-VACCINATED CATS

3rd Vaccination

- Pre-Challenge

- - --- - ---- -- ---


147 185
0199 *205




----- ------ -*------


200 *
0175s

m 150-
0-125*
C(O0
0 100*
75
50 -
C 25
0



200
0 175
150
r- 125
0 100.
75.
D 50o
LL.
CW 25
0



200
0 175.
r' 150-
125.
0 100 .
75-
LL 50
W 25.
0


jCj- FIVpet p24 VACCINATED CATS 0 E 195
- FEL-O-VAX FIV- VACCINATED CATS 17* 183

3rd Vaccination
- - Pre-Challenge
-- ------- ---------- --- -


IT- -f -- -- -- -- -


Hpl Hp2 Hp3 Hp4 Hp5 Hp6 Hp7 Hp8 Hp9 Hp10 lHp11 Hp12 Hp13 Hp14 Hp15 Hp16 Hp17 Hp18 UCD1 N20 Pet IWV
2/4 34 414 p24 p24 p2 4


] HIV-1N20 p24 -VACCINATED CATS -
Li *175 *187
3rd Vaccination
Pre-Challenge -201 207



------ ---- ------ -- ---- -------------- ----- -------- --- ---




Hpl Hp2 Hp3 Hp4 Hp5 Hp6 Hp7 Hp8 Hp9 Hp10 Hp11 Hp2 Hp13 Hp14 Hp15 Hp16 Hp17 Hpl8 UCD1 N20 Pet IWV
p24 p24 p24 2/4
2/4


CD


Cr,
0Z


Hpl Hp2 Hp3 Hp4 Hp5 Hp6 Hp7 Hp8 Hp9 Hp10 Hpll Hpl2 Hpl3 Hpl4 Hp15 Hpl6 Hpl7 Hp18 UCD1 N2 Pet WV
2/5 p24 : p24 p24 45
2HIV-1 p24 PEPTIDE POOL (Hp#) 15 p24 PROTEIN WV (6-8 g poo or 4 pg protein well) 2
HIV-1 p24 PEPTIDE POOL (Hp#) / p24 PROTI / IWV (6-8 pg pool or 4 lag protein /Iwell),


m=l I









Figure 3-9. FIV p24-specific IFNy responses of vaccinated cats from Vaccination Study 2. IFNy
responses to overlapping peptide pools of FIV p24 (Fpl-Fpl7) (A, B, C) were
determined by feline IFNy ELISpot assay. The IFNy responses of the PBMC from
HIV-IUCD1 p24 (panel A), HIV-1N20 p24 (panel B), FIVpet p24 (panel C, cats #163,
#173, #195), and Fel-O-Vax FIV (panel C, cats #171, #183) vaccinated cats are
shown. The virus-specific positive controls included IFNy responses to recombinant
HIV-lUCD1 p24 (UCD1 p24), HIV-1N20 p24 (N20 p24), and FIVpet p24 (Pet p24)
proteins, and inactivated dual-subtype FIV whole-viruses (IWV). The PBMC from
all vaccinated cats had moderate-to-high levels of responses to T-cell mitogen
stimulation with ConA (data not shown). ELISpot assays to both HIV-1 and FIV
peptides were performed at the same time. As a result, the positive control values for
set 1 and set 2 of each vaccination group are identical. No IFNy responses to LPS
and FeT-J cell lysate were detected in the PBMC from vaccinated cats, with the
exception of those from Fel-O-Vax FIV-vaccinated cats, which responded to cell
lysate. The IWV-stimulation results of PBMC from Fel-O-Vax FIV-vaccinated cats
are shown as SFU values after subtracting out the SFU values for FeT-J cell lysate
stimulation. SFU values (bars) above or at the dashed red line at 50 SFU (stringent
threshold) were considered significant.













- HIV-1UCD1 p24-VACCINATED CATS
- 3rd Vaccination
-------- Pre-Challenge


Fpl Fp2 Fp3 Fp4 Fp5 Fp6 Fp7 Fp8 Fp9 FplO0 Fp11 Fp12 Fp13 Fp14 Fp15 Fp16 Fp17 UCD1

3/4


175 B HIV-1N20 p24 -VACCINATED CATS--- 175 187
6 175 187
150 3rd Vaccination 0201 *207

100

50 ------------------------ -
25 -*
0 ., .0I I I .I I n I


Fpl Fp2 Fp3 Fp4 Fp5 Fp6


Fp7 Fp8 Fp9 Fp10 Fp11 Fpl2 Fpl3 Fpl4 Fp15 Fpl6 Fpl7 UCD1
p24
214


200?nn. -- -


.1 I


FIVpet p24-VACCINATED CATS
FEL-O-VAX FIV-VACCINATED CATS
3rd Vaccination
Pre-Challenge


1-I'I Cl


Fp8 Fp9
2/5 _
___


0 163 173 E 195
S171 *183





"IF J


U ~ _____________________ L.A.


Fpl Fp2 Fp3 Fp4
i 35 '1


Fp5 Fp6 Fp7 '
I 2/5 -
I___1


Fp13 iFp14i: Fp15 Fp16
'2/5 '2/15
I -- II- __


FplO0 Fp11 Fp12


Fp17 iUCD1; N20
p24 p24
5/2


FIV p24 PEPTIDE POOL (Fp#) / p24 PROTEIN / IWV (6-8 pg pool or 4 pg protein / well)


200
0175
i150-
0- 125
CO
o100*
S75
so50.
LC/ 25
CO 25


4


147 E 185
0199 *205


r---------- ----
----------- -- -

-- - ------ --- -- - -- ----- -
1, ,,,,, ,,,, ,


Pet IWV
p24 24


Pet IWV
p24 21


CD

-
II
,


J


'









CHAPTER 4
FINAL DISCUSSION

The long-term goal of our studies is the development of a highly efficacious HIV-1

vaccine. In our initiative, our laboratory seeks to develop a blueprint to this objective by

understanding the mechanism of our HIV-1 p24 vaccine efficacy. Our dual-subtype model using

inactivated whole-virus, though highly successful, presents several safety concerns (i.e.,

incomplete inactivation) which limit its use in HIV-1 vaccines for humans. Understanding the

mechanism of protection of our HIV-1 p24 vaccine provides a novel opportunity to establish the

minimum epitopic requirements necessary for a protective vaccine. This is important since

several safety issues prevent the use of a full virus construct.

HIV-1 p24 vaccine was able to provide protection against FIV, while FIV p24 vaccine

had minimal efficacy. This result seems counter-intuitive, since there is considerably more

sequence identity and homology between FIV p24 and FIV challenge virus than HIV-1 p24 and

FIV challenge virus. This result indicates that current vaccine dogma of including conserved

epitopes may not be sufficient for the successful design of an HIV-1 vaccine. Identifying and

omitting the immunodominant non-protective epitopes, which may detract the establishment of

an effective antiviral immune response, also may be necessary.

Core protein may be only a part of the overall vaccine equation. Establishment of an

efficient HIV-1 vaccine may require the utilization of multiple virus proteins in a subunit or

vector vaccine. Currently, our laboratory is engaged in examination of the cellular immune

response of cats to HIV-1 p24 and other conserved HIV-1 p24 proteins (RT and MA). The aim is

to identify the epitopes that are associated with cross-protection and are recognized by both cats

and humans. The results show that both cats and humans recognize the KK10 epitope of HIV-1.

If the feline immune response is similar to that of humans then HIV-1/FIV-cat model provides us









with a means of selecting desirable epitopes for inclusion in an HIV-1 vaccine. It is not yet clear

if this epitope is protective. Though our results are promising, more studies will have to be

performed in order to identify the specific protective epitopes.

The findings from current studies demonstrated that protection of the HIV-1/FIV vaccine

is based on cellular immunity. The exact immune cells involved in this protection are still

unknown. The cells capable of IFNy responses include not only CTL but T-helper (TH1) and NK

cells [64]. It is necessary to perform studies with enriched populations of CTL, TH, and NK cells

to determine which cells are primarily responsible for the vaccine efficacy and which peptides

are recognized. This could also provide us with the means to determine if the epitopes involved

in FIV protection and improved HIV-1 prognosis are the same.

The current studies illustrate that it is possible to develop an efficient FIV vaccine using

HIV-1 p24. These vaccines were consistently able to outperform vaccines based on homologous

virus p24. This presents the possibility of developing a second-generation FIV vaccine for

veterinary medicine, which has broader efficacy than our commercial FIV vaccine and addresses

the problem of conflict with currently available diagnostics faced by our commercial vaccine

(Fel-O-Vax FIV vaccine).

The findings of the passive-transfer study (Chapter 1) indicated that the antibody

immunity to HIV-1 based FIV vaccines was insufficient to provide protection against FIV

challenge. This implies that the protection observed with our HIV-1 p24 based vaccine was the

result of the host cellular response. Further examination of this response is required to fully

understand the mechanism of the vaccine. These findings of cross-species protection of FIV

provide an opportunity to utilize the cat model to determine if the immunodominant hypothesis is










valid and if so the mechanism of utilizing this selection criteria for the formulation of an HIV-1


vaccine.









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[60] Hohdatsu T, Okada S, Motokawa K, Aizawa C, Yamamoto JK ,Koyama H. Effect of
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[63] Burkhard MJ ,Dean GA. Transmission and immunopathogenesis of FIV in cats as a
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BIOGRAPHICAL SKETCH

Marcus Martin earned his B.S. degree at Morgan State University; Baltimore Maryland.

He worked for a time in biomedical research before entering the graduate program at UF where

he earned his PhD.





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1 SUBTYPE-A AND SUBTYPE-B HIV-1 P24 BASED FIV VACCINES: EFFICACY AND MECHANISMS OF CROSS-PROTECTION By MARCUS MAKESI MARTIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Marcus Makesi Martin

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3 To my loving family and dear friends; through th eir love and support, I was able to pursue my dreams

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4 ACKNOWLEDGMENTS I would first like to thank th e department of Microbiology and Cell Science for providing me with the opportunity to fulf ill my dream of becoming a research scientist. I am eternally grateful to Dr. Yamamoto who invited me in to her lab and provided me with exceptional mentorship. It was wonderful to learn from some one who also sees no limitations. I would like to specially thank Dr. Johnson, who provided continual support, and was instrumental in helping me to achieve my research objectives; Dr. Mar uniak who provided advice on several life issues inside and outside of gradua te instruction; Dr. Hoffmann who was very supportive throughout my studies and Dr. Uhl whose interest in my ove rall wellbeing was a constant comfort. Also I would like to specially thank Dr. Eiji Sato w ho advised me on the molecular biology aspect of my work, and Dr. Pu who assisted me immeasurab ly with the animal ha ndling during my studies. I would like to especially acknowledge the essential support provided by my family, relatives, my eternally supportiv e uncle Chick, and my grandparents Norman and Josephine who recently passed on. Peers help to provide support and Johnny Davi s, Laurence Flowers, Karen Viera, Tiffany Snipe, Carla Phillips and Gareth Jordan were ther e to share the journey. It was also my privilege to have the pleasure of working with our lab team; James Coleman, Mayuko Omori,.Blerina Hysi, Samantha Hass, Melissa Voltz, Taishi Tana be, and Maki Tanabe who all provided research camaraderie.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 LIST OF ABBREVIATIONS........................................................................................................10 ABSTRACT....................................................................................................................... ............12 CHAPTER 1 INTRODUCTION..................................................................................................................13 Background..................................................................................................................... ........13 Human Immunodeficiency Virus...........................................................................................13 HIV-1 Viral Infectivity Factor (Vif)................................................................................14 HIV-1 Viral Protein U (Vpu)..........................................................................................14 HIV-1 Viral Protein R (Vpr)...........................................................................................14 HIV-1 Negative Regulatory Factor (Nef)........................................................................15 HIV-1 Trans-Activator of Transcription (Tat)................................................................15 HIV-1 Regulator of Vi ral Protein (Rev)..........................................................................15 HIV-1 Classification........................................................................................................15 HIV-1 Treatment.............................................................................................................16 Feline Immunodeficiency Virus.............................................................................................16 FIV Group Associated Antigen (Gag).............................................................................17 FIV Envelope (Env)........................................................................................................17 FIV Polymerase (Pol)......................................................................................................18 FIV Protease (PR)............................................................................................................18 FIV Reverse Transcriptase (RT).....................................................................................18 FIV Deoxyuridine Triphosphate (DU)............................................................................18 FIV Integrase (IN)...........................................................................................................19 FIV Viral Infectivity Factor (Vif)....................................................................................19 FIV Open Reading Frame 2 (Orf2).................................................................................19 FIV Regulator of Viral Protein (Rev)..............................................................................19 HIV/FIV Vaccine Model........................................................................................................19 Rationale and Goals for the Proposed Studies........................................................................21 Specific Aims.................................................................................................................. ........21 2 THE ROLE OF ANTIBODY IMMUNITY IN THE PROTECTION CONFERRED BY HIV-1UCD1 P24 VACCINE.................................................................................................30 Introduction................................................................................................................... ..........30 Methods........................................................................................................................ ..........31

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6 Animals........................................................................................................................ ....31 Serum Collection for B-cell Ep itope Mapping by FIV ELISA....................................... 31 Overlapping FIV P24 Peptides for ELISA...................................................................... 32 Immunoblot Analysis......................................................................................................33 Passive-transfer Studies w ith An tibodies from Vaccinated Cats .................................... 33 Antibody Preparation.......................................................................................................34 Ammonium sulfate precipitation..............................................................................35 Caprylic acid purification.........................................................................................35 Virus isolation..........................................................................................................36 VNA assay................................................................................................................36 Results........................................................................................................................ .............37 B-cell Epitope Analysis...................................................................................................37 Passive-transfer Studies: The Analysis of Purified Antibody Preparations for Transfer....................................................................................................................... .37 Passive-transfer Studies w ith Serum and Antibodies from Vaccinated Cats .................. 39 Discussion..................................................................................................................... ..........41 3 SUBTYPE A HIV-1 P24 AS AN FIV VACCINE IMMUNOGEN.......................................61 Introduction................................................................................................................... ..........61 Methods........................................................................................................................ ..........63 Virus Selection................................................................................................................63 Co-culturing for Virus Amplification.............................................................................. 63 Isolation of Proviral DNA...............................................................................................64 Sequencing and Expression of HIV P24......................................................................... 64 HIV-1N20 primers......................................................................................................65 Restriction enzyme digestion...................................................................................66 Purification...............................................................................................................67 Statistical Analyses..........................................................................................................70 Results........................................................................................................................ .............71 Analysis of HIV-1N20 P24 for Vaccine..........................................................................71 HIV-1 and FIV Sequence Analyses................................................................................71 Vaccination Studies 1 a nd 2............................................................................................ 72 IFN ELISpot Analysis of PBMC from Vaccination Study 2......................................... 73 Discussion..................................................................................................................... ..........74 4 FINAL DISCUSSION............................................................................................................94 LIST OF REFERENCES............................................................................................................. ..97 BIOGRAPHICAL SKETCH.......................................................................................................102

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7 LIST OF TABLES Table page 1-1 Current drugs availabl e to treat HIV infection a................................................................24 2-2 Current HIV vaccine trials.................................................................................................25 2-1 Passive-Transfer Study 1 with sera from vaccinated and non-vaccinated cats.................44 2-3 Passive-Transfer Study 3 with purified antibodies from vaccinated and nonvaccinated cat sera............................................................................................................ .46 2-4 Summary of the results from Passive-Transfer Studies 1-3...............................................47 2-5 VNA titers of highly purified antib odies and corresponding pooled serum......................48 3-1 Vaccination Study 1 with HIV-1UCD1 p24 vaccine and commercial Fel-O-Vax FIV vaccine........................................................................................................................ .......80 3-2 Vaccination Study 2 with HIV-1 p24, FIV p24, and Fel-O-Vax FIV vaccines.................81

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8 LIST OF FIGURES Figure page 1-1 Proviral genomic organization of FIV, HIV-1, and HIV-2. FIV resembles HIV by having LTR-gag-pol-env-LTR genomic organization, while missing few regulatory genes ( vpu, vpx, nef ). The orf-2 gene was initially reported to express.............................26 1-2 Comparative gag phylogenetic tree of FIV, HIV1, HIV-2, and SIV. An unrooted phylogenetic tree was created using th e BLOSUM matrix and neighbor-joining algorithm based on the Kimura two-para meter correction[44], [45]. Support..................27 1-3 FIV p24 cross-reactivity of the sera from HIV-1 p24-vaccinated cats. FIVBang p24 strip (200 ng/strip) was reacted with individual cat serum at 1:100 dilution. Tested sera were pre(pr) and post-3rd i mmunization(pre-challe nge) sera of cats......................29 2-1 Schedule of vaccination and challenge fo r passive-transfer studies. Nave SPF cats were transfused with antiserum or pur ified antibodies equivalent to 30% of recipients total blood volume (TBV). First transfusion was given on day -1..................49 2-2 Schematic of antibody preparation for passi ve-transfer studies. The figure illustrates the procedure of three separate passive im munity-transfer studies. In the first study four groups of two cats each were transfused with IWV ( inactivated..............................50 2-3 B-cell epitope analyses of sera and PBMC from HIV-1 p24-vaccinated cats. Sera from HIV-1 p24-vaccinated cats (n = 15) and FIV p24-vaccinated cats (n= 14) at 3 weeks after the 3rd vaccination before ch allenge and from FIV-infected control............51 2-4 Amino acid sequence alignment of HIV1 and FIV strains, and B-cell peptide sequence alignment Sequence homology analysis between HIV-1UCD1 and FIVBang is shown with () for amino acid homology and (: ) for amino acid identity according to.....52 2-5 CBB-stained gel and immunoblot of pur ified antibody preparations for PassiveTransfer Study 3. Purified antibodies from HIV-1UCD1 p24 (panel A, lanes 1,2,5,6), HIV-1N20 (panel A, lanes 3,4,8,9) Fel-O-Vax FI V (panel B, lanes 1,2,5,6), FIV p24.......54 2-6 Immunoblot of purified antibody preparati ons used in the six groups from PassiveTransfer Study 3. Each lane contained 2 mg of purified preparation. The antibodies were detected using goat anti-cat IgG heavy and light chain antibodies...........................56 2-7 IgG concentrations of purified antibody preparations from Passive-Transfer Study 3. The IgG concentrations of pre-purificati on pooled serum (Pre) and post-purification antibody preparations (Post) were de termined by commercial feline IgG .......................57 2-8 IgA concentrations of purified antibody preparations from Passive-Transfer Study 3. The IgG concentrations of pre-purificati on pooled serum (Pre) and post-purification antibody preparations (Post) were de termined by commercial feline IgG........................58

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9 2-9 IgM concentrations of pur ified antibody preparations fr om Passive-Transfer Study 3. The IgG concentrations of pre-purificati on pooled serum (Pre) and post-purification antibody preparations (Post) were de termined by commercial feline IgG........................59 2-10 HIV-1 and FIV p24 reactivity of purif ied antibody preparations from PassiveTransfer Study 3. The p24 reactivity was based on immunoblot analysis using HIV1UCD1 p24 (A), HIV-1N20 p24 (B), FIVPet whole-virus (C), and uninfected FeT-J cell...60 3-1 Amino acid alignment of HIV-1N20 p24 (N20) with a subtype-A consensus sequence (ConsA) and with HIV-1 p24. The stars under ea ch amino acid pair indicate identical bases.......................................................................................................................... .........82 3-2 Schedule for vaccination and challeng e for vaccination studies. Vaccinated cats received three of vaccine at 3-week intervals and rece ived FIV challenge 3-weeks after the last boost. These animal s were challenged IV with FIVFC1.................................83 3-3 The full length nucleotide sequence of HIV-1N20 gag The sequence of HIV-1N20 gag was amplified from proviral DNA and seque nced by direct sequencing methods. The forward primer sequence used to amplify the p24 gene is the 18-base sequence......84 3-4 Purity of PCR product and insert product of HIV-1N20 p24 gene. In panel A, PCR product amplified from proviral DNA are shown at 0.04 (lane 2), 0.06 (lane 3), 0.08 (lane 4), and 0.1 (lane 5) g along with negative contro l (lane 6) and HIV-1-.................85 3-5 Schematic of p24 gene expression in E.coli M15 cells.....................................................86 3-6 HIV-1N20 p24 sequence with adapter sequences. HIV-1N20 p24 sequence after PCR amplification (sequence without bold) was identical to the p24 sequence of the gag shown in Figure 3-3. The adaptors ligate d to the gene create the restriction...................87 3-7 Immunoblot and silver-stained gel of the HIV-1N20 p24 protein used for Vaccination Study 2. The immunoblots were reacted with either sheep antiHIV-1 p24 antibodies (panel A) or normal sheep serum (panel B). The immunoblot.........................................88 3-8 HIV-1 p24-specific IFN responses of vaccinated cats from Vaccination Study 2. IFN responses to overlapping peptide pools of HIV-1 (Hp1-Hp18) (A, B, C) were determined by feline IFN ELISpot assay. The IFN responses of the PBMC................90 3-9 FIV p24-specific IFN responses of vaccinated cats from Vaccination Study 2. IFN responses to overlapping peptide pools of FIV p24 (Fp1-Fp17) (A, B, C) were determined by feline IFN ELISpot assay. .......................................................................92

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10 LIST OF ABBREVIATIONS HIV: Human Immunodeficiency Virus HIV-1UCD1: HIV subtype-B strain HIV-1N20: HIV subtype-A strain HIV1LAV: HIV subtype-B strain FIV: Feline Immunodeficiency Virus FIVBang: FIV-Bangston (subtype-A gag-pol-envv1-v2, B envv3-v9) FIVPet: FIV-Petaluma (subtype-A) FIVFC1: FIV-FC1 (subtype-B) Fel-O-Vax: Commercial FIV vaccine, Fort Dodge Animal Health, Fort Dodge, Iowa. IWV: Inactivated whole virus SPF: Specific pathogen free cats LTNP: Long term non-progressor KK10: HIV epitope associated with LTNPs Gag: Group specific antigen Pol: Polymerase Env: Envelope Vif: Viral Infectivity Factor Vpu: Viral Protein U Vpr: Viral Protein R Nef: Negative Regulatory Factor Tat: Trans-Activator of Transcription Rev: Regulator of Viral Protein RT: FIV Reverse Transcriptase PR: Protease

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11 PRIs: Protease inhibitors IN: Integrase DU: Deoxyuridine triphosphate HAART: Highly active an tiretroviral therapy ART: Anti-retroviral therapy MA: Matrix CA: Capsid NC: Nucleocapsid HVTN: HIV Vaccine Trials Network Orf2: Open Reading Frame 2 LANL: Los Alamos National Laboratories NRTI: Nucleoside Reverse Transcriptase Inhibitor NNRTI: Non-nucleoside Reverse Transcriptase Inhibitors PI: Protease Inhibitor FI: Fusion Inhibitors VNA: Virus-neutralizing antibody SC: Vaccinated subcutaneously RID: Radial immunodiffusion FeT-J: Feline T-cell line developed By J.K. Yamamoto EU: Endotoxin TBV: Total blood volume SFU: Spot forming units ELISpot: Enzyme-linked immunosorbent spot pQE30: Expression vector is based on the T5 promoter transcription-translation system

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12 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SUBTYPE-A AND SUBTYPE-B HIV-1 P24 BASED FIV VACCINES: EFFICACY AND MECHANISMS OF CROSS-PROTECTION By Marcus Makesi Martin August 2007 Chair: Janet K. Yamamoto Major: Microbiology and Cell Science Published data shows HIV-1UCD1 p24 based vaccine protects cats from FIV infection. Understanding the mechanism of vaccine prot ection could provide a blueprint to the development of an HIV-1 vaccine for humans. The studies were performed by evaluating the protective ability of the vaccine -induced antibody response in three separate passive-transfer studies. The first involving vaccine antiserum, the second involving ammonium sulfate purified antibodies, and the third using antibodies purified using ammoni um sulfate precipitation and caprylic acid fractionation. The data suggested that the protective efficacy of HIV-1UCD1 p24 (subtype-B) based vaccine was not mediated by vaccine-induced antibody immunity but most likely mediated by vaccine-induced cellular immunity. In addition protective efficiency varied between cats vaccinated with subtype-B HIV-1UCD1 p24 (67% protection ) and subtype-A HIV1N20 p24 (25% protection). In addition, IFN ELISpot analysis using separate peptide pools of overlapping 15mers of FIV and HIV-1 p24 showed cross reactivity between FIV peptides and HIV-1 p24-vaccinated cat sera and between HIV-1 peptides and sera from either FIV p24vaccination a Fel-O-Vax FIV-vaccinated cats. Some vaccinated cats reacted to peptides corresponding to the KK10 HIV-1 epitiope in huma ns, which is associated with long term nonprogressor (LTNP) HIV-1 infected individuals.

