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Immunogenicity and protective efficacy evaluation of canarypoxvirus (ALVAC)-based FIV vaccines in cats

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Immunogenicity and protective efficacy evaluation of canarypoxvirus (ALVAC)-based FIV vaccines in cats
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Tellier, Maria C., 1968-
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English
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xi, 121 leaves : ill. ; 29 cm.

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Antibodies ( jstor )
Cats ( jstor )
Feline immunodeficiency virus ( jstor )
HIV ( jstor )
HIV 1 ( jstor )
Immunity ( jstor )
Immunization ( jstor )
Infections ( jstor )
Polymerase chain reaction ( jstor )
Vaccinations ( jstor )
Avipoxvirus ( mesh )
Cats ( mesh )
Department of Veterinary Medicine thesis Ph.D ( mesh )
Dissertations, Academic -- College of Veterinary Medicine -- Department of Veterinary Medicine -- UF ( mesh )
Feline Acquired Immunodeficiency Syndrome -- prevention & control ( mesh )
Immunodeficiency Virus, Feline -- genetics ( mesh )
Immunodeficiency Virus, Feline -- immunology ( mesh )
Lentivirus Infections -- immunology ( mesh )
Lentivirus Infections -- prevention & control ( mesh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1996.
Bibliography:
Includes bibliographical references (leaves 104-120).
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Also available online.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Maria C. Tellier.

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University of Florida
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Copyright Maria C. Tellier. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION OF
CANARYPOXVIRUS(ALVAC)-BASED FIV VACCINES IN CATS













By


MARIA C. TELLIER














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 1996





























Luctor et Emergo




























For Ella and Bram














ACKNOWLEDGMENTS


This thesis would not have been written without the influence, presence, and support of many people. I am grateful to my family, especially "Pa" and "Ma", for their care and support of me and my endeavors over the years. Further, I would like to recognize my supervisor Janet Yamamoto, who has struck me with her infinite commitment to research. I thank her for letting me pursue this project in her laboratory and giving me the opportunity to become acquainted with the World of Science and Bureaucracy. Special thanks go to Dr. Pu for his expertise in feline phlebotomy, his advice and his the anecdotes from the old China. Further, I was fortunate to have David Pollock as my "right hand". I highly appreciate his meticulous work during the countless virus isolation assays and his chit-chat during the endless fycoll sessions. I am especially indebted to Dr. Paoletti and Dr. Tartaglia at Virogenetics and Dr. Desmettre at Rhone Merieux for providing me the opportunity to work with the ALVAC vector in my studies. I also wish to acknowledge the members of my supervisory committee, Dr. Condit, Dr. Schiffenbauer, Dr. Johnson and Dr. Schuster for their time and advice.

At the homefront, I would like thank Nicole, Mienke, Mirian, Chantal and Simone for their friendship and, Dr. Paul and Dr. Frank, for staying in touch and keeping me updated on the latest from the low-lands. Warm thanks go to Sue Watsana, who was a motivating source at frustrating times. Special thanks go to Susie for all the exciting adventures and for the good times we have had in our lovely shack on NW 2nd street. Last but not least, I would like to thank Dan for his patience and great care of me, Tamika and the Kalahari's. Finally, I would like to thank all my friends in and out of Gainesville for making every day life a little more exciting.



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TABLE OF CONTENTS

page

ACKNOW LEDGM ENTS ....................................................................... iv

LIST OF TA BLES ............................................................... ......... vi

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

LIST OF ABBREVIATIONS..............................................viii

ABSTRACT............................................... ..................................... x

CHAPTER

I LENTIVIRUSES AND VACCINE DEVELOPMENT

Introduction............................. 1

II IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION
OF CANARYPOXVIRUS(ALVAC)-BASED FIV VACCINE AGAINST
HOMOLOGOUS FIV CHALLENGE

Introduction............................. 36
Materials and M ethods ............................................................. 38
R esu lts............................. .......................................... ... 54
D iscussion ......................................................................... 85

III EFFICACY EVALUATION OF CANARYPOXVIRUS(ALVAC)
BASED FIV VACCINE COMBINED WITH INACTIVATED FIV-CELL
VACCINE AGAINST HETEROLOGOUS FIV CHALLENGE

Introduction............................. 94
M aterial and M ethods.............................................................. 97
R esu lts............................. ........................................... ... 98
D iscussion..................................................... 100

IV SUMMARY AND FUTURE STUDIES

Synopsis.......................................................104


R EFER EN CE S............ ... .................................................................107


BIOGRAPHICAL SKETCH ....................................................................121









V















LIST OF TABLES


Table page 1.1 The lentiviruses........................................... .......................... 3

1.2 Conventional inactivated whole virus FIV vaccine trials.......................... 19

1.3 Conventional inactivated FIV-infected cell vaccine trial s ............................. 20

1.4 Subunit and vector based FIV vaccine trials.................................. 21

1.5 Immunogenicity and prophylactic efficacy of ALVAC-based vaccines.......... 29

1.6 Immunogenicity and prophylactic efficacy of ALVAC-based vaccines
against retroviral pathogens....................... ............... 35

2.1 Grouping and immunization......................... ...................... 44

2.2 CD4/CD8 ratios after immunization and challenge ..................... .......... 63

2.3 Proliferation to T-cell mitogens (ConA and SEA) ................................. 64

2.4 FIV-specific antibody titers after immunization and challenge.................... 70

2.5 Viral neutralizing antibody titers after immunization and challenge.............. 71

2.6 T-helper responses to FIV after immunization and challenge..................... 73

2.7 FIV-specific CTL activity in peripheral blood after immunizations................. 74

2.8 NK activity postchallenge......................... ...... .................. 75

2.9 Virus isolation (RT and PCR) on PBMC and WB data pre- and
postchallenge........................................................................... ....77

2.10 Virus isolation on PBMC and tissue samples and WB and ELISA data ......... 79 2.11 Statistical analysis.................................................................... 84

3.1 Immune parameters and viral-status pre- and postchallenge ..................... 99






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LIST OF FIGURES


Figure page

1.1 Electron micrograph of FIV x70,000 ............................................................. 6

2.1 Schematic representation of ALVAC constructs ....... ................... ........... 40

2.2 PCR products of CrFK cells infected with ALVAC-env recombinants ............. 55

2.3 PCR products of CrFK cells infected with ALVAC-gag/prot recombinants....... 56 2.4 RT-PCR products of CrFK cells infected with ALVAC-FIV recombinants ........57

2.5 Immunofluoresence on CrFK cells infected with ALVAC-FIV recombinants..... 59 2.6 FACS analysis of CD4 and CD8 staining of PBMC............................... 62

2.7 Immunoblot of ALVAC-specific humoral responses..............................67

2.8 Immunoblot of FIV-specific humoral responses ....................................... 69

2.9 Immunoblot of FIV-specific humoral responses in kittens........................... 83

3.1 Phylogenetic relationship between FIV-isolates......................... 96



















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LIST OF ABBREVIATIONS



ADCC Antibody dependent cell-mediated cytotoxicity AIDS Acquired immunodeficiency syndrome BM Bone marrow BSA Bovine serum albumin CTL Cytotoxic T-lymphocyte DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetate ELISA Enzyme-linked immunosorbent assay FACS Fluorescence-activated cell sorting FITC Fluorescein isothiocyanate FIV Feline immunodeficiency virus HIV Human immunodeficiency virus LN Lymph node MHC Major histocompatibility complex PBL Peripheral blood lymphocytes PBMC Peripheral blood mononuclear cells PBS Phosphate-buffered saline PCR Polymerase chain reaction RNA Ribonucleic acid RT Reverse transcriptase SDS Sodium dodecyl sulfate SIV Simian immunodeficiency virus



viii










TAE Tris, EDTA buffer TEMED N,N,N',N'-tetramethylethylenediamine THY Thymus Tris Tris(hydroxymethyl)aminomethan VN viral neutralizing WB western blot









































ix














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

IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION OF
CANARYPOXVIRUS (ALVAC)-BASED FIV VACCINES IN CATS By

Maria C. Tellier

December, 1996

Chairman: Dr. J.K. Yamamoto
Major Department: Veterinary Medicine


The infection of cats with the feline immunodeficiency virus (FIV) provides a valuable animal model for the assessment of therapeutic and vaccine strategies against human immunodeficiency virus (HIV) in man. A promising candidate vaccine tested presently in human volunteers is the recombinant canarypoxvirus vector ALVAC. This vector has also been used with some success against HIV infection in the macaque and chimpanzee models. Herein, the efficacy of ALVAC-based vaccines was evaluated against experimental FIV infection in cats. Two approaches were evaluated which included ALVAC-based FIV vaccines alone or in combination with conventional inactivated FIVinfected cell vaccine (ICV).

Immunization schemes employing ALVAC-FIV recombinants alone effectively induced FIV-specific cytotoxic T-cell responses. However, these schemes failed to induce humoral responses including viral neutralizing antibody responses. Immunization schemes employing ALVAC-FIV recombinants combined with conventional inactivated FIVinfected cell vaccine induced FIV-specific cytotoxic T-cell responses and FIV-specific humoral responses but lacked detectable viral neutralizing antibody responses.


x









Cats immunized with the ALVAC recombinants encoding the FIV core (Gag) protein were protected from challenge exposure with 50 ID50 FIV Petaluma, a subtype A FIV isolate highly related to the Ville franche isolate basis of the ALVAC-FIV vaccine. In contrast, ALVAC recombinants expressing the FIV envelope alone or both the FIV envelope and Gag proteins failed to induce such protection. Cats immunized with ALVACFIV recombinants and boosted with ICV were also protected from a FIV Petaluma challenge exposure. In addition, these cats were partially protected from challenge with 75 ID50 FIV Bangston (subtype B), a distinctly heterologous isolate, given eight months after the initial challenge without any intervening booster.

In conclusion, vaccine protocols employing recombinant ALVAC-based FIV vaccines alone or in combination with conventional inactivated FIV-infected cell vaccine can prevent establishment of FIV infection in cats. This immunity may even protect or delay infection with FIV isolates of other subtypes than those used to generate the vaccine. It remains unclear as to what constituted protective immunity in the protected animals. The obtained data suggest a role for cell-mediated responses. However, a role for FIV specific humoral responses including viral neutralizing antibody responses can not be excluded.






















xi














CHAPTER I
LENTIVIRUSES AND VACCINE DEVELOPMENT Introduction



Lentiviruses comprise a group of viruses known to cause life-long chronic infections in a number of species (Table 1.1). The most prominent member of this group is the human immunodeficiency virus (HIV), the causative agent of an acquired immunodeficiency syndrome (AIDS) in man. HIV was first isolated from young male homosexual patients in 1983 (Barre-Sinousi et al. 1983; Gallo et al. 1984). These patients presented with a high incidence of a pneumonia caused by Pneumocystis carinii thus far only known to cause disease in immuno-compromised people and a rare cancer, Kaposi sarcoma (Ammann et al. 1983; Gyorkey et al. 1984; Selik et al. 1984; Weissler 1990). More important, it was noticed that the numbers of circulating lymphocytes, in particular those of the CD4' T-helper phenotype, were severely reduced in these patients, a phenomenon which is now considered one of the main hallmarks of HIV infection in man (Huet et al. 1990). Many years prior to the discovery of HIV, a number of viruses, much later classified as lentiviruses, had been described as pathogens in animals. This included the equine infectious anemia virus (EIAV) which was identified in 1904 as the causative agent of a disease in horses characterized by recurrent episodes of fever and hemolytic anemia (Vallee and Carre 1904). This also included the maedi-visna virus (MVV) which was first isolated from sheep in Iceland that presented with severe chronic pneumonia (maedi), wasting and paralysis (visna) (Gislason 1947; Narayan et al. 1977). Based on the long incubation period and the fact this virus could manifest its effects over a long period,





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MVV was named a lenti- (slow) virus (Sigurdsson 1954). This virus has since become the prototype of the lentivirus genus. In 1974, a virus similar to MVV was identified in goats, the caprine arthritis encephalitis virus (CAEV). CAEV infection presents as chronic inflammation of the joints in adult goats and progressive encephalopathy in younger goats (Clements et al. 1980; Cork 1974).

Thus, several animal lentiviruses had been long known before HIV. However, it was the discovery of HIV that resulted in an increased interest in these viruses and led to the search and isolation of lentiviruses in other species. For example, a number of lentiviruses were isolated from monkeys. These viruses have collectively been named the simian immunodeficiency viruses (SIV) and are specified by the particular monkey species they have been isolated from (Table 1.1) (Huet et al. 1990; Kanki et al. 1985; Ohta et al. 1988; Tjusimoto et al. 1988; Hirsch et al. 1993; Peeters et al. 1992; Fultz et al. 1986). In contrast to HIV infection of man, SIV infection of natural host African monkey species, is relatively nonpathogenic. However, the exception to this is SIV infection in Asian macaques (Daniel et al 1985; Benvisti et al. 1986). Infected Asian macaques develop an acquired immunodeficiency syndrome similar to that of HIV infected individuals (Murphey-Corb et al. 1986). Interestingly, most of the nonpathogenic SIV strains have been isolated from monkeys in the wild. SIV., however, has only been isolated from macaques held in captivity and has never been isolated from this species in its natural habitat. Since these monkeys are of Asian origin whereas other SIV infected monkeys are of African origin it has been suggested that this virus was transmitted to macaques during captivity from an African monkey species, most likely the mangebeys (Murphey-Corb et al. 1986).

The second human lentivirus, now known as HIV-2, was first isolated in 1986 (Clavel et al. 1986). HIV-2 is predominantly found in West-African prostitutes and also causes AIDS although milder in its pathogenesis as marked by a longer incubation period and a lower rate of transmission (Marlink et al. 1994). Much like SIV infection of Asian





3


macaques, the emergence of HIV in humans is thought to be caused by cross-species

transmission. This is supported by the fact that HIV-2 genetically closely resembles the

SIV.- and SIVm-isolates, and HIV-1 more closely resembles the SIVcz-isolate (Hirsch

and Johnson 1994; Franchini et al. 1987).


Table 1.1 The Lentiviruses



Virus subtypes species Clinical signs of disease


EIAV horse anaemia fever
weight loss

Maedi-visna sheep encephalomyelitis wasting
pneumonia

CAEV goats arthritis encephalomyelitis wasting

BIV cows lymphadenopathy lymphocytosis
wasting
FIV cats immunodeficiency opportunistic infections neurological disorders
HIV HIV-1 human immunodeficiency lymphadenopathy neurological syndrome opportunistic infections

HIV-2 human immunodeficiency lymphadenopathy opportunistic infections
SIV SIVm macaques immunodeficiency neurological disease SIVm sooty mangabey
SIV African green monkey
SIVmd mandrill no obvious clinical signs
SIVsyk Sykes monkey of disease
SIVz chimpanzee






4



In addition to the primate lentiviruses, a virus similar in morphology and genetic composition, the bovine immunodeficiency virus (BIV), was isolated from cows in 1985. Although, the pathogenesis of BIV is poorly defined, infection in calves has been associated with lymphocytosis and lymphadenopathy (Gonda et al. 1987, 1994).

The feline homologue of HIV, feline immunodeficiency virus (FIV) was first isolated in 1986 from a cattery in California (Pedersen et al. 1987). In this cattery, several cats presented with a loss in immune function after the introduction of a sentinel cat. The loss of immune function could not be linked to feline leukemia virus (FeLV), another member of the Retroviridae, already known to cause immunosupression in cats (Jarret et al. 1964). This led to the discovery of a novel retrovirus that differed from FeLV and more closely resembled HIV in morphology and the Mg2*- rather than Mn2*-dependence of its reverse transcriptase (Yamamoto et al. 1988a). Subsequent genetic analysis demonstrated that this virus belonged to the lentivirus family.

The seven known members of the lentivirus family as listed above are commonly divided into two groups based on differences in cell tropism and disease manifestation. Those affecting the ungulate species EIAV, MVV, and CAEV are predominantly macrophage-tropic and cause immune-mediated diseases that target specific organs. Those affecting primates, HIV and SIV, are tropic for lymphocytes and macrophages and cause a major loss of immune function that results in an increased susceptibility to opportunistic pathogens. FIV resembles the primate viruses in cell tropism and disease manifestation but is genetically more closely related to the nonprimate lentiviruses (EIAV and MVV) (Olmsted et al. 1989b).

Common to all lentiviruses is the long incubation period, the ability to affect multiple organs, and most importantly the persistence in the face of host-immune responses. The ability to escape from the host immunity is in part explained by a high mutation rate of the lentiviral genome resulting in continuous antigenic variation (Rigby et





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al. 1993). As such, lentiviruses present a major challenge to the development of therapeutic strategies and vaccines.


The Feline Immunodeficiency Virus



The FIV virion, like other members of the Retroviridae, is enveloped and approximately 100-125nm in diameter (Pedersen et al. 1987). A electron micrograph of a mature lentivirion is depicted in Figure 1.1. The FIV core, composed of the capsid protein (CA, p24), has the physical appearance of a cone, typical of all lentiviruses. The core encloses the viral genome which consists of two identical single stranded RNA molecules of approximately 9kb (Elder et al. 1993). Associated with the viral genome is a small nucleocapsid protein (NC, p8), which is thought to play a role in viral assembly and disassembly (Elder et al. 1993; Aldovini and Young 1990). Also packaged within the virion are the enzymes essential for replication, the reverse transcriptase (RT), the integrase

(IN) and the deoxyuridine triphosphatase (dUTPase; DU)(Elder et al. 1993). The RT is responsible for transcription of the viral RNA genome into DNA. The integrase functions as an endonuclease which cuts the cellular genome to allow integration of the provirus. The dUTPase is unique to the nonprimate viruses and its function in the FIV life cycle still remains unknown. However, it is speculated that the dUTPase is required for replication of FIV in nondividing macrophages or resting T-lymphocytes (Miyazawa et al. 1993). Additionally, it has been observed that lack of functional FIV dUTPase results in an increased mutation frequency of FIV propagated in macrophages (Lerner et al. 1995). Surrounding the viral core is a matrix protein layer (MA, p15) which is closely associated with the viral envelope. The viral envelope is derived from the cellular membrane by budding of the viral particles and consists of lipids, inserted viral proteins and cellular proteins (Gelderblom et al. 1987). The virally derived envelope glycoprotein (Env) is composed of two subunits; the transmembrane protein (TM, gp40) and the surface protein





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(SU, gp120). The TM protein protrudes through the viral membrane and noncovalently anchors the outer SU protein, which appears as a knob-like structure on the viral surface.































Figure 1.1 Electron micrograph of FIV x70,000.


FIV genomic organization and regulation of gene expression


Lentiviral genomes are among the smallest of the known viruses and are more complex in their genomic organization than other members of the retrovirus family. The FIV genome is a positive stranded polyadenylated RNA of approximately 9kb. It contains three major open reading frames (ORFs) encoding the structural and enzymatic proteins (Env,Gag and Pol) necessary for the viral life cycle (Olmsted et al. 1989a, 1989b;





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Miyazawa 1993). These genes are organized in the order of 5'-gag-pol-env-3', typical of all replication-competent retroviruses. In addition, lentiviruses contain several small open reading frames (ORFs) that encode for auxiliary proteins. At least 4 ORFs have been identified for FIV which may encode for regulatory proteins similar to those described for the primate lentiviruses (Miyazawa et al. 1993; Olmsted et al. 1989b). Flanking the proviral genome are long terminal repeat (LTR) regions which are crucial for integration of the proviral DNA into the cellular genome. These regions also contain enhancer and promoter elements which are required for efficient transcription of the retroviral genome, as well as, a polyadenylation signal sequence (Phillips et al. 1992; Talbott et al. 1989).

Transcription of the integrated proviral DNA genome initiates at the 5'LTR to generate full length viral mRNA transcripts (Miyazawa et al. 1993). Initially, these transcripts will undergo multiple splicing and give rise to small mRNAs that encode for regulatory proteins such as Tat and Rev. The Tat protein facilitates gene expression from the 5'LTR. Tat activity is essential for replication of all primate lentiviruses. In contrast, Tat is not essential for replication of FIV in T-lymphoblast cells (Sparger et al. 1992; Miyazawa et al. 1993). The Rev protein, encoded by all members of the lentiviridae, is responsible for shifting viral gene expression from early regulatory proteins to that of structural and enzymatic proteins. It accomplishes this by binding to the Rev-responsive element (RRE) in the env coding region of single spliced and unspliced full length mRNAs. In doing so, it promotes the stability and transport of incompletely spliced mRNAs from the nucleus to the cytoplasm (Phillips et al. 1992; Cochrane et al. 1990; Hammarskjold et al. 1989; Stephens et al. 1992). The single spliced messages give rise to the Env precursor protein which is cleaved into the transmembrane glycoprotein (TM) and the outer surface glycoprotein (SU) by cellular proteases (Stephens et al. 1991; Talbott et al. 1989).

The unspliced full length mRNA serves as both a template for the Gag and Gag-Pol proteins and as genome that is packaged into the viral core. Regular translation of full length message gives rise to the Gag precursor protein which is cleaved into the mature





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capsid (CA), matrix (MA) and nucleocore (NC) proteins by the viral protease encoded within the pol gene (Elder et al. 1993). Expression of the Gag-Pol precursor protein from the unspliced full length mRNA is accomplished by a ribosomal frameshift that prevents termination of translation at the gag stop codon (Morikawa and Bishop 1992). The efficiency of this shift is about 5%, so that the production of Gag protein is 20 fold higher than that of Gag-Pol polyprotein. The Gag-Pol precursor protein is proteolytically processed into the viral protease (PR), the reverse transcriptase (RT), the deoxyuridine triphosphatase (DU) and the integrase (IN) proteins (Elder et al. 1993).


FIV replication


The first step in the replication of FIV is the attachment of the virus to the cell receptor. For HIV, the major receptor has been identified as the CD4 molecule, present on T-helper lymphocytes (Dagleish et al. 1984; Klatzman et al. 1984). In addition, the CD26 molecule and the recently described fusin and CKR-5 molecules have been reported to play a role in HIV attachment and fusion (Callebaut et al. 1993; Feng et al. 1996; Alkhatib 1996). The fusin is thought to act as a coreceptor for T cell line-tropic HIV strains whereas the CKR-5 is thought to act as a coreceptor for macrophage-tropic HIV strains (Feng et al. 1996; Alkhatib 1996). The target receptor(s) for FIV, however, is still unknown. Nonlymphoid feline cell lines transfected with cDNA encoding the feline CD4 (fCD4) protein failed to support productive infection indicating that the fCD4 alone is not sufficient (Norimine et al. 1993). Others have proposed a putative role for a receptor homologous to the human CD9 molecule that is expressed on both haematopoietic and nonhaematopoietic cells (Willet et al. 1994; Hosie et al. 1993; Boucheix and Beiot 1988). Anti-CD9 antibodies effectively block replication of FIV infection on lymphoid cells and ectopic expression of CD9 on feline lymphoma cells causes an enhancement of viral infection with cell culture





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adapted FIV strains. However, ectopic expression of CD9 on these cells does not seem to render these cells more susceptible infection with primary FIV isolates (Willetet al. 1996).

Following attachment, the FIV enters the cell either by receptor mediated endocytosis or by fusion of the viral envelope with the cellular membrane. Upon entry the viral genome is released into the cellular cytoplasm. Subsequent, the viral RNA genome is transcribed into double-stranded proviral DNA by the viral reverse transcriptase packaged within the virion. Similar to HIV, initiation of first strand cDNA synthesis is primed by cellular tRNA'Y' (Olmsted et al. 1989b; Talbott et al. 1989). Integration of the provirus into the host cellular genome is facilitated by the viral Integrase and occurs at random sites. The integrated provirus can stay quiescent or give rise to progeny viral particles. During productive infection, the proviral DNA is transcribed into viral mRNAs by cellular RNA polymerase II. These mRNAs are translated into the Gag precursor (p55), the Gag-Pol precursor (p160) and the Env precursor proteins (gp160). The Gag precursor is further processed by proteolysis to give rise to the capsid (CA), matrix (MA), and nucleocapsid

(NC) proteins. Proteolysis is mediated by the viral protease which also facilitates its own cleavage (Elder et al. 1993). Similar to HIV, the FIV matrix protein is myristolated. Myristolation is the attachment of a C14 fatty acid and is required for proper targeting of the MA protein to the cellular membrane (Elder et al. 1993). The Env precursor protein is cleaved into the mature SU and TM proteins by cellular proteases and further processed by glycosylation. For FIV, 22 possible N-linked glycosylation sites have been identified; 18 in the SU and 4 in the TM protein (Stephens et al. 1991; Elder et al. 1993). The final step in the FIV replication cycle involves the assembly of the virions and their release from the cell by budding.





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FIV cell tropism



FIV has a broad cell tropism and infects cells of both lymphoid and monocyte/macrophage origins. In contrast to HIV, which is thought to primarily replicate in CD4' T-lymphocytes and not in CD8' T-lymphocytes, FIV productively infects both CD4' and CD8 T-lymphocytes (Brown et al. 1991). Additionally, FIV has been shown to replicate in B-cells, thus further supporting the view that the feline CD4 receptor is not the primary cell receptor for FIV, as it is for HIV (English et al. 1993; Norimine et al. 1993). The macrophage/monocyte cell types supporting FIV replication include peritoneal macrophages, Kupffer cells in the liver, microglial cells, astrocytes, and endothelial cells in the central nervous system (Steffan et al. 1994; Dow et al. 1990; Martin et al. 1995; Brunner and Pederson 1989). Furthermore, a number of FIV isolates have been shown to infect cells of nonlymphoid origin including Crandell feline kidney cells (CrFK) and feline tongue cells (Fc3Tg) (Yamamoto et al. 1988a).


FIV epidemiology and pathogenesis



FIV has been isolated from cats worldwide. The virus infects domestic cats (Felis catus) and is species specific (Yamamoto et al. 1988b, 1989). FIV-related viruses have been isolated from several wild felids including the African lion (Panthera leo), and the Pallas cat (Felis manul) (Barr et al. 1989, 1995; Poli et al. 1995; Brown et al. 1994). Furthermore, serologic surveys in African and Asian lions revealed that the serum of the majority of these animals reacted positively with FIV (Brown et al. 1994).

The prevalence of FIV varies throughout the world. In North America the average incidence is estimated at 1.4% in healthy animals and 7.4% in diseased cats (Yamamoto et al. 1989; Shelton et al. 1990). The incidence of infections is the highest in free roaming, outdoor male cats. Since FIV is shed in the saliva, the major route of transmission is most





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likely due to biting between male cats as part of territorial behavior (Yamamoto et al. 1989). In addition to saliva, FIV can be recovered from blood, serum, plasma, and cerebrospinal fluid of infected cats (Pedersen et al. 1987; Dow et al. 1990). Horizontal transmission through contact alone appears to be inefficient (Pedersen et al. 1987; Yamamoto et al. 1988b). Vertical transmission in utero or postpartum via the milk has been reported and was found to occur most frequently in queens that became viremic during pregnancy (Callanan et al. 1991; Wasmoen et al. 1992; O'Neil et al. 1996). Although high rate of perinatal transmission has been reported for queens that had been infected with a highly pathogenic FIV strain 4 to 30 months prior to conception (Ueland and Nesse 1992; O'Neil et al. 1996).

Similar to HIV, five clinical stages can be defined for FIV infection in cats. The acute viremic phase, 2 to 4 weeks after infection, is characterized by fever, neutropenia, and generalized lymphadenopathy. These symptoms vary in duration and severity between individual cats and are mostly recognized in experimentally infected cats but rarely in naturally infected cats (Yamamoto et al. 1988b, 1989). Once a full immune response is established and most of the virus is cleared from the plasma, a period of months to years follows with no obvious clinical signs of disease, defined as the asymptomatic phase. However, during this period changes in lymphocyte counts, such as a decrease in CD4' lymphocytes and CD4/CD8 ratios, takes place (Ackley et al. 1990). This period is followed by a phase equivalent to that of AIDS related complex (ARC) in men. Cats with ARC often present with chronic illness such as stomatitis/gingivitis, lower urinary tract infections, skin disorders and diarrhea (Yamamotoet al. 1989). Finally, cats may develop a stage similar to that of AIDS in men, characterized by severe lymphoid depletion, weight loss and opportunistic infections. Opportunistic pathogens reported in cats suffering from AIDS include toxoplasmosis, cryptococcoses, candidiasis, mycobacteriosis, feline caliciand herpes virus (Lapin et al. 1989; Knowles et al. 1989; Ishida and Tomoda 1989). At this stage, CD4' T-cell counts have dropped dramatically and CD4:CD8 ratios are inversed





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(Ackley et al. 1990). Other immunologic abnormalities reported include hypergammaglobulinemia and reduced T-cell responses to T-independent antigens (Ackley et al. 1990; Torten et al. 1991). The occurrence of neurological abnormalities as seen in AIDS patients has only been reported in a small percentage of cats infected with neurotropic isolates of FIV (Podell et al. 1993; Dow et al. 1990).



FIV as an animal model for HIV



With the emergence of the HIV pandemic and an estimated 12 million infected people, animal models to study antiviral drugs and vaccine strategies have become very important. The search for an appropriate animal model for HIV-1 infection in man has, however, been complicated by the host specificity of HIV-1.

To date, HIV-1 has only been shown to infect three other species; pig-tailed macaques, gibbons, and chimpanzees (Agey et al. 1992; Fultz et al. 1986). These animals become viremic and mount specific antibody responses similar to HIV infected humans. However, infection does not result in depletion of CD4' T-lymphocytes, immunosuppression, or opportunistic infections. Furthermore, chimpanzees are an endangered species, therefore limiting the availability of these animals for research purposes. Consequently, the infection of macaques with SIV has become the most prevalent animal model used. Both the SIV,- and SIV.- isolates have been shown to cause an AIDS-like disease in rhesus-, cynomolgus-, and stumptail macaques (MurpheyCorb et al. 1986). Initial infection results in viremia and is characterized by fever, diarrhea and lymphadenopathy. Like HIV-1 in humans, monkeys in the end-stages of disease present with opportunistic infections and show decreased CD4* T-lymphocyte counts and inversed CD4:CD8 ratios. Additional promising models include the infection of baboons and pig-tailed macaques with specific isolates of HIV-2 (Barnett et al. 1994; Novembre et al. 1994). A large percentage of these monkeys become persistently infected and show





13


depletion of CD4' T-lymphocytes and susceptibility to opportunistic pathogens. In addition, several alternative models have been developed with some success. For example, transient HIV infection of SCID mice reconstituted with human lymphocytes or peripheral blood mononuclear cells (PBMC) and the infection of rabbits with HIV-1 (Reina et al. 1993; Mosier et al. 1991; Namikawa et al. 1988). These models, however, are highly artificial and the relevance to HIV pathogenesis in humans should be interpreted with caution.

The infection of FIV in domestic cats offers several advantages over the models discussed above. First, FIV is a natural pathogen of cats and the pathogenesis closely resembles that of HIV in man. Obvious advantages of the feline model include the availability and costs which allow the use of larger study groups. Especially relevant to vaccine development is the availability of a wide variety of FIV subtypes which is lacking in the SIV model. FIV isolates have been grouped into 4 subtypes (A-D) versus 7 for HIV (Sodora et al. 1994; Kakinuma et al. 1995; Rigby et al. 1993). This grouping is primarily based on antigenic diversity in the Env and Gag proteins. Hence, the FIV model provides a means to evaluate the protective efficacy of vaccine strategies against multiple subtypes and as such has implications to the development of multiple subtype HIV vaccines. Further, FIV like HIV replication is sensitive to antiviral drugs such as AZT and protease inhibitors (North et al. 1989, 1990). Thus, FIV infection in cats also provides a model to assess the efficacy of these and other newly developed drugs.



Vaccine development


Vaccine development initiated with the work of Dr. Edward Jenner in 1798. He observed that milkmaids which had recovered from cowpox did not contract the more virulent smallpox. Based on this observation he postulated that smallpox infection of man could be prevented by prior exposure to cowpox. He successfully proved this hypothesis





14


by demonstrating that a boy, injected with material from a cowpox pustula, failed to develop disease upon exposure to smallpox. This technique became known as vaccination (Jenner 1798).

Though the underlying mechanisms were not known at the time, it is now understood that success of vaccination lies in the ability of the immune system to generate long lasting immunity. This immunity is mediated by memory B and T lymphocytes which are capable of rapid anamnestic responses upon exposure to foreign antigens. Responses mediated by B cells include the production of specific antibodies that could prevent the entry of pathogens into host cells by interfering with microbial attachment or fusion upon attachment to host cells. These antibodies, in respect to viral pathogens, have been defined as viral neutralizing (VN) antibodies. Further, antibodies may directly destroy microbes by complement mediated lysis or promote phagocytosis by macrophages and natural killer cells through opsonization. T-lymphocyte responses include those mediated by T-helper cells and T-cytotoxic lymphocytes (CTLs). T-helper cells are of the CD4' phenotype and recognize exogenously produced antigens presented in the context of MHC-class II found predominantly on B-lymphocytes and macrophages. Upon recognition these cells produce interleukins to facilitate the activation of macrophages and maturation of B cells into antibody producing plasma cells. Cytotoxic T-lymphocytes of the CD8' phenotype function by the direct destruction of infected cells displaying foreign antigens in association with MHC-class I molecules. Class I MHC molecules are found on the majority of cells and present endogenously synthesized antigens. As such CTL responses are especially critical to the clearance of intracellular bacteria and virally infected cells. Together, humoral and cell mediated responses are capable of preventing the invasion of pathogens.

Several different types of vaccines have been developed. Most commonly used vaccines against viral pathogens are composed of live attenuated viruses, inactivated whole virus, or inactivated virus infected cells. The majority of these vaccines induce VN antibody responses and some, in particular attenuated live viruses, also induce cell-





15


mediated responses including CTL reponses. More recent developments in the vaccine field involve the use of viruses, bacteria or naked DNA as vaccine vectors. These vectors are genetically engineered to carry and express foreign genes encoding immunogens of pathogens. Upon inoculation of these vectors into the host, the inserted immunogen is expressed and presented to the host immune system. In fact, viral antigens encoded within these vectors are presented to the host immune system in a manner simulating natural infection. Furthermore, vector based vaccines are thought to be more effective in eliciting CTL responses than conventional inactivated vaccines. As such, vaccine strategies employing vector based vaccines may be especially useful against viral and intracellular bacterial pathogens.

To date, success of viral vaccines has been limited to a confined group of viruses. These viruses display constant antigenic specificity and consist of a single or limited number of serotypes. Furthermore, spontaneous recovery has usually been observed shortly after natural infection with these viruses. Lentiviruses, including FIV, do not fall into this category. Lentiviruses are subject to continuous antigenic variation and consists of many serotypes. For FIV a total of 4 subtypes have been defined based on genetic differences in the env and gag coding regions (Sodora et al. 1994; Kakinuma et al. 1995; Rigby et al. 1993). Spontaneous recovery upon infection with lentiviruses has not been reported. Moreover, these viruses integrate into the host cellular genome and can stay latent without the expression of viral proteins. Latently infected cells serve as a reservoir and fail to be recognized by the immune system, allowing the virus to persist. Thus, the development of effective vaccines against lentiviruses faces additional challenges.


FIV vaccine development


An optimal FIV vaccine should induce long-lasting protective immunity. This immunity should be effective against a wide range of FIV strains within as well as across





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subtypes (A-D). In addition, this vaccine should induce protective immunity against cellfree and cell-associated virus and against various routes of infection. Since FIV is predominantly transmitted through biting, protection should be directed in particular against this route of exposure.

In order to properly evaluate the immunogenicity and protective efficacy of FIV vaccines, several factors should be taken into consideration. One factor is the method used to assess the induction of VN antibody responses as these are often used as a parameter for the effectiveness of viral vaccines. The ability of antibodies to neutralize lentiviral infection in vitro may however differ with the specificity of the target cell line used in the assay. For example, it has been found that vaccine induced antibodies capable of neutralizing FIV infection on a feline Crandell kidney cell line (CrFK) failed to neutralize FIV infection on feline thymocytes (Siebelink et al. 1993).

Likewise, the assessment of protective efficacy is influenced by several factors. As for viruses in general, these include the route of infection and the dose of challenge inoculum. In most FIV vaccine trials, the challenge inoculum is given either intravenously or intraperitoneally. Experimental infection through these routes is obtained more readily than through mucosal exposure, e.g. requires less viral particles. Furthermore, the origin of the vaccine virus and challenge virus inoculum play an important role in the outcome of vaccine efficacy. Lentiviruses are enveloped and incorporate cellular antigens into the viral envelope when budding from the host cell. These host derived proteins present in the lentiviral envelope may play a role in protective efficacy of inactivated virus and infected cell vaccines, in particular. For example, monkeys immunized with uninfected human cells were shown to be protected from challenge with SIV grown on the identical human cell line. Protected monkeys had significantly higher levels of antibodies directed to host-cell major histocompatibility complex (MHC) antigens than monkeys that were not protected. In fact, protection against SIV has been obtained with vaccines composed of MHC molecules solely (Chan et al. 1992; Stott 1991; Langlois et al. 1992). Likewise, protection





17



against feline leukemia virus (FeLV), another feline retrovirus, has been afforded in the presence of antibody responses directed against cat-cell antigens (Lee et al. 1982). Thus, the cellular origin of the vaccine virus and the challenge virus should be taken into consideration when evaluating protective efficacy.

In the majority of FIV vaccine trials, the challenge inoculum virus is produced from FIV strains adapted to cell cultures in the laboratory. In vitro cell culturing, however, can lead to changes in virulence, cell tropism, and sensitivity to VN antibodies. To overcome this, FIV isolated freshly from infected cats have been used. A relatively new approach is the use of molecularly cloned FIV. The viral genome is incorporated into a plasmid and as such used to infect animals.



FIV vaccine trials



Early FIV vaccine trials utilizing inactivated whole virus failed to demonstrate protective immunity (Table 1.2 and Table 1.3). These trials included the immunization of specific pathogen free (SPF) cats with inactivated FIV UK8 purified from T cells and incorporated into immune stimulating complexes (ISCOMs) (Morein et al. 1984; Hosie 1994). Similarly, an inactivated virus vaccine produced from Crandell feline kidney cells (CrFK) infected with the FIV UT 113 isolate and adjuvanted with aluminum hydroxide-oil, failed to protect cats against low dose homologous challenge (Hosie 1994) (Table 1.2).

Initial trials involving inactivated FIV-infected cell vaccines were also unsuccessful. Cats immunized with inactivated FIV UK8 infected T-cells or helper T-cells (Q201) became readily infected upon a homologous challenge with 20 ID50 (Hosie 1994, Hosie et al. 1992) (Table 1.3). Comparable results were obtained with a vaccine consisting of inactivated CrFK cells infected with the FIV UT113 isolate (Verschoor et al. 1995). In contrast, partial protection (3 out of 5) was observed in cats immunized with a cell vaccine consisting of FIVt1,3-infected thymocytes. However, one of three control cats immunized with





18


uninfected thymocytes alone also remained virus-negative (Verschoor et al. 1994). Thus, immune responses against cellular antigens and not viral antigens may have been responsible for the observed protection in this study.

Other unsuccessful FIV vaccine trials include those based on FIV subunit proteins and synthetic peptides corresponding to FIV epitopes (Table 1.4). Vaccines composed of nonglycosylated FIV Env produced in Escherichiacoli and glycosylated Env produced in a baculovirus system failed to protect cats against low dose challenge (Lutz et al. 1995). Similar results were obtained with vaccines consisting of bacterial produced Env fragments fused to galactokinase or glutathione-S-transferase (Verschoor et al. 1996). Also unsuccessful were vaccines composed of synthetic peptides corresponding to the V3 region of the FIV surface envelope protein (SU) (Lombardi et al. 1994). This region resembles the V3 loop of the HIV-1 Env surface protein which is thought to contain the principal neutralizing determinant (PND) of HIV (Pancino et al. 1994). Following immunization, all cats developed V3-specific antibodies however no protection against low dose challenge was observed (Lombardi et al. 1994). Interestingly, immunized animals showed enhancement of infection compared to controls as indicated by a higher virus load in the peripheral blood. Enhancement of infection as a result of immunization was also observed in cats immunized with a FIV envelope produced by recombinant vaccinia virus (Siebelink et al. 1995). In addition, subunit vaccines consisting of recombinant Gag protein p24 or native purified p24, lacked prophylactic efficacy despite the presence of high anti-p24 antibody titers (Hosie et al. 1992).