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13 CHAPTER 1 INTRODUCTION Background Human Immunodeficiency (HIV) and feline i mmunodeficiency virus (FIV) are members of the lentiviral family Retroviradae [1],[2]. These viruses are characte rized by their ability to infect terminally differentia ted, non-dividing cells such as macrophages; by their genomic organization of group specific antigen ( gag ) polymerase ( pol ), and envelope ( env ) in addition to regulatory genes (Figure 1-1); and by their utiliza tion of the enzyme revers e transcriptase (RT) to synthesize DNA using their RNA genome as a temp late [1]. The error-prone nature of RT accounts for the high mutation rate observed among these viruses [2]. Human Immunodeficiency Virus HIV-1 infection was first reporte d in 1983 as a retrovi ral infection of humans [3]. HIV-1 is the etiological agent of acquired immune deficiency syndrome (AIDS) which causes a progressive deterioration of the host immune system, characterized by the loss of CD4+ T-helper lymphocytes. The CD4 receptor is the primary recep tor of viral entry into the cell. Other major receptors utilized by the virus include CCR5 and CXCR4 [4]. Its genome contains three major genes, consisting of gag, pol and env The primary p160 mRNA transcript of HIV-1 is translated into Gag and Pol proteins. Gag p55 is further cl eaved into p24 core protein, p17 matrix protein, p9 nucleocapsid, and proline-rich p6 protein involved in viral assembly. Pol precursor protein is cleaved RT, protease, and integrase [5]. Th e HIV-1 genome also encodes two regulatory proteins, Tat and Rev, which regu late transcription and viral R NA transport, respectively. The virus also contains four accessory genes, vif, vpu, vpr and nef which are implicated in viral pathogenesis [6]. The accessory and regulator y genes are described in detail below.

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14 HIV-1 Viral Infectivity Factor (Vif) Vif-like proteins are associated with all known lentiviruses with the exception of equine infectious anemia virus (EIAV) [7]. Vif appears to be needed at th e late stage of viral replication and may suppress antiviral activit y of T lymphocytes and macrophages, which are the main cells infected in humans and cats [8,9]. Macrophages also serve as viral reservoirs of infection even after the acute stage of in fection. It is believed that Vif is important for the retention of viral infectivity because it acts as an inhibiter to the antiviral pathway involving APOBEC3G, a messenger RNA editing enzyme [10] Vif may prevent the editing of the early single strand product of reverse transcription. It is also believed that this activ ity results in the prevention of many mutations, which could compromise the key structural proteins, re gulatory proteins, and enzymes and as a result, aids in retaining viral infectivity. HIV-1 Viral Protein U (Vpu) Vpu protein promotes the degred ation of the CD4 antigen in th e host cell, and this prevents the binding of CD4 molecules to viral gp 120 in the infected cel ls [11]. Vpu also causes the formation of ion channels in the surface membrane of the infected cells. The ability of Vpu to form these channels seems to correlate with the cells ability to release the virus [12]. HIV-1 Viral Protein R (Vpr) Vpr protein appears to be involve d in viral pathogenesis and is important in the infection of macrophages [13]. Vpr is also involved, to a lesse r extent in the infection of other cells. This protein causes host cell division to stop at the G2 stage, and indu ces the apoptosis of infected cells. Vpr acts as a shuttle protein from the cytopl asm to the nucleus by faci litating the transfer of the pre-interaction complex through the nuclear pores.

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15 HIV-1 Negative Regulatory Factor (Nef) Nef protein is synthesized very early in HIV inf ection and promote vi ral fitness through multiple mechanisms [14]. First, in order to prevent super-infection of the infected cells, Nef causes the incorporation and destruction of surf ace CD4 in the lysosomes. Second, this protein causes the down regulation of the major hist ocompatability complex-I (MHC-I) expression, which affects antigen presentation, and thereby reduces the destruction of infected cells by cytotoxic T lymphocytes. Third, Nef increases viral infectivity by inducing the infected macrophages to secrete MIP-1 al pha and MIP-1 beta. These chemokines have a chemotaxic effect on resting CD4+ T lymphocytes, causing the migration of the cells to the site of infection. This process in turn increases the chance for thes e cells to become infected, since free virions do not retain infectivity very long wh en circulating in the blood [7,15]. HIV-1 Trans-Activator of Transcription (Tat) Tat protein is a spliced gene product of 14 kD in size [2].Tat protein interacts with tat responsive element, an RNA loop structure on the 3 end of the viral long terminal repeat (LTR), to upregulate HIV gene expression [16]. HIV-1 Regulator of Viral Protein (Rev) Rev protein is 19 kD and is the product of doubly spliced mRNA. Rev protein is involved in the regulation of vira l RNA expression [2]. HIV-1 Classification There are two types of HIV presently known to exist: HIV-1 and HIV2 [Figures 1-1 and 1-2] [17]. Based on the genetic sequences, HIV-1 has been classified into three groups: the M (major) group, O (outlier) group, and the N ( non-outlier) group. HIV-1 M group is further divided into nine subtypes (A-D, F-H, J, and K) [18]. The major ity of HIV infections worldwide

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16 are the result of M group viruses [18]. There is also an increasi ng amount of infection caused by interand intra-subtype recombinants [19]. HIV-1 Treatment When HIV-1 was first identified over two decades ago, there were no effective treatments available. There were also few treatments for the secondary opportunistic infections, which are associated with HIV-1 infection. Presently th ere are several drug options for HIV-1-positive individuals. A number of dr ugs first approved for HIV-1 th erapy were nucleoside analogs, inhibitors of RT activity. Subsequent classes of drugs included prot ease inhibitors and nonnucleoside RT inhibitors followe d by more recent drugs shown in Table 1-1. The use of two or more of these drugs together was initially cal led highly active antiretr oviral therapy (HAART) and more recently renamed as anti-retroviral ther apy (ART) [20]. ART has b een very effective in prolonging the life expectancy of infected indivi duals and can reduce the amount of circulating virus to nearly undetectable levels. During dr ug induced remission, the virus may still remain dormant in viral reservoirs within the host, such as the testes, brain, a nd retina [21]. Financial constraint has been the major limiting factor for these drugs to have a major global impact. With most of the HIV-infected individuals residing in developing countries, a desirable strategy to arrest the viral spread would be by the devel opment of an effective prophylactic vaccine. Feline Immunodeficiency Virus FIV is a lentivirus of domestic cats, which causes an immunodeficiency syndrome strikingly similar to HIV infecti on in humans [22]. This simila rity between these two viruses extends to the genome organization of 5LTR-gag-pol-env-LTR-3 and the presence of select regulatory genes (Figure 1-1) [9]. The level of FIV infection of domestic cats worldwide has been estimated to be between 1% to 28% [23]. FIV variants have also been classified into five subtypes A-E [23]. FIV

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17 infection usually results in CD4+ T lymphocyte reduction [24]. Si milar to HIV strains, the virulence of FIV strains varies [ 23]. Like HIV-infected individuals, there is also the presence of long term non-progressor (LTNP) subjects among FIV-infected cats [23]. The LTNP cats remain disease free for a period of time, which is well over the mean disease onset of acute and moderate progressors. FIV has a broad tropism by infecting both CD4+ and CD8+ T lymphocytes, B cells, circulating monocytes, and monocytes of the CNS [25]. The tropism of th e virus is determined by the viral envelope characteri stics. FIV has the same genomic organization as most other lentiviruses with coding sequences of the gag, pol, and env, in addition to accessory genes flanked by open reading frames (ORFs) [7]. FIV Group Associated Antigen (Gag) The Gag polyprotein is approximately 50 kD a nd is produced by translation of unspliced viral genomic RNA transcript. This polyprotein is cleaved by viral protease, into three proteins matrix (MA, 15kD), capsid (CA, 24 kD), and nuc leocapsid (NC, 7 kD) [7]. The importance of including Gag proteins into a potential HI V vaccine is now recognized throughout the HIV vaccine community. The current trials in th e HIV Vaccine Trials Network (HVTN) all incorporate gag gene or Gag protein (T able 1-2). The presence of many conserved epitopes coupled with the strong immune response stimulated by this protein support its use as a vaccine immunogen [26]. Many T-cell epitop es are located within the Ga g region. These epitopes would be important to facilitate the rem oval of infected cells in order to stem the spread of the infection. FIV Envelope (Env) FIV Env is the product of a singly spliced 4.4 kD strand of mRNA [7], [27,28]. The Env glycoprotein precursor is composed of SU (95 kD ) and TM 39 (kD), and these two glycoproteins are assembled on the viral membrane as non-cova lently bonded surface glycoprotein. As is the

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18 case with HIV, FIV SU is the most divergent of the viral proteins [9], and mutations of this glycoprotein can affect the tropism of the virus. FIV Polymerase (Pol) The pol gene region of the ORF encodes a polyp rotein, which is cleaved by FIV protease (PR) into four proteins: PR, RT, integrase (I N), and deoxyuridine triphosphate (DU) [7]. The cleavage order of these proteins affect the num ber of infectious viri ons produced [29]. FIV Protease (PR) Protease is a viral enzyme, which processes th e Gag and Gag-Pol polyproteins to the final viral components. Comparison between FIV and HIV-1 PRs have revealed common amino acid (aa) recognition sites, facilitating the utilization of experimental protease inhibitors against both lentiviruses [30]. However, the majority of co mmercial HIV-1 protease in hibitors (PRIs) do not work against FIV PR. FIV Reverse Transcriptase (RT) FIV RT is composed of a 61kD polypeptide an d is the product of cleavage of the Gag-Pol polyprotein by FIV protease [7]. There is a significant sequen ce homology between HIV-1 RT and FIV RT, and FIV RT is also sensitive to the nucleoside analogs utiliz ed in the treatment of HIV-1 [31]. FIV RT may also underg o mutations, which result in re sistance to these drugs [31]. FIV Deoxyuridine Triphosphate (DU) FIV DU is a protein trimer consisting of 14.3kD subunits [32]. DU exists in both eukaryotic and prokaryotic cells, facilitating the hydr olysis of dUTP to dUMP to prevent the miss-incorporation of deoxyuridin e during the synthesis of DNA. Low levels of DU in the infected non-dividing cells can reduce FIV replication. The virus overcomes this obstacle by producing its own DU and thereby enhancing its fitness [7].

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19 FIV Integrase (IN) FIV IN is roughly 32kD and is derived from th e C-terminus of the precursor Pol protein [7]. The C-terminus of IN is important to th e interaction of the enzyme with DNA. It is responsible for site-specific cleavage of the ends of FIV DNA, and for the transfer and disintegration of DNA strands [33]. FIV Viral Infectivity Factor (Vif) Vif is produced from spliced RNA about 5.2 kB long [7]. Vif gene is found in all lentiviruses except EIAV. The size and genome lo cation are also well conserved. This gene affects the infectivity of the vi rions by mechanisms thought to be similar to those of HIV-1 [34]. FIV Open Reading Frame 2 (Orf2) FIV Orf2 is the transactivating factor of FI V [7] [35]. It is a 9. 3 kD protein product of multiply spliced strands of mRNA. Orf2 contains an N-terminus cystene-rich region but lacks the basic domains which are essential for HIV-1 Tat activity. Recent studies suggest that Orf-2 may also serve as Vpr [36]. FIV Regulator of Viral Protein (Rev) Rev protein facilitates the trigge ring of late gene e xpression in the virus life cycle [37]. It promotes nuclear export of mRNAs enabling thei r translation. Similar to HIV-1, FIV Rev is mediated by the rev responsive element, which is located on the 3 end of target mRNAs. Rev is involved in nuclear localization a nd the transfer of genetic materi al in and out of the nucleus. HIV/FIV Vaccine Model FIV is similar to HIV in morphology, genetic se quence, and pathogenesi s [9]. In light of this, vaccine approaches, which are effective agains t one of these viruses, are likely to provide valuable insights into the other. Both HIV a nd FIV have common challenges of high mutation rate due to the error prone nature of viral RT interand intra-subtype recombinations, and the

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20 need to generate broad vaccine protection agains t a range of different subtypes [38]. Cytotoxic T lymphocytes (CTL) responses to HIV and FIV are mediated by CD8+ Tcells, which target epitopes on the whole virus. Several of these ep itopes target have been identified on Gag and Env [23]. The period of time until the onset of cl inical symptoms may vary between strains and between subjects. There is however a significantl y greater numbers of LTNP cats infected with FIV. This may be due to the fact that HIV is believed to only ha ve been discovered in the human population for about two decades, while FIV ha s evolved with its host much longer in evolutionary time [5,9]. HIV Gag protein appears to be less dive rgent phylogenetically than FIV and simian immunodeficiency virus (SIV) (Figure 2). The epitopic characte rs of the attenuated strains may well provide insights into the ideal ta rgets for vaccine design. Currently the average survival time for infected individuals has incr eased considerably causing the CDC to launch new studies to recalculate this figure [39]. In 1993 at University of California at Davis, researchers in the School of Veterinary Medicine were able to elicit a protective immune response in 96% of cats immunized with whole-cell or cell-free FIV vaccines and subseque ntly challenged with heterologous FIV [40]. This was the first instance of an effective vacci ne formulation against an AIDS lentivirus. The exact nature of the protective response was not certain. The next step was to determine the mechanism of the protective response. The protec tion observed initially appeared to correlate with a strong humoral response, including in the generation of virus neutralizing antibodies(VNAs) [41]. However, the VNA titer of the study animals did not correlate completely with protection. One of the first viral proteins to be recogni zed by the host immune system is the core p24. There are also several CTL ep itopes found on core p24 [42]. This protein is the structural

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21 component of the viral capsid a nd is relatively conserved compar ed to the viral Env. HIV-1 and FIV p24 have 31% aa identity and 63% aa homol ogy based on length adjusted analysis. The identity observed with p24 was mu ch higher than those for SU, TM, and MA. Therefore, it was a good target for a vaccine immunogen to explore the concept of evolutionarily conserved epitopes among lentiviruses. The use of HIV-1 p24 as a v accine immunogen facilita ted the formation of cross-reactive antibodie s to FIV (Figure 1-3), despite ha ving only 63% homology. Thus, the possible utility of HIV-1 p24 pr otein as vaccine immunogen agains t FIV was noted, and this led to the following evaluations. Rationale and Goals for the Proposed Studies Following immunization with a subtype-B HIV-1UCD-1 p24 vaccine, 82% of specific pathogen free (SPF) cats were able to elicit a successf ul protective immune response to challenge with FIV [38]. The 31% aa identity between HIV1 and FIV p24 sequences in combination with the cross-protective ability of the HIV-1 p24 vaccine suggested that there may be some protective epitopes within the re gions of homology, and these may be evolutionarily conserved between the two lentiviruses. Ba sed on these observations, our hypothesis is that some of these epitopes conserved among the lentiviruses may serve as potential v accine immunogens against FIV. This hypothesis will be tested by performing the studies for the following specific aims: 1) determine if humoral immunity is respons ible for the protection conferred by HIV-1UCD1 p24 vaccine against FIV and 2) determine if cross-protection against FIV is conserved among different subtypes of HIV-1. Specific Aims Determine if humoral immuni ty is responsible for the protection conferred by HIV1UCD1 p24 vaccine against FIV: Cross-reactive antibodies to HI V-1 proteins were generated in cats infected with FIV [38] (Figure 3). This implied that there were some common epitopes

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22 conserved between both viruses despite their evol ution in different host species. The strongest area of cross reactivity observed by HIV-1 wholevirus western blot was core p24 protein. Hence the role that HIV-1 p24 antibodies play in cr oss-protection against FIV was evaluated. The following two experimental approaches we re undertaken to achieve this goal. A. B-cell mapping of cross-reac tive antibodies to FIV p24. B. Protective efficacy of the antibodies from FIV p24-vaccinated cats was determined in vivo by passive-transfer studies against FIV challenge. In order to identify the cross-reactive B-cell ep itopes, sera from HIV-1 p24-vaccinated cats were analyzed by FIV p24 peptide-based ELISA. Th e substrates for the ELISA consisted of 2830mer peptides that spanned FIV p24 with 1113 aa overlaps. The pub lished B-cell and T-cell epitopes available from Los Alamos National La boratories (LANL) were compared to those on the vaccine p24 antigen and the challenge virus. Since the p24 capsid of the virus is not expressed on its surface such as its Env, it is un likely that it would elicit a protective B-cell response. However, this arm of the immune syst em could be used to identify T-cell epitopes, such as T-helper epitopes for B-cell re sponses and possibly for CTL activity. Determine if p24 from subtype-A HIV-1 wi th a different epitope repertoire can be utilized as a vaccine antigen: The hypothesis proposed for this di ssertation is that some of the p24 epitopes conserved among HIV1 subtypes may work togeth er as a vaccine immunogen against FIV. Whereas, the central hypothesis of our laboratory is that some of the crossprotective HIV-1epitopes, prevalent among viral st ructural and enzymatic proteins, against FIV should be useful as vaccine ep itopes against HIV-1 in humans. As of yet, there has been no studies demonstrating the epitopes found on HIV-1 p24 proteins are all protective against FIV. For this reason, p24 from subtype-A HIV-1 will be used to test our immediate hypothesis by identifying the protective p24 epitopes, which are conserved among subtypes A and B of HIV-1.

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23 Furthermore, the studies proposed for this dissert ation are the first step towards answering the central hypothesis of our laborator y. Hence, the effort to identify cross-protective HIV-1 epitopes against FIV may provide a blueprint for the deve lopment of HIV-1/AIDS vaccines against HIV1 subtypes prevalent worldwide. Overall, our st udies will not only bene fit the development of second-generation FIV vaccines for veterinary medicine but will also contribute to the development of a HIV-1 v accine for human medicine.

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24 Table 1-1. Current drugs availa ble to treat HIV infection a Drug Class Drug Name Drug Mechanism Nucleoside Reverse Transcriptase Inhibitor (NRTI) Lamivudine (3TC), Zidovudine (AZT), Zalcitabine (ddC), Stavudine (d4T), Abacavir (ABC), Tenofovir (TDF) Didanosine (ddI) Nucleotide or nucleoside analogues lacking a hydroxyl group at the 3' end. Non-nucleoside Reverse Transcriptase Inhibitors (NNRTI) Efavirenz (EFV), Nevirapine (NVP), Delaviridine (DLV) Binds to HIV-1 RT and alters the active site to reduce nucleotide binding. Protease Inhibitor (PI) Nelfinavir (NFV), Amprenavir (APV), Fosamprenavir (FPV), Saquinavir (SQV), Lopinavir (LPV/r), Ritonavir (RTV),Atazanavir (ATZ), Indinavir (IDV)Tipranavir (TPV) Mimics Gag-Pol cleavage site and competes with protease, causing the production of immature non-infectious virions. Fusion Inhibitors (FI) Enfuvirtide (EFV) Binds to gp41 and blocks the fusion of the virus and cellular membrane. CCR5 Antagonists Maraviroc (FDA approved April 24, 2007) Blocks binding to CCR5 a Adapted from Sierra S. et al.2005 and update d with newly approved anti-HIV drugs [43].

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25 Table 2-2. Current HIV vaccine trials Prime Formulation Constituents Phase / Subtype Producer Product Name Gag Pol Env Nef Tat Rev Vpr Vpu PR RT I/B Sanofi Pasteaur ALVAC vCP1452 + + + + + I/B Chiron Gag and Env DNA/PLG Microparticles + + I/B Merck MRKAd5 HIV-1 Gag + I/B Therion TBC-M358 TBC-M335 I/B Wyeth GENEVAX gag-2962 + + + + + + I/B Wyeth GENEVAX gag-2962 + I/B PharmexaEpimmune EP-1043 + EP HIV-1090 + + + + + + I/B GeoVax HIVB DNA pGA2/JS7 #2 + + + + + + + I/Ba NIH VRC VRC-ADV014/ VRCADV-009 + + + I/C AlphaVax AVX-201 + + + + Ib PharmexaEpimmune EP HIV-1233 + + + + + + I/B UP c PENNVAX-B + + + I/B Merck MRKAd5 + + + I/A NIH VRC VRCHIVDNA-044 + II/Bb NIH VRC VRCHIVDNA-016 + + + + II/B Merck MRKAd5 Trivalent + + + II/B Merck MRKAd5 Trivalent + + + a Also contains clade A,B,C env. b Includes multiple specific epitopes. c University of Pennsylvania.