FIV vaccine trials using recombinant vector based vaccines have also been unsuccessful (Table 1.4). This included a trial in which the efficacy of a replicative defective adenovirus engineered to express the env gene of FIV was evaluated. After immunization, Env-specific antibody responses could not be detected and all cats became infected upon challenge (Gonin et al. 1995). Likewise, vaccine protocols involving











Table 1.2 Conventional inactivated whole virus FIV vaccine trials


Challenge Inoculum
Type of Immunization Cellular Origin Vaccine Virusa Vaccine Dose Vaccination Type of Cellular Origin& Dose (ID50) Protection (Vaccination Route) (FIV subtype) (mg) Protocol(wks) Adjuvantb Route & Strain rate

(Hosie et al. 1992)
Whole-virus feline T-cell(s.c.) UK-8 (A) 10c 0,5,18 iscom PBMC (i.p.) 20UK-8 0/5 (0%) Unvaccinated (-) (-) (-) (-) (-) PBMC (i.p.) 20UK-8 1/4 (25%)
(V ersc hoor,unpublished)
Whole virus CrFK(s.c.) UT113(A) 100 0,6 AluOH-oil Thymocytes(s.c.) 10UT113 0/5 (0%)
(Yamamoto et al. 1991b)
Whole-virus FL-4 (s.c.) PET (A) 200 0,2,4,6 CFA/IFA FeT1 (i.p.) IOPET 3/3(100%) Whole-virus FL-4 /Fetd(s.c.) PET (A) 200/107 0,2,4,6 CFAIIFA FeT1 (i.p.) O1PET 2/3 (67%) Adjuvant alone (-) (-) (-) 0,2,4,6 CFA/IFA FeTI (i.p.) 10PET 0/3 (0%)
(Yamamoto et al.1993)
Whole-virus FL-4 (s.c.) PET (A) 250 0,2,5 A-MDP FeT1 (i.p.) 10PET 13/15 (87%) Adjuvant alone (-) (-) (-) 0,2,5 A-MDP FeT1 (i.p.) 10PET 0/10 (0%) Whole-virus(boost) FL-4 (s.c.) PET (A) 250 38e A-MDP FeT1 (i.p.) 10DIX(A) 13/13(100%) Adjuvant alone (-) (-) (-) 38e A-MDP FeTI (i.p.) 10DIX 0/5 (0%)
(Hosie et al. 1995)
Pelleted-virus FL-4 (s.c.) PET (A) 250 0,2,4,7,10,17 T-MDP FeT1 (i.p.) 10PET 5/6 (83%) Control (-) (-) (-) (-) (-) FeTI (i.p.) 10PET 0/6 (0%) Pelleted-virus FL-4 (s.c.) PET (A) 250 0,2,4,7,10,17 T-MDP Q201(i.p.) 5UK-8 0/5 (0%) Control (-) (-) (-) (-) (-) Q201(i.p.) 5UK-8 0/5 (0%) Gradient-purified virus FL-4 (s.c.) PET (A) 250 0,3,6 T-MDP FeTI (i.p.) 10PET 5/5 (100%) Control (-) (-) (-) (-) (-) FeT1 (i.p.) 10PET 0/5 (0%) Gradient-purified virus FL-4 (s.c.) PET (A) 250 0,3,6 T-MDP Q201 (i.p.) IOPET 4/5 (80%) Adjuvant alone (-) (-) (-) 0,3,6 T-MDP Q201(i.p.) O1PET 0/5 (0%) Gradient-purified virus FL-4 (s.c.) PET (A) 250 0,3,6 T-MDP Q201 (i.p.) 10UK-8 1/5 (20%) Adjuvant alone (-) (-) (-) (-) T-MDP Q201 (i.p.) 10UK-8 0/5 (0%)
(Johnson et al. 1995)
Whole-virus FL-4 (s.c.) PET (A) 250 0,2,4,6,8 T-MDP FeTI (nasal) 10PET 3/5 (60%) Adjuvant alone (-) (-) (-) 0,2,4,6,8 T-MDP FeT1 (nasal) 10PET 0/5 (0%) Whole-virus FL-4 (s.c.) PET (A) 250 0,2,4,6 T-MDP FeTI (i.p.) 10SHI (D) 0/2 (0%) Adjuvant alone (-) (-) (-) 0,2,4,6 T-MDP FeTI (i.p.) 10SHI 0/7 (0%)
a UK-8, United Kingdom-8; PET, Petaluma; DIX, Dixon; SHI, Shizuoka.
b isom, immune stimulating complex;; A-MDP, adenyl-muramyldipeptide; CFA, complete Freund's adjuvant; IFA, incomplete Freund's; T-MDP, threonyl-muramyldipeptide. c Vaccine dose was 10ug of P17 and P24
d Vaccine consisted of 200mg /dose of inactivated whole-virus mixed with 1x107 cells/dose of uninfected FeT- 1 cells e Vaccinated cats protected from FIVpet challenge were boosted 38 wks after the first immunization and challenged 3 weeks after the boost with FIVdix strain.











Table 1.3 Conventional inactivated FIV-infected cell vaccine trials

Challenge Inoculum
Type of Immunization Cellular Origin Vaccine Virus' Vaccine Dose Vaccination Type of Cellular Origin& Dose (IDs0) Protection (Vaccination Route) (FIV subtype) (mg/PFU) Protocol(wks) Adjuvantb Route & Strain rate

(Hosie et al. 1992)
Infected cell feline T-cell(s.c.) UK-8 (A) 2x10' 0,3,6,9,12,15 QuilA PBMC (i.p.) 20 UK-8 0/5 (0%) Uninfected cell (-)(s.c.) (-) 2x10' 0,3,6,9,12,15 QuilA PBMC (i.p.) 20 UK-8 1/5 (20%)

(Hosie,unpublished)
Infected cell Q201(s.c.) UK-8 (A) 1x107 0,3,6 QuilA Q201 (i.p.) 20 UK-8 0/4 (0%) Adjuvant alone (-)(s.c.) (-) (-) 0,3,6 QuilA Q201 (i.p.) 20 UK-8 0/4 (0%) Control (-)(s.c.) (-) (-) 0,3,6 (-) Q201 (i.p.) 20 UK-8 1/4 (25%)

(Verschoor et al. 1995)
Infected cell CrFK(i.m.) UT-113 (A) 2.5x107 0,3,6 alu/MDP PBMC (i.p.) 10UT-113 0/5(0%) Uninfected cell CrFK(i.m.) (-) 2.5x107 0,3,6 alu/MDP PBMC (i.p.) 10 UT-113 0/3 (0%) Infected cell Thymocytes(i.m.) UT-113 (A) 1.5x107 0,3,6 alulMDP PBMC (i.p.) 10 UT-113 2/5(40%) Uninfected cell Thymocytes(i.m.) (-) 1.5x107 0,3,6 alu/MDP PBMC (i.p.) 10 UT-113 1/3 (33%) Control (-) (-) (-) (-) (-) PBMC (i.p.) 10 UT-113 0/2(0%)

(Yamamoto et al. 1991b)
Infected cell Fetl(s.c.) PET (A) lxl10 0,2,4,6,8,16 T-MDP PBMC (i.p.) 10 PET 4/5 (80%) Infected cell FL4(s.c.) PET (A) 1x107 0,2,4,6,8,16 T-MDP PBMC (i.p.) 10 PET 2/4 (50%) Uninfected FeTI (-)(s.c.) 1x107 0,2,4,6,8,16 T-MDP PBMC (i.p.) 10 PET 0/5 (0%) Adjuvant alone (-)(s.c.) (-) (-) 0,2,4,6,8,16 T-MDP PBMC (i.p.) 10 PET 0/5 (0%)

(Yamamoto et al. 1993)
Infected cell FL4 (s.c.) PET (A) 2.5x107 0,2,5 A-MDP Fetl (i.p.) 10 PET 15/15(100%) Adjuvant alone (-)(s.c.) (-) (-) 0,2,5 A-MDP Fetl (i.p.) 10 PET 0/10 (0%) Uninfected Fetl FL4 (s.c.) PET (A) 2.5x107 38 A-MDP Fetl (i.p.) 10 DIX 14/15 (93%) Adjuvant alone (-)(s.c.) (-) (-) 38 A-MDP Fetl (i.p.) 10 DIX 0/5 (0%)

(Matteucci et al. 1995)
Infected cell MBM(s.c.) M-2 (B) 3x107 0,3,6,9,12,15 IFA plasma (i.p.) 10 M-2 5/6 (83%) Uninfected cell MBM(s.c.) (-) 3x107 0,3,6,9,12,15 IFA plasma (i.p.) 10 M-2 0/3 (0%) Control (-)(s.c.) (-) (-) 0,3,6,9,12,15 (-) plasma (i.p.) 10 M-2 0/6 (0%)

(Johnson et al. 1994)
Infected cell Fl-4(s.c.) PET (A) 2.5x107 0,2,4,6,8 A-MDP Fetl (nasal) 10 PET 2/5 (40%) Infected cell Fl-4(s.c.) PET (A) 2.5x107 0,2,4,6 A-MDP Fetl (i.p.) 20 SHI 1/8 (12%)

a UK-8, United Kingdom 8; UT113, Utrecht 113; PET, Petaluma; M-2, Milan-2; SHI, Shizuoka. b T-MDP, threonyl-muramyldipeptide; A-MDP, adenyl-muramyldipeptide; IFA, incomplete Freund's adjuvant; QuilA, saponin.











Table 1.4 Subunit and vector-based FIV vaccine trials

Challenge Inoculum
Type of Immunization Cellular Origin Vaccine Virus' Vaccine Dose Vaccination Type of Cellular Origin& Dose (ID50) Protection (Vaccination Route) (FIV subtype) (mg/PFU) Protocol(wks) Adjuvantd Route & Strain rate

(Hosie, et al. 1992)
p24 E. coli(s.c.) UK-8 (A) 50 0,3,5,7 iscom PBMC (i.p.) 20 UK-8 0/4 (0%)

(Lutz et al. 1995)
gpl00(denatured) Insect cell (i.m.)' Z2 (A) 100 0,2,4,8 AIOH/QS21 PBMC (i.p.) 20 Z2 0/5 (25%) gpl00(native) Insect cell (i.m.) Z2 (A) 100 0,2,4,8 AIOH/QS21 PBMC (i.p.) 20 Z2 1/5 (20%) gpl00(highly purified) Insect cell (i.m.) BANG (B) 100 0,2,4,8 AIOH/QS21 PBMC (i.p.) 20 Z2 1/5 (20%) gpl00(native ) Insect cell (i.m.) BANG (B) 100 0,2,4,8 AIOH/QS21 PBMC (i.p.) 20 Z2 0/5 (0%) gpl00(denatured) E..Coli (i.m.) BANG (B) 100 0,2,4,8 AIOH/QS21 PBMC (i.p.) 20 Z2 1/5 (25%) Control(Ovalbumin) (-) (-) 100 0,2,4,8 (-) PBMC (i.p) 20 Z2 2/7 (29%)

(Osterhaus et al. 1996)
Env(cleavage) NA(s.c.) AM19 (A) 100 0,4,10 iscom PBMC (i.m) 20AM19 0/6 (0%) Env(no cleavage); NA(s.c.) AM19 (A) 100 0,4,10 iscom PBMC (i.m.) 20AM19 0/6 (0%) Env(no cleavage)' NA(s.c.) AM19 (A) 100 0,4,10 QuilA PBMC (i.m.) 20AM19 0/6 (0%) Env-b Gal NA(s.c.) AM19 (A) 100 0,4,10 QuilA PBMC (i.m.) 20AM19 0/6 (0%) Control(PBS) (-)(s.c.) (-) (-) 0,4,10 (-) PBMC (i.m.) 20AM19 0/6 (0%)

(Flynn et al. 1995)
V3-peptide synthetic (s.c.) UK-8 (A) 100 0,3,6 QuilA/AlOH PBMC (i.p.) 10 UK-8 0/15(0%) Adjuvant alone (-) (s.c.) (-) 100 0,3,6 QuilA/AlOH PBMC (i.p.) 10 UK-8 0/5 (0%) (Lombardi, 1994)
V3-peptide synthetic(s.c.) PET(A) 500 0,2,4,6,8 CFA F14(i.v.) 20-PET 0/3 (0%) Control (-) (-) (-) (-) (-) F14(i.v.) 20-PET 0/3 (0%)

(Verschoor et al. 1996)
V3-fusion protein IP E..Coli(s.c.) UT113 (A) 100 0,4,6,8,10 AIOH NA(s.c.) 10-20UT113 0/5(0%) V3-fusion protein I E..Coli(s.c.) UT113 100 0,6,10 QuilA NA(s.c.) 10-20UTl13 0/5 (0%) Control(PBS) (-)(s.c.) (-) (-) 0,6,10 (-) NA(s.c.) 10-20UT113 0/5 (0%) Feline Herpes-Env (-)oronasal/s.c. UT113 10SPFU 0 (-)
Boost V3-peptide E. Coli (i.m.) UT113 100 4,8 AIOH NA(s.c.) 10-20UT 113 0/5(0%) Feline Herpes-Env (-)oronasal/s.c. UT113 10'PFU 0 (-)
Boost V3 peptide E.Coli (i.m.) UT113 100 4,8 QuilA NA(s.c.) 10-20UT113 0/5 (0%) Feline Herpes-b Gal. (-)oronasal/s.c. (-) 10sPFU 0 (-) Boost PBS (-)(s.c.) (-) (-) 4,8 (-) NA(s.c.) 10-20UT113 0/5(0%)

(Gonin et al. 1995)
Adenovirus-env (-)(i.m.) Wo (A) 11.8-9.2PFU 0,4,30 ISA206 NA(NA) 20 Wo 0/4 (0%) Adeno-psuedorabies(control) (-)(i.m.) (-) 11.8-9.2PFU 0,4,30 ISA708 NA(NA) 20 Wo 0/4 (0%)

a UK-8, United Kingdom; Z2, Zurich; BANG, Bangston; AM19, Amsterdam 19; PET, Petaluma.
b Recombinant vaccinia virus expressed
c Baculovirus expression
d iscom, immune stimulating complex; AIOH/QS21, aluminumhydroxide and non-toxic fraction from Quillaja saponaria; ISA206, water/oil adjuvant;ISA708, water/oil adjuvant. e V3-fusion protein was composed of the FIV V3 region fused to galactokinase.
f Deletion of the cleavage site between the envelope surface(SU) and transmembrane protein(TM).






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priming with a feline herpes virus engineered to express the FIVenv gene followed by booster immunizations with bacterial Env-fusion proteins failed to induce protection against low dose challenge (Verschoor et al. 1996).

Successful FIV vaccine protocols include the use of inactivated cells infected with the FIV Petaluma isolate (FIVe,,; subtype A) or inactivated cell free FIV,,, (Yamamoto et al. 1991b, 1993). These vaccines were produced from either feline lymphoid cells productively infected with FIVp,, (FL-4) or an IL-2 dependent feline lymphoid cell line (FeT) infected with FIVpet (Yamamoto et al. 1991a). Using these vaccines, a protection rate of 70%-90% has been observed against low dose experimental challenge with homologous FIV,, and slightly heterologous FIV Dixon (FIVDix; subtype A) (less than 9% divergence in the env coding region) (Yamamoto et al. 1991b, 1993; Hosie et al. 1995). Further, these vaccines afforded protection against FIV challenge inoculum virus propagated on different cell lines including FeT 1, FL-4, and allogeneic PBMC. Protection was achieved against intraperitoneal challenge and oral-nasal challenge in a small number of animals tested (Yamamotoet al. 1991b, 1993; Johnson et al. 1994). These same vaccines, however, failed to induce protection against a high challenge dose of 5x104 IDs0 with the homologous FIVp,, isolate. Furthermore, these vaccines failed to induce protection against experimental challenge with a moderate heterologous FIV UK8 isolate (subtype-A) and a distinctly heterologous FIV Shizouka (FIVShi; subtype D) isolate. The Env amino acid sequences of these isolates differ from the FIVPet Env sequence by 11% and 21%, respectively. In addition, immunization schemes employing a similar vaccine produced from MBM lymphoid cells infected with the Italian isolate FIV M2 have been shown to induce protective immunity against a homologous plasma derived virus inoculum (Mattuecci et al. 1996).

In summary, conventional inactivated vaccines are capable of inducing protective immunity against low dose homologous FIV challenge and slightly heterologous FIV challenge. Similar vaccine approaches have also been successful in other animal models





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such as SIV in macaques and HIV in chimpanzees (Desrosiers et al. 1989; Fultz et al. 1992; Murphey-Corb et al. 1989). FIV subunit vaccines consisting of either Env or Gag proteins as well as recombinant vector based vaccines expressing Env did not elicit protective immunity against low dose homologous challenge. In contrast, protective immunity has been obtained against homologous SIV challenge in macaques immunized with Env subunit vaccines and against homologous and heterologous HIV challenge in chimpanzees immunized with Env subunit vaccines (Hu et al. 1992; Girard et al. 1995).



Mechanisms of protection



Protection obtained with conventional inactivated whole virus and infected cell vaccines produced with the FIV,, isolate, correlated most with high levels of envelopespecific and VN antibodies (Yamamotoet al. 1991b, 1993). Protection obtained with these vaccines did not correlate with anti-MHC antibodies as could be concluded from the lack of protection in cats that were immunized with uninfected cells alone (Yamamotoet al. 1991b, 1993) (Table 1.3). Similar vaccines produced with FIV isolates grown in feline T cells, thymocytes and CrFK cells failed to induce protective immunity (Verschoor et al. 1995; Hosie et al. 1992; Hosie 1994) (Table 1.3). However, these vaccines elicited lower levels of envelope-specific antibodies and VN antibodies. In fact, only the FIV vaccines produced from infected feline T-cell lines FL-4 and FeT1 cells, induced VN antibody titers similar to those observed in infected cats (Tozzinni et al. 1992). This could be due to the larger quantities of envelope protein produced by FL-4 and FeT 1 cell lines and the fact that the envelope protein present on FL-4 and FeT1 cells is well preserved following purification. Additional contributing factors may have been the choice of inactivating agent. Inactivation of successful vaccines was accomplished by paraformaldehyde which is thought to be more effective in maintaining antigenicity of immunogens when compared to other inactivating agents i.e. 1-propriolactone (Allison and Byars 1991; Warren et al.





24


1986). Furthermore, the type of adjuvant may have affected the protective efficacy, as it influences the proportion and intensity of humoral vs. cell-mediated responses upon immunization (Byars and Allison 1987).

Additional support for a role of Env-specific and VN antibody responses in the protection obtained with the FIVPt FL-4/FeT1 vaccines comes from passive immunization studies. In these studies, cats were passively immunized with pooled sera from cats immunized with inactivated FIV, infected T-cells (FL-4) or sera from cats experimentally infected with FIVp,, (Hohdatsu et al. 1993). Control cats received either phosphate buffered saline (PBS) or pooled sera from cats immunized with inactivated uninfected FeT1 cells (related to FL-4 cell line) or uninfected 3201 cells (allogeneic feline T-cell line). Upon low dose homologous challenge with 5 ID50, all control cats became infected whereas 3 out of 3 cats passively immunized with FIVpet (FL-4) vaccine sera and 4 out of 4 cats immunized with FIV infected cat sera did not. Protected cats showed VN antibody titers averaging between 100-200 whereas uninfected cell or PBS control sera had no VN antibody titers. These findings resemble those reported for SIV. Cynomolgus monkeys passively immunized with sera from SIV, infected and HIV-2 vaccinated monkeys were protected in the presence of high titer antiviral antibodies against homologous challenge at 10 to 100 ID50 (Putkonen et al. 1991). In contrast, passive immunization studies in macaques immunized with SIV. indicated that the levels of anti-cellular and not anti-viral antibodies correlated mostly with protection (Rosenthal et al. 1992).

Findings from studies evaluating protective immunity in kittens born to queens vaccinated with FIV,, FL-4/FeT1 vaccines also imply a role for VN responses (Pu et al. 1995). In these studies, kittens received colostrum/milk from either vaccinated or sham vaccinated queens and were challenged shortly after birth with low dose homologous challenge of 5 ID50. It was found that only those kittens born to and nursed by vaccinated queens were protected. Furthermore, protected kittens showed high levels of VN titers (500-5000) at five days postparturition. The role of transplancental maternal antibody





25


reponses was evaluated in kittens born to vaccinated queens and nursed by sham immunized queens. These kittens became infected and showed significantly lower titers of VN (10-100) postparturition as compared to littermate controls receiving colostrum from vaccinated queens. Nevertheless, these kittens showed lower levels of viremia as compared to littermate controls which were born to and nursed by unvaccinated queens (Pu et al. 1995). As such, these data suggest that VN maternal antibodies transferred via colostrum/milk or placenta play a role in preventing establishment of FIV infection. Supporting this are additional studies on kittens born to queens infected for various lengths of time. It was found that kittens born to queens infected for more than 7 months were protected in the presence of high VN titers in the colostrum. In contrast, kittens born to short term (< 2mo.) infected queens became infected in the presence of low VN titers in colostrum (Pu et al. 1995).

Opposing a role for Env specific and VN responses in protection against FIV infection are findings from a number of subunit vaccines trials. Vaccines composed of the FIV envelope or envelope fragments alone failed to induce protective immunity. This included nonglycosylated, glycosylated, native, and denatured whole FIV Env and Env fragment vaccines in combination with different adjuvants (Lutz et al. 1995; Verschoor et al. 1996; Lombardi et al. 1994). The majority of these vaccines, however, effectively induced Env-specific antibody responses as well as VN antibody responses. Therefore, it could be suggested that the tertiary structure of the envelope protein is crucial as it affects the presentation of the Env to the immune system or that other epitopes besides Env are required to obtain protective immunity. A subunit vaccines composed of Gag proteins alone, however, failed to induce protection despite the induction of Gag specific humoral responses (Hosie et al. 1992). Interestingly, several of the envelope subunit vaccines caused enhancement of infection upon challenge (Hosie et al. 1992; Siebelink et al. 1995; Osterhaus et al. 1996). Similar enhancement of viremia has been observed in horses immunized with a recombinant envelope vaccine against equine infectious anemia virus





26



(EIAV), another lentivirus (Wang et al. 1994). This phenomenon has also been observed with other nonlentiviral vaccines in particular macrophage and monocyte-tropic viruses (Halstead and O'Rourke 1977). It is thought to be mediated by viral specific antibodies elicited upon immunization that can facilitate viral transport to susceptible host cells by binding of the Fc portion of antibodies to the surface of macrophages.

The role of VN antibodies as a factor in preventing FIV infection is not clear. Although, a majority of cats immunized with FIVp FL-4/FeT1 vaccines showed high levels of VN antibody responses at challenge, some cats were protected in their absence. In addition vaccine trials employing inactivated MBM cells infected with the FIV M2 isolate, a vaccine similar to the FIVet FL-4/FeT 1 vaccines, induced protective immunity, in the absence of detectable VN antibody titers (Mattuecci et al. 1996).

In summary, protection against low dose experimental challenge with FIV may in part be mediated by antiviral humoral responses. However, cell-mediated responses such as CTL or underlying immune effector activities i.e. chemokines may also play a role. In fact, recent studies demonstrated the induction of FIV specific CTL responses directed against Env and Gag epitopes in cats immunized with the FIV,, (Fl-4) inactivated infected cell vaccine (Flynn et al. 1995a). The importance of these responses in protective immunity however remains to be established. Induction of FIV specific CTL responses was also observed in cats immunized with a synthetic peptide vaccine corresponding to the FIV V3 region. These cats, however, became infected upon challenge even though V3specific CTL responses were detected at the time of challenge (Flynn et al. 1994, 1995b).


Recombinant poxvirus-based vaccines


Members of the poxvirus family comprise a group of large viruses that infect a number of species. Poxviruses are enveloped and contain a single double stranded DNA genome of 130 to 300kbp. These viruses encode their own enzymes required for viral





27


DNA replication and mRNA synthesis, and are unique in the fact that they replicate within the cytoplasmic compartment of infected cells (Moss 1990).

The use of poxviruses as vaccine vectors was preceded by advances in the field of molecular biology that allowed the manipulation of viruses in such a way that foreign genes could be inserted and expressed (Piccini et al. 1987; Perkus et al. 1989). Poxviruses have since become candidate vaccine-vectors for a wide variety of pathogens (Perkus et al. 1995). In comparison to other candidate vector-viruses, poxviruses are exceptionally wellsuited due to their physical stability, low production costs, and ease of administration. Furthermore, poxviruses have large genomes that allow the insertion of multiple genes.

The most widely used member of the poxvirus family is the vaccinia virus, prototype of the Orthopoxviruses (Esposito 1991). This virus has been engineered to express antigens of bacterial and viral pathogens and shown to induce protective immunity in vivo (Perkus et al. 1995). Immune-responses induced upon inoculation with vaccinia based vaccines include both humoral responses and cell mediated responses directed against the inserted foreign antigens. There are however some concerns about the safety of vaccinia when used in a large population. As vaccinia exhibits a broad host-range there is a potential risk of spread to the general environment. Moreover, vaccinia has been shown to cause disseminated infection in immuno-compromised people (Fulgitini et al. 1968).

For these reasons, the development of poxviruses as vector vaccines has been extended to attenuated poxviruses and poxviruses with a more restricted host-range. One such example is the NYVAC vector. This vector was derived from the Copenhagen vaccinia strain by the selective deletion of 18 open reading frames, encoding genes involved in host-specificity and virulence (Tartaglia et al. 1992). These deletions resulted in a virus which replication is highly impaired on cell lines from several species including human cells. Furthermore, this virus lacks virulence in immuno-compromised animal models. The MVA vaccinia strain is another example of an attenuated vector strain used in vaccine development. This strain was derived by extensive passaging of the Ankara





28


vaccinia strain on primary chick embryo-fibroblast. As a result, the MVA virus is severely attenuated and lacks replication on nonavian cell lines (Sutter et al. 1994).

An alternative to the use of attenuated vaccinia viruses is the use of poxviruses that exhibit species specificity (Baxby and Paoletti 1992). These include the suipoxviruses, capripoxviruses and avipoxviruses. Avipoxviruses only productively infect cells of avian origin and were originally developed as vaccine vectors for the poultry industry. Unexpectedly, it was found that nonavian cells inoculated with avipoxvectors expressed the inserted antigen despite the absence of vector replication (Taylor et al 1992b). Moreover, it was found that these vectors when administered to nonavian species were capable of eliciting protective immunity. It is now understood that these viruses undergo abortive infection in nonavian cells resulting in expression of early gene and inserted gene products.

Two avipoxviruses, the fowlpoxvirus and canarypoxvirus, have been developed as vaccine vectors (Plotkin et al. 1995). ALVAC represents a vector derived from an attenuated canarypoxvirus strain originally used to immunize canaries. The protective potential of recombinant ALVAC vectors has been tested against several viral pathogens including rabies, measles, Japanese encephalitis virus (JEV), cytomegalovirus (CMV), and equine influenza virus (EIV) (Table 1.5) (Cadoz et al. 1992; Konishi et al. 1994; Taylor et al. 1992a, 1992b; Gonczol et al. 1995). The majority of these ALVAC-based vaccines were shown to elicit antigen-specific antibody responses, including VN antibody responses specific for the inserted antigens. More important, cell-mediated immunity was also induced in individuals immunized with these vaccines. In one study, lymphocytes from human volunteers immunized with an ALVAC-rabies recombinant had proliferative responses to the rabies antigens, demonstrating the induction of antigen specific T-helper cells (Cadoz et al. 1992; Taylor et al. 1991). This same recombinant vaccine was used to evaluate the ability of ALVAC-based vaccines to elicit memory immune responses in dogs.








Table 1.5 Immunogenicity and prophylactic efficacy of ALVAC-based vaccines



Pathogen genus Test Humoral Cell-mediated Protection species responses responses


Rabiesa Rhabdoviridae mice + ND + dog + ND + cats + ND + squirrel monkeys + ND ND rhesus macaques + ND ND chimpanzees + ND ND humans + ND ND Cytomegalovirusb Herpesviridae mice + + ND guinea pigs + + ND

Canine distemper virus Paramyxoviridae dogs + ND +

Japanese encephalitis virusd Flaviviridae mice + ND +

a Cadoz et al. 1992; Taylor et al. 1991. b Gonczol et al. 1995.
STaylor et al. 1992a, 1992b.
d Konishi et al. 1994.
ND=-not determined





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It was found that dogs could be protected from a rabies challenge given 36 weeks after a single ALVAC-rabies immunization (Cadoz et al. 1992).

The efficacy of ALVAC-based vaccines has also been tested against a number of retroviral pathogens (Table 1.6). Immunization with ALVAC recombinants expressing the Envelope and Gag proteins of the feline leukemia virus (FeLV) protected cats against experimental infection with FeLV (Tartaglia et al. 1993). Protection was afforded in the absence of detectable VN antibody responses. Interestingly, protected animals developed FeLV-specific VN antibody titers at 9-12 weeks postchallenge whereas control animals failed to develop VN antibody titers. The data presented in this study should be interpreted with some caution since no analysis of FeLV by PCR was performed on tissues of the protected animals (Tartaglia et al. 1993). Thus, immunization may not have resulted in sterilizing immunity but resulted in a reduced viral load explaining the development of VN titers at 9-12 weeks postchallenge. Further, the evaluation of cell-mediated responses was not included in this study.

ALVAC-based vaccines have protective efficacy against human T cell leukemia/lymphoma virus type I (HTLV-1), the causative agent of adult T cell leukemia and tropical spastic paraparesis in humans (Barre-Sinoussi et al. 1983). In the rabbit model used, it was found that two inoculations with ALVAC-based vaccines encoding the HTLV-1 envelope protein (ALVAC-gp65), protected rabbits against challenge infection with HTLV-1 infected cells. Protective immunity was afforded in the absence of HTLV-1 VN antibody responses. Again, this study lacked the evaluation of cell-mediated reponses. Interestingly, rabbits boosted with baculovirus expressed envelope protein (gp65) in addition to ALVAC-gp65 immunizations failed to be protected (Franchini et al. 1995b).

The rhesus macaque model was used to assess the prophylactic efficacy of ALVAC-based vaccines against HIV-2 challenge (Franchini et al. 1995a). It should be kept in mind that rhesus macaques can be infected with HIV-2 but do not develop an AIDS like syndrome. Rhesus macaques were given two immunizations with ALVAC





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recombinants expressing HIV-2 Env and Gag/Prot followed by two immunizations with recombinant Env (gpl60) and a final ALVAC-HIV-2 Env, Gag/Prot boost. Upon challenge with 100 ID50 HIV-2, both macaques were free of viremia and tested negative for the virus by virus isolation and virus specific PCR. Prior to challenge, HIV-2-specific CTL responses were detected. In addition, one of the macaques had a transient VN antibody titer prior to challenge. After challenge both animals developed significant VN antibody titers as such resembling findings in the ALVAC-FeLV trials (Franchini et al. 1995a; Tartagliaet al. 1993). The macaques were given a second HIV-2 challenge of 100 IDso at 7 months post the primary challenge without any intervening boosters. At this time, both macaques became infected despite the presence of VN antibody titers at the time of challenge. Cytotoxic T-lymphocyte responses were not measured at the time of the second challenge, however, both animals were positive for HIV-2 specific CTL activity at one month following the second challenge.

To evaluate the strain specificity of the immunity generated upon immunization with ALVAC-based vaccines, rhesus macaques were immunized with an ALVAC-recombinant expressing Env, Gag/Prot of the HIV-I~ isolate and challenged with a distinctly heterologous HIV-2 isolate (> 40% difference) (Abimiku et al. 1995). Two rhesus macaques received a combination of primary ALVAC-HIV-IM immunization and subsequent subunit boosts with either p24 and gpl60 or a tandem V3-peptide. Both animals developed virus-specific CTL and VN antibodies after immunization. Upon challenge, the animal boosted with the V3-peptide was considered partially protected, based on the low VN titers and the absence of virus by virus isolation and PCR at six months postchallenge. The other animal exhibited a delay by two months and had lower VN antibody titers as compared to control animals. All control animals became positive at 1 month after challenge and remained virus positive throughout the study. Perhaps the most interesting observation made in this study was that the VN antibody responses generated upon immunization did not cross react with the HIV-2 isolate used in the challenge





32



inoculum. Based on this it was speculated that cell mediated responses attributed to the partial protection observed in these animals. However, the assessment of cross-reactive CTL responses to the HIV-2 isolate was not included in this study.

In addition to the macaque model, the chimpanzee model has been used to assess the prophylactic efficacy of ALVAC-based vaccine protocols against HIV infection. In one study, the efficacy of an ALVAC recombinant expressing both the HIV-1L Env and Gag (ALVAC-HIV, gpl20TM, Gag/Prot) was evaluated against cell-associated HIV-1ma challenge in two chimpanzees. Each animals received a total of five immunizations and were challenged one month after the final immunization. HIV-specific antibody responses including VN antibodies were detected after the 4th and 5th immunization in both animals. Upon challenge, one animal remained virus negative and the other, including a naive control animal, became infected. Interestingly, the protected animal had a higher VN antibody titer at the time of challenge than the nonprotected animal (Van der Ryst et al. 1996). Importantly, these same vaccine protocols failed to induce protective immunity against challenge with the heterologous HIV-DH12 isolate (personal communication).

Further, the vaccine efficacy of ALVAC-HIVL gpl20TM, Gag/Prot recombinants was evaluated against mucosal challenge in chimpanzees. One group consisting of two animals was immunized by the intra-muscular (i.m.), cervico-vaginal, and rectal route simultaneously. Another group was immunized by the i.m., oral and nasal route and one animal was immunized by the i.m. route. All animals were challenged intra-cervically with 2500 TCID50 of HIV-1l passaged in chimpanzees. All vaccinated animals were free from virus infection whereas the nonvaccinated control animals were infected. HIV-specific antibody responses, including VN antibody titers, were low or undetectable at the time of challenge, thus implying that mucosal protection occurred through mechanisms other than VN antibody responses (Girard et al. 1996).

Another study reported on the efficacy of ALVAC-based vaccine strategies against heterologous HIV challenge in chimpanzees. Chimpanzees were immunized with an






33



ALVAC recombinant vaccine encoding Env of the HIV-I~ strain and boosted with recombinant envelope glycoproteins (gpl60) of the HIV-1I and HIV-1,, both classified as subtype B strains. The animals were challenge intravenously with HIV-1,, which is also classified as a subtype B virus but differs significantly from the HIV-1A strains. Upon challenge, virus was isolated from both vaccinated animals. However, in comparison to the infected control animals the vaccinated animals had a lower viral load (Girard et al. 1995)

In summary, ALVAC-based recombinants were effective against lentiviral infection in several animal models. However, it is not clear as to what constituted vaccine protection. Some of these studies suggest a role for VN antibody responses. On the other hand, other studies showed protection in the absence of detectable VN antibody responses. Conflicting results may also stem from the fact that only a limited number of animals was used in these trials. Furthermore, the assessment of immune responses, in particular cellmediated responses, was frequently omitted in these trials. Nevertheless, the data obtained from these animal trials are promising and studies on the safety and immunogenicity of these vectors in human volunteers have been initiated. Thus far, no adverse effects have been reported in human subjects immunized with an ALVAC-recombinant expressing HIV Env (gpl60), ALVAC-HIVgp160, followed by a boost with recombinant gpl60 (Clements et al. 1996, Lawrence et al. 1996). Further, it was found that immunization with this ALVAC recombinant alone failed to induce VN antibody responses. However, VN antibodies were detected in most subjects after the rgpl6Om Q boost. Env-specific Tcell proliferative responses were detected in a small percentage of subjects after ALVAC immunizations and in all subjects following rgpl6 io~ boosts. The presence of HIV Env-specific CTL activity was detected in some of the subjects, even without the subunit boost (Pialoux et al. 1995). In a similar study, immune responses elicited with ALVACHIVgpl60 alone was compared to those elicited by ALVAC-HIVgp160 priming followed by a HIV-1 rgpl20sF2 boost. Boosting with envelope protein significantly





34


enhanced VN antibody responses, ADCC, and CTL responses as compared to immunization with either ALVAC- HIVmgpl60 or rgpl20F2, alone (Clements et al. 1996).

In a follow-up study, volunteers received immunizations with an ALVAC recombinant expressing both HIV-1 Env and Gag and a boost with recombinant Env (Pialoux et al. 1995; Clements et al. 1996). The immunogenicity resembled that of the previous studies (Lawrence et al. 1996). Interestingly, Gag-specific CTL responses were detected in the majority of subjects whereas Env-specific CTL-responses were only detected in a small percentage of the subjects. Together, these studies demonstrate that immunization schemes involving ALVAC-based HIV vaccines, in combination with whole protein boosts, are safe and can elicit both humoral and cell-mediated responses specific for the inserted immunogens.










Table 1.4 Subunit and vector-based FIV vaccine trials
Challenge Inoculum
Type of Immunization Cellular Origin Vaccine Virus' Vaccine Dose Vaccination Type of Cellular Origin& Dose (IDs0) Protection (Vaccination Route) (FIV subtype) (mg/PFU) Protocol(wks) Adjuvantd Route & Strain rate
(Hosie, et al. 1992)
p24 E. coli(s.c.) UK-8 (A) 50 0,3,5,7 iscom PBMC (i.p.) 20 UK-8 0/4 (0%)
(Lutz et al. 1995)
gp100(denatured) Insect cell (i.m.) Z2 (A) 100 0,2,4,8 AIOH/QS21 PBMC (i.p.) 20 Z2 0/5 (25%) gpl00(native) Insect cell (i.m.) Z2 (A) 100 0,2,4,8 AIOH/QS21 PBMC (i.p.) 20 Z2 1/5 (20%) gpl0O(highly purified) Insect cell (i.m.) BANG (B) 100 0,2,4,8 AlOH/QS21 PBMC (i.p.) 20 Z2 1/5 (20%) gpl00(native ) Insect cell (i.m.) BANG (B) 100 0,2,4,8 AIOH/QS21 PBMC (i.p.) 20 Z2 0/5 (0%) gpl00(denatured) E..Coli (i.m.) BANG (B) 100 0,2,4,8 AIOH/QS21 PBMC (i.p.) 20 Z2 1/5 (25%) Control(Ovalbumin) (-) (-) 100 0,2,4,8 (-) PBMC (i.p) 20 Z2 2/7 (29%)
(Osterhaus et al. 1996)
Env(cleavage) NA(s.c.) AM19 (A) 100 0,4,10 iscom PBMC (i.m) 20AM19 0/6 (0%) Env(no cleavage)9 NA(s.c.) AM19 (A) 100 0,4,10 iscom PBMC (i.m.) 20AM19 0/6 (0%) Env(no cleavage)5 NA(s.c.) AM19 (A) 100 0,4,10 QuilA PBMC (i.m.) 20AM19 0/6 (0%) Env-b Gal NA(s.c.) AM19 (A) 100 0,4,10 QuilA PBMC (i.m.) 20AM19 0/6 (0%) Control(PBS) (-)(s.c.) (-) (-) 0,4,10 (-) PBMC (i.m.) 20AM19 0/6 (0%)
(Flynn et al. 1995)
V3-peptide synthetic (s.c.) UK-8 (A) 100 0,3,6 QuilA/AIOH PBMC (i.p.) 10 UK-8 0/15(0%) Adjuvant alone (-) (s.c.) (-) 100 0,3,6 QuilA/AIOH PBMC (i.p.) 10 UK-8 0/5 (0%) (Lombardi, 1994)
V3-peptide synthetic(s.c.) PET(A) 500 0,2,4,6,8 CFA Fl4(i.v.) 20-PET 0/3 (0%) Control (-) () (-) (-) (-) F4(i.v.) 20-PET 0/3 (0%)
(Verschoor et al. 1996)
V3-fusion protein I E..Coli(s.c.) UT113 (A) 100 0,4,6,8,10 AIOH NA(s.c.) 10-20UT113 0/5 (0%) V3-fusion protein I E..Coli(s.c.) UT113 100 0,6,10 QuilA NA(s.c.) 10-20UT113 0/5(0%) Control(PBS) (-)(s.c.) (-) (-) 0,6,10 (-) NA(s.c.) 10-20UT113 0/5(0%) Feline Herpes-Env (-)oronasal/s.c. UT 113 105PFU 0 (-)
Boost V3-peptide E. Coli (i.m.) UT113 100 4,8 AIOH NA(s.c.) 10-20UT113 0/5 (0%) Feline Herpes-Env (-)oronasal/s.c. UT113 105PFU 0 () Boost V3 peptide E.Coli (i.m.) UT113 100 4,8 QuilA NA(s.c.) 10-20UT113 0/5 (0%) Feline Herpes-b Gal. (-)oronasal/s.c. (-) 10PFU 0 (-) Boost PBS (-)(s.c.) (-) (-) 4,8 (-) NA(s.c.) 10-20UT113 0/5(0%)
(Gonin et al.1995)
Adenovirus-env (-)(i.m.) Wo (A) 11.8-9.2PFU 0,4,30 ISA206 NA(NA) 20 Wo 0/4 (0%) Adeno-psuedorabies(control) (-)(i.m.) (-) 11.8-9.2PFU 0,4,30 ISA708 NA(NA) 20 Wo 0/4 (0%)
a UK-8, United Kingdom; Z2, Zurich; BANG, Bangston; AM19, Amsterdam 19; PET, Petaluma.
b Recombinant vaccinia virus expressed
c Baculovirus expression
d iscom, immune stimulating complex; AIOH/QS21, aluminumhydroxide and non-toxic fraction from Quillaja saponaria; ISA206, water/oil adjuvant;ISA708, water/oil adjuvant. e V3-fusion protein was composed of the FIV V3 region fused to galactokinase.
f Deletion of the cleavage site between the envelope surface(SU) and transmembrane protein(TM).















CHAPTER II
IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION OF
CANARYPOXVIRUS (ALVAC)-BASED FIV VACCINES AGAINST HOMOLOGOUS FIV CHALLENGE


Introduction



The feline immunodeficiency virus (FIV) is the causative agent of an immunodeficiency syndrome in cats (Pedersen et al. 1987). The immunological and pathological changes observed in FIV infected cats closely resemble those observed in humans infected with HIV the causative agent of AIDS (Yamamoto et al. 1988b; Ackley et al. 1990). Based on these similarities FIV infection in cats has become a valuable model for the evaluation of vaccine and prophylactic strategies. Similar to HIV, it is still unknown what constitutes protective immunity against FIV. However, vaccine protection against experimental FIV infection in cats has been achieved with conventional vaccines such as inactivated wholevirus and inactivated FIV-infected cell vaccines (Yamamoto et al. 1991, 1993; Hosie et al. 1995; Verschoor et al. 1995). This vaccine approach has also been successful against lentiviral infection in other animal models, such as SIV in macaques and HIV in chimpanzees (Murphey-Corb et al. 1986; Fultz et al. 1992). The use of such vaccine approach in humans, however, may not be feasible because of safety issues, such as incomplete inactivation of vaccine virus which could potentially lead to infection of immunized subjects.

An alternative to the use of conventional vaccines, is the use of viral vectors based vaccines which can be engineered to encode specific components of these viruses. One




36





37


vector evaluated in current trials of HIV vaccines, is the canarypoxvirus vector, ALVAC (Baxby et al. 1992; Perkus et al. 1995).Canarypoxvirus (ALVAC)-based vaccines are considered safe due to their restricted host-range and their inability to undergo fullreplication in cells of non-avian origin (Baxby et al. 1992). The efficacy of ALVAC-based vaccines against lentiviruses has been tested in several animal models. Rhesus macaques immunized with ALVAC-recombinants expressing Env and Gag of HIV-2 were protected from infection with homologous HIV-2 (Franchini et al. 1995a). Additionally, ALVACHIV-1 vaccines have been proven effective against experimental infection with homologous HIV- 1 in a small number of chimpanzees tested (Girard et al. 1995, 1996; Van der Ryst et al. 1996). Although, the number of animals in these trials was small, these findings suggest that ALVAC-based vaccines can induce protective immunity against lentiviral infection.

Hence, the main objective of this study was to assess the immunogenicity and protective efficacy of recombinant canarypoxvirus (ALVAC)-vectored FIV vaccines alone or in combination with conventional inactivated FIV-infected cell vaccines. Four distinct ALVAC-FIV recombinants were tested. This included recombinants that encoded the FIV Env (ALVAC-env), the FIV Gag and Prot (ALVAC-gag/prot) or both the FIV Env, Gag and Prot (ALVAC-env,gag/prot). Also included was a recombinant that encoded the FIV Gag and a modified FIV Env from which a putative immunosuppressive region had been deleted (ALVAC-97TMG).


This study was conducted to address the following specific aims:


I. In vitro evaluation of ALVAC-FIV recombinant expression.


Determine if ALVAC-FIV recombinants are able to infect non-avian, non-permissive, feline cells and properly express the inserted FIV gene constructs in these cells.






38


II. In vivo evaluation of ALVAC-FIV recombinant vaccines.


a) Identify the immune responses, both humoral and cell-mediated, elicited in cats after immunization with ALVAC-FIV recombinants alone or after priming with ALVAC-FIV recombinants followed by boosting with inactivated FIV-infected cell vaccine (ICV).


b) Assess the protective efficacy of immunization protocols employing ALVAC-env, ALVAC-gag/prot, ALVAC-97TMG and ALVAC-env,gag/prot against experimental infection with the homologous subtype FIV Petaluma (FIV,,,) isolate in cats.


c) Assess the efficacy of a combination prime-boost protocol consisting of ALVACenv,gag/prot priming followed by inactivated FIV-infected cell boost, against experimental infection with the homologous subtype FIVp,, isolate in cats.



Materials and Methods


Construction of ALVAC-recombinants


ALVAC is a canarypoxvirus vector derived from a canarypoxvirus vaccine strain used to immunize canaries, Kanapox (Rhone-Merieux, Lyon, France). The ALVAC-FIV recombinants were generated using standard procedures similar to those used by Virogenetics to generate ALVAC-based FeLV recombinants (Piccini et al. 1987; Tartaglia et al. 1993). Briefly, the coding region of the FIVvie frche isolate (subtype A) env, gag and prot were amplified by polymerase chain reaction (PCR) and fused in a precise ATG to ATG fashion with vaccinia early/late promotors H6 or I3L, respectively ( Figure 2.1). The protease gene was included in the constructs to ensure proper proteolytic cleavage of the Gag precursor protein into the mature matrix protein (MA, p15), capsid protein (CA, p24)






39



and nucleocapsid protein (NC, p7). The promotor-FIV gene constructs were then cloned into the pC6L donor plasmid that contained ALVAC C6 flanking regions to enable insertion of the FIV promotor-gene constructs into the ALVAC non-essential C6 genetic locus. Prior to insertion into the donor plasmid, T5NT sequence elements were removed from the FIV coding sequences without altering the amino acid sequence. TsNT motifs are recognized by the poxvirus transcriptional apparatus as termination signals at early times postinfection and their retention is known to result in diminished expression of foreign gene products (Yuen and Moss 1987; Earl et al. 1990). The donor plasmid was then transfected into ALVAC-infected chicken embryo fibroblast cells to generate full ALVACrecombinants by homologous recombination. Recombinants were subjected to several rounds of plaque purification and analyzed by nucleotide sequence analysis to ensure proper insertion.

ALVAC-FIV recombinants generated for this trial included ALVAC-env containing the whole FIV env coding region, ALVAC-gag/prot containing the FIV gag and prot coding regions and ALVAC-env,gag/prot containing the FIV env, gag and prot coding regions. The ALVAC-97TMG recombinant contained the FIV gag and prot gene and a modified env coding region, of which a 714bp fragment encoding a putative immunosupressive element, had been deleted (Figure 2.1).



In vitro Expression of the FIV Env and Gag



The ability of ALVAC-FIV recombinants to infect non-permissive non-avian cells was evaluated on CrFK cells, a feline fibroblastic kidney cell line. Petri dishes seeded with a monolayer of CrFK cells were inoculated with ALVAC-FIV recombinants or ALVAC vector alone at a multiplicity of infection (m.o.i.) of 10 for 1 h at 37C in a humidified atmosphere containing 5% CO2. The ALVAC inoculum was removed and cells were incubated with fresh medium for an additional 48 h at 37oC. At 48 h, cells were removed









H6 ATG env 2571bp (857 aa) TAA
A. B( ALVAC-env


13L ATG gag/prot 1710bp (570 aa) TMA
B. M M ALVAC-gag/prot



H6 ATG env 2571bp TAA 13L ATG gag/prot 1710bp TAA
C. h11 1/////7f ALVAC-env,gag/prot




TAA gag/prot 1710bp GTA 13L H6 ATG Aenv 1857bp (619 aa) T
D. /////// ALVAC-97TMG
A
IS


Figure 2.1 Schematic representation of ALVAC constructs






41


by scraping and washed three times in phosphate-buffered saline (PBS). DNA was extracted from the cells by resuspending in lysis buffer consisting of 0.021 M Tris (pH 7.5), 0.029 M EDTA (pH 8.0), 0.1 M NaCl, 1% SDS and proteinase K (10 mg/ml). Cells in lysis buffer were incubated at 56C for 3-4 h followed by a 20 min incubation at 95oC to inactivate Proteinase K activity. The presence of FIV-specific DNA encoded by the ALVAC-recombinants in the obtained DNA samples was determined by PCR using FIV env- and gag-specific primers [FIV env specific primers 5 '-GAAATGTATAATATIGCIGG- '3 and 5'-GAATIrGATITGATTACATCC- '3; 5'-GGTAGGAGAGATTCrACA- '3 and 5'CTGCATTCITGCTGGTGC- '3 FIV gag specific primers]. PCR reactions were carried out in a final volume of 50 ll containing: 1.0 Ltg DNA, 50 mM KCI, 10 mM Tris-HCI (pH 8.3), 2.5 mM MgCl, 0.2 mM of each dNTP (dATP, dCTP, dTTP and dGTP), 20 pmol of each primer, and 2.5 units of Taq DNA polymerase. The reaction mixtures were incubated for 5 min at 94C and cycled 30 times through 1 min at 94oC, 1 min at 55oC, 1.5 min at 72oC, followed by 10 min incubation at 72oC. Finally, the PCR reaction products were separated by electrophoresis on a 1% agarose gel and visualized by staining with ethidium bromide.


Detection of FIV-specific mRNA transcripts by RT-PCR


The expression of messenger RNA corresponding to the FIV genes encoded within the ALVAC-recombinants was evaluated in non-permissive CrFK cells. Monolayers of CrFK cells were inoculated with ALVAC-FIV recombinants or ALVAC vector alone as described above. At 48 h postinoculation, messenger RNA was isolated using the MicroFast track mRNA isolation kit (Invitrogen, San Diego, CA). The isolated mRNA was then reverse transcribed into cDNA. Briefly, 1 pig of isolated mRNA was mixed with 7 pl DEPC-treated H20 and 2 il pdN6 and incubated at 650C for 5 min. Subsequently, the samples were mixed with 4 p1l 5X buffer, 1.3 pl DTT, 0.7 [d RNase inhibitor (40 U/l), 1 pl dNTPs (10 mM dATP, dGTP, dTTP, dGTP), 2.0 pl acetylated BSA and 2 ld M-MLV





42


reverse transcriptase (Superscript RT 200 U/ptl, Rnase H, Gibco BRL, Gaithersburg, MD) and incubated at 42oC for 1 h, followed by 10 min at 95oC. The generated cDNA served as a template for PCR reactions using env and gag specific primers. PCR was performed as described above.


Indirect immunofluorescence


Protein expression of the ALVAC encoded FIV env and gag genes was analyzed by indirect immunofluorescence on CrFK cells inoculated with ALVAC-FIV recombinants. CrFK were seeded at a density of 5x105 cells per 35mm' dish on sterile glass coverslips and infected at a m.o.i. of 10 with ALVAC-FIV recombinants or the ALVAC vector alone. Indirect immunofluorescence was performed at 48 h postinoculation. Cells were fixed in 4% paraformaldehyde for 10 min, washed in PBS, and permeabilized in PBS containing 0.2% Triton X-100. Cells were then incubated with pooled FIV-positive serum for 30 min, washed, and incubated with FITC-labeled anti-cat IgG. All serum and antibody dilutions were made in PBS containing 3% bovine serum albumin (BSA). Finally, cells were washed and counterstained with Evans Blue (0.5% in PBS) for 10 min, observed under a microscope and photographed.


ALVAC vaccine production and titering


Both ALVAC vector and ALVAC-FIV recombinants were amplified on permissive primary chicken embryo fibroblast (CEF) and titered in vitro by measuring the number of plaque forming units (PFU). Vaccines were produced from clarified lysates of infected CEF cells in serum free medium and aliquoted at 1X 108 PFU per dose.





43



Inactivated-cell vaccine preparation and titering



The inactivated FIV-infected cell vaccine (ICV) was produced from an IL-2 independent feline lymphoid cell line (FL-4) chronically infected with the FIV Petaluma isolate (subtype A). This cell line was cloned from an IL-2 dependent feline T-cell line (FeT1) infected with FIV,t and stains positive for CD8,CD4 and PanT surface markers and negative for IgM heavy and light chains (Yamamoto et al. 1991a). The ICV vaccine was generated by inactivation of FL-4 cells with 1.25% paraformaldehyde for 24 h, followed by extensive dialysis against PBS. A single vaccine dose consisted of 2.5x107 fixed infected cells mixed with 250ptg SAF/muramyl dipeptide (MDP) (Chiron Corporation).



Animals



A total of 36 specific pathogen free (SPF) cats (Felis catus, domestic short hair), 12 weeks of age, were purchased from Liberty Research Inc. (Waverly, NY). The animals were housed at the Infectious Disease complex of Animal Resource Services and cared for in accordance with the policies set by the Environmental Health and Safety division (EH&S) and the Animal Care Committee of the University of Florida. All cats received a combination vaccine against feline herpes virus, calicivirus and panleukopeniavirus (Fel-OVax, Ford Dodge laboratories, Mason City, IA). Animals were not vaccinated against feline leukemia virus (FeLV). Prior to immunization, all animals tested negative for Toxoplasma gondii FeLV and FIV by immunoblot analysis.



Grouping and Immunization Protocol



Cats were divided into 7 groups, with equal numbers of males and females in each group (Table 2.1). Littermates were evenly spread over all groups. Cats were immunized





44



Table 2.1 Grouping and Immunization



Group Cat ID# sex Vaccine(Number of Immunizations)


QH4 F PY1 M Group A QO1 F ALVAC-env (3X) QC1 M QUI M QL2 F

QQ1 M QA5 F Group B QU2 F ALVAC-gag/prot (3X) QX3 M QI1 M QL3 F

QH5 F PY3 M Group C QS4 F ALVAC-env,gag/prot (3X) QC3 M QG3 F QE2 M

QQ2 M PY5 F Group D Q02 F ALVAC-97TMG (3X) QX4 M QI2 M QLA F

QH2 M PY2 M Group E QA4 F ALVAC (3X) QC4 M QG5 F QE3 F

QH3 M Group F PY4 M ALVAC-env,gaglprot (2X) QA6 F ICV (lX)

QC5 F Group G QG4 F ALVAC (2X) QE4 M & ICV (1X)

F=Female M=Male





45



a total of three times at monthly intervals. The ALVAC vaccine was administered intramuscularly at 1x108 PFU/cat. The inactivated FIV-infected cell vaccine (ICV) was mixed with 250 tlg SAF/MDP adjuvant and was administered subcutaneously.


Challenge



The challenge inoculum consisted of cell-free culture fluid from PBMC infected with FIV,, previously titered in vivo in SPF cats. The challenge inoculum of 50 ID50 was given intraperitoneally (i.p.) four weeks after the final immunization.


FIV immunoblot assay



Sucrose gradient purified FIVpet from chronically infected FL-4 lymphoid cells was separated by a 10% SDS-polyacrylamide gel (SDS-PAGE). Proteins were transferred to nitrocellulose sheets (pore diameter of 0.45 rpm) by wet blotting. After transfer, the sheets were blocked for 1-2 h at 37oC in gelatin buffer (PBS containing 3% gelatin and 0.02% sodium azide) and cut into strips. Serum samples of immunized and non-immunized cats were diluted at 1:100 in Buffer 3 (0.05 Tris at pH7.4 containing 0.15 M sodium chloride, 0.001 M EDTA, 0.05 % Tween-20, and 1 % BSA) and incubated with immunoblot strips for4 h at37oC. The reactions were stopped with ddiH20O and strips were washed 3 times with ELISA buffer (see ELISA protocol). The strips were incubated with biotinylated anticat IgG (Southern Biotechnology) for 1 h at 370C followed by three washes with ELISA buffer. Strips were then incubated with streptavidin conjugated to horseradish peroxidase for 1 h at 37oC. The reactions were stopped and washed 3 times with ELISA buffer. Finally, strips were incubated with fresh substrate solution (0.1 M Tris at pH7.4 containing 0.05% diamino benzidine, 400 mg/ml of NiC12 and 0.01% H202). Upon appearance of visible bands, reactions were stopped with an excess of ddiH20.





46


The liter of FIV-specific antibodies, if detected, was determined by testing 10-fold serial dilutions of serum samples (1:100 to 1:1000000) as described above and defined as the reciprocal of the highest dilution (in '1Log) at which FIV-specific bands could be visualized.



ALVAC immunoblot assay



ALVAC immunoblots were generated similar to that described for FIV immunoblots, using ALVAC derived from clarified lysates of ALVAC-infected primary chicken embryo fibroblasts. Serum samples obtained from ALVAC immunized animals and control non-immunized animals were tested at a serum dilution of 1:100 in Buffer 3. Reaction were carried out as described (see FIV immunoblot assay).


Enzyme-linked immunosorbent assays (ELISA)



Synthetic peptides corresponding to both conserved and variable regions in the FIVot envelope surface(SU) and transmembrane(TM) protein were coated on 96 well Immunolon microtiter plates at 250 ng/well with bicarbonate buffer (pH 9.6).[ V3-1 (SKWEEAKVKFHCQRTQSQPGS), V3-2 (GSWFRAISSWKQRNRWEWRDF), V3-3 (DFESKKVKISLQCNSTKNLFA) and TM (QLEGCNQNQFFCKI)]. The plates were washed with Buffer 3 immediately prior to use and blocked with 5% dry non-fat milk in HO. Serum samples were diluted 1:200 in Buffer 3 containing 5% newborn-calf serum and incubated in the coated wells for 30 min at 37oC, washed 3 times with ELISA wash solution (0.05 % Tween-20 in 0. 15M sodium chloride), and incubated with biotinylated anti-cat IgG (Vector laboratories, Burlingame, CA) for 30 min at 37 oC. Subsequently, the wells were washed three times and incubated with streptavidin conjugated to horse radish peroxidase (Vector laboratories, Burlingame, CA), washed 3 times with ELISA wash






47


solution, followed by incubation with ELISA substrate solution (0.005 % tetramethylbenzidine and 0.015 % H20, in 0.96 % citrate solution). The reactions were stopped with 0.1 M sulfuric acid upon establishment of visible reaction color. The plates were read in a ELISA reader at 414 nm.



Assessment of viral neutralizing (VN) antibodies


The presence of FIV specific VN antibodies was evaluated using a standard assay (Yamamotoet al. 1991). Serum samples obtained preimmunization, postimmunization and after challenge were diluted at various concentrations (1:5 to 1:100) and incubated at 56oC for 30 minutes to inactivate complement. The diluted sera were then incubated with 100 TCID50 (tissue culture cell infective doses of FIVp,) for 45 min at 370C in a 24-well microtiter plate. Subsequently, peripheral blood mononuclear cells (PBMC) were added to this mixture at 1x106 cells/well. After three days of culturing, cells were washed to remove residual virus from the culture and resuspended in fresh culture media (see RT media). Virus infection was monitored by Mg"2 dependent reverse transcriptase (RT) activity (see RT assay) in culture fluid harvested on Day 6, 9, 12, 15 and 18 of culturing. The VN antibodies titers were defined as the reciprocal of the highest final dilution which gave >50% reduction in reverse transcriptase activity as compared to the reverse transcriptase activity detected in fluids from control cell cultures that contained SPF serum and virus.


Assessment of FIV-specific proliferative T-cell responses


FIV-specific proliferative responses were evaluated using a '[H]-thymidine incorporation assay (Yamamotoetal. 1991). Freshly isolated PBMC were cultured in 96well microtiter plates in a final volume of 200 Rl in RPMI1640 media supplemented with 5% heat inactivated fetal calf serum, 10mM HEPES buffer, 50 mg/ml gentamycin, 5X10s





48


M 2-mercaptoethanol at a final concentration of 1x106 cells/mi (2xl05cells/well). Triplicate cultures were stimulated with inactivated FIV (5 Rg/well) and incubated at 370C for 4 days in a humidified atmosphere containing 5% CO,. On Day 4, cells were pulsed with 1 RCi 3[H]-thymidine (Amersham, Indianapolis, IN) per well for 18 h. Cells were harvested onto filter paper using a cell harvester. The discs were air-dried and 3[H]-thymidine incorporation was assessed by liquid scintillation counting. Results of triplicate samples were expressed as the stimulation index (S.I.), calculated as the mean incorporation in the presence of inactivated FIV divided by the mean incorporation in the absence of inactivated FIV.


Assessment of cytotoxic T-lymphocyte (CTL) responses


PBMC were tested for their ability to lyse autologous lymphoblastoid cells infected with FIVe,. Freshly isolated PBMC were cultured at 2x106 cells/ml in RPMI1640 medium containing 10% FBS and stimulated with Con A (5 mg/ml) for three days. On Day 3, 3.6 x 106 Con A lymphoblasts were removed, infected with FIVt and cultured for 5 days. The remaining cells (effector cells) were maintained in RPMI1640 medium supplemented with 10% FBS and IL-2 (100 U/ml) for 5 days. After5 days, 1-5x106 of the FIV-infected cells were inactivated by UV treatment and added as antigen-presenting cells(APC) to the effector cells at a ratio of 1:15 (APC:effector cells). Effector cells and FIV-infected APC cells were cocultured for an additional 5-7 days in no IL-2 medium. Effector cells were assayed for cytolytic activity against autologous FIV-infected target cells by a standard s5Cr release assay (Song et al. 1992). Target cells were labeled with 51Cr for 2 h at 370C and washed three times prior to use in the assay. Effector and s5Cr -labeled target cells were then mixed at effector to target ratios ranging from 50:1 to 10:1 and incubated for 4 h at 370C without IL-2. After 4 h, 100 tl of supernatant was removed from each well and 51Cr-specific activity was measured in a y-counter. Results are shown as the percentage of





49


specific cytotoxicity for triplicate assays. Maximum release was obtained by repeated freeze-thawing of labeled target cells. Spontaneous release was obtained from s5Cr labeled target cell cultures in the absence of effector cells. The percentage of FIV-specific release was calculated as 100 x (mean cpm test release mean spontaneous release)/(mean cpm maximum release mean cpm spontaneous release). The spontaneous release did not exceed 20% of the maximum release. Specific lysis values equal or greater than 10% were considered positive for CTL activity.



Assessment of natural killer cell activity



The level of natural killer (NK) cell activity was determined using a 4 h S5Cr release assay similar to that described for the CTL assay. Various target cell types were used including FeT-J (feline lymphoid cell line), FL-4 (feline lymphoid cell line chronically infected with FIVpt), non-autologous PBMC, and autologous PBMC. Target cells were labeled with S5Cr for 2 h at 37oC and washed three times prior to use in the assay. Freshly isolated PBMC effector cells were cocultured at effector to target cell ratios ranging between 100:1 to 10:1 for 4 h at 37oC in a humidified atmosphere containing 5% CO2.. After 4 h, 100 [l of supernatant was removed from each well and S5Cr-specific activity was measured in a y-counter. Results are shown as the percentage of cytotoxicity for triplicate assays. Maximum release was obtained by repeated freeze-thawing of labeled target cells. Spontaneous release was obtained from 5'Cr labeled target cell cultures in the absence of effector cells. The percentage of NK activity was calculated as 100 x (mean cpm test release mean cpm spontaneous release)/ (mean cpm maximum release mean cpm spontaneous release). The spontaneous release did not exceed 20% of the maximum release.






50


Viral reverse transcriptase (RT) assay


Freshly isolated PBMC were either stimulated with ConA (5mg/ml) or co-cultured and cultured for 4 weeks in RPMI1640 medium containing 5% heat inactivated FCS, 10mM HEPES buffer, 50 mg/ml gentamycin, 5X10s5 M 2-mercaptoethanol and 100 U/ml human recombinant IL-2, or cocultured with ConA lymphoblasts from SPF cats. Culture supernatants were collected every 3-4 days and assayed for the presence of viral reverse transcriptase (RT) activity. The virus was pelleted from the supernatants by ultracentrifugation (1 h at 17,000rpm). The virus pellet was then incubated with an RT cocktail containing 100 mM Tris (pH8.3), 150 mM KCI, 10 mM MgC,, 4 mM diethiothreitol (DTT), 0.6 Units of Poly (rA), oligo(dT), and 60 taCi of 3[H]TTP per ml. After incubation at 37oC for lh, the cDNA was spotted onto filter paper discs that had been prewashed with 0.1 M sodium pyrophosphate. Discs were washed in the following sequence: twice in 10% cold trichloroacetic acid (TCA), once in 5% TCA, once in 5% TCA containing 0.5 % SDS, and once in ethanol. The filter discs were air-dried and placed in scintillation vials with 3 ml scintillation fluid. 3[H]-TTP incorporation was measured in using a liquid scintillation counter. Supernatants were considered positive for RT activity if cpm in test samples were equivalent or higher than 3 times the cpm of the negative control sample (supernatants from SPF control cats).




Detection of proviral DNA by polymerase chain reaction (PCR)


Proviral DNA (latent infection) was monitored by env specific PCR on DNA extracted from PBMC, bone marrow (BM) cells, and lymph node (LN) cells after culturing for 4 weeks with FIV-free ConA lymphoblasts. BM cells were obtained from 1-2 ml of aspirates taken from the femur. LN cells were obtained from the popliteal lymph nodes.






51


DNA was extracted as described. In the PCR reaction, the following FIV env specific primer sets were used: 5'-GAAATGTATAATATTGCTGG- 3 and 5'GAATTGATFTGATTACATCC-'3. The PCR reactions were carried out in 50 pl reaction mixtures containing 1.0 plg genomic DNA, 50 mM KCI, 10 mM Tris-HCI (pH 8.3), 2.5 mM MgClI, 0.2 mM of each dNTP (dATP, dCTP, dTTP and dGTP), 20 pmol of each primer, and 2.5 units of Taq DNA polymerase. The reaction mixture was incubated for 5 min at 94oC and cycled 30 times through 94oC for 1 min, 55C for 1 min, 720C for 1.5 min, followed by 10 min at 72oC. The specificity of the PCR-amplified 455bp product was verified by nucleotide sequence analysis.


DNA sequencing



DNA sequencing was performed using the Amplicycle sequencing kit (Perkin Elmer, Norwalk, CT). Primers (approximately 10 [pM) were labeled at the 5'-end with 20 pCi y-32P ATP (6000Ci/mmol) and 20 U Polynucleotide kinase in a final reaction volume of 6.2 pl for 10 min at 37oC. The reactions were terminated by incubation at 900C for 5 min. For the sequencing reactions, four separate reaction mixture were prepared containing: 1 pl labeled primer, 4 pl 10X reaction buffer and approximately 10-50 fmol of PCR template and 2 pl of either G, A, T, or C termination mix. Reaction mixtures were then incubated at 95oC for 15 seconds, cycled 25 times at 950C for 1 min, 68oC for 1 min, followed by 7 min at 780C. Upon cycling, 4 pl of formamide stop solution containing 0.05% bromophenol blue and xylene cyanol were added. The reactions were analyzed on a 6% polyacrylamide gel containing 7 M urea and IX TBE.






52


In vivo assessment of viral-status



Three SPF kittens (#DH2, #DH5, #DE4), 12 weeks of age, were transfused intravenously with a total of 1.1 x 108 cells obtained 8 months after the FIVp, challenge from either ALVAC-gag/prot immunized cats (#QQ1 and #QX3) or FI V-infected ALVACcontrol cat (#PY2). Cells consisted of 3 x 10' of PBMC isolated by ficoll hypaque density centrifugation, 7 x 107 BM cells and lx107 LN cells. Prior to transfusion, these cells were washed in sterile PBS and resuspended in 2 ml PBS. Cat #DH2 received cells from infected control cat #PY2, cat #DH5 received cells from ALVAC-gag/prot immunized cat #QQ1 and cat #DE4 received cells from ALVAC-gag/prot immunized cat #QX3.


General parameters



Throughout the trial, all cats were monitored for hematological changes (complete blood count, differential leukocyte count and total protein count) and abnormal clinical manifestations (diarrhea, vomiting, lymphadenopathy, weight loss, elevated rectal temperature and neurological signs).


CD4/CD8 ratios



CD4/CD8 ratios were determined by indirect immunofluorescence staining and flow cytometry. Briefly, 5X105 PBMC isolated by ficoll hypaque density centrifugation, were washed in FACS buffer (PBS containing 0.325% sodium azide and 2.5% BSA) and incubated with feline CD4 or CD8 specific monclonal antibodies at 370C for 1 h. Subsequently, cells were washed in FACS buffer and incubated with secondary antibody, FITC labeled goat F(ab')2 anti-mouse IgG (H+L) (Southern Biotechnology Associates, Inc.) for 1 h at 37oC. Finally, cells were washed and analyzed by flow cytometry on a





53


Becton Dickinson FACSSORT. Monoclonal antibodies to feline CD4 and CD8 were kindly provided by N. Gengozian, University of Tennessee.


T-cell mitogen proliferative responses


T-cell mitogen proliferative responses were measured by 3[IH]-thymidine uptake assays (Ackley et al. 1990). Freshly isolated PBMC were resuspended at 2x105 cells/mi and stimulated with either Con A (5 mg/ml) or SEA (1 mg/ml). Cells were cultured for 48 h and then pulsed with ltCi 3[H]-thymidine/well. After 18 h, cells were harvested using a cell harvester onto filter paper discs. The filter discs were air-dried and "[H]-thymidine incorporation was assessed by liquid scintillation counting. The results were expressed as the stimulation index (S.I.), calculated as incorporation (mean cpm of triplicate samples) in the presence of mitogen divided by incorporation (mean cpm of triplicate samples) in the absence of mitogen.


Statistical analysis



The statistical significance of the data was evaluated by a Fisher's exact test, which is a modification of the chi square test. This test should be used when comparing two sets of discontinuous, quantal (all or none) data. The analysis was set up as follows:


Vaccinated Unvaccinated

Infected A B


Uninfected C D






54


The P (Probability) for a one-tailed test was calculated as (A+B)!(C+D)!(A+C)!(B+D)! / N!A!B!C!D! combined with the P value of stronger combinations. The obtained P value tells if the groups differ significantly and the degree of significance. In this study, a Pvalue equal or less than 0.05 was considered significant.


Results


In vitro assessment


The ability of ALVAC-recombinants to infect non-permissive feline cells was demonstrated by PCR analysis on DNA extracted from feline CrFK cells inoculated with ALVAC-env and ALVAC-gag/prot recombinants (Figure 2.2 and 2.3). The obtained PCR products corresponded in size to PCR-products obtained with DNA extracted from FL-4 cells, a lymphoid cell line chronically infected with FIVp,. The correct nucleotide sequence was verified by DNA sequencing (data not shown). No FIV-specific PCR products could be detected in CrFK cells infected with ALVAC vector alone or FeT-J, a FIV-negative feline lymphoid cell line.

Similarly, the expression of messenger RNA (mRNA) specific for FIVenv and gag products was demonstrated in CrFK cells inoculated with the ALVAC-env and ALVACgag/prot recombinants (Figure 2.4). RT-PCR on mRNA extracted from CrFK cells infected with ALVAC-env revealed a 450bp band and from those infected with ALVACgag/prot revealed a 700bp band consistent with the bands obtained from the FIV-infected FL-4 cell line. No RT-PCR products could be detected in cells infected with ALVAC vector alone and FeT-J control cells (FIV negative feline lymphoid cell line). In addition, no PCR products were obtained PCR reactions in which mRNA extracts were used as template, indicating that the obtained RT-PCR products were not the result of DNA contamination of mRNA extracts (data not shown).





55












Cell F14 Crfk FetJ
FIV+ Alvac- FIVCtrI Env Gag

lane M 1 2 3 4 5 S+ -+










450 bp











Figure 2.2 FIV-env specific PCR on CrFK inoculated with ALVAC-recombinants. Photograph of PCR products following electrophoresis on a 1.0 % agarose gel in Tris acetate-buffer (40mM Tris-acetate, 1 mM EDTA, pH 7.6). Lane M, X DNA EcoR/Hindll marker (1pg/lane). Lane 1, positive control, FIV-env specific PCR on DNA extracted from FL4 (FIV') cells. Lane 2, FIV-Env specific PCR on CrFK cells inoculated with ALVAC-control. Lane 3, FIV-env specific PCR on CrFK cells infected with ALVAC-env. Lane 4, FIV-env specific PCR on CrFK cells infected with ALVAC-gag/prot. Lane 5, negative control, FIV-Env specific PCR on Fet-J (FIV)cells.





56











Cell FetJ Crfk F14
FIV- Alvac- FIV+
CtrI Env Gag

lane M 1 2 3 4 5 S -- + +










700 bp












Figure 2.3 FIV-gag specific PCR on CrFK inoculated with ALVAC-recombinants. Photograph of PCR products following electrophoresis on a 1.0 % agarose gel in Tris acetate-buffer (40mM Tris-acetate, 1 mM EDTA, pH 7.6). Lane M, marker EcoR/Hindlll cut DNA (1 jpg). Lane 1, negative control, FIV-gag specific PCR on DNA extracted from Fet-J (FIV) cells. Lane 2, FIV-gag specific PCR on CrFK cells inoculated with ALVACcontrol. Lane 3, FIV-gag specific PCR on CrFK cells infected with ALVAC-env. Lane 4, FIV-gag specific PCR on CrFK cells infected with ALVAC-gag/prot.. Lane 5, positive control, FIV-gag specific PCR on Fl4 (FIV')cells.





57










Cell F14 CRFK FIV+ AlvacCtrl. Env Gag
primers EG E G E G lane M 1 2 3 4 5 6







700bp

450bp













Figure 2.4 RT-PCR on mRNA extracted from CrFK inoculated with ALVACrecombinants. Photograph of PCR products following electrophoresis on a 1.0 % agarose gel in Tris acetate-buffer (40mM Tris-acetate, 1 mM EDTA, pH 7.6). Lane M, X DNA EcoRI/HindIII marker (1g/lane). RT-PCR on mRNA extracted from FIV' FL-4 cells shows specific amplification of FIV-env (Lane 1) and FIV-gag (Lane 2). RT-PCR on mRNA extracted from CrFK cells infected with ALVAC-control show no amplification of FIV-env (Lane 3) or FIV-gag (Lane 4). Lane 5, FIV-env specific RT-PCR on mRNA extracted from CrFK cells infected with ALVAC-env. Lane 6, FIV-gag specific RT-PCR on mRNA from CrFK cells infected with ALVAC-gag/prot.































Figure 2.5 Indirect immunofluorescence analysis on permeabilized CrFK cells infected with ALVAC-recombinants at a m.o.i. of 10. Expression of FIV Env glycoprotein and Gag proteins was detected using pooled serum from FIV-infected cats as the primary antibody and fluorescein isothiocyanate-conjugated mouse anti-cat IgG as the secondary antibody. Panel A, control uninfected CrFK cells. Panel B, control CrFK cells infected with ALVAC-control. Panel C, CrFK cells infected with ALVAC-env. Panel D, CrFK cells infected with ALVAC-gag/prot. Panel E, CrFK cells infected with ALVACenv,gag/prot. Panel F, CrFK cells infected with ALVAC-97TMG.





59





60


Further, the expression of the ALVAC encoded FIV Env as a membrane associated protein and of the FIV-Gag core protein as intracellular protein was demonstrated by indirect immunofluorescence on CrFK cells inoculated with the ALVAC-FIV recombinants (Figure 2.5). At 48 h postinfection, cells infected with ALVAC-env showed fluorescence predominantly at the surface (panel C) whereas cells infected with ALVAC-gag/prot, ALVAC-env,gag/prot and ALVAC-97TMG showed strong fluorescence in the cytoplasm (see Figure 2.5, panel D-F). Control cells, uninfected CrFK and CrFK infected with ALVAC vector alone, did not show immunofluorescence (panel A and B).


In vivo assessment



A total of 36 SPF cats were used to evaluate the prophylactic efficacy of immunizations protocols employing ALVAC-FIV recombinants alone or in combination with ICV. Cats were divided into 7 groups (A-G) and inoculated three times at monthly intervals with either ALVAC-env (n=6), ALVAC-gaglprot (n=6), ALVAC-env,gaglprot (n=6), ALVAC-97TMG (n=6) or ALVAC vector alone (n=6). Cats in group F and G were immunized twice with ALVAC-env,gaglprot (n=3) or ALVAC (n=3), respectively, followed by a boost with inactivated FIV-infected cell vaccine (see Table 2.1). All cats were challenged 4 weeks after the final immunization with 50 ID50 HVp,. The FIV,et isolate is a subtype A virus, and differs 3% in the Env and 1% in the Gag protein coding region from the FIVvin, f. isolate (subtype A) used to generate the ALVAC recombinants.


General immunologic parameters


No adverse effects and no significant changes in blood chemistry (CBC, HgB, PCV, TPP) were noticed in any of the cats upon immunization with ALVAC-FIV recombinants or after boosting with inactivated FIV-infected cell vaccine (data not shown).





























Figure 2.6 Representative FACS analysis of binding of anti-fCD4 and anti-fCD8 monoclonal antibodies to PBMC. Expression of fCD-4 and fCD-8 was detected by using anti-fCD4 and anti-fCD8 antibodies as the primary antibody and fluorescein isothiocyanateconjugated goat anti-mouse IgG as the secondary antibody. Panel A, scattered dot-plot of PBMC isolated from cat #QG3. The depicted histograms respresents cells gated under gate 1 (predominantly lymphocytes) and gate 2 (negative control, cells of the macrophage and monocyte lineages). Panel B, histogram of CD4 and CD8 staining of cells in gate 1. Panel C, histogram of CD4 and CD8 staining of cells in gate 2.





62





A






gatgatee I



gate I WON ..







ssc-H\sc-Height ->
B
CD4 CD8








54 18s 2a s se da5 ee 18' 20e
FL-HEIGHT FL-HEIGHT


C
CD4 CD8







.e .8 15 25 12151e

FL-HEIGHT FL-HEIGHT






63


Table 2.2 CD4/CD8 ratios before and after immunization and challenge


months
Immunizations post-challenge Cat ID# Before After
-4 -1 +3 +5 +6 +7 +8 +10 +12 QH4 4.7 4.4 3.2 3.0 3.3F PY 1 4.3 4.2 2.4 2.4 2.5F QO1 3.9 2.6 3.5F QC1 3.3 3.3 2.9 2.5 1.9" QUI ND 3.8 3.6 3.3 2.5F QL2 3.6 3.3 2.2 1.9 1.7F

QH5 6.7 4.2 3.8 2.6 2.5F PY3 5.1 4.9 4.3 3.3 2.6F QS4 6.0 4.8 2.6 2.3 2.3F QC3 3.2 2.6 3.6 2.1 2.0O QG3 5.0 4.0 3.6 3.2 2.4F QE2 3.1 ND 3.0 2.0F

QQ1 4.9 2.7 2.1 1.9 1.6 2.3 QA5 4.3 2.2 2.6 2.5 2.9 2.5 QU2 4.0 2.6 2.4 2.0 2.3 2.9 QX3 2.0 2.5 2.0 1.8 1.8 2.3 QI1 6.1 3.9 3.9 3.3 3.3 3.9 QL3 4.9 2.0 2.6 1.9 2.0 2.2

QQ2 2.7 3.4 2.7 2.0 2.2F PY5 5.1 3.8 2.5 2.1 1.9" Q02 3.3 4.0 2.4F QX4 1.3 ND 1.5 1.1 1.0 QI2 4.9 2.5 2.1 2.0 1.9F QL4 4.4 4.1 3.8 2.3 2.5F

QH2 3.9 3.5 4.6F PY2 3.3 2.9 3.5 2.7 3.8 2.5 QA4 2.9 2.6 2.4 1.7 1.8F QC4 4.2 3.1 2.7 2.4 1.6F QG5 4.0 2.5 2.7F QE3 2.1 2.3 2.7 1.7 2.OF

QH3 5.5 4.1 4.0 3.4 2.0 4.3 PY4 2.8 4.0 3.1 2.6 2.6 2.4 QA6 4.3 3.6 2.9 2.5 2.1 2.7 QC5 2.8 2.0 2. 1F QG4 5.1 3.5 2.9" QE4 2.6 3.1 1.8 2.0 1.6 2.5

F=Final testing of CD4/CD8 ratio before animal was euthanized.








Table 2.3 Proliferation responses to T-cell mitogens (ConA and SEA).




Stimulation Index

Vaccine Cat ID# Number of immunizations Post
Pre- 1X 3X Challengea Immunization

ConA SEA ConA SEA ConA SEA ConA SEA ALVAC-env QH4 2.8 26.7 7.9 17.4 22.5 24.9 2.5 3.1 PY1 4.3 29.0 6.6 12.6 4.0 4.0 3.2 3.1 ALVAC-gag/prot QA5 7.5 16.9 32.1 77.4 30.3 14.4 22.7 23.2 QI1 50 93.8 2.2 7.5 25.8 28.2 18.3 17.8 ALVAC-env, QS4 6 29 3.1 6.7 35.0 35.2 37.1 36.7 gag/prot QG3 2.9 6.9 1.0 2.3 38.1 35.0 30.7 22.7 ALVAC-97TMG QQ2 2.7 9.2 1.0 2.0 16.8 18.0 17.2 12.5 QL4 78.4 99.0 9.6 20.3 29.4 27.2 18.8 17.2 ALVAC-env, PY4 13.4 26.8 4.0 15.9 20.5 19.7 63.2 36.7 gag/prot &ICV
ALVAC QH2 4.2 7.2 2.4 5.5 9.5 9 28.7 26.2 QA4 46 65 5.5 9.3 44.3 52.8 15.2 17.3 ALVAC QG4 4 8 12.5 23.5 7.4 7.4 25.4 23.5 &ICV

'3 weeks post challenge





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CD4/CD8 ratios were monitored prior to immunization, postimmunization and after challenge, a representation of a typical FACS analysis is depicted in Figure 2.6. Prior to immunization, most cats displayed CD4/CD8 ratios considered normal for three month old kittens (average ratio 4.0 + 1.2) except for cat #QX3 which displayed a ratio of 1.3 which is considered low. A littermate of this cat (#QX4) also displayed a relatively low ratio, suggesting that genetic factors attributed to this. After immunization, most cats showed an average CD4/CD8 ratio of 3.3 + 0.8. The observed decline in ratio is expected with the increase in age. No inversion (<1) in CD4/CD8 ratios were noticed after challenge (Table 2.2). This was as expected since the FIV,, at the challenge dose used generally does not cause inversion of CD4/CD8 ratio until 1.5-2 years postchallenge.

To determine if ALVAC vaccinations influenced lymphocyte function, we evaluated lymphocyte proliferation upon exposure to concanavalin A (ConA) and staphylococcal enterotoxin A (SEA) at various times in selected cats. Following immunizations and challenge no abnormalities in T-cell proliferative responses were detected in any of the cats tested (Table 2.3).



Humoral responses



The generation of ALVAC-specific antibodies was evaluated before and after immunization in serum samples taken from cat #QS4 immunized with ALVACenv,gaglprot and cat #PY2 immunized with ALVAC vector alone by immunoblotting. ALVAC specific humoral responses were detected in both cats upon a single immunization and additional immunizations resulted in increased titers of these antibodies (Figure 2.7). Serum samples obtained from SPF and FIV infected control cats tested negative.

Next, we tested if the immunization schemes used were able to generate FIVspecific antibody responses. Immunization with ALVAC-FIV recombinants alone (#QC3) failed to induce detectable FIV-specific antibody responses even after three immunizations
























Figure 2.7 ALVAC-specific immunoblot. Serum samples taken from ALVAC-control immunized cat #PY2 and ALVAC-env,gag/prot immunized cat #QS4 prior to immunization (pre-) and after the first, second and third immunization and 17 weeks postchallenge (17pc) were diluted 1:100 in Buffer 3 and incubated with ALVAC-specific westernblot strips. Negative controls: pooled sera from FIV-infected cats and pooled sera from FIV-negative SPF cats.









Immunoblot ALVAC





Cat # : PY2 QS4
Vaccine: Alvac-control Alvac-env,gag/prot Controls pre 1st 2nd 3rd 17pc pre 1st 2nd 3rd 17pc FIV- FIV+ 83 kDa










18 kDa







serum dilution: 1:100

























Figure 2.8 FIV-specific immunoblot. Serum samples from ALVAC-env,gaglprot immunized cat #QC3, ALVAC-env,gaglprot combined with ICV immunized cat #QA6 and ALVAC combined with ICV immunized cat #QC5, obtained before and after immunizations, were diluted 1 to 100 in Buffer 3 and incubated with FIV-specific westernblot strips. The positive control was incubated with pooled serum from FIV-infected cats. The negative control was incubated with serum from a SPF cat.










Immunoblot FIV



Cat # QC3 QA6 QC5
Vaccine Alvac-Env,Gag/pro(3X) Alvac-Env,Gag/pro(2X) Alvac-control(2X) Controls ICV(1X ) ICV(1 X) pre 1st 2nd 3rd pre 1st 2nd 3rd pre 1st 2nd 3rd +








24





p24--





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Table 2.4 FIV-specific antibody titers before and after immunizations and challenge.