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26 Figure 1-1. Proviral genomic orga nization of FIV, HIV-1, and HIV-2. FIV resembles HIV by having LTR-gag-pol-env-LTR genomic organization, while missing few regulatory genes ( vpu, vpx, nef ). The orf-2 gene was initially reporte d to express 79 aa Tat-like protein. However, recent studies sugges t that this gene may also serve as vpr based on its gene product promoting the transport of the DNA pre-integration complex into the cell nuclei. Diagram adapted from Sauter, S.L, et al. 2001 [7]. vpr vpx tat rev nef nef vpu vpr vpx FIV RRE env rev vif orf-2 pol gag LTR LTR HIV-1 tat rev HIV-2

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27 Figure 1-2. Comparative gag phylogenetic tree of FIV, HIV1, HIV-2, and SIV. An unrooted phylogenetic tree was created using th e BLOSUM matrix and neighbor-joining algorithm based on the Kimura two-paramete r correction[44], [45]. Support at each internal node was assessed using 100 0 bootstrap samplings and each tree was visualized using Tree View. Branch lengths, drawn to scale, are based on number of synonymous substitutions per site. The gag tree is based on sequences from NCBI GenBank (FIV accession numb ers NC_001482, AY684181, M36968, D37820, DQ365596, AY13911, AY139112, AY139110, D37819, D37821, D37823, D37824, AF474246, AY679785, D37822, AB027302, AB027304, AB027303; SIV accession numbers AF301156, L06042, AF468 659, AF075269, AF131870, M27470, AF328295, 349680, M30931, L40990, M66437, U58991, AF334679, AF077017, M80194, U72748, U79412, M32741, M33262, M19499, M83293, AJ271369, AF103818, U42720; HIV-1 accession numbers L39106, M62320, M38429, M93258, M17449, L31963, AF004394, U43096, U23487, AY679786, AF321523, AB023804, M22639, K03454, U88826, AF005496, AJ006022, L20587, AJ302647, L20571; HIV-2 accession numbers M31113, AF082339, M30502, D00835, X52223, J03654, M30895, Z48731, X05291, J04498, U22047, X62240, U27200, L07625, U75441, AF208027). HIV-1 distribution is shown in major groups (Group O for outlier group, Group M for main group; Group N for non-M group); while HIV-2 distribution is shown in subtypes (subtypes C, D, and E missing). SIVcol, SIVSYK, SIVGSN, SIVHOEST, SIVSUN, SIVMND, SIVRCM, SIVSM, and SIVAGM are SIV species isolated from Colobus, Sykes, greater-spot-nosed, LHoest, s un-tailed, mandril, red-capped mangabeys, sooty mangabeys, and African green monkeys, respectively. SIVmac belongs to SIV SM group and are isolates from captive macaques [46]. The gag phylogenetic tree shows close relationship between SIVSM/SIVmac and HIV-2 and between SIVcpz and HIV-1, but distant relationship between FI V and primate AIDS lentiviruses. This figure was adapted from Yama moto, J.K, et al. 2005.

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28

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29 Figure 1-3. FIV p24 cross-reactivity of the se ra from HIV-1 p24-vaccinated cats. FIVBang p24 strip (200 ng/strip) was reacted with individual cat serum at 1:100 dilution. Tested sera were pre(pr) and post-3rd immuni zation(pre-challenge) se ra of cats vaccinated with either HIV1LAV or HIV1UCD1 p24 proteins. In addi tion, serum reactivities of post-1st and 2nd vaccinations are show n for cats #ID4 and #626. Based on immunoblot and ELISA analyses, approximate ly 80% of HIV-1 p24-vaccinated cats developed cross-reactive antibodies to FIV p24. +/+ + + +/+/+ p24 + + + + + + + Cat #: FIV + ID4 626 R99 G9F JB6 Pr V1 V2 V3 Pr V1 V2 V3 Pr V3 Pr V3 Pr V3 HIV-1LAI/LAV p24 HIV-1UCD1 p24 Vaccine: Post Vaccination V1= 1st V2 = 2nd V3 = 3rd

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30 CHAPTER 2 THE ROLE OF ANTIBODY IMMUNITY IN THE PROTECTION CONFERRED BY HIV1UCD1 P24 VACCINE Introduction Many of the challenges faced during the deve lopment of an FIV vaccine are common to the development of an HIV-1 vaccine [23]. Therefore, th e insights derived from the development of an FIV vaccine could also lead to clues in developing an effective HIV-1 vaccine. Identifying the epitopes for vaccine pr otection and the immunity required for such protection are the two major aims of the pr oposed project on subtype -A HIV-1 p24-based FIV vaccine. This section initiates the examination of the role of vaccine -induced antibody immunity in p24 vaccine protection. Antibodies generated by vaccination were examined for patterns of FIV and HIV-1 p24 epitope recognition among protec ted/vaccinated cats, unprotected/vaccinated cats, and infected control cats. The vaccine immunogens consisted of subtype-A HIV-1 p24, subtype-B HIV-1 p24, FIV p24, and as a control, in activated dual-subtype FIV whole-virus (FelO-Vax FIV vaccine, Fort Dodge Animal Health, Fort Dodge, Iowa). The reactivity of these sera was evaluated by western blot analysis for antige n specificity, ELISA for level of affinity to native immunogen and peptides, VNA assay for bi ological activity, and B-cell epitope mapping to identify vaccine epitopes of protection. Overall, the structural and func tional analyses of the B-cell epitopes on the HIV-1 p24 were performe d to determine the significance of HIV-1 p24 vaccine-induced antibodies in cr oss-protection against FIV. In addition to the in vitro functional analysis the vaccine-induced antibodies were tested for in vivo efficacy against FIV challenge using passive-transfer studies (Table 2-1, 2-2, and 23). Passive-transfer studies consisted of intrav enous transfusion with v accine-induced antibodies into nave SPF cats one-day prior to and one da y after FIV challenge (Figure 2-1). These cats were then monitored for the levels of infec tion and FIV-specific antibody development. Three

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31 separate passive-transfer studies were perfor med. The first study was performed using pooled serum from vaccinated donor cats as the transfer material. The second study involved the use of donor antibodies, which were pa rtially purified by ammonium sulfate precipitation method. The third study was performed using material purif ied by both ammonium su lfate precipitation and caprylic acid fractionation methods (Figure 2-2). The purity and act ivity of these antibodies were tested prior to transfusion. In order to identify the B-cell epitopes responsible for the cr oss-reactive antibodies, B-cell epitope mapping was performed using sera deri ved from both protecte d and unprotected cats, which were vaccinated with HIV-1 p24. The intent of these studies was to examine the FIV p24 epitope reactivity of these differe nt groups of animals and to dete rmine if there was a particular profile, which was associated with in fection or vaccine protection [38]. Methods Animals Specific pathogen free (SPF) cats were obtaine d from Liberty Res earch (Waverly, New York) or Harlan Sprague Dawley, Inc. (Indianap olis, Indiana). All of these animals tested negative for FIV, toxoplasma, and feline leukemia virus, and they were maintained at the University of Florida experimental animal car e facility under SPF conditions during vaccination or prior to FIV infection. Animals for FIV-challe nge studies were transferred to BSL-2 facilities before FIV inoculation. Serum Collection for B-cell Epitope Mapping by FIV ELISA The animals were vaccinated subcutaneously (SC) at 3-week intervals with FIV-1UCD1 p24 or FIV-Petaluma (FIVPet) p24 vaccine. The vaccine consisted of the p24 protein (200-250 g/dose) in Ribi adjuvant supplemented with 5 g/dose of human IL-12. In addition, sera from FIV-Bangston (FIVPBang)-infected cats at 18 weeks post-cha llenge (wpc) were used as positive

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32 control sera, while negative control sera we re from uninfected SPF cats. Whole blood was centrifuged at 2000 rpm for 20 minutes, and the se rum was collected and stored at -20C until use. Overlapping FIV P24 Peptides for ELISA Overlapping 28-30mer peptides of FIV p24 w ith 11-13 aa overlaps were designed using LANL PeptGen program and synthesized by SynP rep Corp. (Dublin, California) based on the p24 aa sequence of FIVBang, with the exception of four pep tides designated FB1, FB2, FC2, and FB4, which were derived from FIVFC1 (Figure 2-3). These were pr e-screened at the company by HPLC for purity and were determined to be > 95 % pure. The peptides were diluted in coating buffer (0.35 M sodium bicarbonate solution, pH 9.4). ELISA was performed by standard laboratory method [40]. ELISA plat es were coated with 400 ng of the appropriate peptide in 50 L of coating buffer per well and incubated overnight at 4oC. These wells were subsequently blocked for 1 hour with 200 L of blocking soluti on (5% milk protein in PBS), washed 3X with wash buffer (150 mM NaCl and 0.05% Tween 20) and incubated with 1:200 dilution of cat serum for 1 hour at 37oC. The wells were then washed 3X with wash buffer and incubated with 100 L of biotinylated anti-cat IgG (Vector La boratories, Burlingame California), diluted 1:5000 in Buffer 3 (3% BSA/PBS with 0.05% Tw een-20) with 5% milk for 1 hour at 37oC. After incubation the plates were washed 3X, in wash buffer and incubated for 1 hour at 37oC with 100 L per well strepavidin-conjugate d horseradish preoxidase (Vector Laboratories). The plates were then washed 3X and 100 L of substr ate solution (0.005% tetr amethylbenzidine and 0.015% H2O2 in 0.96% citric acid solution) was added to each well and incubated for 15 minutes. The reaction was stopped by the addi tion of 100 L per we ll of 0.12 % hydrogen fluoride solution.

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33 Immunoblot Analysis The sera from vaccinated and unvaccinated cats before and after FIV challenge were tested for reactivity to immunoblot strips, each contai ning 200 ng of virus proteins. Immunoblot strips were incubated individually with 1:50 dilution of cat serum in Buffer 3 with 5% milk. The control sera consisted of serum from an SPF cat for negative control and serum from an FIVinfected cat for positive cont rol. The remaining immunoblot method was performed using laboratory protocol [41]. Passive-transfer Studies with An tibodies from Vaccinated Cats Three separate passive-transfer studies we re undertaken. In Passive-Transfer Study 1, four groups of four cats were IV transfused w ith saline or pooled sera from either HIV-1UCD1 p24-vaccinated cats, Fel-O-Vax FIV-vaccinated (Fel-O-Vax-vaccinated) cats, or non-vaccinated cats. These sera were collect ed from the SC-vaccinated dono r cats 2-3 weeks after the 3rd vaccination and before FIV challenge (vaccinated donors in Chapter 3). All pooled sera were inactivated by incubation at 56C for 40 minutes. In order to prevent serum reaction, the pooled serum was cross-matched with each recipient cat s blood. Each cat received a cross-matchnegative serum equivalent to 30% of the recipien ts total blood volume. The amount of volume, including serum protein level, wa s too large to administer in a single transfusion. For this reason, cats received 20% volume in the firs t transfusion and 10% volume in the second transfusion. The first transfusion was given on day -1 with FIV challenge on day 0 and second transfer on day 1 (Figure 2-1). Thes e cats were challenged IV with FIVPet (10 mean cat infectious dose, CID50). In Passive-Transfer Study 2, the antibodi es were purified from pooled serum by ammonium sulfate precipitation, wh ich partially purifies for IgG. SPF cats were transfused with saline or partially-pur ified antibodies of pool ed serum from HIV-1UCD1 p24-vaccinated cats,

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34 FIVPet p24-vaccinated cats, Fel-O-Vax-vaccinated cat s, or non-vaccinated cats. In PassiveTransfer Study 3, the antibody purification was taken further using a two-step purification method involving caprylic acid fractionation follow ed by ammonium sulfat e precipitation. After these procedures were completed, the anti bodies were dialyzed and sterilized by 0.45 m filtration. Tests for biological activity, purity, and toxicity were performed on the material prior to passive transfer. The vaccine specificity of the antibodies in Study 3 was identical to those of Study 2, except for the additional antibody group derived from uninfected FeT-J cell/lysateimmunized (FeT-J cell-immunized) cats. FeT-J cells were the cells used to produce the viruses for the Fel-O-Vax FIV vaccine. These cats received a combined cell and cell lysate immunization using SC (2x106 cells/dose), intradermal (250 g/dose cell lysate), intranasal (100 g/dose cell lysate), and transcutaneous (100 g/dose cell lysate) routes (see Chapter 3 for additional detail). All immunizations were admini stered at 3-week intervals. The passivetransfer schedule and transfusion procedure for both Studies 2 and 3 were identical to the one in Study 1. Cats in Studies 2 and 3 in cluded groups challenged IV with FIVFC1 (Study 2, 15 CID50; Study 3, 10 CID50) and another set of groups challenged IV with FIVPet (25 CID50). Antibody Preparation Blood was collected from HIV-1UCD1 p24, HIV-1N20, FIV p24, and FeT-J cell lysateimmunized donor cats and from SPF cats in either serum or hepari nized collection tubes. The whole blood was centrifuged at 200 0 rpm for 20 minutes and the serum or plasma was collected. The sera were pooled according to immuniza tion group and purified for IgG by ammonium sulfate precipitation alone (Study 2) or in combination with ca prylic acid fractionation (Study 3) (Figure 2-2). The pooled serum and purified an tibody preparations were tested by immunoblot and radial immunodiffusion (RID) assay to determine the reactiv ity to the target immunogens

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35 and to determine the immunoglobulin levels (IgG, IgA, IgM), respective ly. Commercial RID assay for feline IgG, IgA, and IgM (Bethyl La boratories, Montgomery, Texas) were performed according to the manufacturers protocol. Ammonium sulfate precipitation The supernatant from caprylic acid purification was cooled to 4oC then placed into a container and stirred slowly using a magnetic sti rring bar. Ammonium sulf ate was added to each sample 0.65 g/mL (Fisher Scientific Pittsburg PA) to achieve 100% saturation. Solid ammonium sulfate was slowly added to the antibodi es and the mixture was stirred at 4 oC overnight. Following overnight incubation, the sample was centrifuged (20g) for 20 minutes at 4oC. The solid fraction was pooled and dissolved in an equal volume of H2O. The fractions were then dialyzed against PBS using 600-800 MW Spectra / Por (Sprectra Laboratories, Inc., Rancho Dominguez, California) [47]. Caprylic acid purification Serum samples were pooled for each group, tran sferred to a clean container, and mixed with 60 mM of sodium acetate (Sigma-Aldrich USA, St. Louis, Missouri) solution at pH 4. Each pool of antibodies was mixed with 0.75 mL caprylic acid (Sigma Aldrich USA) per 10 mL of original sample volume. This mixture was st irred for 30 minutes and then centrifuged at 5000g for 10 minutes. The supernatant was decanted and subsequently dialyzed against PBS using 600800 MW Spectra/Por (Sprectra Labora tories. Inc.) [47]. The antibodies were then concentrated to half the previous volume using a Bio-Rad U ltra concentrator. The final product was filter purified using a 0.45 m filter. The product was te sted by western blot, ELISA, and gel analysis using Coomassie Brilliant Blue (CBB) stain and silver stain to determine purity, and by VNA assay to determine retention of biological activity [48].

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36 Virus isolation Whole blood was collected from the cats in passiv e-transfer studies at 3-4 week intervals. The peripheral blood mononuclear cells (PBMC) pur ified by ficoll-hypaque gradient technique [8] were used to determine the FIV infection status. These cells (1.5x106-3x106 cells) were cocultured with indicator cells (3x106 cells) in 3 mL of culture medi a for approximately 3 weeks. The cells were recultured every 3 days and the spent culture fluid from each passage day was collected. The culture fluid was then tested for RT activity [7]. The indicator cells consisted of PBMC from SPF cats, which were stimulated with 5 g/mL of concanavalin A (Con A) for 3 days and recultured every 3 days in fresh culture media for 6-12 days before use in the assay. VNA assay Blood from the cats in passive-transfer studies were collected prea nd post-challenge at 34 week intervals. The serum from blood wa s separated by centrifugation at 2000 rpm for 20 minutes, and the serum was collected and fr ozen until the day of assay. VNA assay was performed in accordance with established laborat ory protocol [6]. On the day of assay, the serum was incubated at 56C for 30 minutes to inac tivate the complement. Serial dilutions of the serum in culture media were incubated with 200 TCID50 of virus for one hour before addition of 0.25 mL mixture to 0.5x105 indicator cells in 0.25 mL/well. The final serum dilutions ranged from 1:10 to 1:10,000, and the final virus titer was at 100 TCID50. The cells were recultured every 3 days in fresh culture media for 18-21 da ys, while the spent culture fluid was collected and later tested for FIV levels by RT assay. The indicator cells were prepared the same way as those used in the virus isolation assay.

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37 Results B-cell Epitope Analysis ELISA analysis of the reactivity of HIV-1 p24-vaccinated cat serum to the overlapping 28 30mer peptides of FIVBang p24 with 11 aa overlaps showed highe st reactivity to peptide 71-100 (FB4) followed by peptide 197-223 (FB11). Less reac tivity was also observe d to peptide 53-81 (FB3), 161-188 (FB9), and 178-207 (FB10) (Figure 23). This indicates th at at least 5 crossreactive B-cell epitopes are indu ced by vaccination of cats with HIV-1 p24. However, these cross-reactive peptides did not correspond to the major homology regions (MHR) found among FIV, SIV, and HIV [49-52] (Fi gure 2-4). As expected, FIV p24vaccinated cats had the most serum reactivity to FIV p24 peptides (11 of 12 peptides), while FIV-infected cats had a moderate-to-high frequency of serum reactivity to three FIV p24 peptides (FB1, FB4, FB11). FB4 and FB11 peptides, which reacted with the highest frequency to sera from infected cats, also reacted with highest frequency to sera from HIV-1 p24-vaccin ated cats. This observation suggests that both vaccination wi th HIV-1 p24 and infection with FIV stimulate a similar B-cell repertoire to FIV p24 epitopes. No VNA activity was observed with antibodies generated to HIV-1 p24 and to FIV p24 (data not shown). In contrast, VNA activity was observed with sera from dual-subtype FIV-vaccinated cats (both prototype and commerci al dual-subtype FIV vaccines tested, data not shown). Passive-transfer Studies: The Analysis of Purified Antibody Preparations for Transfer Greater than 90% of the contaminating seru m proteins were removed with the use of caprylic acid/ammonium sulfate precipitation proce dure (Figure 2-5). This is evident from both the CBB-gel and immunoblot analyses of the purified antibodies and the corresponding prepurification sera from each vaccination group. Album in was used as a measure of contamination in the purified antibody prepara tions. Albumin levels were de termined by immunoblot analysis

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38 using commercial polyclonal antibodies to feline albumin (Bethyl Laboratories). Purified antibody preparations contained <95% albumin when compared to the amounts in corresponding pre-purification sera (Figure 25). Thus, >90% of the contaminating proteins were removed according to the minor albumin contamination remaining and few weak bands of other contaminating proteins in the CBB-gels. Furt hermore, the immunoblots show heavy and light chains of the cat antibodies at the predicted molecular weight sizes of 50 kD and 25 kD, respectively (Figure 2-6). The intensity of the bands for heavy and light chains suggests that high levels of antibodies were retained after pur ification. Analysis for IgG levels using RID assay indicates that most of the IgG in the pur ified antibody preparations (average recovery of 75%; recovery range of 67%-82%) we re retained after the purifica tion (Figure 2-6 and 2-7). The purified antibody preparations also contained lower amounts of Ig A (average recovery of 36%; recovery range of 19-62%) and IgM (average r ecover of 53%; recovery range of 48-55%) than those in the pre-purification sera (Figure 2-8 and 2-9). The loss of IgA and IgM was anticipated since caprylic acid/ammonium sulfate precipit ation procedure has been reported to be a purification procedur e for IgG [47]. The retention of biological ac tivity was illustrated by the re activity of the purified antibody preparations to the vaccine immunogen used on the donors of their pre-pu rification pooled serum (Figure 2-10). Furthermore, no si gnificant loss in VNA titers to FIVPet were detected after purification of pooled serum from Fel-O-Vax-va ccinated cats (Table 2-5; 1000 VNA titers of preand post-purification) The VNA titers to FIVPet and FIVFC1 were not detected in pooled serum from either HIV-1 p24or FIV p24-vaccinated cats. All purified antibodies were resuspended to the original volume to simulate the concentrations equivalent to those in the prepurification pooled sera. The same dilution of

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39 1:200 was used for the purified preparation a nd pre-purification serum in the immunoblot analysis. These adjustments allowed for the direct comparison between the preand postpurification materials. Therefore, the intens ity of the bands was used as a measure for comparing specific-antibody titers in purified preparations to thos e in pre-purification sera. Purified antibody preparations from pooled sera of HIV-1UCD1and HIV-1N20 p24-vaccinated cats reacted strongly to HIV-1UCD1and HIV-1N20 p24 immunoblots (Figure 2-10A, 2-10B). They reacted weakly to p24 of FIV whole-viru s immunoblot (Figure 2-10 C). However, their weak p24 reactivity in the FIV w hole-virus immunoblot was slight ly stronger than their prepurification pooled serum (Figure 2-10C). The purified antibody pr eparation from pooled serum of FIV p24-vaccinated cats reacted to HIV-1 p24 i mmunoblots at a similar weak intensity as that of corresponding pre-purificati on serum (Figure 2-10A, 2-10B). As expected, purified antibody preparations from sera of HIV-1 and FIV p24-v accinated cats had no reaction to uninfected FeTJ cell-lysate immunoblot (Fi gure 2-10D). Whereas, purifie d antibody preparation and prepurification pooled serum from Fe l-O-Vax-vaccinated cats reacted weakly at similar bands (50 and 75kD on FeT-J immunoblots). These reactions to cellular components were anticipated since Fel-O-Vax FIV vaccine, which is composed of inactivated infected cells and viruses, should also induce anti -cellular antibodies. Passive-transfer Studies with Serum a nd Antibodies from Vaccinated Cats In Passive-Transfer Study 1, 3 of 4 (75%) cats transfused with HIV-1UCD1 p24-vaccinated cat serum became infected with FIVPet (10 CID50) (Table 2-1). Whereas, Fel-O-Vax-vaccinated cat serum was able to protect 4 of 4 (100%) cats from this group. Furthermore, no protection was observed in all four control cats, which received saline or pooled serum from non-vaccinated cats.