FIV-specific antibody titera

Cat ID# Vaccine Boost pre- post Mo.postchallenge immunizations 2 8


QA6 ALVAC-env,gag/prot ICV( X) <2 5-6 4-5 4-5 QH3 ALVAC-env,gag/prot ICV( X) <2 5 3-4 4 PY4 ALVAC-env,gaglprot ICV(1X) <2 5 4-5 4-5 QG4 ALVAC(2X) ICV(1X) <2 4-5 5 ND QC5 ALVAC(2X) ICV(1X) <2 3-4 5-6 ND QE4 ALVAC(2X) ICV(1X) <2 3-4 5-6 5-6 QS4 ALVAC-env,gag/prot (3X) <2 <2 5-6 ND QC3 ALVAC-env,gaglprot (3X) <2 <2 <2 ND PY3 ALVAC-env,gag/prot (3X) <2 <2 <2 ND PY2 ALVAC(3X) <2 <2 5-6 5-6 FIV-specific titers expressed as the reciprocal of the highest dilution (in 'Olog) at which FIV specific bands could be detected by immunoblotting. ND- not determined




(Figure 2.8). In contrast, all cats boosted with ICV (group F and G) developed detectable FIV-specific antibody responses. Interestingly, cats primed with ALVAC-env,gag/prot (#QA6) developed approximately 10-fold higher antibody titers than those primed with ALVAC vector alone (#QC5) (Figure 2.8) upon the ICV boost (see Table 2.4)

Selected cats of each group were also tested for the presence of antibody responses to peptides corresponding to the V3 region of the FIV surface envelope glycoprotein by ELISA. This region is thought to be equivalent to the V3 region of HIV which contains the principal neutralizing domain (Pancino et al. 1994). None of the immunized cats exhibited significant levels of antibody titers to the three V3 peptides tested even after three immunizations (data not shown).





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Viral neutralizing antibody responses were measured in selected cats before and

after immunization. The neutralization assay was performed using ConA lymphoblast as

FIV-susceptible cells and FIV, propagated on a feline lymphoid cell line (FL-4) as the

virus inoculum. VN antibody responses were absent before immunization and after

immunization in all cats tested including those boosted with ICV (Table 2.5).







Table 2.5 Viral neutralizing antibody titers after immunization and challenge


VN titera
Vaccine
pre- post- Months postCat ID# immunizations challenge
3 12

ALVAC-env QU1 <5 <5 <5 NT PY1 <5 <5 >100 NT

ALVAC-gag/prot QX3 <5 <5 <5 <5 QQ1 <5 <5 <5 <5 QI1 <5 <5 <5 <5 QL3 <5 <5 <5 <5

ALVAC-env,gag/prot QS4 <5 <5 >100 NT PY3 <5 <5 <5 NT

ALVAC-env,gag/prot QH3 <5 <5 5-20 <5 &ICV QA6 <5 <5 5-20 <5 PY4 <5 <5 5-20 5-20

ALVAC QC4 <5 <5 >100 NT PY2 <5 <5 >100 >100 QA4 <5 <5 <5 NT QE3 <5 <5 <5 NT

ALVAC QG4 <5 <5 >100 NT
&ICV QC5 <5 <5 >100 NT QE4 <5 <5 >100 >100

VN Titer expressed as the reciprocal of the highest final dilution which gave > 50% reduction in reverse transcriptase activity as compared to reverse transcriptase activity observed in control cultures which contained SPF serum. NT= not tested.





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T-helper Lymphoproliferative Responses



Selected cats from each group were tested after the second and third immunization and challenge for lymphocyte proliferation in response to inactivated FIV (Table 2.6). The inactivated FIV preparation used in this assay, included significant amounts of Gag allowing the detection of proliferative responses to both FIV Env and Gag. After two immunizations, no significant levels of FIV-specific lymphoproliferative responses were detected in any of the tested cats. After the third immunization lymphoproliferative responses were detected in two cats. However, the observed levels were low as compared to those detected in cats immunized multiple times with ICV vaccines (S.I. 4-6). This included cat #QC3 immunized with ALVAC-env,gag/prot and cat #PY4 immunized with ALVAC-env,gagprot and boosted with ICV. Upon challenge, lymphoproliferative responses were absent in all of the tested cats even in cats that became viremic except for cat #QX4 which showed low levels of FIV-specific proliferative responses (Table 2.6).



Cvtotoxic T-cell responses



Since viral-vector based vaccines are thought in general to be effective in eliciting CTL responses, FIV-specific CTL responses were measured after each immunization in the peripheral blood of selected cats. PBMC isolated were cultured in the presence of FIV antigen-presenting cells and assayed for their ability to lyse autologous PBMCs infected with FIVr,. FIV-specific CTL activity was detected in one of two cats tested after a single immunization with ALVAC-env,gag/prot (see Table 2.7). After the second and third immunization, CTL activity was detected in some cats of each group immunized with ALVAC-FIV recombinants alone and those immunized with ALVAC-env,gag/prot and boosted with ICV. No major variance between the different immunization schemes, with respect to intensity of CTL activity or percentage of cats displaying CTL activity within





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Table 2.6 T-helper responses to FIV upon immunization and challenge



Stimulation Index

Vaccine Cat ID# Number of Immunizations Post1X 3X challenges


ALVAC-env QL2 1.1 1.2 ND QH4 ND ND 1.0 QC1 ND ND 0.7

ALVAC-gaglprot QX3 1.0 1.3 1.4

ALVAC-env,gag/prot QG3 0.9 1.6 ND QC3 ND 2.6 1.3 PY3 ND 1.6 ND QH5 ND ND 0.7


ALVAC-97TMG Q12 1.1 1.7 ND QX4 ND ND 2.1

ALVAC QC4 1.2 ND ND QE3 ND 1.1 ND QH2 ND ND 1.3

ALVAC-env, PY4 ND 2.4 ND gag/prot &ICV QH3 ND ND 0.7

ALVAC QC5 0.7 1.3 0.7 &ICV QG4 ND 1.0 ND

a 4 weeks post-challenge ND= not determined.





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Table 2.7 FIV specific CTL activity in peripheral blood after immunizations


% specific 51Cr release"

Vaccine Cat ID# Number of Immunizations

IX 2X 3X


QH4 ND 3.9 9.5 ALVAC-env QC1 ND ND 22.9 PY1 ND ND 3.7

QQ1 ND ND 22 ALVAC-gag/prot QA5 ND ND 4 QI1 ND ND 8

QC3 0.1 ND 18.4 ALVAC-env,gag/prot PY3 25 9.9 36.7 QS4 ND 22 7 QH5 ND ND 1

QQ2 ND 25.6 19 ALVAC-97TMG PY5 ND ND 12.5 QI2 ND ND 9.0

QH3 ND 0.4 55.2 ALVAC-env,gaglprot QA6 ND ND 27.3
&ICV PY4 ND ND 8.0

ALVAC&ICV QG4 ND ND 10

QA4 ND ND 7 ALVAC QH2 ND 1.3 ND PY2 ND ND 0.0 QC4 ND ND 10

aPercentage specific release as observed at an average effector to target cell ratio of 1 to 20-30.
ND- not determined





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each group, was observed. Further, cats primed with ALVAC-env,gag/prot and boosted with ICV displayed similar levels of CTL activity as detected in those immunized with ALVAC-env,gag/prot alone. Control cats immunized with ALVAC vector alone or immunized with ALVAC (2X) and boosted with ICV failed to demonstrate significant levels of CTL activity (< 10%).

The detected CTL activity was found to be MHC restricted as effector cells were only capable of lysing autologous FIV-infected target cells and failed to lyse nonautologous FIV-infected target cells (data not shown), thus implying that the detected activity was due to CTL, as opposed to NK cell activity. Further, no lysis was observed using uninfected autologous target cells (data not shown).




Table 2.8 NK activity postchallenge


% specific 5Cr release

Target cell

Vaccine Cat ID# autologous heterologous FL-4 Fet-J PBMC PBMC


ALVAC-env QCL 0 0 10.1 20.7 ALVAC-gag/prot QC3 ND 0 12.5 18.8 ALVAC-env, QX3 0 0 8.7 22.8
gag/prot

ALVAC-97TMG QX4 ND 0 10.7 16.9 ALVAC QC5 ND 0 5.5 17.8
&ICV

ALVAC QC4 ND 12.2 6.5 8.2 ND= not determined





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NK cell activity


NK cells are the principal effector cells in clearance of viral infections early in the course of infection. For this reason, we evaluated levels of NK activity in selected cats at 3.5 weeks postinfection (Table 2.8). NK activity was measured using various target cells including autologous PBMC as negative controls. All cats displayed normal levels of NK activity and no significant differences were observed between the infected and non-infected ALVAC-immunized cats.


Protective efficacy


The presence or absence of FIV following challenge was measured at monthly intervals using several methods. This included the assessment of viral reverse transcriptase

(RT) activity and FIV-specific PCR analysis of cultured PBMC. These assays were also used to test LN and BM cells for the presence of FIV, as these organs function as major reservoirs for the virus. In addition, FIV infection was determined by comparing the level of FIV-specific antibody responses in the serum before and after FIV challenge. FIVspecific immunoblotting (WB) was performed as well as ELISA using a peptide corresponding to the transmembrane (TM) region of the FIV Env. Sera from FIV, infected cats have shown to react strongly to this peptide. Further, VN antibody responses were measured in selected cats before and after FIV challenge. In general, the induction and persistent elevation of FIV-specific antibody responses and VN antibodies are indicative of an active viral infection.

All control cats (n=3) immunized with ALVAC vector alone and boosted with ICV became viremic as assessed by RT for infectious virus and FIV-specific PCR for proviral DNA in peripheral blood and tissue samples (BM and LN cells) (Table 2.9b and Table

2.10). Further, these cats developed high titer VN (>100) responses indicative of active









Table 2.9a Virus isolation (RT and PCR) on PBMC and immunoblot analysis.



Immunizations Months post-challenge pre- postVaccine Cat ID# -3 -1 +1 +2 +3 +4 +8 +12 WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR

QH4
PY1 --- -++ +++ +++ +++ ALVAC-env QO1 --- - + + + + + + + +
QC1 QUl
QIL2 --- -++ +++ +++ +++

QQ1 QA5
ALVAC-gag/prot QU2 --- --- --- --- - --- --- .
QX3 QI1
QL3

QH5 PY3
ALVAC-env, QS4 - - + + + + + + + + + + +
gag/prot QC3
QG3
QE2 --- --- --- ++++ +-+

WB= western blot (FIV-specific), RT= reverse transcriptase, PCR using FIV-specific primers.








Table 2.9b Virus isolation (RT and PCR) on PBMC and immunoblot analysis.


Immunization Months post-challenge pre- postVaccine Cat ID# -3 -1 +1 +2 +3 +4 +8 +12 WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR

QQ2
PY5
ALVAC-97TMG Q02 -++ + + + + + +
QX4 --- --- -++ +++ +++ + QI2 ---+ + ++ + + + + ++
QLA - -

QH2 --- -++ +++ + + +
PY2 --- ++ +++ + ++ +++ +++ ALVAC QA4
QC4 --- --- -+ +++ ++ +
QG5 -- --- -++ +++ ++
QE3

ALVAC-env, QH3 - +- + -- + +-- +-- +
gag/prot&ICV PY4 + - + - + - + - + - +
QA6 --- +-- +-- +-_ +-- +-- +

ALVAC QC5 +-- + + + ++ + + +++ &ICV QG4 -+ -- + + + + + + +++ +++ QFA --- +-- +-- +++ +++ +++ +++ +++

WB= western blot (FIV-specific), RT= reverse transcriptase, PCR using FIV-specific primers.






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Table 2.9 Virus isolation on PBMC and tissue samples and WB and ELISA data



Tissue
Vaccine Cat# Weeks
p.c. PBMC BM LN THY WB/ELISA RT PCR RT PCR RT PCR RT PCR
QH4 27 - + + + -PY1 24 + + - -+ Alvac-env Q01 10 + + + + + + + + ND
QC1 28 -- - -- ND QUi 28 -- -- - ND QL2 24 + + -- ++ ++ -QQ1 29 -- -- - ND QA5 29 -- -- -- ND
Alvac-gag/prot QU2 29 - - - ND
QX3 29 -- -- - ND QI1 29 -- -- - ND QL3 29 -- -- - ND

QH5 28 -- PY3 27 -- -- - ND Alvac-env, QS4 24 + + - + + + -gag,prot QC3 28 -- -
QG3 28 -- -- --
QE2 28 + + -- -+ -+ -+

QQ2 27 - - + -PY5 28 -- Alvac-97TMG QO02 10 + + + + + + + + ND
QX4 25 + +/- -- -- QI2 25 + -- -- --
QL4 27

QH2 10 + +/- + + + + + + ND PY2 39 + + + + - ND Alvac-control QA4 28 - - - -
QC4 26 + + + + + + + + + +
QG5 10 + + + + + + ND QE3 28 - - - -

Alvac-env, QH3 36 + -.. -- - ND gag/prot&ICV PY4 36 + -- -- -- ND
QA6 36 + -- -- -- ND

Alvac-control QC5 10 + + + + - + + ND
&ICV QG4 10 + + + + + + ND
QE4 39 + + -- + + ND ND

WB=western blot, PBMC= peripheral blood mononuclear cells, BM= bone marrow, LN=lymph node, Thy=thymus, ND= not determined.





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viral infection (Table 2.5). In contrast, only 4 of 6 control cats immunized with ALVAC vector alone became viremic as determined by RT, PCR and development of FIV-specific antibodies including VN antibodies. The two other cats (#QA4 and #QE3) in this group tested negative consistently by RT and PCR analysis in peripheral blood samples taken 1, 2, 3, 4 and 7 mo after challenge. In addition, BM, LN and thymus tissues taken from these two cats 7 mo after challenge were negative for virus by RT and PCR analysis (Table 2.9b). These cats also failed to develop FIV-specific antibody responses as assessed by immunoblot (WB) and ELISA and lacked detectable levels of VN antibody responses, further supporting lack of infection in these cats.

In the group immunized with ALVAC-env two of six cats tested negative for virus by RT, and PCR analysis at all time-points postchallenge (Table 2.9a). In addition, these cats failed to develop detectable levels of FIV-specific and VN antibody responses (Table 2.5). Another cat (#QH4) in this group also tested negative by all these criteria up to 6 mo postchallenge but tested positive for virus by PCR analysis in PBMC, BM and LN cells taken 7 mo postchallenge (Table 2.10). The three remaining cats in this group developed viremia upon challenge at a rate similar to that observed in the ALVAC control cats that became infected. Partial protection was also observed in the groups immunized with ALVAC-env,gag/prot and ALVAC-97TMG. Four out of 6 cats immunized with ALVACenv,gag/prot and 3 out of 6 cats immunized with ALVAC-97TMG resisted infection following challenge. The remaining cats in these groups tested positive for virus by all criteria except for cat #QQ2 which tested positive by PCR analysis only in lymph node tissue taken 7 months after challenge (Table 2.9b and Table 2.10).

In contrast, full protection was observed in the group immunized with ALVACgag/prot All six cats tested negative by RT and PCR in peripheral blood and lymphoid tissues. Further, these cats lacked detectable levels of FIV-specific antibodies and VN antibodies up to one year postchallenge ( Table 2.5). Similarly, cats primed with ALVACenv,gag/prot and boosted with ICV tested negative by RT and PCR analysis of peripheral





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blood and lymphoid tissues throughout the vaccine trial (Table 2.9a and 2.10). FIVspecific humoral responses elicited by vaccination remained following challenge but slowly declined thereafter. Interestingly, serum samples obtained three months postchallenge contained low VN antibody titers (5-20) whereas no VN titers were detected in the sera taken from these cats after the third immunization. At 8 mo postchallenge, VN antibody titers persisted at low level in cat #PY4 and could no longer be detected in cat #QA6 and #QH3. The low titer of VN antibodies detected in this group as compared to those detected in infected control cats and the observed delay suggest that these responses were the result of anamnestic responses to the challenge inoculum rather than active viral infection.


In vivo transfer study


As an additional means of analyzing the viral status of ALVAC-gag/prot immunized cats after challenge, two naive SPF kittens were transfused with PBMC, BM and LN cells isolated from cat #QX3 and #QQ1 at 8 months postchallenge. An additional SPF kitten, which served as a positive control, was transfused with cells from FIV-infected control cat #PY2. Following challenge, kittens were monitored for viral infection at monthly intervals by methods analogous to those described previously. Kitten #DH2, which was transfused with cells from FIV, infected cat #PY2, became readily infected as shown by RT, PCR (data not shown), and immunoblotting (Figure 2.9). In contrast, kittens #DH5 and #DE4 which were transfused with cells from ALVAC-gag/prot immunized cats, were negative for virus by RT and PCR up to 5 months after the transfusion in peripheral blood, LN and BM tissues (data not shown). Further, these cats failed to develop FIV-specific humoral responses as determined by immunoblotting (Figure

2.9).



























Figure 2.9 FIV-specific immunoblot. Serum samples obtained before (pre) and 16 weeks (16pc) after transfusion from kittens #DH5 and #DE4, transfused with cells from ALVAC-gag/prot immunized cats, and cat #DH2 transfused with cells from ALVAC-control immunized cat, were diluted 1 to 100 in Buffer 3 and incubated with FIV-specific westernblot strips. The positive control was incubated with pooled serum from FIV-infected cats. The negative control was incubated with serum from a SPF cat.










Cat # DH2 DH5 DE4 cell PY2 QQ1 QX3 Controls inoculum

pre 16pc pre 16pc pre 16pc +















p24-0





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Statistical analysis



The Fisher's exact test was used to analyze the statistical significance of the protective efficacy data (Table 2.11). The infectivity rate of the ALVAC-FIV recombinant immunized groups was compared to that of the ALVAC (n=6) control group alone or to that of the ALVAC control group combined with the ALVAC/ICV (n=3) immunized control group (total n=9). The infectivity rate of ALVAC-env,gag/protlCV immunized cats was compared to either the ALVAC/ICV-immunized control group (n=3) or to both the ALVAC-immunized and ALVAC/ICV-immunized control groups (n=9). Based on this test, immunization schemes employing ALVAC-gag/prot and ALVAC-env,gag/prot together with ICV, showed significant protection as indicated by a P (Probability) value equal or less than 0.05 (Table 2.11). Protection observed by the other immunization schemes was not significant (P value > 0.05).


Table 2.11 Statistical analysis


Viral status

vaccine control P value significant group group
Vaccine +/- +/- (single-tailed)


ALVAC-env 3/3 4/2 0.5 no 3/3 7/2 0.28 no ALVAC-gag/prot 0/6 4/2 0.0303 yes 0/6 7/2 0.00914 yes ALVAC-env,gag/prot 2/4 4/2 0.28 no 2/4 7/2 0.118 no ALVAC-97TMG 3/3 4/2 0.5 no 3/3 7/2 0.28 no ALVAC-env,gag/prot 0/3 3/0 0.05 yes &ICV 0/3 7/2 0.00914 yes





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Discussion


The aim of this study was to evaluate the immunogenicity and protective efficacy of ALVAC-based FIV vaccines alone or in combination with ICV against experimental FIV challenge in cats. ALVAC recombinants tested in this study included ALVAC constructs encoding the FIV Env, the FIV Gag or both the FIV Env and Gag. Additionally, a recombinant was tested which encoded both the FIV Gag and a modified Env in which a putative immunosupressive region had been deleted.

We demonstrated that these recombinants were able to effectively infect nonpermissive feline cells and express the inserted FIV genes. Upon inoculation in cats, ALVAC-specific humoral responses were readily detected. In contrast, FIV-specific humoral responses were detected only in cats that received a booster immunization with ICV. VN antibody titers were undetectable in all cats prior to challenge, even in those boosted with ICV. These observations are consistent with those of ALVAC-based HIV vaccine candidates in macaques, chimpanzees and humans in which humoral responses were weak or undetectable unless the animals received booster immunizations with subunit proteins (Franchini et al. 1995a; Abimiku et al. 1995; Piaoloux et al. 1995; Clements et al. 1996). Also, the induction of VN antibodies required boosting with HIV Env or peptides corresponding to the V3 region. Humoral responses including VN responses have been detected in chimpanzees immunized with ALVAC-HIV-1 recombinants alone (Van der Ryst et al. 1996; Girard et al. 1996). However, these responses were detected after a minimum of four immunizations only. Other FIV vaccine candidates composed of viral vectors including an attenuated adenovirus and a herpesvirus engineered to express the FIV Env, also failed to elicit FIV-specific humoral responses (Gonin et al. 1995; Verschoor et al. 1996).

The induction of humoral responses after the ICV boost is consistent with previous studies in our laboratory in which ICV vaccines were found to elicit FIV-specific humoral





86


responses but failed to induce VN antibody responses after a single immunization. Interestingly, cats immunized with ALVAC-env,gag/prot showed approximately a 10-fold higher immunoblot antibody titer than cats immunized with ALVAC vector alone after the ICV boost. Therefore it is possible that ALVAC-env,gag/prot induced FIV-specific Thelper responses resulting in a more efficient generation of FIV-specific humoral responses after exposure to ICV. This has been reported in other studies in which chimpanzees were immunized with an ALVAC recombinant encoding the HIV Env and boosted with recombinant Env. Chimpanzees immunized with ALVAC-Env produced antibodies following a single recombinant Env immunization, whereas chimpanzees immunized with recombinant Env alone failed to developed such antibodies after a single immunization (Girard et al. 1995). Similar priming of T-helper responses has also been shown in human volunteers immunized with ALVAC-HIV candidate vaccines (Piaoloux et al. 1995; Clements et al. 1996)(Tartaglia, personal communication)

The effectiveness of ALVAC-based vaccines in priming CTL responses as has been reported previously was further confirmed by findings in this study (Cox et al. 1993). Detectable levels of FIV-specific CTL responses were detected even after a single immunization. Although, the phenotype of the effector cells was not determined it was found that the effector cells reacted in a MHC-restricted manner. This finding excludes NK cells as the effector cell and implies a role for CD8' T-lymphocytes known to react in a MHC class I restricted manner. However, we can not exclude a role for CD4' CTL responses since the target cells (autologous PBMC) used in the assay could have presented FIV antigens in the context of both MHC class I and II. In an attempt to identify the FIV epitope recognized by these effector cells, CTL assays were performed using autologous target cells infected with vaccinia recombinants expressing FIV Env or Gag. These experiments did not demonstrate presence of CTL activity (data not shown), possibly due to technical difficulties of the CTL assay itself. The ability of ALVAC-based vaccines to prime CTL responses has been reported in several studies. Mice immunized with an





87


ALVAC-recombinant encoding the HIV-1 Env were shown to elicit CTL responses, including memory T-cell responses (Cox et al. 1993). The effector cells in these studies were characterized as CD8 T-lymphocytes. In addition, specific CTL responses mediated by CD8' T-lymphocytes were detected in 30% of the human volunteers immunized with an ALVAC recombinant encoding the HIV-1 Env (Pialoux et al. 1995; Egan et al. 1995). Interestingly, volunteers immunized with an ALVAC recombinant encoding both the HIV-1 Env and Gag, mounted CTL responses specific for Gag more often than for Env (Lawrence et al. 1996). We were unable to distinguish if this was the case in our study.

In summary, ALVAC-based FIV vaccines were found to differ from inactivated FIV-infected cell vaccines in their ability to elicit cell-mediated and humoral responses. This can be explained, in part, by a difference in the processing and presentation of ALVAC encoded immunogens. Immunogens encoded within ALVAC, require de novo expression within host cells to be presented to the host immune system. In this study, ALVAC immunizations were given intramuscularly and therefore it is likely that the majority of ALVAC infected muscle cells. These cells are capable of presenting immunogens in association with MHC class I and as such are expected to stimulate primarily CTL responses. Additionally, part of the ALVAC inoculum may have been taken up by macrophages or infected cells of the monocyte lineage, such as dendritic cells. These cells are capable of presenting antigens in association with both MHC class I and class II molecules and could therefore have stimulated the generation of both CTL and T-helper cell responses. This is supported by the low level of proliferative responses detected in some of the cats immunized with ALVAC-FIV recombinants alone. The direct stimulation of Bcell responses by ALVAC-encoded immunogens would require the expression and release of the immunogens from ALVAC-infected host cells. This process may have occurred at low level only, since ALVAC-FIV recombinants failed to induce detectable levels of humoral responses. Further, the nature of the immunogen itself may have played a role since ALVAC-recombinants expressing epitopes of viral pathogens other than retroviruses





88


have been shown to effectively elicit humoral responses including VN antibodies to the inserted immunogens (Taylor et al. 1992a, 1992b, 1991).

The protective efficacy ALVAC-based FIV vaccines was evaluated against 50 IDs0 FIV,, (subtype A). This isolate differs only slightly (3% in Env and 1% in the Gag aminoacid coding region) from the FIVvne if., isolate (subtype A) used to generate the ALVAC recombinants and is identical to the vaccine virus. After challenge, complete protection was obtained in all cats immunized with the ALVAC-gag/prot recombinant. The protection observed in this group correlated significantly with immunization as determined by the Fisher's exact test. In addition, virus was not detected in two naive SPF kittens transfused with cells obtained eight months postchallenge from ALVAC-gag/prot immunized cats whereas a kitten transfused with cells from an infected control cat became virus positive. Full protection was also observed in cats immunized with ALVAC-env,gag/prot and boosted with ICV. No significant protection was observed in cats immunized with ALVAC-env, ALVAC-env,gag/prot, and ALVAC-97TMG as determined by the Fisher's exact test (P>0.05). Important to emphasize at this point is the fact that we failed to obtain 100% infectivity in the control group immunized with ALVAC vector alone. Two out of six cats in this group tested virus negative by all criteria throughout the duration of the study. The infective dose (ID50) of the virus challenge inoculum was titrated in cats obtained from a vendor different than the one used in this study. Therefore the true titer of the challenge inoculum may have been less than 50 ID50 explaining the lack of 100% infectivity in the control group. Further, lack of full infectivity could have been due to a general elevation of immune function by the ALVAC vector itself, although, no indication for such an elevation has been reported in any of the vaccine studies conducted with ALVAC. The inability to obtain full infectivity of control animals has also been observed in other FIV vaccine trials as well as SIV and HIV vaccine trials. In general, this is attributed to differences in genetic background which causes some animals to be more resistant to infection than others. For example, the HLA and HLA related genes have been





89


implemented in influencing the intensity and specificity of host immune-responses (Haynes et al. 1996). Also of some relevance to this issue are recent studies in which individuals with a homozygous defect in the CKR-5 loci encoding a coreceptor for HIV, were less likely found to become infected with HIV than individuals without this defect (Liu et al. 1996). Similar genetic based mechanism(s), yet to be determined, may be behind the observed variance in susceptibility to FIV infection in cats.

What constituted protective immunity in the ALVAC-gag/prot immunized cats is not clear. At the time of challenge and after challenge, the sera of these cats tested negative for Gag-specific and VN antibodies. Therefore, the presence of these antibodies did not appear to be crucial to the protection observed in this group. Moreover, vaccine trials in which cats were immunized with Gag proteins lacked protective efficacy despite the presence of Gag-specific antibodies. In fact, in these studies immunized cats showed enhanced infection. Thus, even if these responses would have been present they may not have played a role in the observed protection. However, we can not exclude a role for antibodies directed against Gag epitopes other than those tested for in the immunoblot and VN assays. Recent studies have demonstrated that HIV Gag proteins are displayed on the surface of infected cells (Ikatu et al. 1989; Shang et al. 1991). Similarly, cells infected with ALVAC-gag/prot may have expressed Gag on the host-cell surface and resulted in the induction of Gag-specific antibody dependent cell-mediated cytotoxic responses (ADCC). ADCC responses have been detected in HIV-infected individuals but these responses were predominantly directed against Env epitopes and attempts to demonstrate Gag-specific ADCC have failed thus far (Koup et al. 1988; OToole and Lowdell 1990). Protection in the ALVAC-gag/prot, may have been accomplished through Gag specific CTL responses. The induction of Gag-specific CTL responses has been reported in infected cats as well as cats immunized with ICV (Song et al. 1992; Flynn et al. 1995a). The importance of these responses in terms of protection, however, is still unknown. A vaccine based on a synthetic peptide containing epitopes of both the FIV Env and Gag was shown to




Full Text
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BIOGRAPHICAL SKETCH
Marinka Tellier was born in the summer of 1968 and grew up on the islands
surrounded by the salty waters of the Noordzee and the Schelde in the Netherlands. At
the age of 4 she was first sent to school by her parents not knowing that this episode would
continue for the next 24 years of her life. In the first eight she visited the Prinsenhoven
School in Middelburg. She then went to the Christelijke Scholengemeenschap Walcheren
from which she graduated in 1986. She continued her education at the College of
Pharmacy at the State University of Utrecht in the Netherlands. After enjoying student life
for a period of five years she graduated with a M.S. degree in pharmacy in 1991.
In the summer of 1993, she started her graduate work under the supervision of Dr.
Yamamoto with no experience in the feline field. In fact, at the time she did not even
realize there was such a thing as feline immunodeficiency virus. In the last three years she
has become impressed by this little creature that encompasses a little more than 9000
basepairs. Now, a little wiser and a little more confused, she will go on and keep on
wondering if our curiosity will do to us what it did to the cat.
121


103
(approx. 2.4 %) than the difference displayed at the Env (approx. 21 %). These findings
resemble those of ALVAC-HIV-1 trials in macaques (Abimiku et al. 1995). Macaques
immunized with ALVAC-HIV-1 recombinants and boosted with subumt proteins were
partially protected and had a delay in infection after challenge with distinctly heterologous
HIV-2. Prior to challenge these macaques exhibited HIV-1 specific cell-mediated
responses and VN responses that effectively prevented HIV-1 infection but failed to
prevent HIV-2 infection in vitro.
In summary, prime/boost protocols involving priming with ALVAC-HV
recombinants followed by boosting with inactivated FIV-infected cells vaccines can elicit
partial protection or delay in infection of distinctly heterologous FIV isolates from
heterologous subtypes. The exact immune-correlates of protection are unclear. Although,
the findings in our study suggest a role for both cell mediated and humoral responses.


28
vaccinia strain on primary chick embryo-fibroblast. As a result, the MVA virus is severely
attenuated and lacks replication on nonavian cell lines (Sutter et al. 1994).
An alternative to the use of attenuated vaccinia viruses is the use of poxviruses that
exhibit species specificity (Baxby and Paoletti 1992). These include the suipoxviruses,
capri poxviruses and avipoxviruses. Avipoxviruses only productively infect cells of avian
origin and were originally developed as vaccine vectors for the poultry industry.
Unexpectedly, it was found that nonavian cells inoculated with avipoxvectors expressed the
inserted antigen despite the absence of vector replication (Taylor et al 1992b). Moreover, it
was found that these vectors when administered to nonavian species were capable of
eliciting protective immunity. It is now understood that these viruses undergo abortive
infection in nonavian cells resulting in expression of early gene and inserted gene products.
Two avipoxviruses, the fowlpoxvirus and canary poxvirus, have been developed as
vaccine vectors (Plotkin et al. 1995). ALVAC represents a vector derived from an
attenuated canarypoxvirus strain originally used to immunize canaries. The protective
potential of recombinant ALVAC vectors has been tested against several viral pathogens
including rabies, measles, Japanese encephalitis virus (JEV), cytomegalovirus (CMV), and
equine influenza virus (EIV) (Table 1.5) (Cadoz et al. 1992; Konishi et al. 1994; Taylor et
al. 1992a, 1992b; Gonczol et al. 1995). The majority of these ALVAC-based vaccines
were shown to elicit antigen-specific antibody responses, including VN antibody responses
specific for the inserted antigens. More important, cell-mediated immunity was also
induced in individuals immunized with these vaccines. In one study, lymphocytes from
human volunteers immunized with an ALVAC-rabies recombinant had proliferative
responses to the rabies antigens, demonstrating the induction of antigen specific T-helper
cells (Cadoz et al. 1992; Taylor et al. 1991). This same recombinant vaccine was used to
evaluate the ability of ALVAC-based vaccines to elicit memory immune responses in dogs.


30
It was found that dogs could be protected from a rabies challenge given 36 weeks after a
single ALVAC-rabies immunization (Cadoz et al. 1992).
The efficacy of ALVAC-based vaccines has also been tested against a number of
retroviral pathogens (Table 1.6). Immunization with ALVAC recombinants expressing the
Envelope and Gag proteins of the feline leukemia virus (FeLV) protected cats against
experimental infection with FeLV (Tartagliaet al. 1993). Protection was afforded in the
absence of detectable VN antibody responses. Interestingly, protected animals developed
FeLV-specific VN antibody titers at 9-12 weeks postchallenge whereas control animals
failed to develop VN antibody titers. The data presented in this study should be interpreted
with some caution since no analysis of FeLV by PCR was performed on tissues of the
protected animals (Tartagliaet al. 1993). Thus, immunization may not have resulted in
sterilizing immunity but resulted in a reduced viral load explaining the development of VN
titers at 9-12 weeks postchallenge. Further, the evaluation of cell-mediated responses was
not included in this study.
ALVAC-based vaccines have protective efficacy against human T cell
leukemia/lymphoma virus type I (HTLV-1), the causative agent of adultT cell leukemia and
tropical spastic paraparesis in humans (Barre-Sinoussi et al. 1983). In the rabbit model
used, it was found that two inoculations with ALVAC-based vaccines encoding the
HTLV-1 envelope protein (ALVAC-gp65), protected rabbits against challenge infection
with HTLV-1 infected cells. Protective immunity was afforded in the absence of HTLV-1
VN antibody responses. Again, this study lacked the evaluation of cell-mediated reponses.
Interestingly, rabbits boosted with baculovirus expressed envelope protein (gp65) in
addition to ALVAC-gp65 immunizations failed to be protected (Franchini et al. 1995b).
The rhesus macaque model was used to assess the prophylactic efficacy of
ALVAC-based vaccines against HIV-2 challenge (Franchini et al. 1995a). It should be
kept in mind that rhesus macaques can be infected with HIV-2 but do not develop an AIDS
like syndrome. Rhesus macaques were given two immunizations with ALVAC


8
capsid (CA), matrix (MA) and nucleocore (NC) proteins by the viral protease encoded
within thepol gene (Elder et al. 1993). Expression of the Gag-Pol precursor protein from
the unspliced full length mRNA is accomplished by a ribosomal frameshift that prevents
termination of translation at the gag stop codon (Morikawa and Bishop 1992). The
efficiency of this shift is about 5%, so that the production of Gag protein is 20 fold higher
than that of Gag-Pol polyprotein. The Gag-Pol precursor protein is proteolytically
processed into the viral protease (PR), the reverse transcriptase (RT), the deoxyundine
triphosphatase (DU) and the integrase (IN) proteins (Elder et al. 1993).
FIV replication
The first step in the replication of FIV is the attachment of the virus to the cell
receptor. For HIV, the major receptor has been identified as the CD4 molecule, present on
T-helper lymphocytes (Dagleish et al. 1984; Klatzman et al. 1984). In addition, the CD26
molecule and the recently described fusin and CKR-5 molecules have been reported to play
a role in HIV attachment and fusion (Callebaut et al. 1993; Feng et al. 1996; Alkhatib
1996). The fusin is thought to act as a coreceptor for T cell line-tropic HIV strains whereas
the CKR-5 is thought to actas a coreceptor for macrophage-tropic HIV strains (Feng et al.
1996; Alkhatib 1996). The target receptor(s) for FIV, however, is still unknown.
Nonlymphoid feline cell lines transfected with cDNA encoding the feline CD4 (fCD4)
protein failed to support productive infection indicating that the fCD4 alone is not sufficient
(Nonmine et al. 1993). Others have proposed a putative role for a receptor homologous to
the human CD9 molecule that is expressed on both haematopoietic and nonhaematopoietic
cells (Willetet al. 1994; Hosie et al. 1993; Boucheix and Beiot 1988). Anti-CD9 antibodies
effectively block replication of FIV infection on lymphoid cells and ectopic expression of
CD9 on feline lymphoma cells causes an enhancement of viral infection with cell culture


LIST OF ABBREVIATIONS
ADCC
Antibody dependent cell-mediated cytotoxicity
AIDS
Acquired immunodeficiency syndrome
BM
Bone marrow
BSA
Bovine serum albumin
CTL
Cytotoxic T-lymphocyte
DNA
Deoxyribonucleic acid
EDTA
Ethylenediaminetetraacetate
EUSA
Enzyme-linked immunosorbent assay
FACS
Fluorescence-activated cell sorting
FITC
Fluorescein isothiocyanate
FIV
Feline immunodeficiency virus
HIV
Human immunodeficiency virus
LN
Lymph node
MHC
Major histocompatibility complex
PBL
Peripheral blood lymphocytes
PBMC
Peripheral blood mononuclear cells
PBS
Phosphate-buffered saline
PCR
Polymerase chain reaction
RNA
Ribonucleic acid
RT
Reverse transcriptase
SDS
Sodium dodecyl sulfate
SIV
Simian immunodeficiency virus


3
macaques, the emergence of HIV in humans is thought to be caused by cross-species
transmission. This is supported by the fact that HIV-2 genetically closely resembles the
SIV_ and SlV^-isolates, and HIV-1 more closely resembles the SIVcpz-isolate (Hirsch
and Johnson 1994; Franchini etal. 1987).
Table 1.1 The Lenti viruses
Virus
subtypes
species
Clinical signs of disease
EIAV
horse
anaemia
fever
weight loss
Maedi-visna
sheep
encephalomyelitis
wasting
pneumonia
CAEV
goats
arthritis
encephalomyeli tis
wasting
BIV
cows
lymphadenopathy
lymphocytosis
wasting
FIV
cats
immunodeficiency
opportunistic infections
neurological disorders
HIV
HIV-1
human
immunodeficiency
lymphadenopathy
neurological syndrome
opportunistic infections
HIV-2
human
i mmunodeficiency
1 y mphadenopathy
opportunistic infections
SIV
SIV
mac
SIV
smm
SlV^m
macaques
sooty mangabey
African green monkey
immunodeficiency
neurological disease
SIVmnd
mandrill
no obvious clinical signs
SIVsyk
SIV^
Sykes monkey
chimpanzee
of disease


93
obtained with ALVAC recombinants, in combination with protein based booster
immunizations is similar to that observed in other animal models. Cynomolgus monkeys
immunized with ALVAC-env,gag/prot (HIV-2) alone failed to be protected whereas those
immunized with ALVAC-env,gag/prot and boosted with recombinant Env were partially
protected from HIV-2 challenge (Biberfeld et al. 1994).
In summary, protective immunity can be obtained with ALVAC-based FIV vaccines
encoding Gag or ALVAC-based FIV vaccines encoding Env and Gag in combination with
ICV. This is the first study to report the induction of protective immunity against
experimental FIV challenge in cats utilizing a viral vector-based vaccine. Protection
obtained with ALVAC-based vaccines encoding Gag may have occurred via cell-mediated
responses including CTL and ADCC, or other mechanisms. Protection in ALVAC-
env,gag/prot and ICV immunized cats may have been mediated by these same mechanisms.
Additionally, humoral responses including low but significant VN antibody titers may have
contributed to the protection observed in this group.


Table 2.3 Proliferation responses to T-cell mitogens (ConA and SEA).
Stimulation Index
Vaccine
Cat ID#
Number of immunizations
Pre- IX
Immunization
3X
Post-
Challenge3
ConA
SEA
ConA
SEA
ConA
SEA
ConA
SEA
ALVAC-ewv
QH4
2.8
26.7
7.9
17.4
22.5
24.9
2.5
3.1
PY1
4.3
29.0
6.6
12.6
4.0
4.0
3.2
3.1
A L V A C-gag/>prot
QA5
7.5
16.9
32.1
77.4
30.3
14.4
22.7
23.2
QI1
50
93.8
2.2
7.5
25.8
28.2
18.3
17.8
ALVAC-env,
QS4
6
29
3.1
6.7
35.0
35.2
37.1
36.7
gag/prot
QG3
2.9
6.9
1.0
2.3
38.1
35.0
30.7
22.7
ALVAC-97TMG
QQ2
2.7
9.2
1.0
2.0
16.8
18.0
17.2
12.5
QL4
78.4
99.0
9.6
20.3
29.4
27.2
18.8
17.2
ALVAC-env,
PY4
13.4
26.8
4.0
15.9
20.5
19.7
63.2
36.7
gag/prot &ICV
ALVAC
QH2
4.2
7.2
2.4
5.5
9.5
9
28.7
26.2
QA4
46
65
5.5
9.3
44.3
52.8
15.2
17.3
ALVAC
QG4
4
8
12.5
23.5
7.4
7.4
25.4
23.5
&ICV
3 weeks post challenge


55
Cell
lane
PI4 Crfk FetJ
FIV+ Alvac- FI V-
Ctrl Env Gag
Ml 2 3 4 5
X + + "
450 bp
Figure 2.2 FIV-env specific PCR on CrFK inoculated with ALVAC-recombmants.
Photograph of PCR products following electrophoresis on a 1.0 % agarose gel in Tris
acetate-buffer(40mMTris-acetate, 1 mM EDTA, pH 7.6). LaneM, \ DNA EcoRI/Hindlll
marker (lpg/lane). Lane 1, positive control, FIV-env specific PCR on DNA extracted
from FL4 (FIV+) cells. Lane 2, FIV-Env specific PCR on CrFK cells inoculated with
ALVAC-control. Lane 3, FI\-env specific PCR on CrFK cells infected with ALVAC-env.
Lane 4, FIV-cnv specific PCR on CrFK cells infected with ALVAC-gag/prot. Lane 5,
negative control, FIV-Env specific PCR on Fet-J (FlV)cells.


IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION OF
CANARYPOXVIRUS(ALVAC)-BASED FIV VACCINES IN CATS
By
MARIA C. TELLIER
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
1996


37
vector evaluated in current trials of HIV vaccines, is the canarypoxvirus vector, ALVAC
(Baxby et al. 1992; Perkus et al. 1995).Canarypoxvirus (ALVAC)-based vaccines are
considered safe due to their restricted host-range and their inability to undergo full-
replication in cells of non-avian origin (Baxby et al. 1992). The efficacy of ALVAC-based
vaccines against lentiviruses has been tested in several animal models. Rhesus macaques
immunized with ALVAC-recombinants expressing Env and Gag of HIV-2 were protected
from infection with homologous HIV-2 (Franchini et al. 1995a). Additionally, ALVAC-
HIV-1 vaccines have been proven effective against experimental infection with homologous
HIV-1 in a small number of chimpanzees tested (Girard et al. 1995, 1996; Van der Ryst et
al. 1996). Although, the number of animals in these trials was small, these findings
suggest that ALVAC-based vaccines can induce protective immunity against lentiviral
infection.
Hence, the main objective of this study was to assess the immunogenicity and
protective efficacy of recombinant canarypoxvirus (ALVAC)-vectored FIV vaccines alone
or in combination with conventional inactivated FIV-infected cell vaccines. Four distinct
ALVAC-FIV recombinants were tested. This included recombinants that encoded the FIV
Env (ALVAC-env), the FIV Gag and Prot (ALVAC-gag/prot) or both the FIV Env, Gag
and Prot (ALVAC-e/iv,gag/prot). Also included was a recombinant that encoded the FIV
Gag and a modified FIV Env from which a putative immunosuppressive region had been
deleted (ALVAC-977MG).
This study was conducted to address the following specific aims:
I. In vitro evaluation of ALVAC-FIV recombinant expression.
Determine if ALVAC-FIV recombinants are able to infect non-avian, non-permissive, feline
cells and properly express the inserted FIV gene constructs in these cells.


For Ella and Bram


TAE
Tris, EDTA buffer
TEMED
N,N,N\N-tetramethylethylenediamine
THY
Thymus
Tris
T ris(hydroxymethyl)aminomethan
VN
viral neutralizing
WB
western blot
IX


98
Results
In this study, ALVAC-env,gag/prot/lCV immunized cats, which were previously
shown to resist challenge with FIVPet a homologous subtype strain (subtype A), were
given a second challenge with 75 IDS0 FIVBang, a subtype B virus. Additionally, two
FlVPet-infected control cats were challenged with FIVBang to determine if active infection
could prevent superinfection with the FIVBang isolate. As presented in Table 3.1, all
nonimmunized/noninfected control cats became readily viremic after FI VBang challenge. In
contrast, ALV AC-env, gag!protHCV immunized cat #QA6 remained virus negative as
determined by virus isolation (RT) and PCR in peripheral blood up to four months
postchallenge. Cats #PY4 and #QH3 remained virus negative as determined by virus
isolation (RT) in peripheral blood but tested positive by PCR at three months and four
months postchallenge, respectively. Nucleotide sequence analysis of the amplified PCR
product from PBMC revealed HVBang-specific sequences only (data not shown). The two
FIVPct -infected control cats (#QE4 and #PY2) tested positive by RT and PCR before and
after the FlVBang challenge. However, nucleotide sequence analysis of the FlV-specific
PCR products obtained from these cats only verified FlVPet-specific sequences up to 4
months after the FIVBang challenge (data not shown).
Prior to challenge, FIV-specific antibodies, as determined by immunoblot, were
present in all ALVAC-env,gag/prot/lCV immunized cats. Similarly, sera from FIVPet -
infected control cats (#PY2 and #QE4) contained FIV-specific antibodies which were
slightly higher in titer than those detected in the ALV AC-env,gag/prot/\CV immunized
group (Table3.1). As expected, sera from the naive control cats (#EJ2, #DH3, #GU5)
did not contain detectable levels of FIV-specific antibodies before the FIVBang challenge
(Table 3.1). Viral neutralizing antibodies to either FIVPet or FIVBang were absent in all
naive control cats and in two of three ALV AC-env,gag/protflCV-imm\m7£d cats (#QA6,
#QH3). In contrast, sera of the FlVPet-infected control cats (#PY2 and #QE4) had high


Figure 2.8 FIV-specific immunoblot. Serum samples from ALVAC-env,gag/prot immunized cat #QC3, ALVAC-env,gag/prot
combined with ICV immunized cat #QA6 and ALVAC combined with ICV immunized cat #QC5, obtained before and after
immunizations, were diluted 1 to 100 in Buffer 3 and incubated with FIV-specific westemblot strips. The positive control was incubated
with pooled serum from FI V-infected cats. The negative control was incubated with serum from a SPF cat.


71
Viral neutralizing antibody responses were measured in selected cats before and
after immunization. The neutralization assay was performed using ConA lymphoblast as
FIV-susceptible cells and FIVPet propagated on a feline lymphoid cell line (FL-4) as the
virus inoculum. VN antibody responses were absent before immunization and after
immunization in all cats tested including those boosted with ICY (Table 2.5).
Table 2.5 Viral neutralizing antibody titers after immunization and challenge
VN titer1*
Vaccine
pre- post- Months post-
Cat ID# immunizations challenge
3
12
ALVAC-cnv
QU1
<5
<5
<5
NT
PY1
<5
<5
>100
NT
ALV AC-gag/prot
QX3
<5
<5
<5
<5
QQi
<5
<5
<5
<5
QIl
<5
<5
<5
<5
QL3
<5
<5
<5
<5
ALV AC-env, gag/prot
QS4
<5
<5
>100
NT
PY3
<5
<5
<5
NT
ALVA C-env, gag/prot
QH3
<5
<5
5-20
<5
&ICV
QA6
<5
<5
5-20
<5
PY4
<5
<5
5-20
5-20
ALVAC
QC4
<5
<5
>100
NT
PY2
<5
<5
>100
>100
QA4
<5
<5
<5
NT
QE3
<5
<5
<5
NT
ALVAC
QG4
<5
<5
>100
NT
&ICV
QC5
<5
<5
>100
NT
QE4
<5
<5
>100
>100
a VN Titer expressed as the reciprocal of the highest final dilution which gave > 50%
reduction in reverse transcriptase activity as compared to reverse transcriptase activity
observed in control cultures which contained SPF serum. NT= not tested.


60
Further, the expression of the ALVAC encoded FIV Env as a membrane associated
protein and of the FIV-Gag core protein as intracellular protein was demonstrated by
indirect immunofluorescence on CrFK cells inoculated with the ALVAC-FIV recombinants
(Figure 2.5). At 48 h postinfection, cells infected with ALVAC-env showed fluorescence
predominantly at the surface (panel C) whereas cells infected with ALVAC-gag/prot,
ALVAC-env,gag/prot and ALVAC-97TMG showed strong fluorescence in the cytoplasm
(see Figure 2.5, panel D-F). Control cells, uninfected CrFK and CrFK infected with
ALVAC vector alone, did not show immunofluorescence (panel A and B).
In vivo assessment
A total of 36 SPF cats were used to evaluate the prophylactic efficacy of
immunizations protocols employing ALVAC-FIV recombinants alone or in combination
with ICV. Cats were divided into 7 groups (A-G) and inoculated three times at monthly
intervals with either ALVAC-env (n=6), ALV AC-gag/prot (n=6), ALVA C-env,gag/prot
(n=6), ALVAC-97TMG (n=6) or ALVAC vector alone (n=6). Cats in group F and G
were immunized twice with ALVA C-env, gag/prot (n=3) or ALVAC (n=3), respectively,
followed by a boost with inactivated FIV-infected cell vaccine (see Table 2.1). All cats
were challenged 4 weeks after the final immunization with 50 IDS0 FIVPet. The FIVPet
isolate is a subtype A virus, and differs 3% in the Env and 1% in the Gag protein coding
region from the FIVvi]]efranche isolate (subtype A) used to generate the ALVAC recombinants.
General immunologic parameters
No adverse effects and no significant changes in blood chemistry (CBC, HgB,
PCV, TPP) were noticed in any of the cats upon immunization with ALVAC-FIV
recombinants or after boosting with inactivated FIV-infected cell vaccine (data not shown).


108
Baxby, D., and Paoletti, E., 1992. Potential use of non-replicating vectors as recombinants
vaccines. Vaccine 10, 8-9.
Becker, Y., 1994. HIV-1 proteins in infected cells determine the presentation of viral
peptides by HLA class I and class II molecules and the nature of the cellular and
humoral antiviral immune responses a review. Virus Genes 8, 249-270.
Benviniste, R.E., Arthur, L.O., Tsai, C.C., Sowder, R., Copeland, T.D., Henderson,
L.E., and Oroszian, S. 1986. Isolation of a lentivirus from a macaque with
lymphoma: comparison with HTLV-III/LAV and other lentiviruses. L Virol. 60,
483-490.
Biberfeld, G., Andersson S., Putkonen, P., Thortensson, R., Matikalo, B., Franchini,
G., Tartagha, J. and Paoletti, E. 1994. Protective immunity in cynomolgous
monkeys immunized with canarypox HIV-2 vaccines. Int. Conf. AIDS, 10(1), 74
(Abstr. no. 247A).
Boucheix, C. and Beoit, P. 1988. CD(9) antigen: will platelet physiology help to explain
the function of a surface molecule during haematopoietic differentation? Nouv.
Rev. Fr. Hematol. 30, 201.
Brown, E.W., Yuhiki, N., Pakcer, C., and OBrien, S.J. 1994. A lion lentivirus related to
feline immunodeficiency virus: epidemiologic and phylogenetic aspects. J. Virol. 68,
5953-5968.
Brown, W.C., Bissey, L., Logan, K.S., Pedersen, N.C., Elder, J.H., and Collison,
E.W. 1991. Feline immunodeficiency infects both CD4+ and CD8+ T lymphocytes.
L Virol. 65, 3359-3364.
Brunner, D., and Pederson, N.C. 1989. Infection of peritoneal macrophages in vitro and
in vivo with feline immunodeficiency virus. L Virol. 63, 5483-5488.
Byars, N.E., and Allison, A.C. 1987. Adjuvant formulation for use in vaccines to elicit
both cell-mediated and humoral immunity. Vaccine 5, 223-228.
Cadoz, M., Styrady, A., Meignier, M., Taylor, J., Tartglia, J., Poaletti, E., and Plotkin,
S. 1992. Immunization with recombinant canarypox virus expressing rabies
glycoprotein. Lancet 339, 1429-1432.
Callanan, J.J., Hosie, M.J., and Jarret, O. 1991. Transmission of feline
immunodeficiency virus infection from mother to kitten. Vet. Rec. 139, 293-295.
Callebaut, C., Krust, B., Jacotot, E., and Hovanessian, A.G. 1993. T cell activation
antigen, CD26, as a cofactor for entry of HIV in CD4+ cells [see comments]. Science
262, 2045-2050.
Chan, W.L., Rodgers, A., Hancock, R.D., Taffs, F., Kitchin, P., Farrar, G., and Liew,
F.Y. 1992. Protection in simian immunodeficiency virus-vaccinated monkeys
correlates with anti-HLA class I antibody response. J. Exp. Med. 176, 1203-1207.
Clavel, F., Greetard, D., Brun-Vezinet, F., Charmaret, S., Rey, M.A., Santos-Ferreria,
M.O., Laurent, A.G., Dauguet, C., Katlama, C., and Rouzioux, C. 1986. Isolation


74
Table 2.7 FI V specific CTL activity in peripheral blood after immunizations
% specific 51Cr release3
Vaccine Cat ID# Number of Immunizations
IX
2X
3X
QH4
ND
3.9
9.5
ALVAC-cnv
QC1
ND
ND
22.9
PY1
ND
ND
3.7
QQl
ND
ND
22
ALV AC-gag/prot
QA5
ND
ND
4
QI1
ND
ND
8
QC3
0.1
ND
18.4
ALV AC-env, gag!prot
PY3
25
9.9
36.7
QS4
ND
22
7
QH5
ND
ND
1
QQ2
ND
25.6
19
ALVA C-97TMG
PY5
ND
ND
12.5
QI2
ND
ND
9.0
QH3
ND
0.4
55.2
ALV AC-env, gag/prot
QA6
ND
ND
27.3
&ICV
PY4
ND
ND
8.0
ALVAC&ICV
QG4
ND
ND
10
QA4
ND
ND
7
ALVAC
QH2
ND
1.3
ND
PY2
ND
ND
0.0
QC4
ND
ND
10
a Percentage specific release as observed at an average effector to target cell ratio
of 1 to 20-30.
ND= not determined


80
viral infection (Table 2.5). In contrast, only 4 of 6 control cats immunized with ALVAC
vector alone became viremic as determined by RT, PCR and development of FIV-specific
antibodies including VN antibodies. The two other cats (#QA4 and #QE3) in this group
tested negative consistently by RT and PCR analysis in peripheral blood samples taken 1,
2, 3, 4 and 7 mo after challenge. In addition, BM, LN and thymus tissues taken from
these two cats 7 mo after challenge were negative for virus by RT and PCR analysis (Table
2.9b). These cats also failed to develop FIV-specific antibody responses as assessed by
immunoblot (WB) and ELISA and lacked detectable levels of VN antibody responses,
further supporting lack of infection in these cats.
In the group immumzed with ALVAC-e/iv two of six cats tested negative for virus
by RT, and PCR analysis at all time-points postchallenge (Table 2.9a). In addition, these
cats failed to develop detectable levels of FIV-specific and VN antibody responses (Table
2.5). Another cat (#QH4) in this group also tested negative by all these criteria up to 6 mo
postchallenge but tested positive for virus by PCR analysis in PBMC, BM and LN cells
taken 7 mo postchallenge (Table 2.10). The three remaining cats in this group developed
viremia upon challenge at a rate similar to that observed in the ALVAC control cats that
became infected. Partial protection was also observed in the groups immunized with
ALVAC-env,gag/prot and ALVAC-97TMG. Four out of 6 cats immunized with ALVAC-
env,gag/prot and 3 out of 6 cats immunized with ALVAC-97TMG resisted infection
following challenge. The remaining cats in these groups tested positive for virus by all
criteria except for cat #QQ2 which tested positive by PCR analysis only in lymph node
tissue taken 7 months after challenge (Table 2.9b and Table 2.10).
In contrast, full protection was observed in the group immunized with ALVAC-
gag/prot All six cats tested negative by RT and PCR in peripheral blood and lymphoid
tissues. Further, these cats lacked detectable levels of FIV-specific antibodies and VN
antibodies up to one year postchallenge ( Table 2.5). Similarly, cats primed with ALVAC-
env,gag/prot and boosted with ICV tested negative by RT and PCR analysis of peripheral


18
uninfected thymocytes alone also remained virus-negative (Verschoor et al. 1994). Thus,
immune responses against cellular antigens and not viral antigens may have been
responsible for the observed protection in this study.
Other unsuccessful FIV vaccine trials include those based on FIV subunit proteins
and synthetic peptides corresponding to FIV epitopes (Table 1.4). Vaccines composed of
nonglycosylated FIV Env produced in Escherichiacoli and glycosylated Env produced in a
baculovirus system failed to protect cats against low dose challenge (Lutz et al. 1995).
Similar results were obtained with vaccines consisting of bacterial produced Env fragments
fused to galactokinase or glutathione-S-transferase (Verschoor et al. 1996). Also
unsuccessful were vaccines composed of synthetic peptides corresponding to the V3 region
of the FIV surface envelope protein (SU) (Lombardi et al. 1994). This region resembles
the V3 loop of the HIV-1 Env surface protein which is thought to contain the principal
neutralizing determinant (PND) of HIV (Pancino et al. 1994). Following immunization, all
cats developed V3-specific antibodies however no protection against low dose challenge
was observed (Lombardi et al. 1994). Interestingly, immunized animals showed
enhancement of infection compared to controls as indicated by a higher virus load in the
peripheral blood. Enhancement of infection as a result of immunization was also observed
in cats immunized with a FIV envelope produced by recombinant vaccinia virus (Siebelink
et al. 1995). In addition, subunit vaccines consisting of recombinant Gag protein p24 or
native purified p24, lacked prophylactic efficacy despite the presence of high anti-p24
antibody titers (Hosie et al. 1992).
FIV vaccine trials using recombinant vector based vaccines have also been
unsuccessful (Table 1.4). This included a trial in which the efficacy of a replicative
defective adenovirus engineered to express the env gene of FIV was evaluated. After
immunization, Env-specific antibody responses could not be detected and all cats became
infected upon challenge (Gonin et al. 1995). Likewise, vaccine protocols involving


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59


31
recombinants expressing HIV-2 Env and Gag/Prot followed by two immunizations with
recombinant Env (gpl60) and a final ALVAC-HIV-2 Env, Gag/Prot boost. Upon
challenge with 100ID50 HIV-2, both macaques were free of viremiaand tested negative for
the virus by virus isolation and virus specific PCR. Prior to challenge, HIV-2-specific
CTL responses were detected. In addition, one of the macaques had a transient VN
antibody titer prior to challenge. After challenge both animals developed significant VN
antibody titers as such resembling findings in the ALVAC-FeLV trials (Franchini et al.
1995a; Tartagliaetal. 1993). The macaques were given a second HIV-2 challenge of 100
IDS0 at 7 months post the primary challenge without any intervening boosters. At this time,
both macaques became infected despite the presence of VN antibody titers at the time of
challenge. Cytotoxic T-lymphocyte responses were not measured at the time of the second
challenge, however, both animals were positive for HIV-2 specific CTL activity at one
month following the second challenge.
To evaluate the strain specificity of the immunity generated upon immunization with
ALVAC-based vaccines, rhesus macaques were immunized with an ALVAC-recombinant
expressing Env, Gag/Prot of the HIV-1,^ isolate and challenged with a distinctly
heterologous HIV-2 isolate (> 40% difference) (Abimiku et al. 1995). Two rhesus
macaques received a combination of primary ALVAC-HIV-1^. immunization and
subsequent subunit boosts with either p24 and gpl60 or a tandem V3-peptide. Both
animals developed virus-specific CTL and VN antibodies after immunization. Upon
challenge, the animal boosted with the V3-peptide was considered partially protected, based
on the low VN titers and the absence of virus by virus isolation and PCR at six months
postchallenge. The other animal exhibited a delay by two months and had lower VN
antibody titers as compared to control animals. All control animals became positive at 1
month after challenge and remained virus positive throughout the study. Perhaps the most
interesting observation made in this study was that the VN antibody responses generated
upon immunization did not cross react with the HIV-2 isolate used in the challenge


14
by demonstrating that a boy, injected with material from a cowpox pustula, failed to
develop disease upon exposure to smallpox. This technique became known as vaccination
(Jenner 1798).
Though the underlying mechanisms were not known at the time, it is now
understood that success of vaccination lies in the ability of the immune system to generate
long lasting immunity. This immunity is mediated by memory B and T lymphocytes which
are capable of rapid anamnestic responses upon exposure to foreign antigens. Responses
mediated by B cells include the production of specific antibodies that could prevent the
entry of pathogens into host cells by interfering with microbial attachment or fusion upon
attachment to host cells. These antibodies, in respect to viral pathogens, have been defined
as viral neutralizing (VN) antibodies. Further, antibodies may directly destroy microbes by
complement mediated lysis or promote phagocytosis by macrophages and natural killer
cells through opsonization. T-lymphocyte responses include those mediated by T-helper
cells and T-cytotoxic lymphocytes (CTLs). T-helper cells are of the CD4+ phenotype and
recognize exogenously produced antigens presented in the context of MHC-class II found
predominantly on B-lymphocytes and macrophages. Upon recognition these cells produce
interleukins to facilitate the activation of macrophages and maturation of B cells into
antibody producing plasma cells. Cytotoxic T-lymphocytes of the CD8+ phenotype
function by the direct destruction of infected cells displaying foreign antigens in association
with MHC-class I molecules. Class I MHC molecules are found on the majority of cells
and present endogenously synthesized antigens. As such CTL responses are especially
critical to the clearance of intracellular bacteria and virally infected cells. Together, humoral
and cell mediated responses are capable of preventing the invasion of pathogens.
Several different types of vaccines have been developed. Most commonly used
vaccines against viral pathogens are composed of live attenuated viruses, inactivated whole
virus, or inactivated virus infected cells. The majority of these vaccines induce VN
antibody responses and some, in particular attenuated live viruses, also induce cell-


39
and nucleocapsid protein (NC, p7). The promotor-FIV gene constructs were then cloned
into the pC6L donor plasmid that contained ALVAC C6 flanking regions to enable insertion
of the F1V promotor-gene constructs into the ALVAC non-essential C6 genetic locus.
Prior to insertion into the donor plasmid, TsNT sequence elements were removed from the
FIV coding sequences without altering the amino acid sequence. TsNT motifs are
recognized by the poxvirus transcriptional apparatus as termination signals at early times
postinfection and their retention is known to result in diminished expression of foreign
gene products (Yuen and Moss 1987; Earl et al. 1990). The donor plasmid was then
transfected into ALVAC-infected chicken embryo fibroblast cells to generate full ALVAC-
recombinants by homologous recombination. Recombinants were subjected to several
rounds of plaque purification and analyzed by nucleotide sequence analysis to ensure
proper insertion.
ALVAC-FTV recombinants generated for this trial included ALVAC-chv containing
the whole FIV env coding region, ALVAC-gag/prot containing the FIV gag and prot
coding regions and ALVAC-env, gag/prot containing the FIV env, gag and prot coding
regions. The ALVAC-97TMG recombinant contained the FIV gag and prot gene and a
modified env coding region, of which a 714bp fragment encoding a putative
immunosupressive element, had been deleted (Figure 2.1).
In vitro Expression of the FIV Env and Gag
The ability of ALVAC-FIV recombinants to infect non-permissive non-avian cells
was evaluated on CrFK cells, a feline fibroblastic kidney cell line. Petn dishes seeded with
a monolayer of CrFK cells were inoculated with ALVAC-FIV recombinants or ALVAC
vector alone at a multiplicity of infection (m.o.i.) of 10 for 1 h at 37C in a humidified
atmosphere containing 5% COz. The ALVAC inoculum was removed and cells were
incubated with fresh medium for an additional 48 h at 37C. At 48 h, cells were removed


26
(EIAV), another len ti virus (Wang et al. 1994). This phenomenon has also been observed
with other nonlentiviral vaccines in particular macrophage and monocyte-tropic viruses
(Halstead and ORourke 1977). It is thought to be mediated by viral specific antibodies
elicited upon immunization that can facilitate viral transport to susceptible host cells by
binding of the Fc portion of antibodies to the surface of macrophages.
The role of VN antibodies as a factor in preventing FIV infection is not clear.
Although, a majority of cats immunized with FlVPet FL-4/FeTl vaccines showed high
levels of VN antibody responses at challenge, some cats were protected in their absence.
In addition vaccine trials employing inactivated MBM cells infected with the FIV M2
isolate, a vaccine similar to the FlVPet FL-4/FeTl vaccines, induced protective immunity, in
the absence of detectable VN antibody titers (Mattuecci et al. 1996).
In summary, protection against low dose experimental challenge with FIV may in
part be mediated by antiviral humoral responses. However, cell-mediated responses such
as CTL or underlying immune effector activities i.e. chemokines may also play a role. In
fact, recent studies demonstrated the induction of FIV specific CTL responses directed
against Env and Gag epitopes in cats immunized with the FIVPet (Fl-4) inactivated infected
cell vaccine (Flynn et al. 1995a). The importance of these responses in protective
immunity however remains to be established. Induction of FIV specific CTL responses
was also observ ed in cats immunized with a synthetic peptide vaccine corresponding to the
FIV V3 region. These cats, however, became infected upon challenge even though V3-
specific CTL responses were detected at the time of challenge (Flynn et al. 1994, 1995b).
Recombinant poxvirus-based vaccines
Members of the poxvirus family comprise a group of large viruses that infect a
number of species. Poxviruses are enveloped and contain a single double stranded DNA
genome of 130 to 300kbp. These viruses encode their own enzymes required for viral


117
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vectored vaccines. Dev. Biol. Stand. 84, 165-170.
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1993. AIDS-associated encephalopathy with experimental feline immunodeficiency
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1993. Serological, biological, and molecular characterization of new Zealand white
rabbits infected by intraperitoneal inoculation with cell-free human immunodeficiency
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Rigby, M.A., Holmes, E.C., Pistello, M., Mackay, A., Brown, A.J., and Neil, J.C.
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Rosenthal, A., Jenning, M.B., Cotterman, R., Schmerl, S., Yee, J.L., Joye, S., and
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63
Table 2.2 CD4/CD8 ratios before and after immunization and challenge
Cat ID#
Immunizations
Before After
-4 -1
months
post-challenge
+3
+5
+6
+7
+8
+ 10
+ 12
QH4
4.7
4.4
3.2
3.0
3.3f
PY1
4.3
4.2
2.4
2.4
2.5f
QOl
3.9
2.6
3.5f
QC1
3.3
3.3
2.9
2.5
l.SF
QU1
ND
3.8
3.6
3.3
2.5f
QL2
3.6
3.3
2.2
1.9
1.7f
QH5
6.7
4.2
3.8
2.6
2.5F
PY3
5.1
4.9
4.3
3.3
2.6F
QS4
6.0
4.8
2.6
2.3
2.3F
QC3
3.2
2.6
3.6
2.1
2.0F
QG3
5.0
4.0
3.6
3.2
2.4f
QE2
3.1
ND
3.0
2.0f
QQl
4.9
2.7
2.1
1.9
1.6
2.3
QA5
4.3
2.2
2.6
2.5
2.9
2.5
QU2
4.0
2.6
2.4
2.0
2.3
2.9
QX3
2.0
2.5
2.0
1.8
1.8
2.3
QI1
6.1
3.9
3.9
3.3
3.3
3.9
QL3
4.9
2.0
2.6
1.9
2.0
2.2
QQ2
2.7
3.4
2.7
2.0
2.2f
PY5
5.1
3.8
2.5
2.1
l.^
Q02
3.3
4.0
2.4f
QX4
1.3
ND
1.5
1.1
1.0F
QI2
4.9
2.5
2.1
2.0
l.SF
QL4
4.4
4.1
3.8
2.3
2.5f
QH2
3.9
3.5
4.6f
PY2
3.3
2.9
3.5
2.7
3.8
2.5
QA4
2.9
2.6
2.4
1.7
1.8f
QC4
4.2
3.1
2.7
2.4
1.6f
QG5
4.0
2.5
2.7f
QE3
2.1
2.3
2.7
1.7
2.0f
QH3
5.5
4.1
4.0
3.4
2.0
4.3
PY4
2.8
4.0
3.1
2.6
2.6
2.4
QA6
4.3
3.6
2.9
2.5
2.1
2.7
QC5
2.8
2.0
2.1F
QG4
5.1
3.5
2.9f
QE4
2.6
3.1
1.8
2.0
1.6
2.5
F=Final testing of CD4/CD8 ratio before animal was euthanized.


51
DNA was extracted as described. In the PCR reaction, the following FIV env specific
primer sets were used: 5-GAAATGTATAATATTGCTGG-3 and 5-
GAATTGATTTTGATTACATCC-3. The PCR reactions were carried out in 50 pi
reaction mixtures containing 1.0 pg genomic DNA, 50 mM KC1, 10 mM Tris-HCl (pH
8.3), 2.5 mM MgCl2, 0.2 mM of each dNTP (dATP, dCTP, dTTP and dGTP), 20 pmol of
each primer, and 2.5 units of Taq DNA polymerase. The reaction mixture was incubated
for 5 min at 94C and cycled 30 times through 94C for 1 min, 55C for 1 min, 72C for
1.5 min, followed by 10 mm at 72C. The specificity of the PCR-amplified 455bp product
was verified by nucleotide sequence analysis.
DNA sequencing
DNA sequencing was performed using the Amplicycle sequencing kit (Perkin
Elmer, Norwalk, CT). Primers (approximately 10 pM) were labeled at the 5-end with 20
pCi y-32P ATP (6000Ci/mmol) and 20 U Polynucleotide kinase in a final reaction volume
of 6.2 pi for 10 min at 37C. The reactions were terminated by incubation at 90C for 5
min. For the sequencing reactions, four separate reaction mixture were prepared
containing: 1 pi labeled primer, 4 pi 10X reaction buffer and approximately 10-50 fmol of
PCR template and 2 pi of either G, A, T, or C termination mix. Reaction mixtures were
then incubated at 95C for 15 seconds, cycled 25 times at 95C for 1 min, 68C for 1 min,
followed by 7 mm at 78C. Upon cycling, 4 pi of formamide stop solution containing
0.05% bromophenol blue and xylene cyanol were added. The reactions were analyzed on a
6% polyacry lamide gel containing 7 M urea and IX TBE.


Table 3.1 Immune parameters and viral status before and after FIVBang challenge.
virus status
Cat# Vaccine pre-2nd-challengea post-2nd-challengeb RT/PCR
WB VNPet VNBang WB VNPel VnBang (wks p.c.)
2 6 10 14 18
QA6
ALVAC-env,gag/prot & ICV
+(4-5)
<5
<5
+
5-20
<5
-/-
-/-
-/-
-/-
-/-
PY4
ALVAC-env,gag/prot & ICV
+(4-5)
5-20
5-20
+
5-20
<5
-/-
-/-
-/-
+7+
-/+
QH3
ALVAC-env,gag/prot & ICV
+(4)
<5
<5
+
5-20
<5
-/-
-/-
-/-
-/-
-/+
EJ2
_
_
ND
<5
+
ND
<5
-/-
+/+
+/+
+/+
NT
GU5
-
-
ND
<5
+
ND
<5
-/-
+/+
+/+
+/+
NT
DH3
-
-
ND
<5
+
ND
>100
-/-
+/+
+/+
+/+
+/+
PY2
ALVAC
+(5-6)
>100
<5
+
>100
<5
+/+
-/-
+/+
+/+
-/+
QE4
ALVAC&ICV
+(5-6)
>100
<5
+
>100
<5
+/+
-/+ +/+
+/+
+/+
a serum samples taken at the day of challenge.
b serum samples taken 3-4 months post-2nd-challenge.
c weakly positive by RT (2X background cpm)
ND= not determined
NT= not tested, animal euthanized.


13
depletion of CD4+ T-lymphocytes and susceptibility to opportunistic pathogens. In
addition, several alternative models have been developed with some success. For example,
transient HIV infection of SCID mice reconstituted with human lymphocytes or peripheral
blood mononuclear cells (PBMC) and the infection of rabbits with HIV-1 (Reina et al.
1993; Mosier et al. 1991; Namikawa et al. 1988). These models, however, are highly
artificial and the relevance to HIV pathogenesis in humans should be interpreted with
caution.
The infection of FIV in domestic cats offers several advantages over the models
discussed above. First, FIV is a natural pathogen of cats and the pathogenesis closely
resembles that of HIV in man. Obvious advantages of the feline model include the
availability and costs which allow the use of larger study groups. Especially relevant to
vaccine development is the availability of a wide variety of FIV subtypes which is lacking
in the SIV model. FIV isolates have been grouped into 4 subtypes (A-D) versus 7 for HIV
(Sodora et al. 1994; Kakinuma et al. 1995; Rigby et al. 1993). This grouping is primarily
based on antigenic diversity in the Env and Gag proteins. Hence, the FIV model provides
a means to evaluate the protective efficacy of vaccine strategies against multiple subtypes
and as such has implications to the development of multiple subtype HIV vaccines.
Further, FIV like HIV replication is sensitive to antiviral drugs such as AZT and protease
inhibitors (North etal. 1989, 1990). Thus, FIV infection in cats also provides a model to
assess the efficacy of these and other newly developed drugs.
Vaccine development
Vaccine development initiated with the work of Dr. Edward Jenner in 1798. He
observed that milkmaids which had recovered from cowpox did not contract the more
virulent smallpox. Based on this observation he postulated that smallpox infection of man
could be prevented by prior exposure to cowpox. He successfully proved this hypothesis


CHAPTER IV
SUMMARY AND FUTURE STUDIES
Synopsis
Comparative studies on the efficacy and immunogemcity of various ALVAC-based
FIV vaccines alone or in combination with inactivated FIV-infected cell vaccines support
the following conclusions:
(1) ALVAC-based FIV vaccines are ineffective in priming B-cell responses but
effective in priming cytotoxic T-cell responses and low level T-helper responses specific
for the inserted FIV antigens.
(2) A booster immunization with ICV following immunization with ALVAC-based
FIV vaccines enhances immunogemcity as determined by induction of detectable FIV-
specific humoral responses.
(3) ALVAC-based FIV vaccines encoding the FIV Gag and Prot can induce
protective immunity against experimental challenge with slightly heterologous FIV isolates.
(4) ALVAC-based FIV vaccines combined with ICV can induce protective
immunity against a slightly heterologous FIV isolate and delay infection with distinctly
heterologous FIV isolates of other subtypes.
The data obtained in this study were generated from a relatively small study group.
Future studies, should include larger numbers of animals to add to the statistical
significance of the obtained data. Further, the duration of the ALVAC-FIV-induced
protective immunity and the efficacy against various routes of challenge, in particular the
104


24
1986). Furthermore, the type of adjuvant may have affected the protective efficacy, as it
influences the proportion and intensity of humoral vs. cell-mediated responses upon
immunization (Byars and Allison 1987).
Additional support for a role of Env-specific and VN antibody responses in the
protection obtained with the FTVPet FL-4/FcT 1 vaccines comes from passive immunization
studies. In these studies, cats were passively immunized with pooled sera from cats
immunized with inactivated FI VPet infected T-cells (FL-4) or sera from cats experimentally
infected with FIVPet (Hohdatsu et al. 1993). Control cats received either phosphate
buffered saline (PBS) or pooled sera from cats immumzed with inactivated uninfected FeTl
cells (related to FL-4 cell line) or uninfected 3201 cells (allogeneic feline T-cell line). Upon
low dose homologous challenge with 5 IDS0, all control cats became infected whereas 3 out
of 3 cats passively immunized with FlVPl;t (FL-4) vaccine sera and 4 out of 4 cats
immunized with FIV infected cat sera did not. Protected cats showed VN antibody titers
averaging between 100-200 whereas uninfected cell or PBS control sera had no VN
antibody titers. These findings resemble those reported for SIV. Cynomolgus monkeys
passively immunized with sera from SI V^ infected and HIV-2 vaccinated monkeys were
protected in the presence of high titer antiviral antibodies against homologous challenge at
10 to 100 IDS0 (Putkonen et al. 1991). In contrast, passive immunization studies in
macaques immunized with SIV^ indicated that the levels of anti-cellular and not anti-viral
antibodies correlated mostly with protection (Rosenthal et al. 1992).
Findings from studies evaluating protective immunity in kittens bom to queens
vaccinated with FIVPet FL-4/FeTl vaccines also imply a role for VN responses (Pu et al.
1995). In these studies, kittens received colostrum/milk from either vaccinated or sham
vaccinated queens and were challenged shortly after birth with low dose homologous
challenge of 5 IDS0. It was found that only those kittens born to and nursed by vaccinated
queens were protected. Furthermore, protected kittens showed high levels of VN titers
(500-5000) at five days postparturition. The role of transplancental maternal antibody


89
implemented in influencing the intensity and specificity of host immune-responses (Haynes
et al. 1996). Also of some relevance to this issue are recent studies in which individuals
with a homozygous defect in the CKR-5 loci encoding a coreceptor for HIV, were less
likely found to become infected with HIV than individuals without this defect (Liu et al.
1996). Similar genetic based mechanism(s), yet to be determined, may be behind the
observed variance in susceptibility to FIV infection in cats.
What constituted protective immunity in the ALVAC-gag/prot immunized cats is not
clear. At the time of challenge and after challenge, the sera of these cats tested negative for
Gag-specific and VN antibodies. Therefore, the presence of these antibodies did not
appear to be crucial to the protection observed in this group. Moreover, vaccine trials in
which cats were immunized with Gag proteins lacked protective efficacy despite the
presence of Gag-specific antibodies. In fact, in these studies immumzed cats showed
enhanced infection. Thus, even if these responses would have been present they may not
have played a role in the observed protection. However, we can not exclude a role for
antibodies directed against Gag epitopes other than those tested for in the immunoblot and
VN assays. Recent studies have demonstrated that HIV Gag proteins are displayed on the
surface of infected cells (Ikatu et al. 1989; Shang et al. 1991). Similarly, cells infected
with ALV AC-gag/prot may have expressed Gag on the host-cell surface and resulted in the
induction of Gag-specific antibody dependent cell-mediated cytotoxic responses (ADCC).
ADCC responses have been detected in HIV-infected individuals but these responses were
predominantly directed against Env epitopes and attempts to demonstrate Gag-specific
ADCC have failed thus far (Koup et al. 1988; OToole and Lowdell 1990). Protection in
the ALVAC-gag/prot, may have been accomplished through Gag specific CTL responses.
The induction of Gag-specific CTL responses has been reported in infected cats as well as
cats immumzed with ICV (Song et al. 1992; Flynn et al. 1995a). The importance of these
responses in terms of protection, however, is still unknown. A vaccine based on a
synthetic peptide containing epitopes of both the FIV Env and Gag was shown to


10
FIV cell tropism
FIV has a broad cell tropism and infects cells of both lymphoid and
monocyte/macrophage origins. In contrast to HIV, which is thought to primarily replicate
in CD4+ T-lymphocytes and not in CD8+ T-lymphocytes, FIV productively infects both
CD4+ and CD8+ T-lymphocytes (Brown et al. 1991). Additionally, FIV has been shown to
replicate in B-cells, thus further supporting the view that the feline CD4 receptor is not the
primary cell receptor for FIV, as it is for HIV (English et al. 1993; Norimine et al. 1993).
The macrophage/monocyte cell types supporting FIV replication include peritoneal
macrophages, Kupffer cells in the liver, microglial cells, astrocytes, and endothelial cells in
the central nervous system (Steffan et al. 1994; Dow et al. 1990; Martin et al. 1995;
Brunner and Pederson 1989). Furthermore, a number of FIV isolates have been shown to
infect cells of nonlymphoid origin including Crandell feline kidney cells (CrFK) and feline
tongue cells (Fc3Tg) (Yamamoto et al. 1988a).
FIV epidemiology and pathogenesis
FIV has been isolated from cats worldwide. The virus infects domestic cats (Felis
catus) and is species specific (Yamamoto et al. 1988b, 1989). FIV-related viruses have
been isolated from several wild felids including the African lion (Panthera leo), and the
Pallas cat (Felis tnanul) (Barr et al. 1989, 1995; Poli et al. 1995; Brown et al. 1994).
Furthermore, serologic surveys in African and Asian lions revealed that the serum of the
majority of these animals reacted positively with FIV (Brown et al. 1994).
The prevalence of FIV varies throughout the world. In North America the average
incidence is estimated at 1.4% in healthy animals and 7.4% in diseased cats (Yamamoto et
al. 1989; Shelton et al. 1990). The incidence of infections is the highest in free roaming,
outdoor male cats. Since FIV is shed in the saliva, the major route of transmission is most


CHAPTER II
IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION OF
CANARYPOXVIRUS (ALVAC)-BASED FIV VACCINES AGAINST HOMOLOGOUS
FIV CHALLENGE
Introduction
The feline immunodeficiency virus (FIV) is the causative agent of an immuno
deficiency syndrome in cats (Pedersen et al. 1987). The immunological and pathological
changes observed in FIV infected cats closely resemble those observed in humans infected
with HIV the causative agent of AIDS (Yamamoto et al. 1988b; Ackley et al. 1990). Based
on these similarities FIV infection in cats has become a valuable model for the evaluation of
vaccine and prophylactic strategies. Similar to HIV, it is still unknown what constitutes
protective immunity against FIV. However, vaccine protection against experimental FIV
infection in cats has been achieved with conventional vaccines such as inactivated whole-
virus and inactivated FI V-infected cell vaccines (Yamamoto et al. 1991, 1993; Hosie et al.
1995; Verschoor et al. 1995). This vaccine approach has also been successful against
lentiviral infection in other animal models, such as SIV in macaques and HIV in
chimpanzees (Murphey-Corb et al. 1986; Fultz et al. 1992). The use of such vaccine
approach in humans, however, may not be feasible because of safety issues, such as
incomplete inactivation of vaccine virus which could potentially lead to infection of
immunized subjects.
An alternative to the use of conventional vaccines, is the use of viral vectors based
vaccines which can be engineered to encode specific components of these viruses. One
36


34
enhanced VN antibody responses, ADCC, and CTL responses as compared to
immunization with either ALVAC- HlV^gplO or rgpl20SF2 alone (Clements et al. 1996).
In a follow-up study, volunteers received immunizations with an ALVAC
recombinant expressing both HIV-1 Env and Gag and a boost with recombinant Env
(Pialoux et al. 1995; Clements et al. 1996). The immunogenicity resembled that of the
previous studies (Lawrence et al. 1996). Interestingly, Gag-specific CTL responses were
detected in the majority of subjects whereas Env-specific CTL-responses were only
detected in a small percentage of the subjects. Together, these studies demonstrate that
immunization schemes involving ALVAC-based HIV vaccines, in combination with whole
protein boosts, are safe and can elicit both humoral and cell-mediated responses specific for
the inserted immunogens.