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40 In Passive-Transfer Study 2 with slig htly higher challenge dose of 25 CID50, all three cats transfused with partially -purified antibodies from pooled serum of HIV-1UCD1 and FIVPet p24vaccinated cats, were not protected against FIVPet (Table 2-2). Three of 4 cats, which received similarly purified antibodies from Fel-O-Vax-va ccinated cat sera, were protected against FIVPet but not against FIVFC1 even at a slightly lo wer challenge dose (15 CID50). As expected, all five recipients of saline or partially-purified antibod ies from non-vaccinated ca ts were all infected with either FIVPet or FIVFC1. These observations suggested th at the antibodies induced by Fel-OVax FIV vaccination can confer protection agai nst homologous FIV challenge but not against heterologous-subtype FIVFC1. Furthermore, antibodies induced by HIV-1 or FIV p24 vaccination conferred no protection against FIVPet. In Passive-Transfer Study 3, 1 of 3 cats tran sfused with highly-purified antibodies from HIV-1UCD1 p24-vaccinated cat sera we re protected against FIVFC1 (10 CID50), while no protection (0 of 4) was observed in reci pients of purified antibodies from HIV-1N20 p24vaccinated cat sera (Table 2-3). Moreover, all f our cats transfused with purified antibodies from either FIV p24-vaccinated (n=2) or Fel-O-Vaxvaccinated (n=2) cat sera were not protected against FIVFC1. Like the results from Study 2, all four cats transfused with purified antibodies from Fel-O-Vax-vaccinated cat sera we re protected against homologous FIVPet. VNA analysis of antibodies removed from the transfused cats 6 da ys post transfusion showed the retention of some VNA activity at an average dilution of 1: 800 (800 VNA titer). All reci pients of saline or purified antibodies from FeT-J cel l-immunized or non-vaccinated ca t sera were infected with either FIVPet or FIVFC1. Hence, the antibodies to HIV1 p24 and FIV p24 do not mediate the protection observed in HIV-1 p24-vaccinate d cats and FIV p24-vaccinated cats.

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41 Discussion As summarized in Table 2-4, current studies de monstrated that only 1 of 8 cats transfused with serum or antibodies to HIV-1UCD1 p24, 0 of 3 cats transfused with antibodies to FIV p24, and 11 of 12 cats transfused with antibodies to Fel-O-Vax antigen were protected against FIVPet challenge. In contrast, 1 of 3 cats transfused with antibodies to HIV1UCD1 p24 and none of the cats transfused with antibodies to HIV-1N20 p24, FIV p24, or Fel-O-Vax immunogen were protected against FIVFC1 challenge. These challenge doses re sulted in infection of all control cats, which received saline, antibodies to FeT-J cells, or antibodies from SPF cats. Hence, no significant levels of passive-tran sfer protection were observed in cats transfused with antibodies to HIV-1 p24 or FIV p24. However, passive-trans fer protection was conferred with antibodies from Fel-O-Vax-vaccinated cats against homologous FIVPet challenge but not against FIVFC1 challenge. These results taken t ogether suggested that cross-prot ection, previously observed in HIV-1 p24-vaccinated cats against both FIVPet and FIVFC1 and in Fel-O-vax-vaccinated cats against FIVFC1 was not mediated by vaccine-induced antibodies. Antibodies to HIV-1 p24 and FIV 24 conferre d no passive-transfer protection. This finding was anticipated since VNAs to p24 have yet to be reported. Only antibodies to SU and TM, which are exposed on the viral su rface, have been reported [53]. The core p24 is masked by the viral lipid envelope and blocked from inte raction with the antibod ies to p24. This also explained why only antibodies induced by Fel-O-Va x FIV vaccination, which had high levels of antibodies to FIV SU and TM, conferred passive-transfer protection against FIVPet. High VNA titers to FIVPet, but not to FIVFC1, were observed only in the seru m or purified antibodies from Fel-O-Vax-vaccinated cats (Table 2-5). This observation suggested a correlation of passivetransfer protection with high vaccine-induced VNAs to the challenge strain. Previous studies using sera from inactivated FIVPet (single-strain)-vaccinated cats and inactivated sera from

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42 FIVPet-infected cats demonstrated positive correlation between passive-transfer protection and transfusion with sera co ntaining high VNA titers to the challenge virus [54]. Similar observations were made in passive-transfer studies of maca ques with inactivated infect sera against the homologous SIV strain [55] or with human monoclonal VNA to HIV-1 Env against a challenge with SHIV, which had homologous HIV-1 Env [56] Thus, vaccine-induced antibodies may play a role in the protection of Fel-O-Vax-vaccinat ed cats against vaccine-i nduced VNA-sensitive, challenge viruses. The pattern of passive-protec tion observed in Studies 1 and 2 with pooled serum and partially-purified antibodies wa s similar to the protection pa ttern observed in Study 3 with highly-purified antibodies. The highly-purified antibody prep arations were produced by a combination of caprylic acid fractionation a nd ammonium sulfate precipitation. Such purification procedure produced an tibody preparations with high le vels of IgG antibodies (IgG levels by RID assay), biological activities (VNA activity and reactivity to HIV/FIV p24 on immunoblot), and purity (>90% pur ity and <50 EU endotoxin activity ). The removal of serum albumin and other serum proteins were greatly reduced and had no affect on the results of the passive-transfer studies. This observation indicated that the antibodies, and not other serum proteins, were responsible for the passive -transfer protection observed against FIVPet challenge in the recipients of Fel-O-Vax-vaccinated cat antib odies. These studies do not however determine which anti-FIV immunoglobulin isotypes are more important for passive-transfer protection as well as for protection observed after active vaccinati on. The high retention of IgG antibodies and VNA titers in highly-purified preparations su ggest that FIV-specific IgG antibodies play a major role in both passive-transfer protecti on and vaccine protection against homologous FIVPet challenge. This protection against IV homo logous challenge may also extend towards

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43 mucosal/vaginal challenge, since a recent vacci ne study using a combination of Fel-O-Vax and prototype vaccines demonstrated majo r protection against vaginal FIVPet challenge [57]. High levels of IgG, but very low levels of IgA, were found in the vaginal vault of the vaccinated cats. Furthermore, high levels of FIV-specific IgG antib odies, but only low levels of FIV-specific IgA antibodies, were detected in the vaginal vault. Thus, the role of vaccine-induced IgG antibodies appeared to be more important than IgA antibodies at least in the vagina l homologous challenge. The lack of passive-transfer protection with antibodies to HIV-1 p24 and FIV p24 suggested that vaccine protecti on observed in previous studies with HIV-1 p24-vaccinated cats and FIV p24-vaccinated cats [38] ma y be due to vaccine-induced cellular immunity. In previous studies FIV 24-specific IFN responses have been detect ed in PBMC from HIV-1 p24vaccinated cats [38]. Similarly, cellular immunity may have mediated the protection observed in Fel-O-Vax-vaccinated cats against VNA-resistant FIVFC1. Strong FIV-specific cellular immunity has been reported to be present in cats vaccinated with prototype dual-subtype FIV vaccine (prototype to Fel-O-Vax vaccine) [48]. These observations further support the view that HIV-1 p24 vaccination induces cro ss-protective cellular immunity against FIV challenge. The studies in Chapter 3 were performe d to test this concept.

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44 Table 2-1. Passive-Transfer Study 1 with se ra from vaccinated and non-vaccinated cats Group Cat # Passive-Transfer Serum FIV Challenge (CID50) a Weeks Post-Challenge (FIV Ab/VI) b 3 6 9 12 15 17 20 Protection Rate (%) A RT2 RW1 RX3 RV1 HIV1UCD1 p24-vaccinated cat serum HIV1UCD1 p24-vaccinated cat serum HIV1UCD1 p24-vaccinated cat serum HIV1UCD1 p24-vaccinated cat serum Pet (10) Pet (10) Pet (10) Pet (10) -/-/-/-/-/-/-/-/+ +/+ +/+ +/+ +/+ +/+ Eu -/+/+ +/+ +/+ +/+ +/+ Eu -/-/+ +/+ +/+ +/+ +/+ Eu 1/4 ( 25%) B RT5 RW2 EF3 EH2 Fel-O-Vax FIV-vaccinated cat serum Fel-O-Vax FIV-vaccinated cat serum Fel-O-Vax FIV-vaccinated cat serum Fel-O-Vax FIV-vaccinated cat serum Pet (10) Pet (10) Pet (10) Pet (10) -/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/4/4 (100%) C RT4 RX1 RW4 DQ1 Non-vaccinated cat serum Non-vaccinated cat serum Saline Saline Pet (10) Pet (10) Pet (10) Pet (10) +/+ +/+ +/+ +/+ +/+ +/+ +/+ -/-/-/+ -/+ -/+ +/+ +/+ -/+/+/+ +/+ +/+ +/+ +/+ -/-/+/+ +/+ +/+ +/+ Eu 0/4 (0%) a FIV challenge was subtype-A FIVPet (Pet) at 10 CID50. b FIV infection was based on the development of FIV antibodies (FIV Ab) determined by FIV-immunoblot analysis and the presence of viruses determined by viru s isolation (VI) using RTand PCR analyses. Results for the samples collected at 3, 6, 9, 12, 15, 17, and 20 wpc are shown either as positive (+) or negative (-) for FIV antibodies or viruses. Abbreviation is euthanized (Eu).

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45 Table 2-2. Passive-Transfer Study 2 with partia lly-purified antibodies from vaccinated and nonvaccinated cat sera Group Cat # Passive-Transfer Antibody FIV Challenge (CID50) a Weeks Post-Challenge (FIV Ab/VI) b 3 6 9 12 15 17 20 Protection Rate (%) A QC3 QD3 QE4 BDP HIV1UCD1 p24-vaccinated cat Ab HIV1UCD1 p24-vaccinated cat Ab HIV1UCD1 p24-vaccinated cat Ab HIV1UCD1 p24-vaccinated cat Ab Pet (25) Pet (25) Pet (25) Pet (25) -/+ +/+ +/+ +/+ +/+ +/+ +/+ -/+ +/+ +/+ +/+ +/+ +/+ +/+ -/+ +/+ +/+ +/+ +/+ +/+ +/+ -/+ +/+ +/+ +/+ +/+ +/+ +/+ 0/4 (0%) B QC1 QD4 QE7 FIVPet p24-vaccinated cat Ab FIVPet p24-vaccinated cat Ab FIVPet p24-vaccinated cat Ab Pet (25) Pet (25) Pet (25) -/+ -/+ -/+ -/+ +/+ +/+ +/+ -/+ -/+ -/+ -/+ -/+ +/+ +/+ -/+ -/+ -/+ -/+ +/+ +/+ +/+ 0/3 (0%) C QC2 QD2 QE1 BDQ Fel-O-Vax FIV-vaccinated cat Ab Fel-O-Vax FIV-vaccinated cat Ab Fel-O-Vax FIV-vaccinated cat Ab Fel-O-Vax FIV-vaccinated cat Ab Pet (25) Pet (25) Pet (25) Pet (25) -/-/-/-/-/-/-/-/+ -/+ -/+ -/+ -/+ +/+ +/+ -/-/-/-/-/-/-/-/-/-/-/-/-/-/3/4 (75%) D QD5 BDS QE2 Non-vaccinated cat Ab Non-vaccinated cat Ab Saline Pet (25) Pet (25) Pet (25) -/+ +/+ +/+ +/+ +/+ +/+ +/+ -/+/+/+ +/+ +/+ +/+ +/+ -/-/+/+ +/+ +/+ +/+ +/+ 0/3 (0%) E QE2 PQ6 75B Fel-O-Vax FIV-vaccinated cat Ab Fel-O-Vax FIV-vaccinated cat Ab Fel-O-Vax FIV-vaccinated cat Ab FC1 (15) FC1 (15) FC1 (15) -/-/-/+ -/+ -/+/+/+ -/+/+ +/+ +/+ +/+ +/+ +/+ -/+/+ +/+ +/+ +/+ +/+ +/+ 0/3 (0%) F QE6 75C Non-vaccinated cat Ab Saline FC1 (15) FC1 (15) -/-/+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ 0/2 (0%) a FIV challenge was subtype-A FIVPet (Pet) at 25 CID50 and subtype-B FIVFC1 (FC1) at 15 CID50. b FIV infection was based on the development of FIV antibodies (FIV Ab) determined by FIV-immunoblot analysis and the presence of viruses determined by viru s isolation (VI) using RTand PCR analyses. Results for the samples collected at 3, 6, 9, 12, 15, 17, and 20 wpc are shown either as positive (+) or negative (-) for FIV antibodies or viruses.

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46 Table 2-3. Passive-Transfer Study 3 with purified antibodies from vaccinated and non-vaccinated cat sera. Group Cat # Passive-Transfer Antibody FIV Challenge (CID50)a Weeks Post-Challenge (FIV Ab/VI)b 3 6 9 12 15 17 20 Protection Rate (%) A LF3 KA4 KE2 HIV1UCD1 p24-vaccinated cat Ab HIV1UCD1 p24-vaccinated cat Ab HIV1UCD1 p24-vaccinated cat Ab FC1 (10) FC1 (10) FC1 (10) -/-/-/-/-/-/-/-/+/+/+/+ +/+ +/+ +/+ -/-/-/-/-/+ /+ +/+ 1/3 (33%) B LK2 KA6 KE3 KB2 HIV1N20 p24-vaccinated cat Ab HIV1N20 p24-vaccinated cat Ab HIV1N20 p24-vaccinated cat Ab HIV1N20 p24-vaccinated cat Ab FC1 (10) FC1 (10) FC1 (10) FC1 (10) -/-/-/-/-/+ +/+ +/+ -/-/+/+/+ +/+ +/+ +/+ -/-/-/-/+/+/+ +/+ -/-/-/-/-/+/+ +/+ 0/4 (0%) C KE4 LK4 FIVPet p24-vaccinated cat Ab FIVPet p24-vaccinated cat Ab FC1 (10) FC1 (10) -/+/+/+ +/+ +/+ +/+ +/+ -/-/-/-/-/+/+ +/+ 0/2 (0%) D KE5 KA2 Fel-O-Vax FIV-vaccinated cat Ab Fel-O-Vax FIV-vaccinated cat Ab FC1 (10) FC1 (10) -/-/-/-/+ -/+ +/+ +/+ -/-/-/+ -/+ +/+ +/+ +/+ 0/2 (0%) E KE6 LF2 LD5 FeT-J cell-immunized cat Ab Non-vaccinated cat Ab Saline FC1 (10) FC1 (10) FC1 (10) -/-/-/+/+ +/+ +/+ +/+ -/-/-/+/+ +/+ +/+ +/+ -/-/+/+ +/+ +/+ +/+ +/+ 0/3 (0%) F JX2 LE5 KB4 JY1 Fel-O-Vax FIV-vaccinated cat Ab Fel-O-Vax FIV-vaccinated cat Ab Fel-O-Vax FIV-vaccinated cat Ab Fel-O-Vax FIV-vaccinated cat Ab Pet (25) Pet (25) Pet (25) Pet (25) -/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/4/4 (100%) G LE6 KB3 JX1 KB1 FeT-J cell-immunized cat Ab FeT-J cell-immunized cat Ab Non-vaccinated cat Ab Saline Pet (25) Pet (25) Pet (25) Pet (25) -/+/+/+ +/+ +/+ +/+ +/+ -/+/+ +/+ +/+ +/+ +/+ +/+ -/+/+ +/+ +/+ +/+ +/+ +/+ -/+/+ +/+ +/+ +/+ +/+ +/+ 0/4 (0%) a FIV challenge was subtype-A FIVPet (Pet) at 25 CID50 and subtype-B FIVFC1 (FC1) at 10 CID50. b FIV infection was based on the development of FIV antibodies (FIV Ab) determined by FIV-immunoblot analysis and the presence of viruses de termined by virus isolation (VI) using RT and PCR analyses. Results for the samples collected at 3, 6, 9, 12, 15, 17, and 20 wpc are shown either as positive (+) or negative (-) for FIV antibodies or viruses.

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47 Table 2-4. Summary of the results from Passive-Transfer Studies 1-3. a Donor antibodies represent a comb ination of partially purified, highly purified, and unpurified serum antibodies. b Donor antibodies were from non-vaccinated cats. c Recipient cats were infused with salin e and did not receive donor antibodies. Challenge Virus Donor Antibodies FIVPetaluma FIVFC1 HIV-1UCD-1 p24 1/8 1/3 HIV-1N20 p24 ND1 0/4 FIVPet p24 0/3 0/2 Fel-O-Vax FIV 11/12 0/5 FeT-J cells 0/2 0/1 Non-vaccinated b 0/5 0/2 Salinec 0/4 0/2

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48 Table 2-5. VNA titers of hi ghly purified antibodies and corresponding pooled serum Pooled serum for passive transfer Purification a VNA to FIVPet VNA to FIVFC1 HIV1UCD1 p24-vaccinated cat serum Pre Post <10 <10 <10 <10 HIV1N20 p24-vaccinated cat serum Pre Post <10 <10 <10 <10 FIV p24-vaccinated cat serum Pre Post <10 <10 <10 <10 Fel-O-Vax FIV-vaccinated cat serum Pre Post 1000 1000 10 Ub a Pre-purification (Pre) sample s represent pooled serum from SPF cats immunized with the corresponding vaccine. Post-pur ification (Post) preparatio ns represent highly purified antibodies, which were purified by caprylic ac id fractionation followed by ammonium sulfate precipitation. b Retesting or under investigation (U)

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49 Week 0 1 2 Day 0 1 2 3 4 5 6 7 8 9 10 11 12 Passive Transfer of Sera ( Heat & UV Inactivated Abs from Protected Cats ) 1s t Transfer ( 20 ml ) 2nd Transfer ( 10 ml ) FIV Challenge // 17 Figure 2-1. Schedule of vaccination and challeng e for passive-transfer studies. Nave SPF cats were transfused with antiseru m or purified antibodies equivalent to 30% of recipients total blood volume (TBV). First transfusi on was given on day -1 (equivalent to 20% TBV) with FIV challenge on day 0 and a seco nd transfer on day 1(equivalent to 10% TBV). Passive Transfer of Sera or Antibodies FIV Challenge Day -1 0 1

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50 Figure 2-2. Schematic of antibody preparation fo r passive-transfer studies. The figure illustrates the procedure of three separate passive im munity-transfer studies. In the first study four groups of two cats each were transfus ed with IWV ( inactivated whole virus) antiserum, Fel-O-Vax antiserum, SPF serum and saline solution. A ll antibodies were collected prior to the donor cats being expos ed to FIV. The donor and recipients were cross-matched before use of the serum. The serum was inactivated by incubation at 56C for 40 minutes. In the second study, th e antibodies were purified by ammonium sulfate precipitation in order to isolate primarily IgG. Whilst in the third study, this purification was taken further using a twostep purification met hod involving caprylic acid separation followed by ammonium sulfat e precipitation. After these procedures were completed the antibodies were dialyz ed and filtered using a 0.45m filter. Tests for biological activity, purity, and toxicity were performed on the material prior to passive transfer. Blood collection Serum extraction Passive Transfusion of Antibodies Ca p r y lic acid fractionation Pool seru m Ammonium sulfate precipitation Filter sterilization Concentration of antibodies Blood collec t ion Blood collection Study III Study I Serum extraction Serum extraction Pool seru m Pool seru m H eat inactivation Filter sterilization Filter sterilization Collection and dialysis of precipitate Collection and dialysis of precipitate Ammonium sulfate precipitation Concentration of antibodies Study II

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51 0 10 20 30 40 50 60 70 80 90 100 FB1FB2FC2FB3FB4FB5FB6FB7FB8FB9FB10FB11FIV p24 HIV p24 FIV TM HIV-1 p24 (n=15) FIV p24 (n=14) FIV infected (n=5) Figure 2-3. B-cell epitope analyses of sera and PBMC from HIV-1 p24-vaccinated cats. Sera from HIV-1 p24-vaccinated cats (n = 15) and FIV p24-vaccinated cats (n= 14) at 3 weeks after the 3rd vaccination before chal lenge and from FIV-infected control cats (n = 5) at 18 wpc were tested for re activity to 12 overlapping B-cell peptides by ELISA. These overlapping peptides derive d from FIV p24 sequence are shown with their peptide designation. The reactivity of the sera is shown as percent positive (e.g., number of positive sera among total number of sera tested). The B-cell peptide codes (without aa sequence designation) are show n below the corresponding bars the aa sequence designations are shown in Figure 2-4. As positive ELISA substrate controls, serum reactivity to FIVBang p24 protein, HIV-1UCD1 p24 protein, and FIV transmembrane peptide TM (695) were determined. % Positive cats

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52 Figure 2-4. Amino acid sequence alignment of HIV-1 and FIV strains, and B-cell peptide sequence alignment Sequence homology analysis between HIV-1UCD1 and FIVBang is shown with () for amino acid homology and (: ) for amino acid identity according to the GeneStream Align program (A). Only aa residues that are different from HIV1UCD1 and FIVBang are shown for HIV-1LAI/LAV (above HIV-1UCD1 sequence) and FIVFC1 (below FIVBang sequence), respectively. Th e designations of FIV B-cell peptides and HIV-1/FIV T-cell peptides (peptide code with aa position) are shown on the top left (B). Each bar below the aa alignment represents an overlapping 28mer B-cell peptide, which was used in our ELISA analysis for cross-reactive B-cell epitope mapping. The B-cell peptide ove rlaps by 11 aa, except for the four peptides (FB1, FB2, FC2, FB4) on the am ino-terminus. The 26-mer peptide FC2/34 59 is based on FIVFC1 sequence and differs from FIVBang sequence by having Ser48 and Ser57 instead of Pro48 and Ala57. HIV1 and FIV peptide sequences used for Tcell epitopes are shown with red aa. All 18 T-cell peptides (9-mers) are boxed with dotted line. T-cell peptide code is shown immediately above (HIV-1 p24 peptide) or below (FIV p24 peptide) th e dotted box. Overlapping red bars at the carboxyl-terminus represent two overlappi ng T-cell epitopes and their corresponding sequences. HIV-1 T-cell peptides are identical to the HIV-1UCD1 peptide sequences. FIV T-cell peptides, except for the four peptide sequences below the alignment (F4/73, F4.5/122, F5/142, F7/183), are identical to the FIVBang peptide sequences. FIV peptides F4/7 3 and F7/183 are identical to the corresponding FIVFC1 peptide sequences and remaining two FIV peptides, F4.5/122 131 and F5/142, are identical to the FIVShi peptide sequences. The published major homology region (MHR) is boxed w ith a solid line. This figure is a modification of our figure in Coleman, J., et al. 2005 [38].

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53 10 20 30 40 50 60 70 80 90 100 110 LAI I I A S V G T UCD1 PVVQNLQGQMVHQPI SPRTLNAWVK VVEEKAFSPEVIPMFTA LSEGATPQDL NTMLNTVGGHQAAMQMLKETI NEEAAEWDRL H PVHAGPIAPDQMREPRGSDIAGITSTLQEQ :. :...: ..:. .. ... ..: :: :::.: :: :. :.. .. : :..: :.... .::.:: :: : .: ...: :: : ::: BANG PI-QTVNGAPQYVAL DPKMVSIFMEK AREGLGGEEVQLWFTA FSANLTPTDM ATLIMAAPGCAADKEILDESL KQ L TAEYDRTH P----PDGPRPLPYFTAAEIMGIGLT-QEQ FC1 S S T M 10 20 30 40 50 60 70 80 90 100 E IGWMTNNPPIPVGEIY KRWIILGLNK I--VRMYSPTS ILDIRQGPK EPFR DYVDRFYKTL RAEQASQDVKNWMTETLL VQNANPDCK TILKALGPAATLEEMMT ACQGVGGPGH KARVL ... :. : .:.:. .. :: .. ..::: :: ...::.. ::. .:: .. ..: ::: .:: .. : : .:::: ::: ::.::.: ..: ---QAEARFAPARMQC RAWYLEAL G K LAAIKAKSPR AV-Q LR QG A K EDYS SFIDRLFAQI DQEQNTAEVKLYLKQSLS IANAN AE CK KAMSHLKPESTLEEKLR ACQEVGSPGY KMQLL P R K PD R 120 130 140 150 160 170 180 190 200 210 FB11 FB10 FB9 FB8 FB7 FB6 130 140 150 160 170 180 190 200 210 220 230 FB5 FB4 FB3 FC2 FB2 FB1MHR KQMTAEYDRTH IANANPDCK AVQMKQGVK RAWYLEALSK (H2)(H3)(H4) (F2)(F3) (F4) (H4.5) (H5) (H6) (H7) (H7.8) (H8) (F4.5)(F5) (F6) (F7) (F7.8) (F8) 220 FB1 / 1-30FB6 / 107-136 H2 / 16-25 F2 / 15-25 FB2 / 10-37FB7 / 126-153 H3 / 43-53 F3 / 42-51 FC2 / 34-59FB8 / 143-171 H4 / 74-84 F4 / 73-83 FB3 / 53-81FB9 / 161-188 H4.5 / 130-140 F4.5 / 122-131 FB4 / 71-100FB10 / 178-207 H5 / 150-158 F5 / 142-150 FB5 / 90-119FB11 / 197-223 H6 / 162-172 F6 / 155-164 H7 / 191-199 F7 / 183-191 H7.8 / 217-226 F7.8 / 210-219 H8 / 222-230 F8 / 214-223 T-cell Peptides FIV B-cell PeptidesHIV-1 FIV10 20 30 40 50 60 70 80 90 100 110 LAI I I A S V G T UCD1 PVVQNLQGQMVHQPI SPRTLNAWVK VVEEKAFSPEVIPMFTA LSEGATPQDL NTMLNTVGGHQAAMQMLKETI NEEAAEWDRL H PVHAGPIAPDQMREPRGSDIAGITSTLQEQ :. :...: ..:. .. ... ..: :: :::.: :: :. :.. .. : :..: :.... .::.:: :: : .: ...: :: : ::: BANG PI-QTVNGAPQYVAL DPKMVSIFMEK AREGLGGEEVQLWFTA FSANLTPTDM ATLIMAAPGCAADKEILDESL KQ L TAEYDRTH P----PDGPRPLPYFTAAEIMGIGLT-QEQ FC1 S S T M 10 20 30 40 50 60 70 80 90 100 E IGWMTNNPPIPVGEIY KRWIILGLNK I--VRMYSPTS ILDIRQGPK EPFR DYVDRFYKTL RAEQASQDVKNWMTETLL VQNANPDCK TILKALGPAATLEEMMT ACQGVGGPGH KARVL ... :. : .:.:. .. :: .. ..::: :: ...::.. ::. .:: .. ..: ::: .:: .. : : .:::: ::: ::.::.: ..: ---QAEARFAPARMQC RAWYLEAL G K LAAIKAKSPR AV-Q LR QG A K EDYS SFIDRLFAQI DQEQNTAEVKLYLKQSLS IANAN AE CK KAMSHLKPESTLEEKLR ACQEVGSPGY KMQLL P R K PD R 120 130 140 150 160 170 180 190 200 210 FB11 FB10 FB9 FB8 FB7 FB6 130 140 150 160 170 180 190 200 210 220 230 FB5 FB4 FB3 FC2 FB2 FB1MHR KQMTAEYDRTH IANANPDCK AVQMKQGVK RAWYLEALSK (H2)(H3)(H4) (F2)(F3) (F4) (H4.5) (H5) (H6) (H7) (H7.8) (H8) (F4.5)(F5) (F6) (F7) (F7.8) (F8) 220 FB1 / 1-30FB6 / 107-136 H2 / 16-25 F2 / 15-25 FB2 / 10-37FB7 / 126-153 H3 / 43-53 F3 / 42-51 FC2 / 34-59FB8 / 143-171 H4 / 74-84 F4 / 73-83 FB3 / 53-81FB9 / 161-188 H4.5 / 130-140 F4.5 / 122-131 FB4 / 71-100FB10 / 178-207 H5 / 150-158 F5 / 142-150 FB5 / 90-119FB11 / 197-223 H6 / 162-172 F6 / 155-164 H7 / 191-199 F7 / 183-191 H7.8 / 217-226 F7.8 / 210-219 H8 / 222-230 F8 / 214-223 T-cell Peptides FIV B-cell PeptidesHIV-1 FIV B A

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54 Figure 2-5. CBB-stained gel a nd immunoblot of purified anti body preparations for PassiveTransfer Study 3. Purified antibodies from HIV-1UCD1 p24 (panel A, lanes 1,2,5,6), HIV-1N20 (panel A, lanes 3,4,8,9) Fel-O-Vax FIV (panel B, lanes 1,2,5,6), FIV p24 (panel B, lanes 3,4,8,9), and FeT-J cell (panel C, lanes 3,4,7,8) vaccinated cat sera and non-vaccinated cat sera (panel C, lanes 1,2,5,6) are shown. Pre-purification pooled serum (Pre, 1,3,5,7) at 4 mg/lane a nd post-purification antibody preparation (Post, lanes 2,4,6,8) at 2 mg/lane were used in both CBB gels and immunoblots. All purified preparations in the CBB gel had st rong heavy and light chain bands and weak bands at 150 kD and 100 kD, which was most likely due to unreduced and partiallyreduced antibodies. The bands were also pr esent in the immunoblot reacted to antifeline IgG heavy and light chain in Figur e 2-6. The antibodies in the immunoblots were detected using goat an ti-cat albumin antibodies. Bovine serum albumin (BSA) (500 g/lane) was used as positive control for the immunoblots (panels A and B, lane 9). A weak band at 70 kD on lane 9 were those to BSA. Feline albumin contamination was highest in the non-vaccin ated cat antibody preparation. Arrows are placed next to lanes 6 and 8 of immunoblots indicate the minimal amount of feline albumin in the purified product.

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55 Feline Albumin 66-68kD HIVUCD1 p24 HIVN20 p24 HIVUCD1 p24 HIVN20 p24 Vaccine Vaccine Vaccine Vaccine Antibodies Antibodies Antibodies Antibodies ___________________ ___________________ ___________________ _______________ __ Pre Post Pre Post Pre Post Pre Post BSA CBB Immunoblot __________________________________________ _______________________________________ Heavy Chain 50 kD Light Chain 25 kD Feline Albumin 66-68kD Feline Albumin 66-68kD A Fel-O-Vax FIVPET p24 Fel-O-Vax FIVPET p24 Vaccine Vaccine Vaccine Vaccine Antibodies Antibodies Antibodies Antibodies _________________ _________________ _________________ _________________ Pre Post Pre Post Pre Po st Pre Post BSA B Light Chain 25 kD Heavy Chain 50 kD Non-vaccinated Fet-J SPF Fet-J Cat Vaccine Cat Vaccine Antibodies Antibodies Antibodies Antibodies _________________ _______________ _________________ ________________ Pre Post Pre Post Pre P ost Pre Post C Light Chain 25 kD Heavy Chain 50 kD 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 CBB Immunoblot __________________________________________ _______________________________________ CBB Immunoblot _______________________________________ _______________________________________

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56 Figure 2-6. Immunoblot of purifie d antibody preparations used in the six groups from PassiveTransfer Study 3. Each lane contained 2 mg of purified preparation. The antibodies were detected using goat anti-cat IgG hea vy and light chain antibodies (Kirkegaard & Perry Laboratories, Gaithersburg, Maryland ). Purified antibodies from HIV-1N20 p24 (lane 1), HIV-1UCD1 p24 (lane 2), FIV p24 (lane 3), Fe l-O-Vax FIV (lane 4), and FeTJ cell (lane 5) vaccinated cat sera and from non-vaccinated SPF cat sera (lane 6) are shown. The molecular weight marker indi cated that the bands corresponded to the predicted sizes for feline heavy and light chains. All purified antibody preparations had weaker band at 100 kD, which may be the partially-reduced immunoglobulin chain ( 2 heavy chains with disulphide bonds intact). The anticat IgG heavy/light chain antibodies showed no cross-reactivity to sheep serum, mouse serum, or BSA (data not shown). HIV-1N20 HIV-1UCD FIV-1Pet Fel-O-Vax P24 P24 P24 FIV Fet-J Non-vaccinated Vaccine Vaccine Vaccine Vaccine Immunized SPF Antibodies Antibodies Antibodies Antibodies Antibodies Antibodies Heav y Chain Li g ht Chain Lane 1 2 3 4 5 6

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57 0 20 40 60 80 100 120 140 160 180 200Concentration (mg/dl) Ref Std1 Ref Std2 Ref Std3 A B C D E SPFSample Pre Post Std Figure 2-7. IgG concentrations of purified antib ody preparations from Passive-Transfer Study 3. The IgG concentrations of pre-purificati on pooled serum (Pre) and post-purification antibody preparations (Post) were determ ined by commercial feline IgG RID assay. The IgG concentration for pooled preserum (grey bar) and purified antibody preparations (dark grey bar) from Fel-O-Vax FIV (Sample A), HIV-1UCD1 p24 (Sample B), HIV-1N20 p24 (Sample C), FIVPet p24 (Sample D), and FeT-J cell (Sample E) vaccinated cats and non-vaccina ted SPF cats (Sample SPF) are shown with values from reference standards. Commercial reference standards corresponded to 25 (Ref Std3), 100 (Ref Std2), and 200 (R ef Std1) mg/dL. The % recovery for each purified antibody preparations is s hown above the corresponding bar. The average IgG recovery was 75%, indi cating an average loss of 25%. 82% 67% 78% 73% 78% 70%

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58 0 20 40 60 80 100 120 140 160 180 200Concentration (mg/dl) Ref Std1 R ef St d2 R ef Std3 A B C D E S PFSample Pre Post Std Figure 2-8. IgA concentrations of purified antibod y preparations from Passive-Transfer Study 3. The IgG concentrations of pre-purificati on pooled serum (Pre) and post-purification antibody preparations (Post) were determ ined by commercial feline IgG RID assay. The IgG concentration for pooled preserum (grey bar) and purified antibody preparations (dark grey bar) from Fel-O-Vax FIV (Sample A), HIV-1UCD1 p24 (Sample B), HIV-1N20 p24 (Sample C), FIVPet p24 (Sample D), and FeT-J cell (Sample E) vaccinated cats and non-vaccina ted SPF cats (Sample SPF) are shown with values from reference standards. Commercial reference standards corresponded to 25 (Ref Std3), 100 (Ref Std2), and 200 (R ef Std1) mg/dL. The % recovery for each purified antibody preparations is s hown above the corresponding bar. The average IgA recovery was 36%, in dicating an average loss of 64%. 21% 31% 19% 40% 62% 43%

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59 0 20 40 60 80 100 120 140 160 180 200Concentration (mg/dl) Re f Std1 Ref Std 2 Re f Std3 A B C D E S PFSample Pre Post Std Figure 2-9. IgM concentrations of purified antibody pr eparations from Passive-Transfer Study 3. The IgG concentrations of pre-purificati on pooled serum (Pre) and post-purification antibody preparations (Post) were determ ined by commercial feline IgG RID assay. The IgG concentration for pooled preserum (grey bar) and purified antibody preparations (dark grey bar) from Fel-O-Vax FIV (Sample A), HIV-1UCD1 p24 (Sample B), HIV-1N20 p24 (Sample C), FIVPet p24 (Sample D), and FeT-J cell (Sample E) vaccinated cats and non-vaccina ted SPF cats (Sample SPF) are shown with values from reference standards. Commercial reference standards corresponded to 25 (Ref Std3), 100 (Ref Std2), and 200 (R ef Std1) mg/dL. The % recovery for each purified antibody preparations is s hown above the corresponding bar. The % recovery for each purified antibody prepar ations is shown above the corresponding bar. The average IgM recovery was 53% indicating an average loss of 47%. 52% 55% 55% 55% 48% 55%

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60 Env gp 95/ 100 ___________________________ __________________________ HIVN20 HIVUCD FIVPet FOV HIVN20 HIVUCD FIVPet FOV p24 p24 p24 p24 p24 p24 pr po pr po pr po pr po pr po pr po pr po pr po __________________________ ___________________________ HIVN20 HIVUCD FIVPet FOV HIVN20 HIVUCD FIVPet FOV p24 p24 p24 p24 p24 p24 pr po pr po pr po pr po pr po pr po pr po pr po 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 200 ng HIVUCD1 p24/strip 200 ng FIVPET whole virus/strip 200 ng Fet-J cell lysate/strip 200 ng HIVN20 p24/strip Vaccine Antibodies Pre/Post purification Vaccine Antibodies Pre/Post purification Vaccine Antibodies Pre/Post purification Vaccine Antibodies Pre/Post purification 75kD 50kD p24 dimer p24 A B C D p24 dimer p24 Figure 2-10. HIV-1 and FIV p24 reactivity of purified antibody prepar ations from PassiveTransfer Study 3. The p24 reactivity was based on immunoblot analysis using HIV1UCD1 p24 (A), HIV-1N20 p24 (B), FIVPet whole-virus (C), and uninfected FeT-J celllysate (D) as substrates at 200 ng/stri p. Pre-purification serum (pr) and postpurification antibody prepar ation (po) from HIV-1N20 p24 (Pre/Post Strip Pair 1), HIV-1UCD1 p24 (Pair 2), FIV p24 (Pair 3), a nd Fel-O-Vax FIV (FOV, Pair 4) vaccinated cats sera were r eacted at a dilution of 1:200.

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61 CHAPTER 3 SUBTYPE A HIV-1 P24 AS AN FIV VACCINE IMMUNOGEN Introduction One of the major hurdles impeding the develo pment of an effective HIV vaccine is the necessity to elicit an immune response, which pr otects against different subtypes of the virus [58]. Thus far, our work on the development of an improved FIV vaccine shows that this is an achievable goal. With an aim to increase the e fficacy of the FIV vaccine, a combination of two virus strains isolated from LTNP cats was utiliz ed [59,60]. The vaccine was able to induce from moderate to significant levels of protecti on among SPF cats challenged with homologous and heterologous FIV strains. The vaccine induced many antibodies, which were also reactive to HIV-1 p24. The induction of these cross-reactiv e antibodies was expected, since several p24 antigens are conserved in the same viral subfamil y. The principle of crossprotective epitopes as the basis for vaccine design has been utilized previously in the development of smallpox and canine distemper vaccines [61,62]. The studies describe d in this section will utilize this principle to develop an FIV vaccine that is effectiv e against strains from multiple FIV subtypes. Although HIV-1 and FIV p24 proteins have only 31% aa identity, core p24 of these viruses have a number of cross-reactive epitopes [38]. Pr evious studies from our laboratory tested the use of subtype-B HIV-1UCD1 p24 as an immunogen for FIV vaccines. This vaccine conferred protection in 60%-100% of vacci nated cats against strains from multiple FIV subtypes [38]. Based on this observation, current studies were performed to test the following hypothesis. HIV1 p24 has epitopes that are highly immunogenic to cats and some of these immunogenic epitopes that are common to HIV-1 and FIV can serve as conserved vaccine epitopes for protection of domestic cats against FIV. In previous studies, the p24 proteins from two HIV-1 subtype-B strains had cross-protective epitopes, but such epitopes have yet to be identified on p24 from

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62 other HIV-1 subtypes. Therefore, current studies were performed to evaluate the prevalence of cross-protective p24 epitopes on HIV-1 subtype -A. The studies included the sequencing, production, and purification of HIV-1 subtype-A p24; T-cell ep itope mapping; immunogenicity analysis; and pilot vaccine-efficacy trials. HIV-1 subtype-A p24 was sequenced from a subtype-A strain obtained from AIDS Reagent Bank. Upon confirmation of its subtype-A origin, its se quence was compared to HIV-1 subtype-B p24 (Figure 3-1) and FIVFC1 p24 sequences to identify potential differences in TH/CTL epitopes. Since there was no T-cell epitop e database for FIV, LANL database for TH and CTL epitopes on subtype-B HIV-1 p24 was used to identify aa differen ces in T-cell epitopes between subtype-A and subtype-B HIV-1 p24 prot eins. Vaccinated cats received 3 doses of vaccine at 3-week intervals before FIV challenge at 3 weeks after the last vaccination (Figure 32). These animals were challenged IV with FIVFC1 (Table 3-1 and 3-2). SPF cats were immunized with either subtype-A or subtype-B HIV-1 p24 protein and their PBMC were assessed for T-cell responses to FIV and HIV-1. The interferon-gamma (IFN ) ELISpot analysis with overlapping FIV p24 peptid es was used to identif y the cross-reactive Tcell epitopes. Results showing similar epitope recognition between the two vaccinated groups will indicate strong potential for subtype-A p24 vaccine to work as effectively as subtype-B HIV-1 p24 vaccine. However, results showing di sparity in the T-cell epitope responses will suggest potential difference in vaccine efficacy be tween these two vaccines. Thus, these analyses were used to project the outcome of subse quent vaccine-efficacy trials against subtype-B pathogenic FIVFC1. Since the previous report had only one HIV-1 p24 vaccine study using FIVFC1 as challenge virus, the goals of Vaccination Study 1 were to te st the reproducibility of the original findings

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63 and to determine the level of vaccine protecti on observed in the vaccinated donors for PassiveImmunity Study 1 in Chapter 2. The vaccinati on groups in Vaccination Study 1 consisted of subtype-B HIV-1UCD1 p24-vaccinated cats, Fel-O-Vax FIVvaccinated cats, and age-matched non-vaccinated cats. Upon conformation of pr otection with HIV-1 subtype-A p24 vaccine against FIVFC1, Vaccination Study 2 was initiated with v accinated donors from Passive-Immunity Study 3 (Chapter 2). The vaccination groups consisted of subtype-A HIV-1N20 p24, subtype-B HIV-1UCD1 p24-vaccinated cats, FIVPet p24-vaccinated cats, Fel-O-Vax FIV-vaccinated cats, and age-matched non-vaccinated cats. Overall, these vaccination studies were perfor med to determine the prevalence of crossprotective epitopes on HIV-1 subtype-A p24 and to te st the hypothesis on cr oss-reactive epitopes for vaccine prophylaxis. Furthermore, these studies were the first step towa rds our central goal to provide insight into the developmen t of an HIV-1 vaccine for humans. Methods Virus Selection HIV-1 subtype-A isolates were obtained from AIDS Reagent Bank and stored in a secure freezer at -80oC. The isolates were amplified by cell cu lture and then sequenced to identify the strain to be used as the p24 immunogen for subtype-A vaccine. Co-culturing for Virus Amplification Whole blood was collected from a healthy human donor, and PBMCs isolated using the ficoll-hypaque gradient technique. These cells were resuspended in culture media (RPMI 1640, FBS 10%, Gentomycin, and 2-mecaptoethanol) a nd stimulated with 0.1g/mL staphylococcal enterotoxin A for 3 days. The cells were then rea dy to be co-cultured with HIV-1 infected cells. Cells were cultured at 3 day intervals by removing two-third of the culture media and replacing it with fresh culture media. The supernatant from each flask was stored for RT assay analysis. The

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64 cells were monitored daily to observe changes in morphology or the formation of multinucleated giant cells indicative of HIV-1 infection. Wh en the cultures were observed to contain a significant amount of these large multinucleated cells, they were termin ated to harvest the proviral DNA. Isolation of Proviral DNA The cell suspension from the HIV co-cultures were placed into 15-mL tubes and centrifuged at 2000 rpm for 5 minutes. The supernat ant was removed and the cells were washed in PBS. The cell pellet was resuspended in 200 L lysis buffer (0.45%Tween 20, 0.45% NP40, PCR Buffer, 200 g/mL proteina se K) and incubated at 56oC for 24 hours. After heat treatment, an equal volume of phenol-chloroform-isomyl alcohol was added and the suspension gently rocked for 4-18 hours. The sample was subse quently votexed followe d by centrifugation at 13000 rpm for 5 minutes. The upper layer containi ng the proviral DNA was transferred to a new microfuge tube and mixed with 50 l of 3M s odium acetate and 400 L of ethyl alcohol. The supernatant was removed and the pe llet was rinsed in 1 mL of 70% ethanol. The sample was then centrifuged, and the supernatant removed to recover purified DNA. The DNA recovered was resuspended in DNase free H2O and stored at -80oC until its use in PCR amplification for proviral p24 sequencing. Sequencing and Expression of HIV P24 PCR primers were designed based on the c onsensus and published sequences and reacted with the regions upstream and downstream of the p24 gene of HIV-1 (Figure 3-3). PCR conditions were optimized, and the following cond ition was used: the initial denaturing step at 94oC for 2 minutes followed by 30 cycles of 30 seconds at 94oC, 1 minute at 43oC ( gag ) or 52oC ( p24 ), additional 1 minute at 72oC, and the final denaturati on step of 10 minutes at 72oC. The reaction was performed in several 25-L PCR r eactions. The products of these reactions were

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65 pooled and run on an 0.8% tris acetate EDTA gel electrophoresis for 15 minutes to separate the target band from primer dimers and other cont aminating non-target nucleic acids (Figure 3-4). This band was then carefully excised from the ge l, and the product was purified with a Qiagen gel purification kit. The purifie d product was measured (concentr ation of DNA/protein) and set to 0.5 g/L, and then sent to ICBR Sequenci ng Laboratory with accompanying primers. This procedure was performed 3X from start to finish ensuring the accuracy of the sequence result. Both HIV-1 p24 and FIV p24 pr otein were expressed by E. coli M15 cells that were transfected with recombinant co nstruct of lentiviral p24 and pQ E 30 expression vector (Figure 35). This expression system produced p24 tagged w ith a 6X histidine resi due chain. The tag was utilized in the pr otein purification. Altered primer pairs were designed with adap tors on both ends of the gene insert to facilitate restriction enzyme di gestion of the ends of the PCR product and to allow sticky-end ligation of the target gene in th e correct orientation to the promot er of the vector. The histidine tag was placed on the N-terminus of the prot ein product. The endotoxin levels for each p24 protein preparation were measur ed using LAL E-toxate assay (S igma Aldrich USA). All purified proteins were below 50 endotoxin uni ts/dose, which is the endotoxin dose previously tested to be safe for use in cats. HIV-1N20 primers Two pairs of primers were used for sequencing of the whole gag and p24 regions of the proviral genome. The whole gag sequence primer pair consisted of the forward primer HGagF (5GGACTCGGCTTGCTGAAGCGCGC-3) a nd reverse primer HGagR (5ATCATCTGCTCCTGTATCTAATAG-3) (Figure 2). Th e p24 primer pair consisted of forward primer HG1 (5-CAGCATTATCAGAAGGAGCCA C-3) and reverse primer HG2 (5CACTCCCTGACATGCTGTCATCAT-3). Using the PCR conditions mentioned earlier, the