2
MW was named a lenti- (slow) virus (Sigurdsson 1954). This virus has since become
the prototype of the lentivirus genus. In 1974, a virus similar to MW was identified in
goats, the caprine arthritis encephalitis virus (CAEV). CAEV infection presents as chronic
inflammation of the joints in adult goats and progressive encephalopathy in younger goats
(Clements et al. 1980; Cork 1974).
Thus, several animal lentiviruses had been long known before HIV. However, it
was the discovery of HIV that resulted in an increased interest in these viruses and led to
the search and isolation of lentiviruses in other species. For example, a number of
lentiviruses were isolated from monkeys. These viruses have collectively been named the
simian immunodeficiency viruses (SIV) and are specified by the particular monkey species
they have been isolated from (Table 1.1) (Huet et al. 1990; Kanki et al. 1985; Ohta et al.
1988; Tjusimotoetal. 1988; Hirsch etal. 1993; Peeters et al. 1992; Fultz et al. 1986). In
contrast to HIV infection of man, SIV infection of natural host African monkey species, is
relatively nonpathogemc. However, the exception to this is SIV infection in Asian
macaques (Daniel et al 1985; Benvisti et al. 1986). Infected Asian macaques develop an
acquired immunodeficiency syndrome similar to that of HIV infected individuals
(Murphey-Corb et al. 1986). Interestingly, most of the nonpathogenic SIV strains have
been isolated from monkeys in the wild. SIV^, however, has only been isolated from
macaques held in captivity and has never been isolated from this species in its natural
habitat. Since these monkeys are of Asian origin whereas other SIV infected monkeys are
of African origin it has been suggested that this virus was transmitted to macaques during
captivity from an African monkey species, most likely the mangebeys (Murphey-Corb et al.
1986).
The second human lentivirus, now known as HIV-2, was first isolated in 1986
(Clavel et al. 1986). HIV-2 is predominantly found in West-African prostitutes and also
causes AIDS although milder in its pathogenesis as marked by a longer incubation period
and a lower rate of transmission (Marlink et al. 1994). Much like SIV infection of Asian


56
Cell
FetJ
Crfk
FI4
FI V-
Alvac-
FIV+
Ctrl Env Gag
lane
M
1
2 3 4
5
X

- +
+
Figure 2.3 FIV-gag specific PCR on CrFK inoculated with ALVAC-recombinants.
Photograph of PCR products following electrophoresis on a 1.0 % agarose gel in Tris
acetate-buffer (40mM Tris-acetate, 1 mMEDTA,pH 7.6). Lane M, marker EcoRl/Hindlll
cutDNA (1 pg). Lane 1, negative control, FI V-gag specific PCR on DNA extracted from
Fet-J (FIV ) cells. Lane 2, FI V-gag specific PCR on CrFK cells inoculated with ALVAC-
control. Lane 3, FIV-gag specific PCR on CrFK cells infected with ALVAC-env. Lane 4,
FTV-gag specific PCR on CrFK cells infected with ALVAC-gag/prot.. Lane 5, positive
control, FIV-gag specific PCR on F14 (FIV+)cells.


Immunoblot ALVAC
Cat # : PY2
Vaccine; Alvac-control
pre 1st 2nd 3rd 17pc
83 kDa
18 kDa
serum dilution: 1:1 00
Alvac-env,gag/prot


33
ALVAC recombinant vaccine encoding Env of the HIV-1^. strain and boosted with
recombinant envelope glycoproteins (gpl60) of the HIV-1N1N and HIV-1^, both classified
as subtype B strains. The animals were challenge intravenously with HIV-1SF2 which is
also classified as a subtype B virus but differs significantly from the HIV-1^ strains.
Upon challenge, virus was isolated from both vaccinated animals. However, in
comparison to the infected control animals the vaccinated ammals had a lower viral load
(Girard etal. 1995)
In summary, ALVAC-based recombinants were effective against lentiviral infection
in several animal models. However, it is not clear as to what constituted vaccine
protection. Some of these studies suggest a role for VN antibody responses. On the other
hand, other studies showed protection in the absence of detectable VN antibody responses.
Conflicting results may also stem from the fact that only a limited number of ammals was
used in these trials. Furthermore, the assessment of immune responses, in particular cell-
mediated responses, was frequently omitted in these trials. Nevertheless, the data obtained
from these animal trials are promising and studies on the safety and immunogemcity of
these vectors in human volunteers have been initiated. Thus far, no adverse effects have
been reported in human subjects immunized with an ALVAC-recombinant expressing HIV
Env (gpl60), ALVAC-HIVMNgpl60, followed by a boost with recombinant gpl60
(Clements et al. 1996, Lawrence et al. 1996). Further, it was found that immumzation
with this ALVAC recombinant alone failed to induce VN antibody responses. However,
VN antibodies were detected in most subjects after the rgp 160N1N/IjU boost. Env-specific T-
cell proliferative responses were detected in a small percentage of subjects after ALVAC
immunizations and in all subjects following rgpl60N1NiI AI boosts. The presence of HIV
Env-specific CTL activity was detected in some of the subjects, even without the subunit
boost (Pialoux et al. 1995). In a similar study, immune responses elicited with ALVAC-
HlV^.gplO alone was compared to those elicited by ALVAC-HIVN1Ngp 160 priming
followed by a HIV-1 rgpl20SF2 boost. Boosting with envelope protein significantly


91
have resulted in immune responses against Env epitopes other than those generated upon
immunization with ICV and inactivated whole virus.
The lack of protective immunity in the group immunized with ALVAC-env,gag/prot
is somewhat surprising. It is possible that the inclusion of Env interfered with efficient
presentation of Gag and as a result failed to properly prime the immune system. Immune
responses directed against certain Env epitopes have been reported to cause enhancement of
infection and as such could have negated the protective efficacy (Hosie et al. 1992;
Siebelinket al. 1995; Osterhaus et al. 1996). This enhancement, however, was thought to
be mediated by VN antibodies. In our study, VN antibodies were not detected prior to
challenge. Further, virus isolation data did not show differences in the viral load among
infected cats in the ALVAC-env group and infected cats in the control group immunized
with ALVAC vector alone. Alternatively, a putative immunosupressive region in the
transmembrane portion of Env could have interfered with the development of protective
immune responses. However, cats immunized with the AUVAC-97TMG, a recombinant in
which this region had been removed were readily infected at a same ratio as the ALVAC-
env,gag/prot -immunized cats, implying that this region did not play a role. However, other
immunosupressive regions located elsewhere than in the Env transmembrane region could
have attributed to the reduction in immunogemcity of ALVAC-env,gag/prot recombinants.
Similar to our findings, ALVAC recombinants expressing both the Env and Gag of HIV-2
failed to protect cynomolgus monkeys from HIV-2 challenge (Biberfeld et al. 1994). The
challenge in this trial, however, was given by the mucosal route. Opposing our findings are
studies in the chimpanzee model. Chimpanzees immunized with ALVAC recombinants
expressing HIV-1 Env and Gag were protected from homologous HIV challenge (Van der
Ryst et al. 1996; Girard et al. 1996). These animals, however, exhibited HIV-1-specific
humoral responses including VN antibodies prior to challenge. Therefore, it would be of
interest to determine if a higher dose of ALVAC-env,gag/prot or an increased number of
immunizations could elicit such antibody responses in cats and enhance protective efficacy.


88
have been shown to effectively elicit humoral responses including VN antibodies to the
inserted immunogens (Taylor et al. 1992a, 1992b, 1991).
The protective efficacy ALVAC-based FIV vaccines was evaluated against 50 IDS0
FIVPet (subtype A). This isolate differs only slightly (3% in Env and 1% in the Gag amino-
acid coding region) from the FIVviU(. franche isolate (subtype A) used to generate the ALVAC
recombinants and is identical to the vaccine virus. After challenge, complete protection was
obtained in all cats immunized with the ALVAC-gag/prot recombinant. The protection
observed in this group correlated significantly with immunization as determined by the
Fishers exact test. In addition, virus was not detected in two naive SPF kittens transfused
with cells obtained eight months postchallenge from ALVA C-gag/prot immunized cats
whereas a kitten transfused with cells from an infected control cat became virus positive.
Full protection was also observed in cats immunized with ALVAC-env,gag/prot and
boosted with ICV. No significant protection was observed in cats immunized with
ALVAC-env, ALVAC-env,gag/prot, and ALVAC-9777VG as determined by the Fishers
exact test (R>0.05). Important to emphasize at this point is the fact that we failed to obtain
100% infectivity in the control group immunized with ALVAC vector alone. Two out of
six cats in this group tested virus negative by all entena throughout the duration of the
study. The infective dose (IDS0) of the virus challenge inoculum was titrated in cats
obtained from a vendor different than the one used in this study. Therefore the true titer of
the challenge inoculum may have been less than 50 IDS0 explaining the lack of 100%
infectivity in the control group. Further, lack of full infectivity could have been due to a
general elevation of immune function by the ALVAC vector itself, although, no indication
for such an elevation has been reported in any of the vaccine studies conducted with
ALVAC. The inability to obtain full infectivity of control animals has also been observed
in other FIV vaccine trials as well as SIV and HIV vaccine trials. In general, this is
attributed to differences in genetic background which causes some animals to be more
resistant to infection than others. For example, the HLA and HLA related genes have been


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of
Doctor in Philosophy
IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION OF
CANARYPOXVIRUS (ALVAC)-BASED FIV VACCINES IN CATS
By
Maria C. Tellier
December, 1996
Chairman: Dr. J.K. Yamamoto
Major Department: Veterinary'Medicine
The infection of cats with the feline immunodeficiency virus (FIV) provides a
valuable animal model for the assessment of therapeutic and vaccine strategies against
human immunodeficiency virus (HIV) in man. A promising candidate vaccine tested
presently in human volunteers is the recombinant canarypoxvirus vector ALVAC. This
vector has also been used with some success against HIV infection in the macaque and
chimpanzee models. Herein, the efficacy of ALVAC-based vaccines was evaluated against
experimental FIV infection in cats. Two approaches were evaluated which included
ALVAC-based FIV vaccines alone or in combination with conventional inactivated FIV-
infected cell vaccine (ICV).
Immunization schemes employing ALVAC-FIV recombinants alone effectively
induced FIV-specific cytotoxic T-cell responses. However, these schemes failed to induce
humoral responses including viral neutralizing antibody responses. Immunization schemes
employing ALVAC-FIV recombinants combined with conventional inactivated F1V-
infected cell vaccine induced FIV-specific cytotoxic T-cell responses and FIV-specific
humoral responses but lacked detectable viral neutralizing antibody responses.
x


7
Miyazawa 1993). These genes are organized in the order of 5-gag-pol-env-3, typical of
all replication-competent retroviruses. In addition, lentiviruses contain several small open
reading frames (ORFs) that encode for auxiliary proteins. At least 4 ORFs have been
identified for FIV which may encode for regulatory proteins similar to those described for
the primate lentiviruses (Miyazawa et al. 1993; Olmsted et al. 1989b). Flanking the
proviral genome are long terminal repeat (LTR) regions which are crucial for integration of
the proviral DNA into the cellular genome. These regions also contain enhancer and
promoter elements which are required for efficient transcription of the retroviral genome, as
well as, a polyadenylation signal sequence (Phillips et al. 1992; Talbott et al. 1989).
Transcription of the integrated proviral DNA genome initiates at the 5LTR to
generate full length viral mRNA transcripts (Miyazawa et al. 1993). Initially, these
transcripts will undergo multiple splicing and give rise to small mRNAs that encode for
regulatory proteins such as Tat and Rev. The Tat protein facilitates gene expression from
the 5LTR. Tat activity is essential for replication of all primate lentiviruses. In contrast,
Tat is not essential for replication of FIV in T-lymphoblast cells (Sparger et al. 1992;
Miyazawa et al. 1993). The Rev protein, encoded by all members of the lentiviridae, is
responsible for shifting viral gene expression from early regulatory proteins to that of
structural and enzymatic proteins. It accomplishes this by binding to the Rev-responsive
element (RRE) in the env coding region of single spliced and unspliced full length mRNAs.
In doing so, it promotes the stability and transport of incompletely spliced mRNAs from
the nucleus to the cytoplasm (Phillips et al. 1992; Cochrane et al. 1990; Hammarskjold et
al. 1989; Stephens et al. 1992). The single spliced messages give rise to the Env precursor
protein which is cleaved into the transmembrane glycoprotein (TM) and the outer surface
glycoprotein (SU) by cellular proteases (Stephens etal. 1991; Talbott etal. 1989).
The unspliced full length mRNA serves as both a template for the Gag and Gag-Pol
proteins and as genome that is packaged into the viral core. Regular translation of full
length message gives rise to the Gag precursor protein which is cleaved into the mature


LIST OF FIGURES
Figure page
1.1 Electron micrograph of FIV x70,000 6
2.1 Schematic representation of ALVAC constructs 40
2.2 PCR products of CrFK cells infected with ALVAC-env recombinants 55
2.3 PCR products of CrFK cells infected with AlAJ AC-gag/prot recombinants 56
2.4 RT-PCR products of CrFK cells infected with ALVAC-FIV recombinants 57
2.5 Immunofluoresence on CrFK cells infected with ALVAC-FIV recombinants 59
2.6 FACS analysis of CD4 and CD8 staining of PBMC 62
2.7 Immunoblot of ALVAC-specific humoral responses 67
2.8 Immunoblot of FIV-specific humoral responses 69
2.9 Immunoblot of FIV-specific humoral responses in kittens 83
3.1 Phylogenetic relationship between FIV-isolates 96
vii


97
Materials and Methods
Animals and grouping
Eight cats were used in this study including three ALV AC-env,gag/prot/\CV
immunized cats (#PY4, #QH3, #QA6) and two FIVPet infected control cats (#PY2 and
#QE4) (see Chapter II, Materials and Methods). Also included were three age matched
SPF cats (#EJ2, #DH3, #GU5), purchased from Liberty Research Inc., which received
no immunizations prior to the FI VBang challenge.
Challenge inoculum
The challenge inoculum consisted of cell-free culture fluid from PBMC infected
with FIVBang previously titered in SPF cats. The challenge inoculum of 75 IDS0 was given
i.p. 8 months after the initial FI VPet challenge.
Viral status monitoring
Viral infection was monitored by RT activity and FIV-specific PCR and by
evaluating the level of FIV-specific humoral responses, including viral neutralizing (VN)
antibody responses to both the FIVPet and FIVBang isolate, as described previous (see
Chapter II, Materials and Methods).
DNA sequencing
Nucleotide sequencing of the amplified PCR products was performed as described
previously using FIV specific primers: FIVPct (5-TAGTAGTTATAGTGGTACTA-3) and
FIVBang (5-GGGACTACTAGCAATGGAATA-3) (see Chapter II, Materials and Methods).


85
Discussion
The aim of this study was to evaluate the immunogenicity and protective efficacy of
ALVAC-based FIV vaccines alone or in combination with ICV against experimental FIV
challenge in cats. ALVAC recombinants tested in this study included ALVAC constructs
encoding the FIV Env, the FIV Gag or both the FIV Env and Gag. Additionally, a
recombinant was tested which encoded both the FIV Gag and a modified Env in which a
putative immunosupressive region had been deleted.
We demonstrated that these recombinants were able to effectively infect non-
permissive feline cells and express the inserted FIV genes. Upon inoculation in cats,
ALVAC-specific humoral responses were readily detected. In contrast, FIV-specific
humoral responses were detected only in cats that received a booster immunization with
ICV. VN antibody titers were undetectable in all cats pnor to challenge, even in those
boosted with ICV. These observations are consistent with those of ALVAC-based HIV
vaccine candidates in macaques, chimpanzees and humans in which humoral responses
were weak or undetectable unless the animals received booster immunizations with subunit
proteins (Franchini etal. 1995a; Abimikuetal. 1995; Piaoloux et al. 1995; Clements et al.
1996). Also, the induction of VN antibodies required boosting with HIV Env or peptides
corresponding to the V3 region. Humoral responses including VN responses have been
detected in chimpanzees immunized with ALVAC-HI V-l recombinants alone (Van der Ryst
et al. 1996; Girard et al. 1996). However, these responses were detected after a minimum
of four immunizations only. Other FIV vaccine candidates composed of viral vectors
including an attenuated adenovirus and a herpesvirus engineered to express the FIV Env,
also failed to elicit FIV-specific humoral responses (Gonin et al. 1995; Verschoor et al.
1996).
The induction of humoral responses after the ICV boost is consistent with previous
studies in our laboratory in which ICV vaccines were found to elicit FIV-specific humoral


Table 1.2 Conventional inactivated whole virus FI V vaccine trials
Type of Immunization
Cellular Origin
(Vaccination Route)
Vaccine Virus'
(FIV subtype)
Vaccine Dose
(mg)
Vaccination
Protocol(wks)
Type of
Adjuvantb
Challenge Inoculum
Cellular Origin& Dose (IDS0)
Route & Strain
Protection
rate
(Hosie et al. 1992)
Whole-virus
feline T-cell(s.c.)
UK-8 (A)
10c
0,5,18
iscom
PBMC (i.p.)
20UK-8
0/5 (0%)
Unvaccinated
(-)
(-)
(-)
(-)
(-)
PBMC (i.p.)
20UK-8
1/4(25%)
(V erschoor,unpublished)
Whole virus
CrFK(s.c.)
UT113(A)
100
0,6
AluOH-oil
Thymocytes(s.c.)
10UT113
0/5 (0%)
(Yamamoto et al. 1991b)
Whole-virus
FL-4 (s.c.)
PET (A)
200
0,2,46
CFA/IFA
FeTl (i.p.)
10PET
3/3(100%)
Whole-virus
FL-4 /Fetd(s.c.)
PET (A)
200/107
0,2,4,6
CFA/IFA
FeTl (i.p.)
10PET
2/3 (67%)
Adjuvant alone
(-)
(-)
(-)
0,2,46
CFA/IFA
FeTl (i.p.)
10PET
0/3 (0%)
(Yamamoto et al.1993)
Whole-vims
FL-4 (s.c.)
PET (A)
250
0,2,5
A-MDP
FeTl (i.p.)
10PET
13/15(87%)
Adjuvant alone
(-)
(-)
(-)
0,2,5
A-MDP
FeTl (i.p.)
10PET
0/10 (0%)
Whole-viras(boost)'
FL-4 (s.c.)
PET (A)
250
38e
A-MDP
FeTl (i.p.)
10DDC(A)
13/13(100%)
Adjuvant alone
(-)
(-)
(-)
38e
A-MDP
FeTl (i.p.)
10DIX
0/5 (0%)
(Hosie et al. 1995)
Pelleted-virus
FL-4 (s.c.)
PET (A)
250
0,2,4,7,10,17
T-MDP
FeTl (i.p.)
10PET
5/6 (83%)
Control
(-)
(-)
(-)
(-)
(-)
FeTl (i.p.)
10PET
0/6 (0%)
Pelleted-virus
FL-4 (s.c.)
PET (A)
250
0,2,4,7,10,17
T-MDP
Q201(i.p.)
5UK-8
0/5 (0%)
Control
(-)
(-)
(-)
(-)
(-)
Q201(i.p.)
5UK-8
0/5 (0%)
Gradient-purified vims
FL-4 (s.c.)
PET (A)
250
0,3,6
T-MDP
FeTl (i.p.)
10PET
5/5 (100%)
Control
(-)
(-)
(-)
(-)
(-)
FeTl (i.p.)
10PET
0/5 (0%)
Gradient-purified vims
FL-4 (s.c.)
PET (A)
250
0,3,6
T-MDP
Q201 (i.p.)
10PET
4/5 (80%)
Adjuvant alone
(-)
(-)
(-)
0,3,6
T-MDP
Q201(i.p.)
10PET
0/5 (0%)
Gradient-purified vims
FL-4 (s.c.)
PET (A)
250
0,3,6
T-MDP
Q201 (i.p.)
10UK-8
1/5 (20%)
Adjuvant alone
(-)
(-)
(-)
(-)
T-MDP
Q201 (i.p.)
10UK-8
0/5 (0%)
(Johnson et al.1995)
Whole-vims
FL-4 (s.c.)
PET (A)
250
0,2,46,8
T-MDP
FeTl (nasal)
10PET
3/5 (60%)
Adjuvant alone
(-)
(-)
(-)
0,2,46,8
T-MDP
FeTl (nasal)
10PET
0/5 (0%)
Whole-vims
FL-4 (s.c.)
PET (A)
250
0,2,4,6
T-MDP
FeTl (i.p.)
10SHI (D)
0/2 (0%)
Adjuvant alone
(-)
(-)
(-)
0,2,4,6
T-MDP
FeTl (i.p.)
10SHI
0/7 (0%)
a UK-8, United Kingdom-8; PET, Petaluma; DIX, Dixon; SHI, Shizuoka.
b isom, immune stimulating complex;; A-MDP, adenyl-muramyldipeptide; CFA, complete Freunds adjuvant; IFA, incomplete Freunds; T-MDP, threonyl-muramyldipeptide.
c Vaccine dose was lOug of P17 and P24
d Vaccine consisted of 200mg /dose of inactivated whole-virus mixed with lxlO7 cells/dose of uninfected FeT-1 cells
e Vaccinated cats protected from FIVpet challenge were boosted 38 wks after the first immunization and challenged 3 weeks after the boost with FlVdix strain.


45
a total of three times at monthly intervals. The ALVAC vaccine was administered
intramuscularly at lxlO8 PFU/cat. The inactivated FlV-infected cell vaccine (ICV) was
mixed with 250 fig SAF/MDP adjuvant and was administered subcutaneously.
Challenge
The challenge inoculum consisted of cell-free culture fluid from PBMC infected
with FI VPet, previously titered in vivo in SPF cats. The challenge inoculum of 50 IDS0 was
given intraperitoneally (i.p.) four weeks after the final immunization.
FIV immunoblot assay
Sucrose gradient purified FlVPet from chronically infected FL-4 lymphoid cells was
separated by a 10% S DS pol yacryl am i de gel (SDS-PAGE). Proteins were transferred to
nitrocellulose sheets (pore diameter of 0.45 pm) by wet blotting. After transfer, the sheets
were blocked for 1-2 h at 37C in gelatin buffer (PBS containing 3% gelatin and 0.02%
sodium azide) and cut into strips. Serum samples of immunized and non-immunized cats
were diluted at 1:100 in Buffer 3 (0.05 Tris at pH7.4 containing 0.15 M sodium chloride,
0.001 M EDTA, 0.05 % Tween-20, and 1 % BSA) and incubated with immunoblot strips
for 4 h at 37C. The reactions were stopped with ddiHzO and strips were washed 3 times
with ELISA buffer (see ELISA protocol). The strips were incubated with biotinylated anti
cat IgG (Southern Biotechnology) for 1 h at 37C followed by three washes with ELISA
buffer. Strips were then incubated with streptavidin conjugated to horseradish peroxidase
for 1 h at 37C. The reactions were stopped and washed 3 times with ELISA buffer.
Finally, strips were incubated with fresh substrate solution (0.1 M Tris at pH7.4 containing
0.05% diamino benzidine, 400 mg/ml of NiCl2 and 0.01% H202). Upon appearance of
visible bands, reactions were stopped with an excess of ddiH20.


53
Becton Dickinson FACSSORT. Monoclonal antibodies to feline CD4 and CD8 were
kindly provided by N. Gengozian, University of Tennessee.
T-cell mitogen proliferative responses
T-cell mitogen proliferative responses were measured by 3[H]-thymidine uptake
assays (Ackley et al. 1990). Freshly isolated PBMC were resuspended at 2x10s cells/ml
and stimulated with either Con A (5 mg/ml) or SEA (1 mg/ml). Cells were cultured for 48
h and then pulsed with lpCi3[H]-thymidine/well. After 18 h, cells were harvested using a
cell harvester onto filter paper discs. The filter discs were air-dried and 3[H]-thymidine
incorporation was assessed by liquid scintillation counting. The results were expressed as
the stimulation index (S.I.), calculated as incorporation (mean cpm of triplicate samples) in
the presence of mitogen divided by incorporation (mean cpm of triplicate samples) in the
absence of mitogen.
Statistical analysis
The statistical significance of the data was evaluated by a Fishers exact test, which
is a modification of the chi square test. This test should be used when comparing two sets
of discontinuous, quantal (all or none) data. The analysis was set up as follows:
Vaccinated
Unvaccinated
Infected
A
B
Uninfected
C
D


Table 1.3 Conventional inactivated FI V-infected cell vaccine trials
Type of Immunization
Cellular Origin
(Vaccination Route)
V accine Virus1
(FIV subtype)
Vaccine Dose
(mg/PFU)
Vaccination
Protocol(wks)
Type of
Adjuvant6
Challenee Inoculum
Cellular Origin& Dose (ID50)
Route & Strain
Protection
rate
(Hosie et al. 1992)
Infected cell
feline T-cell(s.c.)
UK-8 (A)
2x10*
0,3,6,9,12,15
QuilA
PBMC (i.p.)
20 UK-8
0/5 (0%)
Uninfected cell
(-)(s.c.)
(-)
2x1o6
0,3,6,9,12,15
QuilA
PBMC (i.p.)
20 UK-8
1/5 (20%)
(Hosie,unpublished)
Infected cell
Q201(s.c.)
UK-8 (A)
lxlO7
0,3,6
QuilA
Q201 (i.p.)
20 UK-8
0/4 (0%)
Adjuvant alone
(-)(s.c.)
(-)
(-)
0,3,6
QuilA
Q201 (i.p.)
20 UK-8
0/4 (0%)
Control
(-Xs.c.)
(-)
(-)
0,3,6
(-)
Q201 (i.p.)
20 UK-8
1/4 (25%)
(Verschoor et al. 1995)
Infected cell
CrFK(i.m.)
UT-113 (A)
2.5x107
0,3,6
alu/MDP
PBMC (i.p.)
10 UT-113
0/5 (0%)
Uninfected cell
CrFK(i.m)
(-)
2.5x107
0,3,6
alu/MDP
PBMC (i.p.)
10 UT-113
0/3 (0%)
Infected cell
Thymocyte s(i.m.)
UT-113 (A)
1.5xl07
0,3,6
alu/MDP
PBMC (i.p.)
10 UT-113
2/5 (40%)
Uninfected cell
Thymocytesfi.m.)
(-)
1.5xl07
0,3,6
alu/MDP
PBMC (i.p.)
10 UT-113
1/3 (33%)
Control
(-)
(-)
(-)
(-)
(-)
PBMC (i.p.)
10 UT-113
0/2 (0%)
(Yamamoto et al. 1991b)
Infected cell
Fetl (s.c.)
PET (A)
lxlO7
0,2,4,6,8,16
T-MDP
PBMC (i.p.)
10 PET
4/5 (80%)
Infected cell
FL4(s.c.)
PET (A)
lxlO7
0,2,4,6,8,16
T-MDP
PBMC (i.p.)
10 PET
2/4(50%)
Uninfected FeTl
(-)(s.c.)
(-)
lxlO7
0,2,4,6,8,16
T-MDP
PBMC (i.p.)
10 PET
0/5 (0%)
Adjuvant alone
(-)(s.c.)
(-)
(-)
0,2,4,6,8,16
T-MDP
PBMC (i.p.)
10 PET
0/5 (0%)
(Yamamoto et al. 1993)
Infected cell
FL4 (s.c.)
PET (A)
2.5x107
0,2,5
A-MDP
Fetl (i.p.)
10 PET
15/15(100%)
Adjuvant alone
(-Xs.c.)
(-)
(-) ,
0,2,5
A-MDP
Fetl (i.p.)
10 PET
0/10 (0%)
Uninfected Fetl
FL4 (s.c.)
PET (A)
2.5x107
38
A-MDP
Fetl (i.p.)
10DIX
14/15 (93%)
Adjuvant alone
(-Xs.c.)
(-)
(-)
38
A-MDP
Fetl (i.p.)
10DIX
0/5 (0%)
(Matteucci et al. 1995)
Infected cell
MBM(s.c.)
M-2 (B)
3x107
0,3,6,9,12,15
IFA
plasma (i.p.)
10 M-2
5/6 (83%)
Uninfected cell
MBM(s.c.)
(-)
3xl07
0,3,6,9,12,15
IFA
plasma (i.p.)
10 M-2
0/3 (0%)
Control
(-Xs.c.)
(-)
(-)
0,3,6,9,12,15
(-)
plasma (i.p.)
10 M-2
0/6 (0%)
(Johnson et al. 1994)
Infected cell
Fl-4(s.c.)
PET (A)
2.5x107
0,2,4,6,8
A-MDP
Fetl (nasal)
10 PET
2/5 (40%)
Infected cell
H-4(s.c.)
PET (A)
2.5x107
0,2,4,6
A-MDP
Fetl (i.p.)
20 SHI
1/8(12%)
a UK-8, United Kingdom 8; UT113, Utrecht 113; PET, Petaluma; M-2, Milan-2; SHI, Shizuoka.
b T-MDP, threonyl-muramyldipeptide; A-MDP, adenyl-muramyldipeptide; IFA, incomplete Freunds adjuvant; QuilA, saponin.


46
The titer of FIV-specific antibodies, if detected, was determined by testing 10-fold
serial dilutions of serum samples (1:100 to 1:1000000) as described above and defined as
the reciprocal of the highest dilution (in 10Log) at which FIV-specific bands could be
visualized.
ALVAC immunoblot assay
ALVAC immunoblots were generated similar to that described for FTV
immunoblots, using ALVAC derived from clarified lysates of ALVAC-infected primary
chicken embryo fibroblasts. Serum samples obtained from ALVAC immunized animals
and control non-immunized animals were tested at a serum dilution of 1:100 in Buffer 3.
Reaction were carried out as described (see FI V immunoblot assay).
Enzyme-linked immunosorbent assays (ELISA)
Synthetic peptides corresponding to both conserved and variable regions in the
FIVPet envelope surface(SU) and transmembrane(TM) protein were coated on 96 well
Immunolon microtiter plates at 250 ng/well with bicarbonate buffer (pH 9.6).[ V3-1
(SKWEEAKVKFHCQRTQSQPGS), V3-2 (GSWFRAISSWKQRNRWEWRDF), V3-3
(DFESKKVKISLQCNSTKNLFA) and TM (QLEGCNQNQFFCKI)]. The plates were
washed with Buffer 3 immediately prior to use and blocked with 5% dry non-fat milk in
H20. Serum samples were diluted 1:200 in Buffer 3 containing 5% newbom-calf serum
and incubated in the coated wells for 30 min at 37C, washed 3 times with ELISA wash
solution (0.05 % Tween-20 in 0.15M sodium chloride), and incubated with biotinylated
anti-cat IgG (Vector laboratories, Burlingame, CA) for 30 mm at37C. Subsequently, the
wells were washed three times and incubated with streptavidin conjugated to horse radish
peroxidase (Vector laboratories, Burlingame, CA), washed 3 times with ELISA wash


Figure 2.5 Indirect immunofluorescence analysis on permeabilized CrFK cells infected
with ALVAC-recombinants at a m.o.i. of 10. Expression of FIV Env glycoprotein and
Gag proteins was detected using pooled serum from FIV-mfected cats as the primary
antibody and fluorescein isothiocyanate-conjugated mouse anti-cat IgG as the secondary
antibody. Panel A, control uninfected CrFK cells. Panel B, control CrFK cells infected
with ALVAC-control. Panel C, CrFK cells infected with ALVAC-e/iv. Panel D, CrFK
cells infected with ALVAC-gag/prot. Panel E, CrFK cells infected with ALVAC-
env,gag/prot. Panel F, CrFK cells infected with ALVAC-97TMG.


102
In our studies, cross-reactive VN antibody responses may not have played a key
role in vaccine protection. Poor to the FIVBang challenge, VN antibody titers were
detected in both FIVPct-infected control cats and in the ALV AC-env,gag/prot/lCV-
immunized cat #PY4. Except for the low VN antibody titer detected in cat #PY4, these
VN antibody responses were specific for FIVPet and did not cross-react with FIVBang
Surprisingly, cat #PY4 was the first one of the ALV AC-env,gag/protllCV immunized
group to become positive by PCR analysis, suggesting that the presence of FIVBang-
specific VN antibodies prior to challenge was not beneficial. Again, this resembles the
findings from supennfection studies in which FIVBang-infected cats which developed
FIVBang-specific antibody responses were susceptible to superinfection FlVPel whereas
those that lacked FTVBang-specific VN antibodies resisted superinfection (Okada et al.
1994). Furthermore, cats #QA6 and #QH3 showed a delay in infection (i.e. partial
protection) without detectable VN antibodies at the time of challenge, suggesting that
these antibodies were not responsible for this delay. However, the low titers of VN
antibodies detected alter challenge, similar to those observed after the initial FIVPet
challenge may have contributed, although the mechanisms behind this remains to be
identified.
In addition to humoral immunity, FIV-specific CTL activity may have contributed
to the delay in infection and/or reduction in viral load. In the previous chapter, the
presence of FlVPet-specific CTL activity was reported in the ALVAC-env,gag/prot /ICV
immunized cats. Unfortunately, cross reactivity to FIVBang was not evaluated due to
limited amount of PBMC that could be harvested from these cats. However, the
generation of cross-reactive CTL responses against FIVBang has been detected in cats long
term infected with the FlVPet (unpublished data). Thus, ALV AC-env, gag/prot and ICV
immunizations could have elicited similar cross-reactive CTL responses. These
responses may have been directed against Gag epitopes in particular, since the amino acid
sequence difference between the FIVPet and FIVBang Gag protein is significantly less


Table 2.9b Virus isolation (RT and PCR) on PBMC and immunoblot analysis.
Immunization
pre- post-
Months post-challenge
Vaccine
Cat ID#
-3 -1
+1
+2 +3 +4
+8
+ 12
WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR
QQ2
PY5
ALVAC-97TMG Q02
QX4
QI2
QL4
+ + + + + + + +
+ + + + + + + + +--
+ + + + + + + + + +
QH2
- -
- + +
+
+
+
+
+
+
PY2
- -
- -
+
+
+
+
+
+
+
+
+
+ + +
ALVAC
QA4
- -
- -
-
-
-
-
-
-
-
-
-
QC4
- -
- -
+
+
+
+
+
+
+
+
+
QG5
QE3

- + +
+
+
+
+
+
+
ALVAC-env,
QH3
+ -
+ -
+
+
+
+ -
gag/prot &ICV
PY4
+ -
+ -
+
-
-
+
-
-
+
-
-
+ -
QA6
+ -
+ -
+
-
-
+
-
-
+
-
-
+ -
ALVAC
QC5
+ -
+ + +
+
+
+
+
+
+
+
+
+
&ICV
QG4
+ -
+ + +
+
+
+
+
+
+
+
+
+
QE4
+ -
+ -
+
+
+
+
+
+
+
+
+
+ + +
WB= western blot (FIV-specific), RT= reverse transcriptase, PCR using FIV-specific primers.


11
likely due to biting between male cats as part of territorial behavior (Yamamotoet al. 1989).
In addition to saliva, FIV can be recovered from blood, serum, plasma, and cerebrospinal
fluid of infected cats (Pedersen et al. 1987; Dow et al. 1990). Horizontal transmission
through contact alone appears to be inefficient (Pedersen et al. 1987; Yamamoto et al.
1988b). Vertical transmission in tero or postpartum via the milk has been reported and
was found to occur most frequently in queens that became viremic during pregnancy
(Callanan et al. 1991; Wasmoen et al. 1992; ONeil et al. 1996). Although high rate of
perinatal transmission has been reported for queens that had been infected with a highly
pathogenic FIV strain 4 to 30 months prior to conception (Ueland and Nesse 1992; ONeil
et al. 1996).
Similar to HIV, five clinical stages can be defined for FIV infection in cats. The
acute viremic phase, 2 to 4 weeks after infection, is characterized by fever, neutropenia,
and generalized lymphadenopathy. These symptoms vary in duration and severity between
individual cats and are mostly recognized in experimentally infected cats but rarely in
naturally infected cats (Yamamoto et al. 1988b, 1989). Once a full immune response is
established and most of the virus is cleared from the plasma, a period of months to years
follows with no obvious clinical signs of disease, defined as the asymptomatic phase.
However, during this period changes in lymphocyte counts, such as a decrease in CD4+
lymphocytes and CD4/CD8 ratios, takes place (Ackley et al. 1990). This period is
followed by a phase equivalent to that of AIDS related complex (ARC) in men. Cats with
ARC often present with chronic illness such as stomatitis/gingivitis, lower urinary tract
infections, skin disorders and diarrhea (Yamamotoet al. 1989). Finally, cats may develop
a stage similar to that of AIDS in men, characterized by severe lymphoid depletion, weight
loss and opportunistic infections. Opportunistic pathogens reported in cats suffering from
AIDS include toxoplasmosis, cryptococcoses, candidiasis, mycobacteriosis, feline calici-
and herpes virus (Lapin et al. 1989; Knowles et al. 1989; Ishida and Tomoda 1989). At
this stage, CD4+ T-cell counts have dropped dramatically and CD4:CD8 ratios are inversed


65
CD4/CD8 ratios were monitored prior to immunization, postimmunization and after
challenge, a representation of a typical FACS analysis is depicted in Figure 2.6. Prior to
immunization, most cats displayed CD4/CD8 ratios considered normal for three month old
kittens (average ratio 4.0 + 1.2) except for cat #QX3 which displayed a ratio of 1.3 which
is considered low. A littermate of this cat (#QX4) also displayed a relatively low ratio,
suggesting that genetic factors attributed to this. After immunization, most cats showed an
average CD4/CD8 ratio of 3.3 + 0.8. The observed decline in ratio is expected with the
increase in age. No inversion (<1) in CD4/CD8 ratios were noticed after challenge (Table
2.2). This was as expected since the FIVPct at the challenge dose used generally does not
cause inversion of CD4/CD8 ratio until 1.5-2 years postchallenge.
To determine if ALVAC vaccinations influenced lymphocyte function, we evaluated
lymphocyte proliferation upon exposure to concanavalin A (ConA) and staphylococcal
enterotoxin A (SEA) at various times in selected cats. Following immunizations and
challenge no abnormalities in T-cell proliferative responses were detected in any of the cats
tested (Table 2.3).
Humoral responses
The generation of ALVAC-specific antibodies was evaluated before and after
immunization in serum samples taken from cat #QS4 immunized with ALVAC-
env,gag/prot and cat #PY2 immunized with ALVAC vector alone by immunoblotting.
ALVAC specific humoral responses were detected in both cats upon a single immunization
and additional immunizations resulted in increased titers of these antibodies (Figure 2.7).
Serum samples obtained from SPF and FIV infected control cats tested negative.
Next, we tested if the immunization schemes used were able to generate FIV-
specific antibody responses. Immumzation with ALVAC-FIV recombinants alone (#QC3)
failed to induce detectable FI V-specific antibody responses even after three immunizations


Luctor et Emergo


6
(SU, gpl20). The TM protein protrudes through the viral membrane and noncovalently
anchors the outer SU protein, which appears as a knob-like structure on the viral surface.
Figure 1.1 Electron micrograph of FI V x70,000.
FI V genomic organization and regulation of gene expression
Lentiviral genomes are among the smallest of the known viruses and are more
complex in their genomic organization than other members of the retrovirus family. The
FIV genome is a positive stranded polyadenylated RNA of approximately 9kb. It contains
three major open reading frames (ORFs) encoding the structural and enzymatic proteins
(Env,Gag and Pol) necessary for the viral life cycle (Olmsted et al. 1989a, 1989b;




CHAPTER III
EFFICACY EVALUATION OF CANARYPOXVIRUS (ALVAC)- BASED FIV
VACCINE COMBINED WITH INACTIVATED FI V-CELL VACCINE AGAINST
HETEROLOGOUS FIV CHALLENGE IN CATS
Introduction
Based on genetic variation in the Env and Gag coding regions, HIV isolates
obtained worldwide have been classified into several subtypes or clades (Cheingsong-
Popov etal. 1994; Brodineetal. 1995). Optimally, a vaccine against HIV should induce
immune responses that can cross react with a wide variety of these HIV subtypes. Thus
far, HIV vaccine trials have concentrated mainly on a single subtype, subtype B, as this
represents the predominant type found in Europe and the United States. However, the
emergence of HIV isolates other than subtype B is increasing significantly. Furthermore,
90% of the reported HIV cases are in the developing countries where the HIV epidemic
encompasses multiple subtypes. These countries in particular would benefit from the
development of broad spectrum vaccines since drug treatments, even if they would
become available, would be too expensive.
Similar to HIV, FIV strains have been classified into different subtypes (A-D),
based on aa sequence differences in Env and to a lesser extent in the Gag (Sodora et al.
1994; Kakinuma et al. 1995; Rigby et al. 1993) (Figure 3.1). As such, the FIV model
provides a means to assess the protective efficacy of vaccine strategies against multiple
HIV subtypes. Vaccine protection against homologous and slightly heterologous FIV
strains (within one subtype) has been achieved with inactivated whole virus and
inactivated FIV-infected cell vaccines (ICV) (Yamamoto et al. 1991, 1993; Verschoor et
al. 1995; Johnson et al. 1994; Hosie et al. 1995). These same vaccines,
94


38
II. In vivo evaluation of ALVAC-FIV recombinant vaccines.
a) Identify the immune responses, both humoral and cell-mediated, elicited in cats after
immunization with ALVAC-FIV recombinants alone or after priming with ALVAC-FIV
recombinants followed by boosting with inactivated FI V-infected cell vaccine (ICV).
b) Assess the protective efficacy of immunization protocols employing ALVAC-ewv,
ALVAC-gag/prot, ALVAC-97TMG and ALVAC-env,gag/prot against experimental
infection with the homologous subtype FIV Petaluma (FIVPet) isolate in cats.
c) Assess the efficacy of a combination prime-boost protocol consisting of ALVAC-
env,gag/prot priming followed by inactivated FI V-infected cell boost, against experimental
infection with the homologous subtype FlVPet isolate in cats.
Materials and Methods
Construction of ALVAC-recombinants
ALVAC is a canarypoxvirus vector derived from a canarypoxvirus vaccine strain
used to immunize canaries, Kanapox (Rhone-Merieux, Lyon, France). The ALVAC-FIV
recombinants were generated using standard procedures similar to those used by
Virogenetics to generate ALVAC-based FeLV recombinants (Piccini et al. 1987; Tartaglia et
al. 1993). Briefly, the coding region of the FIVViUefranche isolate (subtype A) env, gag and
prot were amplified by polymerase chain reaction (PCR) and fused in a precise ATG to
ATG fashion with vaccinia early/late promotors FI6 or I3L, respectively ( Figure 2.1). The
protease gene was included in the constructs to ensure proper proteolytic cleavage of the
Gag precursor protein into the mature matrix protein (MA, pl5), capsid protein (CA, p24)


76
NK cell activity
NK cells are the principal effector cells in clearance of viral infections early in the
course of infection. For this reason, we evaluated levels of NK activity in selected cats at
3.5 weeks postinfection (Table 2.8). NK activity was measured using various target cells
including autologous PBMC as negative controls. All cats displayed normal levels of NK
activity and no significant differences were observed between the infected and non-infected
ALVAC-immunized cats.
Protective efficacy
The presence or absence of FIV following challenge was measured at monthly
intervals using several methods. This included the assessment of viral reverse transcriptase
(RT) activity and FIV-specific PCR analysis of cultured PBMC. These assays were also
used to test LN and BM cells for the presence of FIV, as these organs function as major
reservoirs for the virus. In addition, FIV infection was determined by comparing the level
of FIV-specific antibody responses in the serum before and after FIV challenge. FIV-
specific immunoblotting (WB) was performed as well as ELISA using a peptide
corresponding to the transmembrane (TM) region of the FIV Env. Sera from FIVPet
infected cats have shown to react strongly to this peptide. Further, VN antibody responses
were measured in selected cats before and after FIV challenge. In general, the induction
and persistent elevation of FIV-specific antibody responses and VN antibodies are
indicative of an active viral infection.
All control cats (n=3) immunized with ALVAC vector alone and boosted with ICV
became viremic as assessed by RT for infectious virus and FIV-specific PCR for proviral
DNA in peripheral blood and tissue samples (BM and LN cells) (Table 2.9b and Table
2.10). Further, these cats developed high titer VN (>100) responses indicative of active


86
responses but failed to induce VN antibody responses after a single immunization.
Interestingly, cats immunized with ALVAC-env,gag/prot showed approximately a 10-fold
higher immunoblot antibody titer than cats immumzed with ALVAC vector alone after the
ICV boost. Therefore it is possible that ALVAC-env,gag/prot induced FIV-specific T-
helper responses resulting in a more efficient generation of FIV-specific humoral responses
after exposure to ICV. This has been reported in other studies in which chimpanzees were
immunized with an ALVAC recombinant encoding the HIV Env and boosted with
recombinant Env. Chimpanzees immunized with ALVAC-Env produced antibodies
following a single recombinant Env immunization, whereas chimpanzees immunized with
recombinant Env alone failed to developed such antibodies after a single immunization
(Girard etal. 1995). Similar priming of T-helper responses has also been shown in human
volunteers immunized with ALVAC-HIV candidate vaccines (Piaoloux et al. 1995;
Clements et al. 1996)(Tartaglia, personal communication)
The effectiveness of ALVAC-based vaccines in priming CTL responses as has been
reported previously was further confirmed by findings in this study (Cox et al. 1993).
Detectable levels of FIV-specific CTL responses were detected even after a single
immunization. Although, the phenotype of the effector cells was not determined it was
found that the effector cells reacted in a MHC-restricted manner. This finding excludes NK
cells as the effector cell and implies a role for CD8+ T-lymphocytes known to react in a
MHC class I restricted manner. However, we can not exclude a role for CD4+ CTL
responses since the target cells (autologous PBMC) used in the assay could have presented
FIV antigens in the context of both MHC class I and II. In an attempt to identify the F1V
epitope recognized by these effector cells, CTL assays were performed using autologous
target cells infected with vaccinia recombinants expressing FIV Env or Gag. These
experiments did not demonstrate presence of CTL activity (data not shown), possibly due
to technical difficulties of the CTL assay itself. The ability of ALVAC-based vaccines to
prime CTL responses has been reported in several studies. Mice immunized with an


48
M 2-mercaptoethanol at a final concentration of lxlO6 cells/ml (2x10scells/well). Triplicate
cultures were stimulated with inactivated FI V (5 pg/well) and incubated at 37C for 4 days
in a humidified atmosphere containing 5% C02. On Day 4, cells were pulsed with 1 pCi
3 [H]-thymidine (Amersham, Indianapolis, IN) per well for 18 h. Cells were harvested
onto filter paper using a cell harvester. The discs were air-dned and 3[H]-thymidine
incorporation was assessed by liquid scintillation counting. Results of triplicate samples
were expressed as the stimulation index (S.I.), calculated as the mean incorporation in the
presence of inactivated FI V divided by the mean incorporation in the absence of inactivated
FIV.
Assessment of cytotoxic T-lymphocyte(CTL) responses
PBMC were tested for their ability to lyse autologous lymphoblastoid cells infected
with FIVPet. Freshly isolated PBMC were cultured at 2xl06 cells/ml in RPMI1640 medium
containing 10% FBS and stimulated with Con A (5 mg/ml) for three days. On Day 3, 3.6 x
106 Con A lymphoblasts were removed, infected with FIVPet and cultured for 5 days. The
remaining cells (effector cells) were maintained in RPMI 1640 medium supplemented with
10% FBS and IL-2 (100 U/ml) for 5 days. After5 days, l-5xl06 of the FIV-infected cells
were inactivated by UV treatment and added as antigen-presenting cells(APC) to the
effector cells at a ratio of 1:15 (APC:effector cells). Effector cells and FIV-infected APC
cells were cocultured for an additional 5-7 days in no IL-2 medium. Effector cells were
assayed for cytolytic activity against autologous FIV-infected target cells by a standard slCr
release assay (Song et al. 1992). Target cells were labeled with slCr for 2 h at 37C and
washed three times prior to use in the assay. Effector and slCr -labeled target cells were
then mixed at effector to target ratios ranging from 50:1 to 10:1 and incubated for 4 h at
37C without IL-2. After 4 h, 100 pi of supernatant was removed from each well and
slCr-specific activity was measured in a y-counter. Results are shown as the percentage of


54
The P (Probability) for a one-tailed test was calculated as (A+B)!(C+D)!(A+C)!(B+D)! /
N!A!B!C!D! combined with the P value of stronger combinations. The obtained P value
tells if the groups differ significantly and the degree of significance. In this study, a P-
value equal or less than 0.05 was considered significant.
Results
In vitro assessment
The ability of ALVAC-recombinants to infect non-permissive feline cells was
demonstrated by PCR analysis on DNA extracted from feline CrFK cells inoculated with
ALVAC-'/zv and ALVAC-gag/prot recombinants (Figure 2.2 and 2.3). The obtained PCR
products corresponded in size to PCR-products obtained with DNA extracted from FL-4
cells, a lymphoid cell line chronically infected with FIVPrt. The correct nucleotide sequence
was verified by DNA sequencing (data not shown). No FI V-specific PCR products could
be detected in CrFK cells infected with ALVAC vector alone or FeT-J, a FIV-negative
feline lymphoid cell line.
Similarly, the expression of messenger RNA (mRNA) specific for FIWenv and gag
products was demonstrated in CrFK cells inoculated with the ALVAC-env and ALVAC-
gag/prot recombinants (Figure 2.4). RT-PCR on mRNA extracted from CrFK cells
infected with ALVAC-env revealed a 450bp band and from those infected with ALVAC-
gag/prot revealed a 700bp band consistent with the bands obtained from the FIV-infected
FL-4 cell line. No RT-PCR products could be detected in cells infected with ALVAC
vector alone and FeT-J control cells (FIV negative feline lymphoid cell line). In addition,
no PCR products were obtained PCR reactions in which mRNA extracts were used as
template, indicating that the obtained RT-PCR products were not the result of DNA
contamination of mRNA extracts (data not shown).


25
reponses was evaluated in kittens bom to vaccinated queens and nursed by sham
immunized queens. These kittens became infected and showed significantly lower titers of
VN (10-100) postparturition as compared to littermate controls receiving colostrum from
vaccinated queens. Nevertheless, these kittens showed lower levels of viremia as
compared to littermate controls which were bom to and nursed by unvaccinated queens (Pu
et al. 1995). As such, these data suggest that VN maternal antibodies transferred via
colostrum/milk or placenta play a role in preventing establishment of FIV infection.
Supporting this are additional studies on kittens born to queens infected for various lengths
of time. It was found that kittens bom to queens infected for more than 7 months were
protected in the presence of high VN titers in the colostrum. In contrast, kittens bom to
short term (< 2mo.) infected queens became infected in the presence of low VN titers in
colostrum (Pu et al. 1995).
Opposing a role for Env specific and VN responses in protection against FIV
infection are findings from a number of subunit vaccines trials. Vaccines composed of the
FIV envelope or envelope fragments alone failed to induce protective immunity. This
included nonglycosylated, glycosylated, native, and denatured whole FIV Env and Env
fragment vaccines in combination with different adjuvants (Lutz et al. 1995; Verschoor et
al. 1996; Lombardi et al. 1994). The majority of these vaccines, however, effectively
induced Env-specific antibody responses as well as VN antibody responses. Therefore, it
could be suggested that the tertiary structure of the envelope protein is crucial as it affects
the presentation of the Env to the immune system or that other epitopes besides Env are
required to obtain protective immunity. A subunit vaccines composed of Gag proteins
alone, however, failed to induce protection despite the induction of Gag specific humoral
responses (Hosie et al. 1992). Interestingly, several of the envelope subunit vaccines
caused enhancement of infection upon challenge (Hosie et al. 1992; Siebelink et al. 1995;
Osterhaus et al. 1996). Similar enhancement of viremia has been observed in horses
immunized with a recombinant envelope vaccine against equine infectious anemia virus


32
inoculum. Based on this it was speculated that cell mediated responses attributed to the
partial protection observed in these animals. However, the assessment of cross-reactive
CTL responses to the HIV-2 isolate was not included in this study.
In addition to the macaque model, the chimpanzee model has been used to assess
the prophylactic efficacy of ALVAC-based vaccine protocols against HIV infection. In one
study, the efficacy of an ALVAC recombinant expressing both the HIV-1^ Env and Gag
(ALVAC-HIVj^, gpl20TM, Gag/Prot) was evaluated against cell-associated HIV-1IIIB(LAI)
challenge in two chimpanzees. Each animals received a total of five immumzations and
were challenged one month after the final immunization. HIV-specific antibody responses
including VN antibodies were detected after the 4th and 5th immunization in both animals.
Upon challenge, one animal remained virus negative and the other, including a naive
control animal, became infected. Interestingly, the protected animal had a higher VN
antibody titer at the time of challenge than the nonprotected animal (Van der Ryst et al.
1996). Importantly, these same vaccine protocols failed to induce protective immunity
against challenge with the heterologous HI V-DH12 isolate (personal communication).
Further, the vaccine efficacy of ALVAC-HIV^ gpl20TM, Gag/Prot recombinants
was evaluated against mucosal challenge in chimpanzees. One group consisting of two
animals was immunized by the intra-muscular (i.m.), cervico-vaginal, and rectal route
simultaneously. Another group was immumzed by the i.m., oral and nasal route and one
animal was immunized by the i.m. route. All animals were challenged intra-cervically with
2500 TCIDS0 of HIV-1lvi passaged in chimpanzees. All vaccinated animals were free from
virus infection whereas the nonvaccinated control animals were infected. HIV-specific
antibody responses, including VN antibody titers, were low or undetectable at the time of
challenge, thus implying that mucosal protection occurred through mechanisms other than
VN antibody responses (Girard et al. 1996).
Another study reported on the efficacy of ALVAC-based vaccine strategies against
heterologous HIV challenge in chimpanzees. Chimpanzees were immunized with an


95
however, failed to induce protective immunity against distinctly heterologous FV strains
from heterologous subtypes (Yamamoto et al. 1993; Johnson et al. 1994; Hosie et al.
1995). Thus, a modified or different vaccine approach is required to induce immune
responses that will cross-react with a wide variety of FIV subtypes.
Cross-protective immunity has been obtained against heterologous HIV-1
infection in chimpanzees using vaccines composed of whole Env or Env fragments from
multiple isolates (Girard et al. 1995). However, the use of viral vector-based vaccines
may overcome the need for the inclusion of antigenic determinants from multiple isolates.
Viral vector-based vaccines have been shown to effectively prime cell-mediated responses
whereas conventional vaccines have been shown to prime predominantly humoral
responses. The epitopes recognized by cell-mediated responses may be directed against
epitopes that are more conserved among different isolates and as such provide protection
against a wider variety of HIV isolates. In fact, preliminary findings from vaccine trials
in which macaques were immunized with a canarypoxvirus (ALVAC) vectored HIV-1
vaccines suggest that this may be the case (Abimiku et al. 1995). After immunization
these macaques were partially protected from infection with a distinctly heterologous
HIV-2 isolate.
In the previous chapter, we demonstrated that cats immunized with ALVAC-
recombinants encoding FIV Env and Gag {ALV AC-env, gag/prot) and boosted with a
conventional inactivated FIV-infected cell vaccine (ICV), resisted infection w'ith FIV
Petaluma (FIVPet), a subtype A isolate. This isolate was closely related to the isolate used
to generate the ALVAC-FIV recombinant (FIVviUe franche, subtype A) and identical to the
FIV isolate used to generate the ICV vaccine. In this study, we evaluated if these
ALV AC-env, gag/prot/lCV immunized cats could be protected from a second challenge
with a distinctly heterologous FIV isolate, FIV Bangston (FIVBang). The FIVBang isolate is
classified as a subtype B virus and differs from the HVPet isolate (subtype A) by 21% in
the Env and 2.4% in the Gag amino-acid sequence.


84
Statistical analysis
The Fishers exact test was used to analyze the statistical significance of the
protective efficacy data (Table 2.11). The infectivity rate of the ALVAC-FIV recombinant
immunized groups was compared to that of the ALVAC (n=6) control group alone or to
that of the ALVAC control group combined with the ALVAC/ICV (n=3) immunized control
group (total n=9). The infectivity rate of AJJVAC-env.gag/protlCV immunized cats was
compared to either the ALVAC/ICV-immunized control group (n=3) or to both the
ALVAC-immunized and ALVAC/ICV-immunized control groups (n=9). Based on this
test, immunization schemes employing ALVAC-gag/prot and ALVAC-env,gag/prot
together with ICV, showed significant protection as indicated by a P (Probability) value
equal or less than 0.05 (Table 2.11). Protection observed by the other immunization
schemes was not significant (P value > 0.05).
Table 2.11 Statistical analysis
Viral status
vaccine control P value significant
group group
Vaccine +/- +/- (single-tailed)
ALVAC-env
3/3
4/2
0.5
no
3/3
7/2
0.28
no
ALVA C-gag/prot
0/6
4/2
0.0303
yes
0/6
7/2
0.00914
yes
ALVAC-env, gag/pro t
2/4
4/2
0.28
no
2/4
7/2
0.118
no
ALVAC-97TMG
3/3
4/2
0.5
no
3/3
7/2
0.28
no
ALVAC-env, gag/prot
0/3
3/0
0.05
yes
& ICV
0/3
7/2
0.00914
yes


Figure 2.9 FIV-specific immunoblot. Serum samples obtained before (pre) and 16 weeks (16pc) after transfusion from kittens #DH5 and
#DE4, transfused with cells from ALVAC-gag/prot immunized cats, and cat #DH2 transfused with cells from ALVAC-control
immunized cat, were diluted 1 to 100 in Buffer 3 and incubated with FIV-specific westemblot strips. The positive control was incubated
with pooled serum from FI V-infected cats. The negative control was incubated with serum from a SPF cat.


ACKNOWLEDGMENTS
This thesis would not have been written without the influence, presence, and
support of many people. I am grateful to my family, especially Pa and Ma, for their
care and support of me and my endeavors over the years. Further, I would like to
recognize my supervisor Janet Yamamoto, who has struck me with her infinite commitment
to research. I thank her for letting me pursue this project in her laboratory and giving me
the opportunity to become acquainted with the World of Science and Bureaucracy. Special
thanks go to Dr. Pu for his expertise in feline phlebotomy, his advice and his the anecdotes
from the old China. Further, I was fortunate to have David Pollock as my right hand. I
highly appreciate his meticulous work during the countless virus isolation assays and his
chit-chat dunng the endless fycoll sessions. I am especially indebted to Dr. Paoletti and
Dr. Tartagliaat Virogenetics and Dr. Desmettre at Rhone Merieux for providing me the
opportunity to work with the ALVAC vector in my studies. I also wish to acknowledge the
members of my supervisory committee, Dr. Condit, Dr. Schiffenbauer, Dr. Johnson and
Dr. Schuster for their time and advice.
At the homefront, I would like thank Nicole, Mienke, Mirian, Chantal and Simone
for their friendship and, Dr. Paul and Dr. Frank, for staying in touch and keeping me
updated on the latest from the low-lands. Warm thanks go to Sue Watsana, who was a
motivating source at frustrating times. Special thanks go to Susie for all the exciting
adventures and for the good times we have had in our lovely shack on NW 2nd street. Last
but not least, I would like to thank Dan for his patience and great care of me, Tamika and
the Kalaharis. Finally, I would like to thank all my friends in and out of Gainesville for
making every day life a little more exciting.
IV


120
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feline herpesvirus and baculovirus as vaccine vectors for the gag and env genes of
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Warren, H.S., Vogel, F.R., and Chedid, L.A. 1986. Current status of immunological
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Wasmoen, T., Armiger-Lehman, S., Egan, C., Hall, V., Chu, H.-J., Chavez, L. and
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Willet, B.J., Hosie, M.J., Jarret, O., and Neil, J.C. 1994. Identification of a putative
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LIST OF TABLES
Table page
1.1 The lentiviruses 3
1.2 Conventional inactivated whole virus FIV vaccine trials 19
1.3 Conventional inactivated FIV-infected cell vaccine trials 20
1.4 Subunit and vector based FIV vaccine trials 21
1.5 Immunogenicity and prophylactic efficacy of ALVAC-based vaccines 29
1.6 Immunogenicity and prophylactic efficacy of ALVAC-based vaccines
against retroviral pathogens 35
2.1 Grouping and immunization 44
2.2 CD4/CD8 ratios after immunization and challenge 63
2.3 Proliferation to T-cell mitogens (ConA and SEA) 64
2.4 FIV-specific antibody titers after immunization and challenge 70
2.5 Viral neutralizing antibody titers after immunization and challenge 71
2.6 T-helper responses to FIV after immunization and challenge 73
2.7 FIV-specific CTL activity in peripheral blood after immunizations 74
2.8 NK activity postchallenge 75
2.9 Virus isolation (RT and PCR) on PBMC and WB data pre- and
postchallenge 77
2.10 Virus isolation on PBMC and tissue samples and WB and ELISA data 79
2.11 Statistical analysis 84
3.1 Immune parameters and viral-status pre-and postchallenge 99
vi


101
cats had high titer of FIV-specific humoral responses prior to challenge whereas the naive
control cats lacked such responses. On the other hand, ICV vaccines capable of eliciting
high titers of FIV-specific antibody responses were effective against homologous and
slightly heterologous challenges but not against distinctly heterologous FIV isolates
(Yamamotoet al. 1991, 1993; Johnson et al. 1994). It is possible that the presentation of
both exogenous and endogenous FIV antigens in the ALVAC-env,gag/protHCV
immunized cats resulted in the generation of higher titers of antibody responses or
antibodies to a wide variety of FIV epitopes including those shared by FIVPet and FIVBang
isolates. Similarly, active FlVPet infection in the infected control cats could have
broadened humoral responses, resulting in resistance to superinfection. This is consistent
with results from a previous study in which a long-term FIVBang-infected cat was
protected from FIVPet infection in the presence of high titer FIV-specific antibody
responses. Likewise, cross-protection by infection with HIV-2 in high risk woman has
been found to correlated with resistance to infection with HIV-1 which is genetically
highly divergent from HIV-2 (Kanki et al. 1995). Additionally, monkeys infected with
an attenuated macrophage tropic SIV strain were resistant to superinfection with a highly
virulent SIV strain displaying 16% difference in the Env amino-acid sequence (Clements
et al. 1995). In these studies, the length of infection and the concomitant broadening of
humoral immune responses positively correlated with protection. Moreover, naive
monkeys passively immumzed with serum from these long-term infected monkeys were
partially protected from subsequent heterologous challenge. The broadening of humoral
responses in these monkeys, however, also included the broadening of VN antibody
responses capable of cross-reacting with the challenge inoculum virus. In contrast,
chimpanzees immunized with recombinant Env and V3 peptides corresponding to two
HIV-1 isolates, were protected from heterologous subtype challenge in the presence of
VN antibodies that neutralized vaccine strains but not the challenge strain (Girard et al.
1995).


79
Table 2.9 Virus isolation on PBMC and tissue samples and WB and ELISA data
Vaccine Cat#
Weeks
p .c.
WB/ELISA
PBMC
RT PCR
Tissue
BM LN
RT PCR RT PCR
THY
RT PCR
QH4
27
-
-
-
+
-
+
_
+
_
PYl
24
+
+
-
-
-
-
-
+
-
Alvac-env
Q01
10
+
+
+
+
+
+
+
+
ND
QC1
28
-
-
-
-
-
-
-
-
ND
QUl
28
ND
QL2
24
+
+
-
-
+
+
+
+
- -
QQ1
29
ND
QA5
29
-
-
-
-
-
-
-
-
ND
Alvac-gragr/prot
QU2
29
ND
QX3
29
ND
QI1
29
-
-
-
-
-
-
-
-
ND
QL3
29
-
-
-
-
-
-
-
-
ND
QH5
28
PY3
27
-
-
-
-
-
-
-
-
ND
Alvac-env,
QS4
24
+
+
-
-
-
+
+
+
-
gag,prot
QC3
28
-
-
-
-
-
-
-
-
- -
QG3
28
-
-
-
-
-
-
-
-
- -
QE2
28
+
+
-
-
-
+
-
+
- +
QQ2
27
_
_
_
_
_
_
+
PY5
28
Alvac-97TMG
Q02
10
+
+
+
+
+
+
+
+
ND
QX4
25
+
+ /-
-
-
-
-
-
-
- -
QI2
25
+
-
-
-
-
-
-
-
-
QL4
27
-
-
-
-
-
-
-
-
- -
QH2
10
+
+ /-
+
+
+
+
+
+
ND
PY2
39
+
-
+
+
-
+
-
-
ND
Alvac-control
QA4
28
- -
QC4
26
+
+
+
+
+
+
+
+
+ +
QG5
10
+
+
-
+
-
+
+
+
ND
QE3
28
-
-
-
-
-
-
-
-
- -
Alvac-env,
QH3
36
+
_
_
ND
gag/prot&ICV
PY4
36
+
-
-
-
-
-
-
-
ND
QA6
36
+
-
-
-
-
-
-
-
ND
Alvac-control
QC5
10
+
+
+
+
+
+
ND
&ICV
QG4
10
+
+
-
+
-
+
+
+
ND
QE4
39
+
+
-
-
+
+
ND
ND
WB=westem blot, PBMC= peripheral blood mononuclear cells, BM= bone marrow, LN=lymph node,
Thy=thymus, ND= not determined.


100
VN antibody titers (>100). More importantly, these responses were specific for FlVPet
and did not cross-neutralize FIVBang in vitro. Interestingly, serum taken before the FI VBang
challenge from cat #PY4 contained low level VN antibodies(<20) which neutralized both
FlVPet and FIVBang in vitro.
After challenge, all naive control cats developed FIV-specific antibodies as
determined by immunoblotting. In contrast, FlVBang-specific VN antibodies were detected
in only one of the three cats (#DH3). Interestingly, all three cats immunized with
ALV AC-env,gag/protHCV developed low level of VN antibodies to FIVPet similar to that
observed after the initial FI VPet challenge but failed to develop VN antibodies specific for
FIVBang. Similarly, FIVPet-infected control cats failed to develop FIVBang-specific VN
antibodies after challenge whereas FIVPet-specific antibodies persisted at high titers
(>100).
Discussion
In this study, cats immunized with both ALV AC -env,gag/prot and ICV were
partially protected from heterologous FIV subtype challenge given eight months after an
initial homologous FIV challenge. These data should be interpreted with some caution as
they reflect only a 4 month period postchallenge. Nevertheless, immunized cats showed a
delay in infection, as all control cats became viremic within 6 weeks postchallenge
whereas two of the immunized cats became positive based on PCR analysis only at 14
and 18 weeks postchallenge, respectively. Furthermore, throughout the study these cats
were negative for virus isolation, demonstrating a significant reduction in viral load.
The exact immune-mechamsm(s) responsible for the delay in infection or the
reduction in viral load in the ALV AC-env,gag/prot/lCV immunized cats is unclear. It is
possible that FIV-specific humoral responses present prior to the FIVBang challenge played
a role. Both the ALV AC-env, gag!protHCV immunized cats and FIVPet-infected control


Figure 2.6 Representative FACS analysis of binding of anti-fCD4 and anti-fCD8
monoclonal antibodies to PBMC. Expression of fCD-4 and fCD-8 was detected by using
anti-fCD4 and anti-fCD8 antibodies as the primary antibody and fluorescein isothiocyanate-
conjugated goat anti-mouse IgG as the secondary antibody. Panel A, scattered dot-plot of
PBMC isolated from cat#QG3. The depicted histograms respresents cells gated under gate
1 (predominantly lymphocytes) and gate 2 (negative control, cells of the macrophage and
monocyte lineages). Panel B, histogram of CD4 and CD8 staining of cells in gate 1. Panel
C, histogram of CD4 and CD8 staining of cells in gate 2.


50
Viral reverse transcriptase (RT) assay
Freshly isolated PBMC were either stimulated with ConA (5mg/ml) or co-cultured
and cultured for 4 weeks in RPMI1640 medium containing 5% heat inactivated FCS,
lOmMHEPES buffer, 50 mg/ml gentamycin, 5X10 M 2-mercaptoethanol and 100 U/ml
human recombinant IL-2, or cocultured with ConA lymphoblasts from SPF cats. Culture
supernatants were collected every 3-4 days and assayed for the presence of viral reverse
transcriptase (RT) activity. The virus was pelleted from the supernatants by ultra
centrifugation (1 h at 17,000rpm). The virus pellet was then incubated with an RT cocktail
containing 100 mM Tris (pH8.3), 150 mM KC1, 10 mM MgCl2, 4 mM diethiothreitol
(DTT), 0.6 Units of Poly (rA), oligo(dT), and 60 pCi of 3[H]TTP per ml. After incubation
at 37C for lh, the cDNA was spotted onto filter paper discs that had been prewashed with
0.1 M sodium pyrophosphate. Discs were washed in the following sequence: twice in
10% cold trichloroacetic acid (TCA), once in 5% TCA, once in 5% TCA containing 0.5 %
SDS, and once in ethanol. The filter discs were air-dried and placed in scintillation vials
with 3 ml scintillation fluid. 3[H]-TTP incorporation was measured in using a liquid
scintillation counter. Supernatants were considered positive for RT activity if cpm in test
samples were equivalent or higher than 3 times the cpm of the negative control sample
(supernatants from SPF control cats).
Detection of proviral DNA by polymerase chain reaction (PCR)
Proviral DNA (latent infection) was monitored by env specific PCR on DNA
extracted from PBMC, bone marrow (BM) cells, and lymph node (LN) cells after culturing
for 4 weeks with FIV-free ConA lymphoblasts. BM cells were obtained from 1-2 ml of
aspirates taken from the femur. LN cells were obtained from the popliteal lymph nodes.


90
effectively induce Gag-specific CTL responses but failed to protect cats. No efficacy
studies have been undertaken in monkeys to assess protection of ALVAC recombinants
encoding Gag alone. Protocols in these models often involve priming and boosting with
multiple antigenic determinants, making it difficult to resolve which epitope(s) are crucial
for protection. However, there have been reports suggesting the importance of Gag
epitopes in vaccine trials against other retroviruses. Vaccine efficacy of a herpes
recombinant vector expressing FeLV Env was significantly enhanced with the inclusion of
the Gag (Wardley et al. 1992). Furthermore, mice immunized with a vaccinia recombinant
expressing the Gag of the Friends murine leukemia retrovirus (F-MuLV) were protected
against disease upon exposure to F-MuLV (Miyazawa et al. 1992).
Env-expressing ALVAC recombinants in our study lacked protective efficacy,
similar to adenovirus and herpesvirus vectored vaccines encoding FIV Env (Gonin et al.
1995; Verschoor et al. 1996). Similar to ALVAC-chv, these vector vaccines failed to
induce Env-specific humoral responses including VN antibodies. The evaluation of cell-
mediated responses was not included in these studies. In our study, ALVAC-e/zv
immunized cats were shown to elicit FIV-specific CTL responses. However, the presence
of these responses did not correlate with protection. Chimpanzees immunized with an
ALV AC-recombinant encoding the HIV-1 Env and boosted with recombinant Env (gpl60)
also failed to be protected. Lack of protection in these studies correlated with low VN
antibody titers, since, chimpanzees immunized simultaneously with recombinant Env
(gpl60) and boosted with V3 peptide were protected in the presence of high VN antibody
titers (Girard et al. 1995). Further, vaccine trials employing ICV and inactivated whole
FIV in cats indicate that Env-specific humoral responses, including VN antibodies may be
responsible for the observed protection (Yamamoto etal. 1991, 1993). Thus, the inability
to generate such responses by the ALVAC-cnv vaccine could explain the lack of protective
efficacy. Additionally, differences in the processing pathway, as discussed above, may


43
Inactivated-cell vaccine preparation and titering
The inactivated FIV-infected cell vaccine (ICV) was produced from an IL-2
independent feline lymphoid cell line (FL-4) chronically infected with the FIV Petaluma
isolate (subtype A). This cell line was cloned from an IL-2 dependent feline T-cell line
(FeTl) infected with FIVPet and stains positive for CD8,CD4 and PanT surface markers and
negative for IgM heavy and light chains (Yamamoto et al. 1991a). The ICV vaccine was
generated by inactivation of FL-4 cells with 1.25% paraformaldehyde for 24 h, followed
by extensive dialysis against PBS. A single vaccine dose consisted of 2.5xl07 fixed
infected cells mixed with 250pg SAF/muramyl dipeptide (MDP) (Chiron Corporation).
Animals
A total of 36 specific pathogen free (SPF) cats (Felis catus, domestic short hair), 12
weeks of age, were purchased from Liberty Research Inc. (Waverly, NY). The animals
were housed at the Infectious Disease complex of Animal Resource Services and cared for
in accordance with the policies set by the Environmental Health and Safety division
(EH&S) and the Animal Care Committee of the University of Florida. All cats received a
combination vaccine against feline herpes virus, calicivirus and panleukopeniavirus (Fel-O-
Vax, Ford Dodge laboratories, Mason City, IA). Animals were not vaccinated against
feline leukemia virus (FeLV). Prior to immunization, all animals tested negative for
Toxoplasma gondii, FeLV and FIV by immunoblot analysis.
Grouping and Immunization Protocol
Cats were divided into 7 groups, with equal numbers of males and females in each
group (Table 2.1). Littermates were evenly spread over all groups. Cats were immunized


TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iv
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ABBREVIATIONS viii
ABSTRACT x
CHAPTER
ILENTI VIRUSES AND VACCINE DEVELOPMENT
Introduction 1
II IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION
OF CANARYPOXVIRUS( ALVAQ-B ASED FIV VACCINE AGAINST
HOMOLOGOUS FIV CHALLENGE
Introduction 36
Materials and Methods 38
Results 54
Discussion 85
III EFFICACY EVALUATION OFCANARYPOXVIRUS(ALVAC)
BASED FIV VACCINE COMBINED WITH INACTIVATED FIV-CELL
VACCINE AGAINST HETEROLOGOUS FIV CHALLENGE
Introduction 94
Material and Methods 97
Results 98
Discussion 100
IV SUMMARY AND FUTURE STUDIES
Synopsis 104
REFERENCES 107
BIOGRAPHICAL SKETCH 121
v


73
Table 2.6 T-helper responses to FIV upon immunization and challenge
Stimulation Index
Vaccine
Cat ID#
Number
oflmmunizations
Post-
IX
3X
challenge
ALVAC -env
QL2
1.1
1.2
ND
QH4
ND
ND
1.0
QC1
ND
ND
0.7
ALV AC-gag/prot
QX3
1.0
1.3
1.4
ALV AC-env, gag/prot
QG3
0.9
1.6
ND
QC3
ND
2.6
1.3
PY3
ND
1.6
ND
QH5
ND
ND
0.7
ALV A C- 9 7TMG
QI2
1.1
1.7
ND
QX4
ND
ND
2.1
ALVAC
QC4
1.2
ND
ND
QE3
ND
1.1
ND
QH2
ND
ND
1.3
ALVAC-env,
PY4
ND
2.4
ND
gag/prot &ICV
QH3
ND
ND
0.7
ALVAC
QC5
0.7
1.3
0.7
&ICV
QG4
ND
1.0
ND
a 4 weeks post-challenge
ND= not determined.


87
ALVAC-recombinant encoding the HIV-1 Env were shown to elicit CTL responses,
including memory T-cell responses (Cox et al. 1993). The effector cells in these studies
were characterized as CD8+ T-lymphocytes. In addition, specific CTL responses mediated
by CD8+ T-lymphocytes were detected in 30% of the human volunteers immunized with an
ALVAC recombinant encoding the HIV-1 Env (Pialoux et al. 1995; Egan et al. 1995).
Interestingly, volunteers immunized with an ALVAC recombinant encoding both the HIV-1
Env and Gag, mounted CTL responses specific for Gag more often than for Env
(Lawrence et al. 1996). We were unable to distinguish if this was the case in our study.
In summary, ALVAC-based FIV vaccines were found to differ from inactivated
FIV-infected cell vaccines in their ability to elicit cell-mediated and humoral responses.
This can be explained, in part, by a difference in the processing and presentation of
ALVAC encoded immunogens. Immunogens encoded within ALVAC, require de novo
expression within host cells to be presented to the host immune system. In this study,
ALVAC immunizations were given intramuscularly and therefore it is likely that the
majority of ALVAC infected muscle cells. These cells are capable of presenting
immunogens in association with MHC class I and as such are expected to stimulate
primarily CTL responses. Additionally, part of the ALVAC inoculum may have been taken
up by macrophages or infected cells of the monocyte lineage, such as dendritic cells. These
cells are capable of presenting antigens in association with both MHC class I and class II
molecules and could therefore have stimulated the generation of both CTL and T-helper cell
responses. This is supported by the low level of proliferative responses detected in some
of the cats immunized with ALVAC-FIV recombinants alone. The direct stimulation of B-
cell responses by ALV AC-encoded immunogens would require the expression and release
of the immunogens from ALV AC-infected host cells. This process may have occurred at
low level only, since ALVAC-FIV recombinants failed to induce detectable levels of
humoral responses. Further, the nature of the immunogen itself may have played a role
since ALVAC-recombinants expressing epitopes of viral pathogens other than retroviruses


12
(Ackley et al. 1990). Other immunologic abnormalities reported include hypergamma
globulinemia and reduced T-cell responses to T-independent antigens (Ackley et al. 1990;
Tortenetal. 1991). The occurrence of neurological abnormalities as seen in AIDS patients
has only been reported in a small percentage of cats infected with neurotropic isolates of
FIV (Podelletal. 1993; Dow etal. 1990).
FIV as an animal model for HIV
With the emergence of the HIV pandemic and an estimated 12 million infected
people, animal models to study antiviral drugs and vaccine strategies have become very
important. The search for an appropriate animal model for HIV-1 infection in man has,
however, been complicated by the host specificity of HI V-l.
To date, HIV-1 has only been shown to infect three other species; pig-tailed
macaques, gibbons, and chimpanzees (Agey et al. 1992; Fultz et al. 1986). These animals
become viremic and mount specific antibody responses similar to HIV infected humans.
However, infection does not result in depletion of CD4+ T-lymphocytes,
immunosuppression, or opportunistic infections. Furthermore, chimpanzees are an
endangered species, therefore limiting the availability of these animals for research
purposes. Consequently, the infection of macaques with SIV has become the most
prevalent animal model used. Both the SIVniac- and SIVsmm- isolates have been shown to
cause an AIDS-like disease in rhesus-, cynomolgus-, and stumptail macaques (Murphey-
Corb etal. 1986). Initial infection results in viremiaand is characterized by fever, diarrhea
and lymphadenopathy. Like HIV-1 in humans, monkeys in the end-stages of disease
present with opportunistic infections and show decreased CD4+ T-lymphocyte counts and
inversed CD4:CD8 ratios. Additional promising models include the infection of baboons
and pig-tailed macaques with specific isolates of HIV-2 (Barnett et al. 1994; Novembre et
al. 1994). A large percentage of these monkeys become persistently infected and show


70
Table 2.4 FI V-specific antibody titers before and after immunizations and challenge.
FTV-specific antibody titer2
Cat ID# Vaccine Boost pre- post Mo.postchallenge
immunizations 2 8
QA6
ALVAC-env,gag/prot
ICV(IX)
<2
5-6
4-5
4-5
QH3
ALVAC-env,gag/prot
ICV(IX)
<2
5
3-4
4
PY4
ALVAC-env,gag/prot
ICV(IX)
<2
5
4-5
4-5
QG4
ALVAC(2X)
ICV(IX)
<2
4-5
5
ND
QC5
ALVAC(2X)
ICV(IX)
<2
3-4
5-6
ND
QE4
ALVAC(2X)
ICV(IX)
<2
3-4
5-6
5-6
QS4
ALVAC-env,gag/prot (3X)
<2
<2
5-6
ND
QC3
ALVAC-env,gag/prot (3X)
<2
<2
<2
ND
PY3
ALVAC-env,gag/prot (3X)
<2
<2
<2
ND
PY2
ALVAC(3X)
<2
<2
5-6
5-6
2 FI V-specific titers expressed as the reciprocal of the highest dilution (in 10log) at which
FIV specific bands could be detected by immunoblotting.
ND= not determined
(Figure 2.8). In contrast, all cats boosted with ICV (group F and G) developed detectable
FIV-specific antibody responses. Interestingly, cats primed with ALVAC-env,gag/prot
(#QA6) developed approximately 10-fold higher antibody titers than those primed with
ALVAC vector alone (#QC5) (Figure 2.8) upon the ICV boost (see Table 2.4)
Selected cats of each group were also tested for the presence of antibody responses
to peptides corresponding to the V3 region of the FIV surface envelope glycoprotein by
ELISA. This region is thought to be equivalent to the V3 region of HIV which contains the
principal neutralizing domain (Pancino et al. 1994). None of the immunized cats exhibited
significant levels of antibody titers to the three V3 peptides tested even after three
immunizations (data not shown).


44
Table 2.1 Grouping and Immunization
Group
Cat ID#
sex
Vaccine(Number of Immunizations)
QH4
F
PY1
M
Group A
QOl
F
ALVAC-env (3X)
QC1
M
QU1
M
QL2
F
QQl
M
QA5
F
Group B
QU2
F
ALVAC-gag/prot (3X)
QX3
M
QI1
M
QL3
F
QH5
F
PY3
M
Group C
QS4
F
ALVAC-env,gag/prot (3X)
QC3
M
QG3
F
QE2
M
QQ2
M
PY5
F
Group D
Q02
F
ALVAC-97TMG (3X)
QX4
M
QI2
M
QL4
F
QH2
M
PY2
M
Group E
QA4
F
ALVAC (3X)
QC4
M
QG5
F
QE3
F
QH3
M
Group F
PY4
M
ALVAC-env,gag/prot (2X)
QA6
F
ICV(IX)
QC5
F
Group G
QG4
F
ALVAC (2X)
QE4
M
&ICV(1X)
F=Female M=Male


Table 1.4 Subunit and vector-based FIV vaccine trials
Type of Immunization
Cellular Origin
(Vaccination Route)
Vaccine Virus"
(FIV subtype)
Vaccine Dose
(mg/PFU)
Vaccination
Protocol) wks)
Type of
Adjuvant1*
Challenge Inoculum
Cellular Origin& Dose (ID50)
Route & Strain
Protection
rate
(Hosie, et al. 1992)
p24
E. coli(s.c.)
UK-8 (A)
50
0,3,5,7
iscom
PBMC (i.p.)
20 UK-8
0/4 (0%)
(Lutz et al. 1995)
gplOO(denatured)
Insect cell (i.m.)c
Z2 (A)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
0/5 (25%)
gplOO(native)
Insect cell (i.m.)
Z2 (A)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (20%)
gplOOfhighly purified)
Insect cell (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (20%)
gplOO(native )
Insect cell (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
0/5 (0%)
gplOO(denatured)
E..Coli (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (25%)
Control(Ovalbumin)
(-)
(-)
100
0,2,4,8
(-)
PBMC (i.p)
20 Z2
2/7 (29%)
(Osterhaus et al. 1996)
Env(cleavage)
NA(s.c.)
AM 19 (A)
100
0,4,10
iscom
PBMC (i.m)
20AM19
0/6 (0%)
Env(no cleavage)6
NA(s.c.)
AM 19 (A)
100
0,4,10
iscom
PBMC (i.m.)
20AM19
0/6 (0%)
Env(no cleavage)6
NA(s.c.)
AM 19 (A)
100
0,4,10
QuilA
PBMC (i.m.)
20AM19
0/6 (0%)
Env-b Gal
NA(s.c.)
AM19 (A)
100
0,4,10
QuilA
PBMC (i.m.)
20AM19
0/6 (0%)
Control(PBS)
(-XS.C.)
(-)
(-)
0,4,10
(-)
PBMC (i.m.)
20AM19
0/6 (0%)
(Flynn et al. 1995)
V3-peptide
synthetic (s.c.)
UK-8 (A)
100
0,3,6
QuilA/AlOH
PBMC (i.p.)
10 UK-8
0/15(0%)
Adjuvant alone
(-) (s.c.)
(-)
100
0,3,6
QuilA/AlOH
PBMC (i.p.)
10 UK-8
0/5 (0%)
(Lombardi, 1994)
V3-peptide
synthetic(s.c.)
PET(A)
500
0,2,4,6,8
CFA
F14(i.v.)
20-PET
0/3 (0%)
Control
(-)
(-)
(-)
(-)
(-)
F14(i.v.)
20-PET
0/3 (0%)
(Verschoor et al. 1996)
V3-fusion protein F
E..Coli(s.c.)
UT113 (A)
100
0,4,6,8,10
AlOH
NA(s.c.)
10-20UT113
0/5 (0%)
V3-fusion protein I
E..Coli(s.c.)
UT113
100
0,6,10
QuilA
NA(s.c.)
10-20UT113
0/5 (0%)
Control(PBS)
(-)(s.c.)
(-)
(-)
0,6,10
(-)
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-Env
(-)oronasal/s.c.
UT113
10SPFU
0
(-)
Boost V3-peptide
E. Coli (i.m.)
UT113
100
4,8
AlOH
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-Env
(-)oronasal/s.c.
UT113
105PFU
0
(-)
Boost V3 peptide
E.Coli (i.m.)
UT113
100
4,8
QuilA
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-b Gal.
(-)oronasal/s.c.
(-)
10SPFU
0
(-)
Boost PBS
(-)(s.c.)
(-)
(-)
4,8
(-)
NA(s.c.)
10-20UT113
0/5 (0%)
(Gonin et al.1995)
Adenovirus-env
(-)(i.m.)
Wo (A)
11.8-9.2PFU
0,4,30
ISA206
NA(NA)
20 Wo
0/4 (0%)
Adeno-psuedorabies(control)
(Kim.)
(-)
11.8-9.2PFU
0,4,30
ISA708
NA(NA)
20 Wo
0/4 (0%)
a UK-8, United Kingdom; Z2, Zurich; BANG, Bangston; AM19, Amsterdam 19; PET, Petaluma,
b Recombinant vaccinia virus expressed
c Baculovirus expression
d iscom, immune stimulating complex; A10H/QS21, aluminumhydroxide and non-toxic fraction from Quillaja saponaria; ISA206, water/oil adjuvant;ISA708, water/oil adjuvant,
e V3-fusion protein was composed of the FIV V3 region fused to galactokinase.
f Deletion of the cleavage site between the envelope surface(SU) and transmembrane protein(TM).