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66 Gag primer pair produced a fragment that wa s 1.685 kB long, while the p24 primer produced a fragment 542 bp long, which was within the p24 gene. Alternate reacti ons with HGagF/HG2 and HG1/HGagR produced fragments of 1.115 kB and 1050 bp, respectively. The primers used for expression were desi gned to introduce restri ction enzyme sites (RES). The sequences for forward and reverse primers were 5CGCG/GATCCGCG CCTATAGTGCAAAATGCA-3and 5CCCA/AGCTTGGGCTA CAAAACCCTTGCTTTATG-3, respec tively. The underlined bases correspond to the cassette and RE integrat ion sites in the primer (Figure 3-6). Restriction enzyme digestion The restriction enzyme reaction using Buffer E was designed to facilitate the digestion of both the insert DNA and the plasmid DNA, and th e simultaneous digestion of both enzymatic sites with high fidelity. The dige stion reaction of the pQE 30 v ector contained 6 L plasmid DNA, 0.5 L Hind III, 0.5 L BamH1, 1 L Buffer E, 1 L RNase A, and 1 L 10X BSA. The digestion reaction of the HIVN20 p24 insert contained 3 L plasmid DNA, 0.5 L Hind III, 0.5 L BamH1, 1 L Buffer(E), 1 L RNase A, and 1 L 10x BSA. Both reactions were performed at 37oC for 3 hours. Expression and purification of p24 The expression system utilized to produce recombinant HIV-1 p24 protein was the pQE30 expression vector transfected into chemically competent E. coli M15 cells (derived from E. coli K12). This expression system produced p24 prot ein with a 6X histidine tag, which was used later for purification. Altered primer pairs were designed to introduce adaptors on both ends for facilitating restriction enzyme di gestion of the ends of the P CR product and to allow sticky end ligation of the target gene in th e correct orientation to the promoter sequence. The histidine tag was placed on the N-terminus of the protein produc t. The 6X histidine tag facilitated the binding

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67 of the recombinantly expressed p24 protein to a Ni -NTA resin used in affinity purification to remove contaminating E. coli proteins. M15 cells were transfor med with vector plus insert under standard conditions (Sambrook et al. 1989) and plated on selectiv e media containing 25 g/mL of kanamycin and 100 g/mL of ampicillin. After overnight incubation at 37C, the colonies grown on the replica plate were screened by colonydirect PCR to ensure that they contained the target insert. After screening, glyc erol stock was prepared from c onfirmed colonies and stored at -80C. Two flasks, both contai ning 20 mL Lauria Bertani (L B) broth with 100 g/ml of ampicillin and 25 g/mL of kanamycin were inoc ulated with the selected M15 colony and cultured overnight at 37C. Culture flasks, each containing 500 mL LB broth (100 g/mL of ampicillin and 25 g/mL of kanamycin), were inoculated with 1:50 dilution of the overnight culture. The cells were allowed to grow for appr oximately 2.5 hours, until an optical density of 0.6 was reached. The culture was then induced us ing IPTG at a final concentration of 1mM. Throughout 4.5 hours of culturing, 1ml of the sample was collected for analysis. The cultures were then centrifuged at 4000g for 20 minutes, and the pelleted cells stored overnight at -20C. Purification E. coli pellets were thawed on ice, then re -suspend in Lysis Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole) (3 mL/g E. coli ). The cells were then soni cated to disrupt cell walls, which allowed complete lysis. Protease inhib itor was added to the lysate to inhibit the degredation of the expressed protein, and the suspension was centrigufed at 10000g for 30 minutes to remove the cellular debris. Some of supernatant was saved for analysis, and the remaining fraction was purified under native co nditions using Ni-NTA affinity chromotography column according to Ni Super Flow specificatio ns (Qiagen Inc., Valencia, California). The purified products were tested by CBB and silver stain analyses, e ndotoxin test, Bradford protein

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68 assay (BioRad Systems, Inc. Hercules, Califor nia) according to manufacturers protocol and immunoblot analysis[40]. Vaccination Studies 1 and 2 SPF cats from Liberty Research (Vaccination Study 1) and Harlan Sprague Dawley, Inc. (Vaccination Study 2) were immunized 3X at 3-w eek intervals with either HIV-1 p24, FIV p24, or Fel-O-Vax FIV vaccine. Cats were also immunized with PBS or FeT-J cell/lysate combination (as described in Chapter 2 and de tailed below). The p24 vaccines were 200 g of p24 formulated in Ribi (Study 1) or FD-1 (St udy 2) adjuvant supplemented with recombinant feline IL-12 (R&D Systems, Inc., Minnesota, MN ). Fel-O-Vax FIV vaccine is a commercial product formulated in FD-1 adjuvant. FD-1 adju vant generally provided better efficacy results than Ribi adjuvant. Study 1 consisted of HIV-1UCD1 p24-vaccinated (n=4), Fel-O-Vax FIVvaccinated (n=4), and PBS-immunized (n=3) groups, and used an FIVFC1 challenge of 15 CID50. Study 2 consisted of HIV-1UCD1 p24-vaccinated ( n=4), HIV-1N20 p24-vaccinated (n=4), FIVPet p24-vaccinated (n=3), Fel-O-Vax FIV-vaccina ted (n=2), and FeT-J cell-immunized (n=2) groups, and used an FIVFC1 challenge of 10 CID50. The FeT-J cell/lysate-immunized cats rece ived a combined cell and cell-lysate immunization using SC (2x106 cells/dose), intradermal (ID, 250 g/dose cell lysate), intranasal (IN, 100 g/dose cell lysate), and transcutaneous (TC, 100 g/dose cell lysate) r outes. The total number of cells/dose for SC immunization was equi valent to the inactivated infected cells (2x106 cells/dose) and viral proteins (50 g/dose) in the Fel-O-Vax FIV v accine. The total cell-lysate dose for combined ID/IN/TC/SC immunization was 500 g/dose. This amount was equivalent to the amount of inactivated dual-subtype wholeviruses in prototype vaccines, which also contained a moderate level of cellu lar debris. Therefore, this celllysate dose can be used safely

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69 in cats. This high dose was close to 2X the am ount of p24 proteins and was 10X the whole-viral proteins in Fel-O-Vax FIV vaccine. This high ce ll-lysate dose in combination with uninfected cells was used to ensure that sufficient amount of MHC was in the prepar ation to serve as cell control for Fel-O-Vax FIV vaccine. FIV-infect ed FeT-J cells have higher levels of MHC expressed on the cells than uninfected FeT-J ce lls. Furthermore, the FIV virion, much like HIV1, has a concentrated amount of hosts MHC on the viral membrane. Previous inactivated SIV vaccine studies in macaques demonstrated prot ection against SIV with uninfected human cells used to grow the vaccine virus. In this study, the xenogeneic MHC of the human cells present in the inactivated SIV vaccine provided the protection against SIV challenge, which was also grown in human cells. Feline IFN (Fe IFN ELISpot analysis Commercial FeIFN ELISpot (R&D Systems, Inc.) was used to determine HIV-1 p24 and FIV p24 peptide responses of the PBMC from the HIV-1UCD1 p24-, HIV-1N20 p24-, FIV p24-, and Fel-O-Vax FIV-vaccinated cats. Fresh cells (2x105 cells/well) suspended in feline assay media were cultured with peptides at 1 g peptide/well in FeIFN ELISpot plate for 18 hours. FeIFN ELISpot plates were processed according to manufacturers methods (R&D Systems, Inc.) with the exception of the a ssay media used, and analyzed with an ELISpot reader. Feline assay media consisted of AIM-V media (Gibco -Invitrogen Co., Carlsbad, CA), 10% heatinactivated pooled SPF sera, and 25 g/ml gentamycin (Mediatech, Inc., Herndon, VA). All assays included T-cell mitogen, concanavalin A (Con A) (Sigma-Aldrich USA), at 2-4 g/well as the positive control; recombinant HIV-1UCD1, HIV-1N20, and FIVPet p24 proteins and inactivated FIVPet plus FIVShi whole-viruses at 2 g/well as virus-specific immunogens; and FIVBang p24 peptide F8 at 5 g/well as the negative control. In addition, LPS at 5 and 50

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70 EU/well was included as a control for recombinan t products. The LPS used for control was from E. coli M15, which was used to produ ce the recombinant p24 proteins. These products had 1.19 EU per ng, whereas the recombinant vaccine had <50 EU per vaccine dose of 200 g. All results were in duplicates and adjusted to spot forming units (SFU) per 1x106 cells, after subtracting the background derived from non-specific peptide control or media cont rol, whichever was higher in value. The standard deviation of duplicate results for the feline IFN ELISpot analysis was <15% of the median SFU. Overlapping 15mer peptides for HIV-1LAI/LAV (identical to NIH reference subtype-B virus HIV-1HXB2) with 11 aa overlap were produced by S ynpep Corporation, USA). Each peptide pool was designated as HIV-1 peptide pool number 118 (Hp1-Hp18). Peptide-pool Hp1 consisted of the first four overlapping peptides from the amino-terminal, and each peptide pool Hp2 through Hp18 contained three consecutive overlapping peptides starting fr om the amino-terminal. The FIV peptides consisted of overlapping 15mer peptides for FIVBang (FIV subtype-A p24 sequence) with 11 aa overlap and also produced by (Synpe p Corporation, USA). Each FIV peptide pools consisted of 3-4 consecutive peptides and we re designated as FIV peptide pool number 1-17 (Fp1-Fp17). Statistical Analyses Individual immunization gr oups, including protected versus infected cats in each study, were analyzed for statistical significant difference by Mann-Whitney Rank Sum Test using Sigma Stat (Windows Version 3.11). The differe nce was considered stat istically significant when P <0.05.

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71 Results Analysis of HIV-1N20 P24 for Vaccine Silver-stained PAGE gel (Figure 3-7) and CBB-st ained gel (data not show n) analyses showed minimal contamination and a purity of 95% using BSA standard to extrapolate the p24 concentration. The silver-stained gel showed three bands at 24 kD (62.5-500 ng lanes), 20 kD (250-500 ng lanes), and 48 kD (500 ng lane) (panel C). The HIV1 p24 specificity of the 24 kD product and truncated products was detected by immunoblot analysis using sheep anti-HIV-1 p24 antibodies (Cliniqa Corporati on, Fallbrook, California) (panel A). The HIV-1 p24 bands at 24 kD were detectable from 31.25-500 ng, and a highe r band at 48 kD at 500 ng appeared to be p24 dimer. The two lower bands in the imm unoblot at 15 kD and 20 kD were observed at 62.5500 ng and 125-500 ng, respectively, and appeared to be truncated p24 products (panel A). The level of endotoxin in the HIV-1N20 p24 preparation used for vaccine was 1.19 EU/ng (2.38 EU/dose of vaccine), which was safe level for use as vaccine (LPS safety level previously tested as <50 EU/dose). HIV-1 and FIV Sequence Analyses Sequences comparison of FIVFC1 p24 Challenge virus) with HIV-1N20 and HIV-1UCD1 p24 revealed the challenge virus to possess 52.1% homology and 31.3% identity with HIV-1UCD1 p24 compared to 50.8% homology and 30.5% identity with HIV-1N20 p24. The HIV-1 p24 sequence is 231 aa long compared to 223 of FIV p24. This resulted in alignment gap generation of 6.0% and 7.6% gaps for HIV-1UCD1 and HIV-1N20 p24, respectively. HIV-1N20 p24 contained aa substitutions compared to HIV-1UCD1 p24 within the 3 of the 4 published p24 CTL epitopes shown on Figure 3-1, with a P S and I M substitution in the first epitope shown, a K to R substitution in the third and a S T and D E substitution in the fourth. The second epitope was identical between the two sequences.

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72 Vaccination Studies 1 and 2 In Vaccination Study 1 (Table 3-1), 3 of 4 HIV1UCD1 p24-vaccinated cats (Group 1A) and 4 of 4 Fel-O-Vax FIV-vaccinated cats (G roup 1B) were protected against FIVFC1 challenge (15 CID50), which infected all three PBS-imm unized control cats (Group 1C). The unprotected/vaccinated cat (#AA1) and infected control cats ha d a major decrease in CD4+-cell (CD4) counts and CD4+-cell/CD8+-cell (CD4/CD8) ratio when compared to the values from protected/vaccinated cats. A comp arison between infected cats a nd protected/vaccinated cats at 20 wpi showed only statistically significant difference for CD4/CD8 ratio (CD4 count, p =0.14; CD4/CD8 ratio, p =0.001). However, statistical diffe rence was observed between the mean CD4/CD8 ratios of Fel-O-Vax FIV-vaccinated group and PBS-immunized control group at 20 wpi. These results demonstrate immune protecti on in the protected/vaccinated cats and further support the results from FIV antibody and virus isol ation analyses that these cats were clearly not infected. In Vaccination Study 2 (Table 3-2), 2 of 4 HIV1UCD1 p24-vaccinated cats (Group 2A), 1 of 4 HIV1N20 p24-vaccinated cats (Group 2B), and 0 of 3 FIV p24-vaccinated cats (Group 2C) were protected against FIVFC1 challenge (10 CID50). All Fel-O-Vax FIV-v accinated cats (Group 2D, n=2) were also protected, while all control cats immunized with FeT-J cell/lysate (Group 2E, n=2) were infected. Overall, the protected/vaccinat ed cats had higher CD4 counts and CD4/CD8 ratios than unprotected/vaccinated cats and infected control cats. In fact, statistical differences in both the mean CD4 counts ( p =0.019) and the mean CD4/CD8 ratios ( p <0.001) were observed between protected/vaccinated cats a nd infected cats. These statisti cal differences were due to the infection and were not due to the difference in the mean CD4 counts of the pre-challenge groups (Table 3-2; Groups A vs. B vs. C vs. D vs. E: CD4 count, p>0.10; CD4/CD8 ratio, p>0.2). This pilot study confirms the results from Vaccination Study 1 that HIV1UCD1 p24 vaccine and Fel-O-

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73 Vax FIV vaccine are effective against FIVFC1. In contrast, minimal protection was achieved by HIV1N20 p24 vaccination. The level of protection (2 of 4) achieved with HIVUCD1 vaccine in Vaccination Study 1 was slightly lower than Va ccination Study 2 (3 of 4) even though the challenge dose in Study 1 was slightly higher than Study 2. IFN ELISpot Analysis of PBMC from Vaccination Study 2 As a measure for vaccine-induced cellular immunity, FIV-specific IFN responses of PBMC from vaccinated cats were compared. PBMC from HIV-1UCD1 p24-vaccinated cats had more cats responding to overlapping HIV-1LAI/LAV p24 and FIV p24 peptides (Hp3, Hp11, Hp12; Fp9) than PBMC from HIV-1N20 p24-vaccinated cats (Figures 38, and 3-9, Panels A and B). However, the PBMC from both HIV-1UCD1 p24and HIV-1N20 p24-vaccinated cat s (2 of 4 cats from each group) responded to dual-subtype FIV whole-virus (IWV) immunogen. These results clearly indicated that the PBMC from HIV-1 p24-vaccinated cats recognized cross-reactive epitopes on FIV p24. Two HIV-1 p24-vaccinated cats also recognized F p5 although one cat had response slightly below the threshold value (figure 3-9A). The PBMC of a ll protected cats from the HIV-1 p24-vaccinated gr oups (#185, #205, #207) responded to Fp9 peptide pool but the response from cat #207 was way below the thres hold level of significance. Moreover, the PBMC from Fel-O-Vax FIV-vaccinated ca t #171 and FIV p24-vaccinated cat #163 had a significant cross-reactive response to Hp11 (Figure 3-8C). Hence, two-way cross-reactivity was observed, whereby HIV-1 p24-vaccinated cats r ecognized Fp9 epitope, while Fel-O-Vax FIVand FIV p24-vaccinated cats recognized Hp11. The PBMC from the two Fel-O-Vax FIV-vaccinated cats had robust IFN response (Figure 3-9C). One cat (#171) had significant responses (> 50 SFU threshold) to a number of FIV p24 peptide pools, including Fp9, but the other prot ected cat (#183) had no re sponse to Fp9 (Figure

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74 3-9A, C). Both cats had a significant respons e to Fp3, suggesting the potential of multiple epitopes for protection. Thus, 4 of 5 protected cats vaccinated with HIV-1 p24 or Fel-O-Vax FIV vaccine reacted to Fp9, of which 3 responses were above the threshold response, while none of the unprotected/vaccinated cats had significant response to Fp9 (Figure 3-9A, B and C). In any event, the numbers of cats are much too lim ited in the current study to determine the crossprotective epitope(s) solely base d on one parameter (i.e., IFN response) of cellular immune response. Discussion The efficacy of Vaccination Studies 1 and 2 are summarized in Table 3-2 along with results from other published and unpublished studie s of our laboratory. The combined results from current two studies show subtype-B HIV-1UCD1 p24 vaccine conferring the highest protection rate of 62% (5 of 8 cats) among the two HIV-1 p24 vaccines tested against FIVFC1 strain. Only minimal efficacy (1 of 4) was observed with subtype-A HIV-1N20 p24 vaccine in Vaccination Study 2. This observation was somewhat in conflict with the sequence results since considerable sequence identity and homology existed between HIV-1N20 and HIV-1UCD1 p24 sequences. However, the numbers of animals used in each group were much too small to make major conclusion. In fact, the efficacy of HIV-1UCD1 p24 in Study 2 had the lowest rate, along with another study, among the seven vaccination studi es performed so far (Table 3-2). Since the efficacy was also low for HIV-1UCD1 p24 vaccine in Study 2, more studies will be needed to determine if significant difference in efficacy exists between subtype-B HIV-1UCD1 p24 vaccine and subtype-A HIV-1N20 p24 vaccine. One potential reason for lower efficacy in St udy 2 than Study 1 may be due to the SPF cats used. SPF cats from Liberty Research and Harlan Sprague Dawley, Inc. were used in Studies 1

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75 and 2, respectively. In general, cats from Harlan Sprague Dawley. were mo re susceptible to FIV infection and more difficult to confer protec tion with HIV-1 p24 and Fe l-O-Vax FIV vaccines than cats from Liberty Research (R. Pu and J.K. Yamamoto, personal communiqu). Based on MHC-I sequence analysis of one cat from Harl an Sprague Dawley and our semi-inbred cats derived from backcross inbreeding with tom from Liberty Research, cats from Harlan Sprague Dawley had MHC-I sequence lineage different from the predominant lineages of the cats from Liberty Research (E. Sato and J.K. Yamamot o, personal communiqu). Furthermore, according to our findings from Chapter 2, both HIV1 p24 and Fel-O-Vax FIV vaccines conferred protection against FIVFC1 by cellular immunity. Recent a doptive-transfer studies by our laboratory demonstrated protection with protot ype dual-subtype FIV vaccine was mediated by CD4+ CTL and CD8+ CTL [57]. Hence, MHC-re stricted T-cell immunity may play a central role in protection observed with HIV-1 p24 vaccine. The importance of cellular immunity was determ ined in current studies by monitoring the FIV-specific IFN responses of the vaccinated cats. HIV-1UCD1 p24 vaccine was more immunogenic than HIV-1N20 p24 vaccine, but not as immunoge nic as Fel-O-Vax FIV vaccine. In current study, only 1 of 3 cats vaccinated with FIVPet p24 had robust IFN responses (Fp7, Fp8, Fp14, Fp15), while another cat (#195) from the same group had a single significant response to Fp3. The PBMC from both FelO-Vax FIV-vaccinated cats had significant responses to two or more FIV peptide pools. Th us, Fel-O-Vax FIV vaccine appeared to be more immunogenic than FIV p24 vaccine. This obser vation supported the previous study of our laboratory, which showed only 2 of 7 FIV p24-vacci nated cats with low but above the threshold responses to FIV p24 peptide pools (Fp3, Fp7, a nd Fp10). Fp3 and Fp7 were the common FIV

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76 peptide pools recognized by the cats from both st udies. Furthermore, all cats, which responded to these peptide epitopes, were from the same vendor (Harlan Sprague Dawley). The PBMC from HIV-1UCD1 p24-vaccinated cats had IFN responses to Hp3 (2 of 4), Hp11 (3 of 4), and Hp12 (4 of 4), while the PB MC from Fel-O-Vax FIV-vaccinated cats and FIV p24-vaccinated cats had a significant response to Hp11 (2 of 5). In the previous unpublished studies of our laborator y, the PBMC from HIV-1UCD1 and HIV-1LAI/LAV p24-vaccinated cats had significant responses to Hp3 (1 of 3), Hp6 (4 of 8), Hp11 (6 of 8) Hp12 (2 of 8), Hp14 (3 of 8), and Hp17 (2 of 8). Current study had lower numbe rs of peptide epitopes recognized by the HIV1 p24-vaccinated cats than the previous studies This may be due to the more diverse MHC patterns present in the cats from previous studies, since previous studies used three sources of cat vendors. Only 2 of the 8 cats were from the sa me vendor as current study. Moreover, the one cat that responded significantly to all Hp3, Hp11, and Hp12 in the previous study was from the same vendor as the current study. Whereas, the othe r cat from the same vendor in previous study responded slightly below the threshold value to Hp11 and Hp12, but had no responses to other peptide epitopes. In current study, the PBMC fr om the two Fel-O-Vax FIV-vaccinated cats had a significant response to Fp3. In the previous study, the PBMC from 8 of 8 cats vaccinated with prototype vaccine of Fel-O-Vax FIV vaccine, ha d significant responses to Fp3. Thus, Fp3 may be the immunodominant epitope for both prot otype and commercial vaccines. Other major epitopes recognized by prototype vaccine were Fp7, Fp10, and Fp13 (5 of 8 each) followed by Fp1 (3 of 8), Fp2 (4 of 8), Fp8 (3 of 8) Fp9 (3 of 8), Fp11 (3 of 8), and Fp15 (4 of 8). Thus, both prototype and commercial v accines induced strong IFN responses to multiple epitopes. Findings from recent adoptive-transfer studies from our laboratory [7] further support the importance of MHC-restricted cellular immun ity in prototype vaccine of the commercial

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77 vaccine. These findings taken together with re sults from HIV-1 p24 vaccine studies and passivetransfer studies in Chapter 2 suggest the impor tance of MHC-restricted cellular immunity in protection conferred by subt ype-B HIV-1 p24 vaccines. FIVPet 24 vaccine conferred no protection (0 of 3) in Vaccination Study 2. This result taken together with other FIV p24 studies demons trated a very minimal protection with FIV p24 vaccine (Table 3-2, 3 of 21, 14%). Moreover, subtype-B HIV-1 p24 vaccines were far more effective against FIV challenge than FIV p24 vaccine (64-67% versus 14%, Table 3-3). A comparison of HIV-1UCD1 and FIVFC1 p24 sequences demonstrated only 31.3% identity and 52.1% homology between them. Two potential e xplanations can be provided. FIV p24 may possess immunodominant epitopes, which restrict the development of immune responses to protective epitopes. Protective epitopes need to stimulate functional activity essential for vaccine protection such as CTL activity to viral antigen. Many CTL epitopes have been identified on HIV-1 p24, including those that are immunodominant [LANL]. However, these CTL epitopes were identified by analyzing the CTL responses to HIV-1 p24 peptides made by the PBMC from HIV-1-positive human subjects. Since cats may not recognize the same epitopes as human, our IFN ELISpot analysis with overlap ping FIV and HIV-1 p24 peptides were the first step towards identifying cr oss-protective CTL and TH epitopes induced by effecticacious vaccines (HIV-1UCD1 p24 and Fel-O-Vax FIV vaccines). The other possibility is that cellular response elicited by HIV-1 p24 is not reacting to the cross-reactive epitopes on the FIV p24 but instead reacting to th e mimotopes elsewhere on the FIV virus. One way to examine this possibility was by performing NCBI Bl ast search on both homologous and divergent segments of the HIV-1UCD1 p24 sequence against other structur al and enzymatic proteins (Env,

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78 Pol, MA) of FIV. Initial analysis suggests no significant mimicry of p24 aa sequences elsewhere on the virus. This study is the first to simultaneously exam ine the cross-protective epitopes of FIV/HIV by IFN ELISpot. The results seem to indicate that there is cro ss-recognition of epitopes with HIV-1 p24-vaccinated cats responding to FIV IWV immunogen and FIV p24 epitope Fp9. Furthermore, both Fel-O-Vax FIV-vaccinate d cat #171 and FIV p24-vaccinated cat #163 responded to HIV-1UCD1 p24 protein and HIV-1 p24 epitope Hp11. Moreover, 3 of 4 HIV1UCD1 p24-vaccinated cats responded to Hp11, with th e response of the fourth cat near the threshold. Overall, these results demonstrated two-way cross-reactivity and also confirmed the existence of conserved epitopes between subtype-B HIV-1 p24 and FIV p24. Though HIV-1N20 and HIV-1UCD1 p24 sequences are 90.5% identical and 95.2% homologous, there was a considerab le difference in the protection rate of the two groups with 62% protection for HIV-1UCD1 p24 vaccinated cats and only 25% efficacy for cats vaccinated with HIV-1N20 p24. Both vaccinated groups generated an tibodies to HIV-1 p24, but the ELISpot result showed less overall reactivity of HIV-1N20 p24-vaccinated cats compared to HIV-1UCD1 p24-vaccinated cats to HIV-1 p24 peptides. Two reasons for this disparity in IFN responses can be speculated. This could also be the result of aa substitutions within the corresponding regions of HIV-1N20 p24. It is also possible that the difference in reactivity could be based on the fact that the peptide pool was designed based on HIV-1HXB2 (identical to HIV-1LAV/LAI), which is more similar to HIV-1UCD1 p24 than to HIV-1N20 p24. One reasons for selecting this sequence was because of its sequence variation within th e CTL epitopes shown in Figure 3-1, which are recognized by both cats and humans. Though the substitutions were similar in charge this change

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79 or the steric alteration of cleavage patterns by flanking non-homologous substitutions may be responsible for the disparity in the immunogenicity and the protection rate of these vaccines. In current studies, Fel-O-Vax FIV vaccine c onferred 100% protection (6 of 6). The high protection rate against heterologous-subtype FIVFC1 was in agreement with previous published and unpublished results (Table 3-2) These data taken together clearly demonstrate that the commercial Fel-O-Vax FIV vaccine provides considerable protection (Table 3-2, 17 of 18) against heterologous-subtype-B virus. Furtherm ore, long-duration vaccin e analyses of Fel-OVax FIV vaccine showed 50-56% protection in cats challenged with FIVBang and FIVFC1 (both 25 CID50), respectively, after 1-year bo ost. Since natural transmission doses are reported to be far lower than the lowest dose (10 CID50) used in current studies [23,57,63], commercial Fel-O-Vax FIV vaccine is likely to be more effective against natural tran smission. A pilot study performed by our laboratory also showed long-term efficacy (67%, 2 of 3) with HIV-1UCD1 p24 vaccine against subtype-A/B recombinant FIVBang challenge administered afte r 1-year boost (Table 3-2). Since single boost 1-year later was sufficient enough to provide 67% protection, the immunity elicited during the first three vacc inations must have sustained, in order for a single 1-year boost to confer such level of protection.

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80 Group Cat # Vaccination FIV Chal. (CID50) a CD4# (CD4/CD8) b Pre Post Weeks Post-Challenge (FIV Ab/VI) c 3 6 9 12 15 17 20 P/L/B c Protection Rate (%) A AA1 MD1 MG1 MF3 HIV1UCD1 p24 HIV1UCD1 p24 HIV1UCD1 p24 HIV1UCD1 p24 FC1 (15) FC1 (15) FC1 (15) FC1 (15) 3.47 (3.6) 2.44 (1.4) 3.47 (4.9) 3.88 (2.5) 3.53 (2.7) 2.90 (1.8) 1.83 (3.4) 1.71 (1.7) 3.07 (3.6) 2.73 (1.8) +/+ +/+ +/+ +/+ +/+ +/+ +/+ ND -/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/3/4 (75%) B AA2 MD2 MG2 MF4 Fel-O-Vax FIV Fel-O-Vax FIV Fel-O-Vax FIV Fel-O-Vax FIV FC1 (15) FC1 (15) FC1 (15) FC1 (15) 3.58 (3.4) 3.56 (2.7) 3.41 (3.8) 2.83 (2.6) 3.61 (2.8) 2.92 (2.2) 1.44 (2.7) 2.13 (2.1) 3.01 (3.2) 2.86 (2.4) -/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/4/4 (100%) C MD3 MG5 MK4 PBS PBS PBS FC1 (15) FC1 (15) FC1 (15) 3.23 (2.5) 2.89 (1.4) 1.99 (2.6) 1.50 (0.8) 3.03 (4.3) 1.62 (1.1) 2.75 (3.1) 2.00 (1.1) -/+/+/+ +/+ +/+ +/+ +/+ +/+/-/-/+/+ +/+ +/+ +/+ +/+ +/-/+ -/-/+/+ +/+ +/+ +/+ +/+ +/-/+ 0/3 (0%) Table 3-1. Vaccinati on Study 1 with HIV-1UCD1 p24 vaccine and commercial Fel-O-Vax FIV vaccine a FIV challenge (FIV chal.) was subtype-B FIVFC1 (FC1) at 15 CID50. b The CD4 counts (CD4#; x1000/ L) and CD4/CD8 ratios (CD4/CD8) determined at -2 (Pre) and 20 (Post) weeks. The average count and rati o are shown in bold italics for each group. c FIV infection was based on the development of FIV antibodies (FIV Ab) determined by FIVimmunoblot analysis and the presen ce of viruses determined by viru s isolation (VI) using RT and PCR analyses. Results for the samples collected at 3, 6, 9, 12, 15, 17, and 20 wpc are shown either as positive (+) or negative (-) for FIV antibodies or vi ruses. Virus isolation was also performed on PBMC (P), lymph node (L), and bone marrow (B) cells at 34-46 wpc. Abbrev iation is not done (ND).

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81 Group Cat # Vaccination Status FIV Challenge (CID50) a CD4# (CD4/CD8) b Pre Post Weeks Post-Challenge (FIV Abs/VI) c 3 6 9 12 15 17 20 P rotection Rate (%) 2 A 147 185 199 205 HIV1UCD1 p24 HIV1UCD1 p24 HIV1UCD1 p24 HIV1UCD1 p24 FC1 (10) FC1 (10) FC1 (10) FC1 (10) 3.85 (5.59) 0.68 (1.98) 2.39 (3.43) 1.80 (2.42) 1.19 (2.27) 1.06 (1.55) 2.31 (3.66) 2.98 (2.25) 2.44 (3.74) 1.63 (2.05) -/-/+/+/+ +/+ +/+ +/+ -/-/-/-/-/-/-/-/-/+ /+/+ +/+ +/+ +/+ -/-/-/-/-/-/-/2/4 (50%) 2 B 175 187 201 207 HIV1N20 p24 HIV1N20 p24 HIV1N20 p24 HIV1N20 p24 FC1 (10) FC1 (10) FC1 (10) FC1 (10) 0.87 (2.09) 0.40 (1.71) 3.05 (4.54) 1.40 (0.88) 0.89 (3.22) 1.55 (1.20) 1.90 (2.72) 2.90 (2.62) 1.68 (3.14) 1.56 (1.60) -/-/+ /+ /+/+/+/+ -/+ /+/+/+ +/+ +/+ +/+ -/-/+/+/+ +/+ +/+ +/+ -/-/-/-/-/-/-/1/4 (25%) 2 C 163 173 195 FIVPet p24 FIVPet p24 FIVPet p24 FC1 (10) FC1 (10) FC1 (10) 1.56 (3.20) 0.56 (1.07) 1.03 (2.78) 0.76 (0.76) 1.72 (2.54) 1.76 (1.58) 1.44 (2.84) 1.03 (1.14) -/+ /+/+/+ +/+ +/+ +/+ -/+ /+/+/+ +/+ +/+ +/+ -/-/-/+/+/+ +/+ +/+ 0/3 (0%) 2 D 171 183 Fel-O-Vax FIV Fel-O-Vax FIV FC1 (10) FC1 (10) 1.22 (2.49) 1.09 (2.44) 1.86 (2.78) 2.51 (2.25) 1.54 (2.64) 1.80 (2.35) -/-/-/-/-/-/-/-/-/-/-/-/-/-/2/2 (100%) 2 E 189 203 FeT-J cell lysate FeT-J cell lysate FC1 (10) FC1 (10) 1.98 (3.22) 1.31 (1.15) 2.57 (3.20) 2.61 (1.60) 2.28 (3.21) 1.96 (1.38) -/-/+ /+/+ +/+ +/+ +/+ -/-/-/-/+ + /+ +/+ +/+ 0/2 (0%) Table 3-2. Vaccination Study 2 with HIV1 p24, FIV p24, and Fel-O-Vax FIV vaccines a FIV challenge was subtype-B FIVFC1 (FC1) at 10CID50. b The CD4 counts (CD4#; x1000/ L) and CD4/CD8 ratios (CD4/CD8) determined at -2 (Pre) and 20 (Post) weeks. The average count and ratio are shown in bol d italics for each group. c FIV infection was based on the development of FIV antibodies (FIV Ab) determined by FIVimmunoblot analysis and the pres ence of viruses determined by vi rus isolation (VI) using RT and PCR analyses. Results for the samples collect ed at 3, 6, 9, 12, 15, 17, and 20 wpc are shown either as positive (+) or negative (-) for FIV antibodies or viruses. Virus isolation was also performed on PBMC (P), lymph node (L), a nd bone marrow (B) cells at 34-46 wpc.

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82 N20 PIVQNAQGQMVHQSMSPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDLNMMLNIVG ConsA PIVQNAQGQMVHQSLSPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDLNMMLNIVG **************:********************************************* N20 GHQAAMQMLKDTINEEAAEWDRVHPVHAGPIPPGQMREPRGSDIAGTTSTLQEQIGWMTS ConsA GHQAAMQMLKDTINEEAAEWDRLHPVHAGPIPPGQMREPRGSDIAGTTSTPQEQIGWMTG **********************:*************************** ********. N20 NPPIPVGEIYRRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQATQE ConsA NPPIPVGDIYKRWIILGLNKIVRMYSPVSILDIKQGPKEPFRDYVDRFFKTLRAEQATQE *******:**:**********************:**************:*********** N20 VKNWMTETLLVQNANPDCKAILKSLGPGATLEEMMTACQGVGGPSHKARVL ConsA VKNWMTETLLVQNANPDCKSILRALGPGATLEEMMTACQGVGGPGHKARVL *******************:**::********************.****** N20 PIVQNAQGQMVHQSMSPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDLNMMLNIVG UCD PVVQNLQGQMVHQPISPRTLNAWVKVVEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVG *:*** *******.:***********:*************:************ *** ** N20 GHQAAMQMLKDTINEEAAEWDRVHPVHAGPIPPGQMREPRGSDIAGTTSTLQEQIGWMTS UCD GHQAAMQMLKETINEEAAEWDRLHPVHAGPIAPDQMREPRGSDIAGITSTLQEQIGWMTN **********:***********:********.*.************ ************. N20 NPPIPVGEIYRRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKT UCD NPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKT **********:****************.*********************** N20 LRAEQATQEVKNWMTETLLVQNANPDCKAILKSLGPGATLEEMMTACQGVGGPSHKARVL UCD LRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVL ******:*:*******************:***:***.****************.****** Figure 3-1. Amino acid alignment of HIV-1N20 p24 (N20) with a subtype-A consensus sequence (ConsA) and with HIV-1 p24. The stars under ea ch amino acid pair indicate identical bases. The number of dots listed below the non-identical pairs indicates the charge similarity of the amino acids with 2 dots indicating a greater similarity in charge signature than 1 dot, with no dots being i ndicative of least charge similarity. The alignment comparison between HIV-1N20 and consensus sequences indicates that HIV-1N20 was a good representative sequence of th e subtype-A, and is not an outlier. Therefore it should contain epitopes typi cal of this subtype. The boxes highlight established CTL epitopes in the Los Alamos Database.

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83 Figure 3-2. Schedule for vaccina tion and challenge for vaccination studies. Vaccinated cats received three of vaccine at 3-week intervals and rece ived FIV challenge 3-weeks after the last boost. These animal s were challenged IV with FIVFC1. Week -9 -6 3 0 3 6 9 12 15 18 21 24 27 Vaccination & Challenge Vaccination FIV Challenge// 52

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84 TTCGCGCTTAACCCTAGCCTTTTAGAAACAGCGGA AGGATGTCAGCAACTAATGGAACAGTT ACAATCAGCTCTCAAGACAGGGTCAGAAG AACTTAAATCATTGTTTAACACCATAGCAACCC TTTGGTGCGTGCATCAAAGGATAGATGTAAAAGACACCAAGGAAGCCTTAGATAAAGTAGA GGAAGTACAGAACAAGAGCAAACAAAAGACACAGCAGGCAGCAGCTGCCACAGGAAGCGG CAGCCAAAATTAC CCTATAGTGCAAAATGCA CAAGGGCAAATGGTACATCAGTCCATGTCA CCTAGGACTTTGAATGCATGGGTGAAGGTAATAGAAGAAAAGGCTTTCAGTCCAGAAGTAA TACCCATGTTTTCAGCATTATC AGAGGGAGCCACCCCACAAG ATTTGAATATGATGCTAAAC ATAGTGGGGGGACACCAGGCAGCAATGCAGATGCTAAAAGATACCATCAATGAGGAAGCTG CAGAATGGGATAGGGTACACCCAGTACATGCAGGGCCTATTCCACCAGGCCAGATGAGGGA ACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAAGAACAAATAGGATGGATG ACCAGCAATCCACCTATCCCAGTGGGAGAAATCTATAGAAGATGGATAATTCTGGGATTAA ATAAAATAGTAAGAATGTATAGCCCTGT CAGCATTTTGGACATAA GACAAGGGCCAAAAGA ACCCTTTAGAGATTATGTAGATCGGTTCTATAAAACTTTGAGAGCTGAACAAGCTACACAGG AAGTAAAAAATTGGATGACAGAAACCTTGCTGGTCCAAAATGCGAATCCAGACTGTAAGGC CATTTTAAAATCATTAGGACCAGGGGGCTACA TTAGAAGAGATGATGACAGCATGTCAGGG AGTAGGGGGACCTAGC CATAAAGCAAGGGTTTTG GCTGAGGCAATGAGTCAAGCACAACA GGCCAACATAATGATGCAGAGGGGCAATTTTAGGGGCCAGAGAACAATAAAATGCTTCAAC TGTGGCAAAGAAGGACATCTAGCCAGAAAT TGCAAGGCCCCTAGAAAAAAGGGCTGTTGGA AATGTGGGAAGGAGGGACA CCAAATGAAGGACTGTACT GAGAGACAGGCTAATTTTTTAGG GAAAATCTGGCCTTCCAGCAAAGGGAGGCCAGGAAATTTTCCTCAGAGCAGACCGGAAACC AGACCGGAACCAACAG Figure 3-3. The full length nuc leotide sequence of HIV-1N20 gag The sequence of HIV-1N20 gag was amplified from proviral DNA and sequenc ed by direct sequencing methods. The forward primer sequence used to amplify the p24 gene is the 18-base sequence in bold/underline, and the reverse primer seque nce is the 18-base sequence in bold (no underline).

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85 Figure 3-4. Purity of PCR produc t and insert product of HIV-1N20 p24 gene. In panel A, PCR product amplified from proviral DNA are shown at 0.04 (lane 2), 0.06 (lane 3), 0.08 (lane 4), and 0.1 (lane 5) g along with negative control (lane 6) and HIV-1-positive control (lane 7) samples at 1 g. The molecular weight marker (MM) is shown on lane 1. In panel B, the p24 insert (lane 3) and pQE30 vector (lane 4) after restriction enzyme digestion are shown, along with lo w (lane 1, l-MM) and high (lane 2, h-MM) molecular weight markers. All samples we re run on 0.8% tris-acetate EDTA gel for 20 minutes. Lane 1 2 3 4 l -MM h-MM Insert Vector Lane 1 2 3 4 5 6 7 MM 0.04 0.06 0.08 0.1 + B A p24 p24 pQE30

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86 Figure 3-5. Schematic of p24 gene expression in E.coli M15 cells. Ligation (T4 DNA Ligase) HindIII BamH1 BamH1 HindIII Products PCR HindIII Reverse primer Forward primer BamH1 Gel extraction pQE-30 (3.4Kb) Gel extraction p24 Transformation into E. coli M15 chemically competent cells Sequence colonies selected to confirm insert and start large scale culture for induction of p24 expression Cut by BamH1, HindIII

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87 BamH1 CGCG/GATCCGCG CCTATAGTGCAAAATGCACAAGGGCAAATGGTACATCAGTCC ATGTCACCTAGGACTTTGAATGCATGGGTGAAGGTAATAGAAGAAAAGGCTTTCAGTCCA GAAGTAATACCCATGTTTTCAGCATTATCAGAGGGAGCCACCCCACAAGATTTGAATATG ATGCTAAACATAGTGGGGGGACACCAGGCAGCAATGCAGATGCTAAAAGATACCATCAAT GAGGAAGCTGCAGAATGGGATAGGGTACACCCAGTACATGCAGGGCCTATTCCACCAGGC CAGATGAGGGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAAGAACAA ATAGGATGGATGACCAGCAATCCACCTATCCCAGTGGGAGAAATCTATAGAAGATGGATA ATTCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTGTCAGCATTTTGGACATAAGA CAAGGGCCAAAAGAACCCTTTAGAGATTATGTAGATCGGTTCTATAAAACTTTGAGAGCT GAACAAGCTACACAGGAAGTAAAAAATTGGATGACAGAAACCTTGCTGGTCCAAAATGCG AATCCAGACTGTAAGGCCATTTTAAAATCATTAGGACCAGGGGCTACATTAGAAGAGATG ATGACAGCATGTCAGGGAGTAGGGGGACCTAGCCATAAAGCAAGGGTTTTGTAGCCC A/AGCTTGGG Lambda Hind III Figure 3-6. HIV-1N20 p24 sequence with adapter sequences. HIV-1N20 p24 sequence after PCR amplification (sequence without bold) was identical to the p24 sequence of the gag shown in Figure 3-3. The adaptors ligated to the gene create the restriction enzyme sites and are shown in bold on eith er ends of the gene sequence.

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88 Figure 3-7. Immunoblot and silv er-stained gel of the HIV-1N20 p24 protein used for Vaccination Study 2. The immunoblots were reacted with either sheep antiHIV-1 p24 antibodies (panel A) or normal sheep serum (panel B). The immunoblot consisted of HIV-1UCD1 p24 (UCD1) at 500 ng (lane 2) and HIV-1N20 p24 (N20) at 500 (lane 3), 250 (lane 4), 125 (lane 5), 62.5 (lane 6), 31.25 (lane 7), a nd 15.62 (lane 8) ng. No non-specific reactivity was observed with normal sheep se rum (panel B). The purity of the HIV1N20 p24 preparation was determined by silve r-stain analysis of the p24 preparation on 12% SDS-PAGE gel (panel C). The gel consisted of HIV-1UCD1 p24 (UCD1) at 500 ng (lane 2) and HIV-1N20 p24 (N20) at 500 (lane 3), 250 (lane 4), 125 (lane 5), and 62.5 (lane 6) ng. The gel also containe d BSA standards at 25 (lane 7), 12.5 (lane 8), 6.25 (lane 9), and 3.12 (lan e 10) ng, which was used to derive the concentration curve. The molecular weight marker (MM) is shown on lane 1 for all panels.

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89 Sheep Anti-HIV p24 Antibodies Normal Sheep Serum HIVN20 p24 HIVN20 p24 HIVN20 p24 BSA standards Lane 1 2 3 4 5 6 7 8 9 kD 50 37 25 20 15 10 kD 50 37 25 20 15 kD 50 37 25 20 15 10 HIVN20 p 24 Immunoblot HIV-1N20 p 24 Silve r -Stain Gel C B A p24 p24 Lane 1 2 3 4 5 6 7 8 9 Lane 1 2 3 4 5 6 7 8 9 UCD1 ng 500 500 250 125 62.5 25 12.5 6.25 3.125 ng 500 500 250 125 62.5 31.25 15.625 ng 500 500 250 125 62.5 31.25 15.625

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90 Figure 3-8. HIV-1 p24-specific IFN responses of vaccinated cats from Vaccination Study 2. IFN responses to overlapping peptide pools of HIV-1 (Hp1-Hp18) (A, B, C) were determined by feline IFN ELISpot assay. The IFN responses of the PBMC from HIV-1UCD1 p24 (panel A), HIV-1N20 p24 (panel B), FIVPet p24 (panel C, cats #163, #173, #195), and Fel-O-Vax FIV (panel C, cats #171, #183) vaccinated cats are shown. The virus-specific posi tive controls included IFN responses to recombinant HIV-1UCD1 p24 (UCD1 p24), HIV-1N20 p24 (N20 p24), and FIVPet p24 (Pet p24) proteins, and inactivated dual-subtype FI V whole-viruses (IWV). The PBMC from all vaccinated cats had moderate-to-high le vels of responses to T-cell mitogen stimulation with ConA (data not shown). ELISpot assays to both HIV-1 and FIV peptides were performed at the same time. As a result, the positive control values for set 1 and set 2 of each vaccination group are identical. No IFN responses to LPS and FeT-J cell lysate were detected in th e PBMC from vaccinated cats, with the exception of those from Fel-O-Vax FIVvaccinated cats, which responded to cell lysate. The IWV-stimulation results of PB MC from Fel-O-Vax FIV-vaccinated cats are shown as SFU values after subtracting out the SFU values for FeT-J cell lysate stimulation. SFU values (bars) above or at the dashed red line at 50 SFU (stringent threshold) were considered significant.

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91 0 25 50 75 100 125 150 175 200Hp1Hp2Hp3Hp4Hp5Hp6Hp7Hp8Hp9Hp10Hp11Hp12Hp13Hp14Hp15Hp16Hp17Hp18UCD1 p24 N20 p24 Pet p24 IWV 147 185 199 205 0 25 50 75 100 125 150 175 200 Hp1Hp2Hp3Hp4Hp5Hp6Hp7Hp8Hp9Hp10Hp11Hp12Hp13Hp14Hp15Hp16Hp17Hp18UCD1 p24 N20 p24 Pet p24 IWV 175 187 201 207 0 25 50 75 100 125 150 175 200 Hp1Hp2Hp3Hp4Hp5Hp6Hp7Hp8Hp9Hp10Hp11Hp12Hp13Hp14Hp15Hp16Hp17Hp18UCD1 p24 N20 p24 Pet p24 IWV 163 173 195 171 183 Threshold of 50 Threshold of 50 Threshold of 50SFU / 106PBMC SFU / 106PBMC SFU / 106PBMCHIV-1 p24 PEPTIDE POOL (Hp#) / p24 PROTEIN / IWV (6-8 g pool or 4 g protein / well) Hp1 Hp2 Hp3 Hp4 Hp5 Hp6 Hp7 Hp8 Hp9 Hp10 Hp11 Hp12 Hp13 Hp14 Hp15 Hp16 Hp17 Hp18 UCD1 N20 Pet IWV p24 p24p242 / 4 3 / 4 4 / 4 3 / 4 2 / 4 Hp1 Hp2 Hp3 Hp4 Hp5 Hp6 Hp7 Hp8 Hp9 Hp10 Hp11 Hp12 Hp13 Hp14 Hp15 Hp16 Hp17 Hp18 UCD1 N20 Pet IWV p24 p24p243 / 4 2 / 4 2 / 4 Hp1 Hp2 Hp3 Hp4 Hp5 Hp6 Hp7 Hp8 Hp9 Hp10 Hp11 Hp12 Hp13 Hp14 Hp15 Hp16 Hp17 Hp18 UCD1 N2 Pet IWV p24 p24p242 / 5 2 / 5 4 / 5 3 / 5 A 3rd Vaccination Pre-Challenge B C 3rd Vaccination Pre-Challenge HIV-1N20p24 -VACCINATED CATS HIV-1UCD1p24 -VACCINATED CATS 3rd Vaccination Pre-Challenge FIVPetp24 -VACCINATED CATS FEL-O-VAX FIV VACCINATED CATS 0 25 50 75 100 125 150 175 200Hp1Hp2Hp3Hp4Hp5Hp6Hp7Hp8Hp9Hp10Hp11Hp12Hp13Hp14Hp15Hp16Hp17Hp18UCD1 p24 N20 p24 Pet p24 IWV 147 185 199 205 0 25 50 75 100 125 150 175 200 Hp1Hp2Hp3Hp4Hp5Hp6Hp7Hp8Hp9Hp10Hp11Hp12Hp13Hp14Hp15Hp16Hp17Hp18UCD1 p24 N20 p24 Pet p24 IWV 175 187 201 207 0 25 50 75 100 125 150 175 200 Hp1Hp2Hp3Hp4Hp5Hp6Hp7Hp8Hp9Hp10Hp11Hp12Hp13Hp14Hp15Hp16Hp17Hp18UCD1 p24 N20 p24 Pet p24 IWV 163 173 195 171 183 Threshold of 50 Threshold of 50 Threshold of 50SFU / 106PBMC SFU / 106PBMC SFU / 106PBMCHIV-1 p24 PEPTIDE POOL (Hp#) / p24 PROTEIN / IWV (6-8 g pool or 4 g protein / well) Hp1 Hp2 Hp3 Hp4 Hp5 Hp6 Hp7 Hp8 Hp9 Hp10 Hp11 Hp12 Hp13 Hp14 Hp15 Hp16 Hp17 Hp18 UCD1 N20 Pet IWV p24 p24p242 / 4 3 / 4 4 / 4 3 / 4 2 / 4 Hp1 Hp2 Hp3 Hp4 Hp5 Hp6 Hp7 Hp8 Hp9 Hp10 Hp11 Hp12 Hp13 Hp14 Hp15 Hp16 Hp17 Hp18 UCD1 N20 Pet IWV p24 p24p243 / 4 2 / 4 2 / 4 Hp1 Hp2 Hp3 Hp4 Hp5 Hp6 Hp7 Hp8 Hp9 Hp10 Hp11 Hp12 Hp13 Hp14 Hp15 Hp16 Hp17 Hp18 UCD1 N20 Pet IWV p24 p24p243 / 4 2 / 4 2 / 4 Hp1 Hp2 Hp3 Hp4 Hp5 Hp6 Hp7 Hp8 Hp9 Hp10 Hp11 Hp12 Hp13 Hp14 Hp15 Hp16 Hp17 Hp18 UCD1 N2 Pet IWV p24 p24p242 / 5 2 / 5 4 / 5 3 / 5 A 3rd Vaccination Pre-Challenge B C 3rd Vaccination Pre-Challenge HIV-1N20p24 -VACCINATED CATS HIV-1UCD1p24 -VACCINATED CATS 3rd Vaccination Pre-Challenge FIVPetp24 -VACCINATED CATS FEL-O-VAX FIV VACCINATED CATS

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92 Figure 3-9. FIV p24-specific IFN responses of vaccinated cats from Vaccination Study 2. IFN responses to overlapping peptide pools of FIV p24 (Fp1-Fp17) (A, B, C) were determined by feline IFN ELISpot assay. The IFN responses of the PBMC from HIV-1UCD1 p24 (panel A), HIV-1N20 p24 (panel B), FIVPet p24 (panel C, cats #163, #173, #195), and Fel-O-Vax FIV (panel C, cats #171, #183) vaccinated cats are shown. The virus-specific posi tive controls included IFN responses to recombinant HIV-1UCD1 p24 (UCD1 p24), HIV-1N20 p24 (N20 p24), and FIVPet p24 (Pet p24) proteins, and inactivated dual-subtype FI V whole-viruses (IWV). The PBMC from all vaccinated cats had moderate-to-high le vels of responses to T-cell mitogen stimulation with ConA (data not shown). ELISpot assays to both HIV-1 and FIV peptides were performed at the same time. As a result, the positive control values for set 1 and set 2 of each vaccination group are identical. No IFN responses to LPS and FeT-J cell lysate were detected in th e PBMC from vaccinated cats, with the exception of those from Fel-O-Vax FIVvaccinated cats, which responded to cell lysate. The IWV-stimulation results of PB MC from Fel-O-Vax FIV-vaccinated cats are shown as SFU values after subtracting out the SFU values for FeT-J cell lysate stimulation. SFU values (bars) above or at the dashed red line at 50 SFU (stringent threshold) were considered significant.

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93 0 25 50 75 100 125 150 175 200Fp1Fp2Fp3Fp4Fp5Fp6Fp7Fp8Fp9Fp10Fp11Fp12Fp13Fp14Fp15Fp16Fp17UCD1 p24 N20 p24 Pet p24 IWV 147 185 199 205 0 25 50 75 100 125 150 175 200Fp1Fp2Fp3Fp4Fp5Fp6Fp7Fp8Fp9Fp10Fp11Fp12Fp13Fp14Fp15Fp16Fp17UCD1 p24 N20 p24 Pet p24 IWV 175 187 201 207 0 25 50 75 100 125 150 175 200 Fp1Fp2Fp3Fp4Fp5Fp6Fp7Fp8Fp9Fp10Fp11Fp12Fp13Fp14Fp15Fp16Fp17UCD1 p24 N20 p24 Pet p24 IWV 163 173 195 171 183 Threshold of 50 Threshold of 50 Threshold of 50SFU / 106PBMC SFU / 106PBMC SFU / 106PBMCFIV p24 PEPTIDE POOL (Fp#) / p24 PROTEIN / IWV (6-8 g pool or 4 g protein / well) Fp1 Fp2 Fp3 Fp4 Fp5 Fp6 Fp7 Fp8 Fp9 Fp10 Fp11 Fp12 Fp13 Fp14 Fp15 Fp16 Fp17 UCD1 N20 Pet IWV p24 p24p243 / 4 2 / 4 p24 p24p242 / 4 2 / 4 p24 p24p243 / 5 2 / 5 2 / 5 2 / 5 2 / 5 2 / 5 4 / 5 3 / 5 Fp1 Fp2 Fp3 Fp4 Fp5 Fp6 Fp7 Fp8 Fp9 Fp10 Fp11 Fp12 Fp13 Fp14 Fp15 Fp16 Fp17 UCD1 N20 Pet IWV Fp1 Fp2 Fp3 Fp4 Fp5 Fp6 Fp7 Fp8 Fp9 Fp10 Fp11 Fp12 Fp13 Fp14 Fp15 Fp16 Fp17 UCD1 N20 Pet IWV A 3rd Vaccination Pre-Challenge B C 3rd Vaccination Pre-Challenge HIV-1N20p24 -VACCINATED CATS HIV-1UCD1p24 -VACCINATED CATS 3rd Vaccination Pre-Challenge FIVPetp24 -VACCINATED CATS FEL-O-VAX FIV VACCINATED CATS 0 25 50 75 100 125 150 175 200Fp1Fp2Fp3Fp4Fp5Fp6Fp7Fp8Fp9Fp10Fp11Fp12Fp13Fp14Fp15Fp16Fp17UCD1 p24 N20 p24 Pet p24 IWV 147 185 199 205 0 25 50 75 100 125 150 175 200Fp1Fp2Fp3Fp4Fp5Fp6Fp7Fp8Fp9Fp10Fp11Fp12Fp13Fp14Fp15Fp16Fp17UCD1 p24 N20 p24 Pet p24 IWV 175 187 201 207 0 25 50 75 100 125 150 175 200 Fp1Fp2Fp3Fp4Fp5Fp6Fp7Fp8Fp9Fp10Fp11Fp12Fp13Fp14Fp15Fp16Fp17UCD1 p24 N20 p24 Pet p24 IWV 163 173 195 171 183 0 25 50 75 100 125 150 175 200Fp1Fp2Fp3Fp4Fp5Fp6Fp7Fp8Fp9Fp10Fp11Fp12Fp13Fp14Fp15Fp16Fp17UCD1 p24 N20 p24 Pet p24 IWV 147 185 199 205 0 25 50 75 100 125 150 175 200Fp1Fp2Fp3Fp4Fp5Fp6Fp7Fp8Fp9Fp10Fp11Fp12Fp13Fp14Fp15Fp16Fp17UCD1 p24 N20 p24 Pet p24 IWV 175 187 201 207 0 25 50 75 100 125 150 175 200 Fp1Fp2Fp3Fp4Fp5Fp6Fp7Fp8Fp9Fp10Fp11Fp12Fp13Fp14Fp15Fp16Fp17UCD1 p24 N20 p24 Pet p24 IWV 163 173 195 171 183 Threshold of 50 Threshold of 50 Threshold of 50SFU / 106PBMC SFU / 106PBMC SFU / 106PBMCFIV p24 PEPTIDE POOL (Fp#) / p24 PROTEIN / IWV (6-8 g pool or 4 g protein / well) Fp1 Fp2 Fp3 Fp4 Fp5 Fp6 Fp7 Fp8 Fp9 Fp10 Fp11 Fp12 Fp13 Fp14 Fp15 Fp16 Fp17 UCD1 N20 Pet IWV p24 p24p243 / 4 2 / 4 p24 p24p242 / 4 2 / 4 p24 p24p243 / 5 2 / 5 2 / 5 2 / 5 2 / 5 2 / 5 4 / 5 3 / 5 Fp1 Fp2 Fp3 Fp4 Fp5 Fp6 Fp7 Fp8 Fp9 Fp10 Fp11 Fp12 Fp13 Fp14 Fp15 Fp16 Fp17 UCD1 N20 Pet IWV Fp1 Fp2 Fp3 Fp4 Fp5 Fp6 Fp7 Fp8 Fp9 Fp10 Fp11 Fp12 Fp13 Fp14 Fp15 Fp16 Fp17 UCD1 N20 Pet IWV A 3rd Vaccination Pre-Challenge B C 3rd Vaccination Pre-Challenge HIV-1N20p24 -VACCINATED CATS HIV-1UCD1p24 -VACCINATED CATS 3rd Vaccination Pre-Challenge FIVPetp24 -VACCINATED CATS FEL-O-VAX FIV VACCINATED CATS

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94 CHAPTER 4 FINAL DISCUSSION The long-term goal of our studies is the development of a highly efficacious HIV-1 vaccine. In our initiative, our laboratory seeks to develop a blueprint to this objective by understanding the mechanism of our HIV-1 p24 v accine efficacy. Our dual-subtype model using inactivated whole-virus, though highly successful, presents several safety concerns (i.e., incomplete inactivation) which limit its use in HIV-1 vaccines for humans. Understanding the mechanism of protection of our HIV-1 p24 vaccine provides a novel opportunity to establish the minimum epitopic requirements necessary for a protective vaccine. This is important since several safety issues prevent the use of a full virus construct. HIV-1 p24 vaccine was able to provide prot ection against FIV, while FIV p24 vaccine had minimal efficacy. This result seems counter-i ntuitive, since there is considerably more sequence identity and homology between FIV p24 and FIV challenge virus than HIV-1 p24 and FIV challenge virus. This re sult indicates that cu rrent vaccine dogma of including conserved epitopes may not be sufficient for the successful design of an HIV-1 vaccine. Identifying and omitting the immunodominant non-protective epitope s, which may detract the establishment of an effective antiviral immune response, also may be necessary. Core protein may be only a part of the ove rall vaccine equation. Establishment of an efficient HIV-1 vaccine may require the utilizatio n of multiple virus proteins in a subunit or vector vaccine. Currently, our laboratory is e ngaged in examination of the cellular immune response of cats to HIV-1 p24 and other conserved HIV-1 p24 proteins (RT and MA). The aim is to identify the epitopes that are associated with cross-protection and are recognized by both cats and humans. The results show that both cats a nd humans recognize the KK10 epitope of HIV-1. If the feline immune response is similar to that of humans then HIV-1/FIV-cat model provides us

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95 with a means of selecting desirable epitopes for in clusion in an HIV-1 vaccine. It is not yet clear if this epitope is protective. Though our results are promising, more studies will have to be performed in order to identify th e specific protective epitopes. The findings from current studies demonstrated that protection of the HIV-1/FIV vaccine is based on cellular immunity. Th e exact immune cells involved in this protection are still unknown. The cells capable of IFN responses include not only C TL but T-helper (TH1) and NK cells [64]. It is necessary to perform studies wi th enriched populations of CTL, TH, and NK cells to determine which cells are pr imarily responsible for the vaccine efficacy and which peptides are recognized. This could also provide us with the means to determine if the epitopes involved in FIV protection and improved HIV-1 prognosis are the same. The current studies illustrate that it is possible to develop an efficient FIV vaccine using HIV-1 p24. These vaccines were consistently able to outperform vaccines based on homologous virus p24. This presents the possibility of de veloping a second-generation FIV vaccine for veterinary medicine, which has broader efficacy than our commercial FI V vaccine and addresses the problem of conflict with currently availabl e diagnostics faced by our commercial vaccine (Fel-O-Vax FIV vaccine). The findings of the passive-transfer study (Chapter 1) indicated that the antibody immunity to HIV-1 based FIV vaccines was in sufficient to provide protection against FIV challenge. This implies that the protection obs erved with our HIV-1 p24 based vaccine was the result of the host cellular response. Further exam ination of this response is required to fully understand the mechanism of the vaccine. These findings of cross-speci es protection of FIV provide an opportunity to utili ze the cat model to determine if the immunodominant hypothesis is

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96 valid and if so the mechanism of utilizing this se lection criteria for the formulation of an HIV-1 vaccine.

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97 LIST OF REFERENCES [1] Clements JE ,Zink MC. Molecular biol ogy and pathogenesis of animal lentivirus infections. Clin Microb iol Rev 1996;9:(1), 100-117. [2] Levy JA. HIV pathogenesis: knowledge gained after two decades of research. Adv Dent Res 2006;19:(1), 10-16. [3] Montagnier L. Historical essay. A history of HIV di scovery. Science 2002;298:(5599), 1727-1728. [4] Sattentau QJ ,Weiss RA. The CD4 antigen : physiological ligand and HIV receptor. Cell 1988;52:(5), 631-633. [5] Levy JA. HIV And the Pathogenesis of AIDS, ASM Press, Herndon, 1998. 588. [6] Emerman M ,Malim MH. HIV-1 regulatory/acc essory genes: keys to unraveling viral and host cell biology. Science 1998;280:(5371), 1880-1884. [7] Sauter SL ,Gasmi M. FIV vector syst ems. Somat Cell Mol Genet 2001;26:(1-6), 99-129. [8] Kramer-Hammerle S, Rothenaigner I, Wol ff H, Bell JE ,Brack-Werner R. Cells of the central nervous system as targets and reserv oirs of the human immunodeficiency virus. Virus Res 2005;111:(2), 194-213. [9] Bendinelli M, Pistello M, Lombardi S, Poli A, Garzelli C, Matteucci D, Ceccherini-Nelli L, Malvaldi G ,Tozzini F. Fe line immunodeficiency virus: an interesting model for AIDS studies and an important cat pathogen. Clin Microbiol Rev 1995;8:(1), 87-112. [10] Malim MH. Natural resistance to HIV inf ection: The Vif-APOBEC interaction. C R Biol 2006;329:(11), 871-875. [11] Wildum S, Schindler M, Munch J ,Kirc hhoff F. Contribution of Vpu, Env, and Nef to CD4 down-modulation and resist ance of human immunodeficiency virus type 1-infected T cells to superinfection. J Virol 2006;80:(16), 8047-8059. [12] Lindwasser OW, Chaudhuri R ,Bonifacino JS Mechanisms of CD4 downregulation by the Nef and Vpu proteins of primate immunodeficiency viruses. Curr Mol Med 2007;7:(2), 171-184. [13] Gummuluru S ,Emerman M. Cell cycleand Vpr-mediated regulation of human immunodeficiency virus type 1 expression in primary and transformed T-cell lines. J Virol 1999;73:(7), 5422-5430. [14] Burtey A, Rappoport JZ, Bouchet J, Basmaci ogullari S, Guatelli J, Simon SM, Benichou S ,Benmerah A. Dynamic intera ction of HIV-1 Nef with the clathrin-mediated endocytic pathway at the plasma membra ne. Traffic 2007;8:(1), 61-76.

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98 [15] Lehmann MH, Walter S, Ylisastigui L, St riebel F, Ovod V, Geyer M, Gluckman JC ,Erfle V. Extracellular HIV-1 Nef increases migration of monocytes. Exp Cell Res 2006;312:(18), 3659-3668. [16] Yang L, Morris GF, Wang Z ,Bohan Morris C. Repression of tumor necrosis factor-beta expression by the human immunodeficiency viru s type-1 tat protein in central nervous system-derived glial cells. Virus Res 1997;50:(2), 195-203. [17] Rowland-Jones SL ,Whittle HC. Out of Af rica: what can we learn from HIV-2 about protective immunity to HIV-1? Nat Immunol 2007;8:(4), 329-331. [18] Perrin L, Kaiser L ,Yerly S. Travel and the spread of HIV-1 genetic variants. Lancet Infect Dis 2003;3:(1), 22-27. [19] Stefani MM, Pereira GA, Lins JA, Alcanta ra KC, Silveira AA, Viegas AA, Maya NC ,Mussi AH. Molecular screening shows exte nsive HIV-1 genetic diversity in Central West Brazil. J Clin Virol 2007. [20] Jones J, Taylor B, Wilkin TJ ,Hammer SM Advances in antiretr oviral therapy. Top HIV Med 2007;15:(2), 48-82. [21] Schrager LK ,D'Souza MP. Cellular and an atomical reservoirs of HIV-1 in patients receiving potent antiretr oviral combination thera py. Jama 1998;280:(1), 67-71. [22] Callanan JJ, Thompson H, Toth SR, O' Neil B, Lawrence CE, Willett B ,Jarrett O. Clinical and pathological findings in feline immunodefici ency virus experimental infection. Vet Immunol Immunopathol 1992;35:(1-2), 3-13. [23] Uhl EW, Heaton-Jones TG, Pu R ,Yamam oto JK. FIV vaccine development and its importance to veterinary and human medici ne: a review FIV vaccine 2002 update and review. Vet Immunol Immunopa thol 2002;90:(3-4), 113-132. [24] de Parseval A, Chatterji U, Sun P ,Eld er JH. Feline immunodeficiency virus targets activated CD4+ T cells by using CD134 as a bi nding receptor. Proc Natl Acad Sci U S A 2004;101:(35), 13044-13049. [25] Vahlenkamp TW, de Ronde A, Schuurman NN, van Vliet AL, van Drunen J, Horzinek MC ,Egberink HF. Envelope gene sequences encoding variable regions 3 and 4 are involved in macrophage tropism of feline im munodeficiency virus. J Gen Virol 1999;80 ( Pt 10), 2639-2646. [26] Kaushik S, Vajpayee M, Wig N ,Seth P. Characterization of HIV-1 Gag-specific T cell responses in chronically infected Indi an population. Clin Exp Immunol 2005;142:(2), 388-397. [27] Verschoor EJ, Hulskotte EG, Ederveen J, Koolen MJ, Horzinek MC ,Rottier PJ. Posttranslational processing of th e feline immunodeficiency virus envelope precursor protein. Virology 1993;193:(1), 433-438.

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102 BIOGRAPHICAL SKETCH Marcus Martin earned his B.S. degree at Mo rgan State University; Baltimore Maryland. He worked for a time in biomedical research be fore entering the graduate program at UF where he earned his PhD.