62
FL-HEIGHT
FL-HEIGHT


116
Olmsted, R.A., Barnes, A.K., Yamamoto, J.K., Hirsch, V.M., Purcell, R.H., and
Johnson, P.R. 1989a. Molecular cloning of feline immunodeficience virus. Proc.
Natl. Acad. Sci. USA 86, 2448-2452.
Olmsted, R.A., Hirsch, V.M., Purcell, R.H., and Johnson, P.R. 1989b. Nucleotide
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Cat #
DH2
cell
inoculum
PY2
pre 16pc
p24
DH5
DE4
QQ1
QX3 Controls
pre 16pc
pre 1 6pc +
oo
CO


112
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5
al. 1993). As such, lentiviruses present a major challenge to the development of
therapeutic strategies and vaccines.
The Feline Immunodeficiency Virus
The FIV virion, like other members of the Retrovindae, is enveloped and
approximately 100-125nm in diameter (Pedersen et al. 1987). A electron micrograph of a
mature lentivirion is depicted in Figure 1.1. The FIV core, composed of the capsid protein
(CA, p24), has the physical appearance of a cone, typical of all lentiviruses. The core
encloses the viral genome which consists of two identical single stranded RNA molecules
of approximately 9kb (Elder et al. 1993). Associated with the viral genome is a small
nucleocapsid protein (NC, p8), which is thought to play a role in viral assembly and
disassembly (Elder et al. 1993; Aldovini and Young 1990). Also packaged within the
virion are the enzymes essential for replication, the reverse transcriptase (RT), the integrase
(IN) and the deoxyundine triphosphatase (dUTPase; DU)(Elder et al. 1993). The RT is
responsible for transcription of the viral RNA genome into DNA. The integrase functions
as an endonuclease which cuts the cellular genome to allow integration of the provirus.
The dUTPase is unique to the nonprimate viruses and its function in the FIV life cycle still
remains unknown. However, it is speculated that the dUTPase is required for replication
of FIV in nondividing macrophages or resting T-lymphocytes (Miyazawa et al. 1993).
Additionally, it has been observed that lack of functional FIV dUTPase results in an
increased mutation frequency of FIV propagated in macrophages (Lemer et al. 1995).
Surrounding the viral core is a matrix protein layer (MA, pl5) which is closely associated
with the viral envelope. The viral envelope is derived from the cellular membrane by
budding of the viral particles and consists of lipids, inserted viral proteins and cellular
proteins (Gelderblom et al. 1987). The virally derived envelope glycoprotein (Env) is
composed of two subunits; the transmembrane protein (TM, gp40) and the surface protein


106
to evaluate which of the Gag proteins, CA, NC or MA are required to induce protective
immunity. Particular attention should be paid to the Gag pl7 protein as vaccine studies
involving HIV-1 pl7 Gag protein have demonstrated both the induction of CTL, T-help
and VN antibody responses. Moreover, SCID mice transfused with PBMC from human
volunteers immunized with a synthetic pl7 were reported to be protected from HIV-1
challenge whereas control mice transfused with cells from nonimmunized subjects became
infected (Goldstein et al. 1993).
Overall, these studies should add to our understanding of the interactions between
lentiviral pathogens and the host immune system and aid in the development of safe and
effective vaccines against FIV as well as HIV. Additionally, these studies may aid in the
development of vaccines against other pathogens that display continuous antigenic variation
and pathogens for which current vaccine approaches have failed.


49
specific cytotoxicity for triplicate assays. Maximum release was obtained by repeated
freeze-thawing of labeled target cells. Spontaneous release was obtained from slCr labeled
target cell cultures in the absence of effector cells. The percentage of FIV-specific release
was calculated as 100 x (mean cpm test release mean spontaneous release)/(mean cpm
maximum release mean cpm spontaneous release). The spontaneous release did not
exceed 20% of the maximum release. Specific lysis values equal or greater than 10% were
considered positive for CTL activity.
Assessment of natural killer cell activity
The level of natural killer (NK) cell activity was determined using a 4 h slCr release
assay similar to that described for the CTL assay. Various target cell types were used
including FeT-J (feline lymphoid cell line), FL-4 (feline lymphoid cell line chronically
infected with FlVPet), non-autologous PBMC, and autologous PBMC. Target cells were
labeled with slCr for 2 h at 37C and washed three times prior to use in the assay. Freshly
isolated PBMC effector cells were cocultured at effector to target cell ratios ranging
between 100:1 to 10:1 for 4 h at 37C in a humidified atmosphere containing 5% C02.
After 4 h, 100 pi of supernatant was removed from each well and slCr-specific activity was
measured in a y-counter. Results are shown as the percentage of cytotoxicity for tnplicate
assays. Maximum release was obtained by repeated freeze-thawing of labeled target cells.
Spontaneous release was obtained from slCr labeled target cell cultures in the absence of
effector cells. The percentage of NK activity was calculated as 100 x (mean cpm test
release mean cpm spontaneous release)/ (mean cpm maximum release mean cpm
spontaneous release). The spontaneous release did not exceed 20% of the maximum
release.


15
mediated responses including CTL reponses. More recent developments in the vaccine
field involve the use of viruses, bacteria or naked DNA as vaccine vectors. These vectors
are genetically engineered to carry and express foreign genes encoding immunogens of
pathogens. Upon inoculation of these vectors into the host, the inserted immunogen is
expressed and presented to the host immune system. In fact, viral antigens encoded within
these vectors are presented to the host immune system in a manner simulating natural
infection. Furthermore, vector based vaccines are thought to be more effective in eliciting
CTL responses than conventional inactivated vaccines. As such, vaccine strategies
employing vector based vaccines may be especially useful against viral and intracellular
bacterial pathogens.
To date, success of viral vaccines has been limited to a confined group of viruses.
These viruses display constant antigenic specificity and consist of a single or limited
number of serotypes. Furthermore, spontaneous recovery has usually been observed
shortly after natural infection with these viruses. Lentiviruses, including FIV, do not fall
into this category. Lentiviruses are subject to continuous antigenic variation and consists of
many serotypes. For FIV a total of 4 subtypes have been defined based on genetic
differences in the env and gag coding regions (Sodora et al. 1994; Kakinuma et al. 1995;
Rigby et al. 1993). Spontaneous recovery upon infection with lentiviruses has not been
reported. Moreover, these viruses integrate into the host cellular genome and can stay
latent without the expression of viral proteins. Latently infected cells serve as a reservoir
and fail to be recognized by the immune system, allowing the virus to persist. Thus, the
development of effective vaccines against lentiviruses faces additional challenges.
FIV vaccine development
An optimal FIV vaccine should induce long-lasting protective immunity. This
immunity should be effective against a wide range of FIV strains within as well as across


16
subtypes (A-D). In addition, this vaccine should induce protective immunity against cell-
free and cell-associated virus and against various routes of infection. Since FIV is
predominantly transmitted through biting, protection should be directed in particular against
this route of exposure.
In order to properly evaluate the immunogenicity and protective efficacy of FIV
vaccines, several factors should be taken into consideration. One factor is the method used
to assess the induction of VN antibody responses as these are often used as a parameter for
the effectiveness of viral vaccines. The ability of antibodies to neutralize lentiviral infection
in vitro may however differ with the specificity of the target cell line used in the assay.
For example, it has been found that vaccine induced antibodies capable of neutralizing FIV
infection on a feline Crandell kidney cell line (CrFK) failed to neutralize FIV infection on
feline thymocytes (Siebelink et al. 1993).
Likewise, the assessment of protective efficacy is influenced by several factors. As
for viruses in general, these include the route of infection and the dose of challenge
inoculum. In most FIV vaccine trials, the challenge inoculum is given either intravenously
or intraperitoneally. Experimental infection through these routes is obtained more readily
than through mucosal exposure, e.g. requires less viral particles. Furthermore, the origin
of the vaccine virus and challenge virus inoculum play an important role in the outcome of
vaccine efficacy. Lentiviruses are enveloped and incorporate cellular antigens into the viral
envelope when budding from the host cell. These host derived proteins present in the
lentiviral envelope may play a role in protective efficacy of inactivated virus and infected
cell vaccines, in particular. For example, monkeys immunized with uninfected human cells
were shown to be protected from challenge with SIV grown on the identical human cell
line. Protected monkeys had significantly higher levels of antibodies directed to host-cell
major histocompatibility complex (MHC) antigens than monkeys that were not protected.
In fact, protection against SIV has been obtained with vaccines composed of MHC
molecules solely (Chan et al. 1992; Stott 1991; Langlois et al. 1992). Likewise, protection


52
In vivo assessment of viral-status
Three SPF kittens (#DH2, #DH5, #DE4), 12 weeks of age, were transfused
intravenously with a total of 1.1 x 108 cells obtained 8 months after the FIVPet challenge
from either ALVAC-gag/prot immunized cats (#QQ1 and #QX3) or FIV-mfected ALVAC-
control cat (#PY2). Cells consisted of 3 x 107 of PBMC isolated by ficoll hypaque density
centrifugation, 7 x 107 BM cells and lxlO7 LN cells. Prior to transfusion, these cells were
washed in sterile PBS and resuspended in 2 ml PBS. Cat #DH2 received cells from
infected control cat #PY2, cat #DH5 received cells from ALVA C-gag/prot immumzed cat
#QQ1 and cat #DE4 received cells from ALVAC-gag/prot immunized cat #QX3.
General parameters
Throughout the trial, all cats were momtored for hematological changes (complete
blood count, differential leukocyte count and total protein count) and abnormal clinical
manifestations (diarrhea, vomiting, lymphadenopathy, weight loss, elevated rectal
temperature and neurological signs).
CD4/CD8 ratios
CD4/CD8 ratios were determined by indirect immunofluorescence staining and flow
cytometry. Briefly, 5X10S PBMC isolated by ficoll hypaque density centrifugation, were
washed in FACS buffer (PBS containing 0.325% sodium azide and 2.5% BSA) and
incubated with feline CD4 or CD8 specific monclonal antibodies at 37C for 1 h.
Subsequently, cells were washed in FACS buffer and incubated with secondary antibody,
FITC labeled goat F(ab)2 anti-mouse IgG (H+L) (Southern Biotechnology Associates,
Inc.) for 1 h at 37C. Finally, cells were washed and analyzed by flow cytometry on a


17
against feline leukemia virus (FeLV), another feline retrovirus, has been afforded in the
presence of antibody responses directed against cat-cell antigens (Lee et al. 1982). Thus,
the cellular origin of the vaccine virus and the challenge virus should be taken into
consideration when evaluating protective efficacy.
In the majority of FIV vaccine trials, the challenge inoculum virus is produced from
FIV strains adapted to cell cultures in the laboratory. In vitro cell culturing, however, can
lead to changes in virulence, cell tropism, and sensitivity to VN antibodies. To overcome
this, FIV isolated freshly from infected cats have been used. A relatively new approach is
the use of molecularly cloned FIV. The viral genome is incorporated into a plasmid and as
such used to infect animals.
FIV vaccine trials
Early FIV vaccine trials utilizing inactivated whole virus failed to demonstrate
protective immunity (Table 1.2 and Table 1.3). These trials included the immunization of
specific pathogen free (SPF) cats with inactivated FIV UK8 purified from T cells and
incorporated into immune stimulating complexes (ISCOMs) (Morein et al. 1984; Hosie
1994). Similarly, an inactivated virus vaccine produced from Crandell feline kidney cells
(CrFK) infected with the FIV UT113 isolate and adjuvanted with aluminum hydroxide-oil,
failed to protect cats against low dose homologous challenge (Hosie 1994) (Table 1.2).
Initial trials involving inactivated FI V-infected cell vaccines were also unsuccessful.
Cats immumzedwith inactivated FIV UK8 infected T-cells or helper T-cells (Q201) became
readily infected upon a homologous challenge with 20IDS0 (Hosie 1994, Hosie et al. 1992)
(Table 1.3). Comparable results were obtained with a vaccine consisting of inactivated
CrFK cells infected with the FIV UT113 isolate (Verschoor et al. 1995). In contrast,
partial protection (3 out of 5) was observed in cats immunized with a cell vaccine consisting
of FIVUT113-infected thymocytes. However, one of three control cats immunized with


119
vaccines: recombinant canarypoxvirus expressing measles virus fusion (F) and
Hemagglutinin (HA) proteins. Virology 187, 321-328.
Tartaglia, J., Jarret, O., Neil, J.C., Desmedre, P., and Paoletti, E. 1993. Protection of cats
against feline leukemia virus by vaccination with a canarypox virus recombinant,
ALVAC-FL. J. Virol. 67, 2370-2375.
Tartaglia, J., Perkus, M, Taylor, J., Norton, E.K., Audonnet, J.C., Cox, W.I., Davis,
S.W., VanderHoeven, J.R., Meignier, B., Riviere, M., Languet, B., and Paoletti,
E. 1992. NYVAC: a highly attenuated strain of vaccinia virus. Virology 188, 217-
232.
Tjusjimoto, H., Cooper, R.W., Kodama, T., Fukasawa, M., Tomoyuki, M., Ohta, Y.,
Ishikawa, I., Nakai, M., Frost, E., Roelants, G.E., Roffi, J., and Hayami, M.
1988. Isolation and characterization of simian immunodeficiency from mandrills in
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Tozzini F., Matteucci D., Bandecchi P., Baldinaotti F., Poli A., Pistello M., Siebelink
K.H., Ceccherini-Nelli L., and Bendinelli, M. 1992. Simple in vitro methods for
titrating feline immunodeficiency virus and FIV neutralizing antibodies. L Virol.
Methods 37, 241-252.
Torten, M., Franchini, M., Barlough, J.E., George, J.W., Mozes, E., Lutz, H., and
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Ueland, K., and Nesse, L.L. 1992. No evidence of vertical transmission of naturally
acquired feline immunodeficiency virus infection. Vet. Immunol. Immunopathol. 33,
301-308.
Vallee, H., and Carre, H. 1904. Nature infectieusede Fanemiedu che val. C.R. Acad. Sci.
139, 331-333.
Van der Ryst, E., Fultz, P.N., Tartaglia, J., Barre-Sinnousi, F., Paoletti, E., Nara, P.,
Meignier, B., Blondeau, C., Muchmore, E., and Girard, M. 1996. Protection from
HIV-1 challenge in chimpanzees by immunization with an canarypox virus
recombinant. XI Int. Conf. AIDS, Vancouver, 1996 (Abstr. We.A.280).
Verschoor, E.J., Van Vliet, A.L., Egbennk, H.F., Hesselink, W., van Alphen, W.E.,
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Verschoor, E.J., Willemse, M.J., Stam, J.G., Vliet van, A.L.W., Pouwels, H.,
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glycoprotein. Virol. 199, 247-251.


92
Interestingly, cats immunized with ALVAC-env,gag/prot but boosted with ICV
were protected. The difference between this group and the ALVAC-env,gag/prot
immunized group was the presence of FIV-specific humoral responses (antibodies detected
by FIV immunoblot analysis) at the time of challenge. Control cats immunized with
ALVAC vector alone and boosted once with ICV became infected despite the presence of
FIV-specific antibody responses. The antibody titers in these control cats, however, were
10-fold lower than those detected in the ALV AC-env,gag/prot-pnmed cats. Based on this
it could be speculated that priming with ALVAC-env,gag/prot enhanced the
immunogenicity of ICV in a such a manner that protective immunity could be elicited after a
single ICV immunization rather than after the three to four immunizations that are usually
required to obtain protection. Interestingly, the protected cats in this group displayed low
VN antibody titers shortly after challenge. These titers (5-20) differed significantly from
those detected in infected control cats (>100). Therefore, VN antibody production may
have been the result of anamnestic responses to the challenge inoculum. This is supported
by the fact that VN antibodies in two of three cats could no longer be detected at 8 mo post
challenge. In the case of active infection, an increase rather than a decrease in VN antibody
titers, is expected. The low level VN titer detected at 8 mo postchallenge in cat #PY4
presented a dilemma as this may have been the result of low level viral infection
undetectable by RT and PCR analysis. Alternatively, this may have been the result of
strong immunity that persisted which is more likely since this cat tested persistently
negative by all other criteria up to one year postchallenge. Moreover, this cat was not able
to resist supennfection with another FIV isolate (see chapter III) whereas two infected
control cats resisted superinfection with this same FIV isolate. The generation of VN
responses upon challenge in this group resembles observations in macaques which had
been immunized with ALVAC recombinants encoding HIV-2 Env and Gag (Franchim et al.
1995a). After challenge, protected animals tested negative for virus but developed
significant levels of viral neutralizing antibodies. Furthermore, the protective efficacy


23
such as SIV in macaques and HIV in chimpanzees (Desrosiers et al. 1989; Fultz et al.
1992; Murphey-Corb et al. 1989). FIV subunit vaccines consisting of either Env or Gag
proteins as well as recombinant vector based vaccines expressing Env did not elicit
protective immunity against low dose homologous challenge. In contrast, protective
immunity has been obtained against homologous SIV challenge in macaques immunized
with Env subunit vaccines and against homologous and heterologous HIV challenge in
chimpanzees immunized with Env subunit vaccines (Hu et al. 1992; Girard et al. 1995).
Mechanisms of protection
Protection obtained with conventional inactivated whole virus and infected cell
vaccines produced with the FIVPet isolate, correlated most with high levels of envelope-
specific and VN antibodies (Yamamotoet al. 1991b, 1993). Protection obtained with these
vaccines did not correlate with anti-MHC antibodies as could be concluded from the lack of
protection in cats that were immunized with u ninfec ted cel Is alone (Yamamoto et al. 1991b,
1993) (Table 1.3). Similar vaccines produced with FIV isolates grown in feline T cells,
thymocytes and CrFK cells failed to induce protective immunity (Verschoor et al. 1995;
Hosie etal. 1992; Hosie 1994) (Table 1.3). However, these vaccines elicited lower levels
of envelope-specific antibodies and VN antibodies. In fact, only the FIV vaccines
produced from infected feline T-cell lines FL-4 and FeTl cells, induced VN antibody titers
similar to those observed in infected cats (Tozzinm et al. 1992). This could be due to the
larger quantities of envelope protein produced by FL-4 and FeTl cell lines and the fact that
the envelope protein present on FL-4 and FeTl cells is well preserved following
purification. Additional contributing factors may have been the choice of inactivating
agent. Inactivation of successful vaccines was accomplished by paraformaldehyde which
is thought to be more effective in maintaining antigenicity of immunogens when compared
to other inactivating agents i.e. fl-propriolactone (Allison and Byars 1991; Warren et al.


75
each group, was observed. Further, cats primed with ALVAC-env,gag/prot and boosted
with ICV displayed similar levels of CTL activity as detected in those immunized with
ALVAC-env, gag/prot alone. Control cats immunized with ALVAC vector alone or
immunized with ALVAC (2X) and boosted with ICV failed to demonstrate significant
levels of CTL activity (< 10%).
The detected CTL activity was found to be MHC restricted as effector cells were
only capable of lysing autologous FIV-infected target cells and failed to lyse non-
autologous FIV-infected target cells (data not shown), thus implying that the detected
activity was due to CTL, as opposed to NK cell activity. Further, no lysis was observed
using uninfected autologous target cells (data not shown).
Table 2.8 NK activity postchallenge
Vaccine
Cat ID#
% specific slCr release
Target cell
autologous
PBMC
heterologous
PBMC
FL-4
Fet-J
ALVAC-env
QC1
0
0
10.1
20.7
ALV AC-gag/prot
QC3
ND
0
12.5
18.8
ALVA C-env,
QX3
0
0
8.7
22.8
gag/prot
ALVAC-97TMG
QX4
ND
0
10.7
16.9
ALVAC
QC5
ND
0
5.5
17.8
&ICV
ALVAC
QC4
ND
12.2
6.5
8.2
ND= not determined


109
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Dagleish, A.G., Beverly, P.C.L., Chapman, P.R., Crawford, M.F., Greaves, M.F., and
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Desrosiers, R.C., Wyand, M.S., Kodama, T., Ringler, D.J., Arthur, L.O., Sengal, P.K.,
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specific cytolytic-host-range restricted canarypox vector (ALVAC) carrying the HIV-
1MN env gene. J. Infect. Pis. 171, 1623-1627.


Table 1.4 Subunit and vector-based FIV vaccine trials
Type of Immunization
Cellular Origin
(Vaccination Route)
Vaccine Virus
(FIV subtype)
Vaccine Dose
(mg/PFU)
V accination
Protocol(wks)
Type of
Adjuvant11
Challenge Inoculum
Cellular Origin& Dose (IDS0)
Route & Strain
Protection
rate
(Hosie, et al. 1992)
p24
E. coli(s.c.)
UK-8 (A)
50
0,3,5,7
iscom
PBMC (i.p.)
20 UK-8
0/4 (0%)
(Lutz et al. 1995)
gplOO(denatured)
Insect cell (i.m.)c
Z2 (A)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
0/5 (25%)
gplOO(native)
Insect cell (i.m.)
Z2 (A)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (20%)
gplOO(highly purified)
Insect cell (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (20%)
gplOO(native )
Insect cell (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
0/5 (0%)
gplOO(denatured)
E..Coli (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (25%)
Control(Ovalbumin)
(-)
(-)
100
0,2,4,8
(-)
PBMC (i.p)
20 Z2
2/7 (29%)
(Osterhaus et al. 1996)
Env(cleavage)
NA(s.c.)
AM19 (A)
100
0,4,10
iscom
PBMC (i.m)
20 AM19
0/6 (0%)
Env(no cleavage)
NA(sc)
AM19 (A)
100
0,4,10
iscom
PBMC (i.m.)
20 AM19
0/6 (0%)
Env(no cleavage)
NA(s.c.)
AM 19 (A)
100
0,4,10
QuilA
PBMC (i.m.)
20AM19
0/6 (0%)
Env-b Gal
NA(s.c.)
AM 19 (A)
100
0,4,10
QuilA
PBMC (i.m.)
20 AM19
0/6 (0%)
Control(PBS)
(-)(s.c.)
(-)
(-)
0,4,10
(-)
PBMC (i.m.)
20AM19
0/6 (0%)
(Flynn etal. 1995)
V3-peptide
synthetic (s.c.)
UK-8 (A)
100
0,3,6
QuilA/AlOH
PBMC (i.p.)
10 UK-8
0/15(0%)
Adjuvant alone
(-) (s.c.)
(-)
100
0,3,6
QuilA/AlOH
PBMC (i.p.)
10 UK-8
0/5 (0%)
(Lombardi, 1994)
V3-peptide
synthetic(s.c.)
PET(A)
500
0,2,4,6,8
CFA
F14 20-PET
0/3 (0%)
Control
(-)
(-)
(-)
(-)
(-)
F14 20-PET
0/3 (0%)
(Verschoor et al. 1996)
V3-fusion protein Ie
E..Coli(s.c.)
UT113 (A)
100
0,4,6,8,10
AlOH
NA(s.c.)
10-20UT113
0/5 (0%)
V3-fusion protein I
E..Coli(s.c.)
UT113
100
0,6,10
QuilA
NA(s.c.)
10-20UT113
0/5 (0%)
Control(PBS)
(-)(s.c.)
(-)
(-)
0,6,10
(-)
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-Env
(-)oronasal/s.c.
UT113
10s PFU
0
(-)
Boost V3-peptide
E. Coli (i.m.)
UT113
100
4,8
AlOH
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-Env
(-)oronasal/s.c.
UT113
105PFU
0
(-)
Boost V3 peptide
E.Coli (i.m.)
UT113
100
4,8
QuilA
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-b Gal.
(-)oronasal/s.c.
(-)
105PFU
0
(-)
Boost PBS
(-)(s.c.)
(-)
(-)
4,8
(-)
NA(s.c.)
10-20UT113
0/5 (0%)
(Gonin et al. 1995)
Adenovirus-env
(-)(i.m.)
Wo (A)
11.8-9.2PFU
0,4,30
ISA206
NA(NA)
20 Wo
0/4 (0%)
Adeno-psuedorabies(control)
(-)(i.m.)
(-)
11.8-9.2PFU
0,4,30
ISA708
NA(NA)
20 Wo
0/4 (0%)
a UK-8, United Kingdom; Z2, Zurich; BANG, Bangston; AM19, Amsterdam 19; PET, Petaluma.
b Recombinant vaccinia virus expressed
c Baculovirus expression
d iscom, immune stimulating complex; A10H/QS21, aluminumhydroxide and non-toxic fraction from Quillaja saponaria; ISA206, water/oil adjuvant;ISA708, water/oil adjuvant,
e V3-fusion protein was composed of the FIV V3 region fused to galactokinase.
f Deletion of the cleavage site between the envelope surface(SU) and transmembrane protein(TM).


105
mucosal route, should be evaluated. In addition, the protective efficacy should be
examined against FIV field isolates, higher challenge doses and other types of challenge
inocula, such as cell-associated and plasma-derived virus. Moreover, studies should be
undertaken to further assess the protective efficacy against FIV strains of heterologous
subtypes. It will be particularly interesting to evaluate if cats immunized with ALVAC-
gag/prot alone can resist heterologous subtype challenge since sequence variability in the
gag gene is significantly lower between different FIV isolates when compared to the
variability observed in the env gene. Therefore, protective immune responses directed
against Gag epitopes may cross-react with a wider range of FIV isolates. If such protection
can not be achieved, ALVAC recombinants encoding Gag of multiple FIV isolates could be
tested for their ability to elicit broad-range protective immunity.
Furthermore, it would be of interest to evaluate immunization protocols involving
priming with ALVAC-gag/prot in combination with inactivated FIV-infected cell vaccine.
This may be a more optimal combination than ALVAC-env,gag/prot in combination with
ICV as ALVAC-env,gag/prot alone lacked protective efficacy.
Although we were able to demonstrate protective immunity upon immumzation with
ALVAC-based FIV vaccines, the mechamsm(s) involved in the observed protection
remains unclear. This important issue needs to be addressed in future studies. To further
delineate the role of cell mediated and humoral responses naive cats could be passively
immunized with serum or cells from ALVAC-FIV immunized cats and subsequently tested
for their ability to resist experimental FIV infection. Further, efforts should be made to
improve the current methods used to assess the induction of cell-mediated responses in
particular CTL responses. At present, these assays require large numbers of cells and
prolonged in vitro culturing which may result in inaccurate representation of what actually
occurs in vivo. Since our study implies a role for Gag in protection the next step would be


Table 2.9a Virus isolation (RT and PCR) on PBMC and immunoblot analysis.
Vaccine
Immunizations
pre- post-
Cat ID# -3 -1 +1
WB RT PCR WB RT PCR WB RT PCR
Months post-challenge
+2 +3 +4 +8 +12
WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR
QH4
PY1 ___ ___ + + + + + + + + + + +
ALVAC-env QOl - + + + + + + + +
QC1
QU1
QL2 ___ ___ + + + + + + + + + + +
QQl
QA5
ALVAC-gag/prot QU2
QX3
QI1
QL3
QH5
PY3
ALVAC-e/iv, QS4
gag/prot QC3
QG3
QE2
- + + + + + + + + + + +
+ -- + + + +- +
-4
-4
WB= western blot (FIV-specific), RT= reverse transcriptase, PCR using FIV-specific primers.


114
Lawrence, C., Weinhold, K., McElrath, J., Excler, J.L., DuliegeA.M., Clements M.L.,
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Liu, R., Paxton, W.A., Choe, S., Ceradini, D., Martin, S.R., Horuk, R., MacDonald,
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CHAPTER I
LENTI VIRUSES AND VACCINE DEVELOPMENT
Introduction
Lentiviruses comprise a group of viruses known to cause life-long chronic
infections in a number of species (Table 1.1). The most prominent member of this group is
the human immunodeficiency virus (HIV), the causative agent of an acquired
immunodeficiency syndrome (AIDS) in man. HIV was first isolated from young male
homosexual patients in 1983 (Barre-Sinousi et al. 1983; Gallo et al. 1984). These patients
presented with a high incidence of a pneumonia caused by Pneumocystis carinii thus far
only known to cause disease in immuno-compromised people and a rare cancer, Kaposi
sarcoma (Ammann et al. 1983; Gyorkey et al. 1984; Selik et al. 1984; Weissler 1990).
More important, it was noticed that the numbers of circulating lymphocytes, in particular
those of the CD4+ T-helper phenotype, were severely reduced in these patients, a
phenomenon which is now considered one of the main hallmarks of HIV infection in man
(Huet et al. 1990). Many years prior to the discovery of HIV, a number of viruses, much
later classified as lentiviruses, had been described as pathogens in animals. This included
the equine infectious anemia virus (EIAV) which was identified in 1904 as the causative
agent of a disease in horses characterized by recurrent episodes of fever and hemolytic
anemia (Vallee and Carre 1904). This also included the maedi-visna virus (MW) which
was first isolated from sheep in Iceland that presented with severe chronic pneumonia
(maedi), wasting and paralysis (visna) (Gislason 1947; Narayan et al. 1977). Based on the
long incubation period and the fact this virus could manifest its effects over a long period,
1


Cats immunized with the ALVAC recombinants encoding the FIV core (Gag)
protein were protected from challenge exposure with 50 ID50 FIV Petaluma, a subtype A
FIV isolate highly related to the Villefranche isolate basis of the ALVAC-FIV vaccine. In
contrast, ALVAC recombinants expressing the FIV envelope alone or both the FIV
envelope and Gag proteins failed to induce such protection. Cats immunized with ALVAC-
FIV recombinants and boosted with ICV were also protected from a FIV Petaluma
challenge exposure. In addition, these cats were partially protected from challenge with 75
IDS0 FIV Bangston (subtype B), a distinctly heterologous isolate, given eight months after
the initial challenge without any intervening booster.
In conclusion, vaccine protocols employing recombinant ALVAC-based FIV vaccines
alone or in combination with conventional inactivated FI V-infected cell vaccine can prevent
establishment of FIV infection in cats. This immunity may even protect or delay infection
with FIV isolates of other subtypes than those used to generate the vaccine. It remains
unclear as to what constituted protective immunity in the protected animals. The obtained
data suggest a role for cell-mediated responses. However, a role for FIV specific humoral
responses including viral neutralizing antibody responses can not be excluded.
xi


110
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47
solution, followed by incubation with ELISA substrate solution (0.005 %
tetramethylbenzidine and 0.015 % H202 in 0.96 % citrate solution). The reactions were
stopped with 0.1 M sulfuric acid upon establishment of visible reaction color. The plates
were read in a ELISA reader at 414 nm.
Assessment of viral neutralizing (VN) antibodies
The presence of FIV specific VN antibodies was evaluated using a standard assay
(Yamamotoet al. 1991). Serum samples obtained preimmunization, postimmunization and
after challenge were diluted at various concentrations (1:5 to 1:100) and incubated at 56C
for 30 minutes to inactivate complement. The diluted sera were then incubated with 100
TCIDS0 (tissue culture cell infective doses of HVpJ for 45 min at 37C in a 24-well
microtiter plate. Subsequently, peripheral blood mononuclear cells (PBMC) were added to
this mixture at lxlO6 cells/well. After three days of culturing, cells were washed to remove
residual virus from the culture and resuspended in fresh culture media (see RT media).
Virus infection was monitored by Mg2+ dependent reverse transcriptase (RT) activity (see
RT assay) in culture fluid harvested on Day 6, 9, 12, 15 and 18 of culturing. The VN
antibodies titers were defined as the reciprocal of the highest final dilution which gave
>50% reduction in reverse transcriptase activity as compared to the reverse transcriptase
activity detected in fluids from control cell cultures that contained SPF serum and virus.
Assessment of FIV-specific proliferative T-cell responses
FIV-specific proliferative responses were evaluated using a 3[H]-thymidine
incorporation assay (Yamamoto etal. 1991). Freshly isolated PBMC were cultured in 96-
well microtiter plates in a final volume of 200 pi in RPMI1640 media supplemented with
5% heat inactivated fetal calf serum, lOmM HEPES buffer, 50 mg/ml gentamycin, 5X10 s


9
adapted FIV strains. However, ectopic expression of CD9 on these cells does not seem to
render these cells more susceptible infection with primary FIV isolates (Willetet al. 1996).
Following attachment, the FIV enters the cell either by receptor mediated
endocytosis or by fusion of the viral envelope with the cellular membrane. Upon entry the
viral genome is released into the cellular cytoplasm. Subsequent, the viral RNA genome is
transcribed into double-stranded proviral DNA by the viral reverse transcriptase packaged
within the virion. Similar to HIV, initiation of first strand cDNA synthesis is primed by
cellular tRNAlys (Olmsted etal. 1989b; Talbott etal. 1989). Integration of the provirus into
the host cellular genome is facilitated by the viral Integrase and occurs at random sites. The
integrated provirus can stay quiescent or give rise to progeny viral particles. During
productive infection, the proviral DNA is transcribed into viral mRNAs by cellular RNA
polymerase II. These mRNAs are translated into the Gag precursor (p55), the Gag-Pol
precursor (pl60) and the Env precursor proteins (gpl60). The Gag precursor is further
processed by proteolysis to give rise to the capsid (CA), matrix (MA), and nucleocapsid
(NC) proteins. Proteolysis is mediated by the viral protease which also facilitates its own
cleavage (Elder et al. 1993). Similar to HIV, the FIV matrix protein is myristolated.
Myristolation is the attachment of a C14 fatty acid and is required for proper targeting of the
MA protein to the cellular membrane (Elder et al. 1993). The Env precursor protein is
cleaved into the mature SU and TM proteins by cellular proteases and further processed by
glycosylation. For FIV, 22 possible N-linked glycosylation sites have been identified; 18
in the SU and 4 in the TM protein (Stephens et al. 1991; Elder et al. 1993). The final step
in the FIV replication cycle involves the assembly of the virions and their release from the
cell by budding.


Figure 3.1. Phylogenetic relationship between FIV-isolates comparing envelope sequences. The four subtypes (A-D) are grouped with
circles.
5% diversity


IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION OF
CANARYPOXVIRUS(ALVAC)-BASED FIV VACCINES IN CATS
By
MARIA C. TELLIER
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
1996

Luctor et Emergo

For Ella and Bram

ACKNOWLEDGMENTS
This thesis would not have been written without the influence, presence, and
support of many people. I am grateful to my family, especially Pa and Ma, for their
care and support of me and my endeavors over the years. Further, I would like to
recognize my supervisor Janet Yamamoto, who has struck me with her infinite commitment
to research. I thank her for letting me pursue this project in her laboratory and giving me
the opportunity to become acquainted with the World of Science and Bureaucracy. Special
thanks go to Dr. Pu for his expertise in feline phlebotomy, his advice and his the anecdotes
from the old China. Further, I was fortunate to have David Pollock as my right hand. I
highly appreciate his meticulous work during the countless virus isolation assays and his
chit-chat dunng the endless fycoll sessions. I am especially indebted to Dr. Paoletti and
Dr. Tartagliaat Virogenetics and Dr. Desmettre at Rhone Merieux for providing me the
opportunity to work with the ALVAC vector in my studies. I also wish to acknowledge the
members of my supervisory committee, Dr. Condit, Dr. Schiffenbauer, Dr. Johnson and
Dr. Schuster for their time and advice.
At the homefront, I would like thank Nicole, Mienke, Mirian, Chantal and Simone
for their friendship and, Dr. Paul and Dr. Frank, for staying in touch and keeping me
updated on the latest from the low-lands. Warm thanks go to Sue Watsana, who was a
motivating source at frustrating times. Special thanks go to Susie for all the exciting
adventures and for the good times we have had in our lovely shack on NW 2nd street. Last
but not least, I would like to thank Dan for his patience and great care of me, Tamika and
the Kalaharis. Finally, I would like to thank all my friends in and out of Gainesville for
making every day life a little more exciting.
IV

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iv
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ABBREVIATIONS viii
ABSTRACT x
CHAPTER
ILENTI VIRUSES AND VACCINE DEVELOPMENT
Introduction 1
II IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION
OF CANARYPOXVIRUS( ALVAQ-B ASED FIV VACCINE AGAINST
HOMOLOGOUS FIV CHALLENGE
Introduction 36
Materials and Methods 38
Results 54
Discussion 85
III EFFICACY EVALUATION OFCANARYPOXVIRUS(ALVAC)
BASED FIV VACCINE COMBINED WITH INACTIVATED FIV-CELL
VACCINE AGAINST HETEROLOGOUS FIV CHALLENGE
Introduction 94
Material and Methods 97
Results 98
Discussion 100
IV SUMMARY AND FUTURE STUDIES
Synopsis 104
REFERENCES 107
BIOGRAPHICAL SKETCH 121
v

LIST OF TABLES
Table page
1.1 The lentiviruses 3
1.2 Conventional inactivated whole virus FIV vaccine trials 19
1.3 Conventional inactivated FIV-infected cell vaccine trials 20
1.4 Subunit and vector based FIV vaccine trials 21
1.5 Immunogenicity and prophylactic efficacy of ALVAC-based vaccines 29
1.6 Immunogenicity and prophylactic efficacy of ALVAC-based vaccines
against retroviral pathogens 35
2.1 Grouping and immunization 44
2.2 CD4/CD8 ratios after immunization and challenge 63
2.3 Proliferation to T-cell mitogens (ConA and SEA) 64
2.4 FIV-specific antibody titers after immunization and challenge 70
2.5 Viral neutralizing antibody titers after immunization and challenge 71
2.6 T-helper responses to FIV after immunization and challenge 73
2.7 FIV-specific CTL activity in peripheral blood after immunizations 74
2.8 NK activity postchallenge 75
2.9 Virus isolation (RT and PCR) on PBMC and WB data pre- and
postchallenge 77
2.10 Virus isolation on PBMC and tissue samples and WB and ELISA data 79
2.11 Statistical analysis 84
3.1 Immune parameters and viral-status pre-and postchallenge 99
vi

LIST OF FIGURES
Figure page
1.1 Electron micrograph of FIV x70,000 6
2.1 Schematic representation of ALVAC constructs 40
2.2 PCR products of CrFK cells infected with ALVAC-env recombinants 55
2.3 PCR products of CrFK cells infected with AlAJ AC-gag/prot recombinants 56
2.4 RT-PCR products of CrFK cells infected with ALVAC-FIV recombinants 57
2.5 Immunofluoresence on CrFK cells infected with ALVAC-FIV recombinants 59
2.6 FACS analysis of CD4 and CD8 staining of PBMC 62
2.7 Immunoblot of ALVAC-specific humoral responses 67
2.8 Immunoblot of FIV-specific humoral responses 69
2.9 Immunoblot of FIV-specific humoral responses in kittens 83
3.1 Phylogenetic relationship between FIV-isolates 96
vii

LIST OF ABBREVIATIONS
ADCC
Antibody dependent cell-mediated cytotoxicity
AIDS
Acquired immunodeficiency syndrome
BM
Bone marrow
BSA
Bovine serum albumin
CTL
Cytotoxic T-lymphocyte
DNA
Deoxyribonucleic acid
EDTA
Ethylenediaminetetraacetate
EUSA
Enzyme-linked immunosorbent assay
FACS
Fluorescence-activated cell sorting
FITC
Fluorescein isothiocyanate
FIV
Feline immunodeficiency virus
HIV
Human immunodeficiency virus
LN
Lymph node
MHC
Major histocompatibility complex
PBL
Peripheral blood lymphocytes
PBMC
Peripheral blood mononuclear cells
PBS
Phosphate-buffered saline
PCR
Polymerase chain reaction
RNA
Ribonucleic acid
RT
Reverse transcriptase
SDS
Sodium dodecyl sulfate
SIV
Simian immunodeficiency virus

TAE
Tris, EDTA buffer
TEMED
N,N,N\N-tetramethylethylenediamine
THY
Thymus
Tris
T ris(hydroxymethyl)aminomethan
VN
viral neutralizing
WB
western blot
IX

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of
Doctor in Philosophy
IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION OF
CANARYPOXVIRUS (ALVAC)-BASED FIV VACCINES IN CATS
By
Maria C. Tellier
December, 1996
Chairman: Dr. J.K. Yamamoto
Major Department: Veterinary'Medicine
The infection of cats with the feline immunodeficiency virus (FIV) provides a
valuable animal model for the assessment of therapeutic and vaccine strategies against
human immunodeficiency virus (HIV) in man. A promising candidate vaccine tested
presently in human volunteers is the recombinant canarypoxvirus vector ALVAC. This
vector has also been used with some success against HIV infection in the macaque and
chimpanzee models. Herein, the efficacy of ALVAC-based vaccines was evaluated against
experimental FIV infection in cats. Two approaches were evaluated which included
ALVAC-based FIV vaccines alone or in combination with conventional inactivated FIV-
infected cell vaccine (ICV).
Immunization schemes employing ALVAC-FIV recombinants alone effectively
induced FIV-specific cytotoxic T-cell responses. However, these schemes failed to induce
humoral responses including viral neutralizing antibody responses. Immunization schemes
employing ALVAC-FIV recombinants combined with conventional inactivated F1V-
infected cell vaccine induced FIV-specific cytotoxic T-cell responses and FIV-specific
humoral responses but lacked detectable viral neutralizing antibody responses.
x

Cats immunized with the ALVAC recombinants encoding the FIV core (Gag)
protein were protected from challenge exposure with 50 ID50 FIV Petaluma, a subtype A
FIV isolate highly related to the Villefranche isolate basis of the ALVAC-FIV vaccine. In
contrast, ALVAC recombinants expressing the FIV envelope alone or both the FIV
envelope and Gag proteins failed to induce such protection. Cats immunized with ALVAC-
FIV recombinants and boosted with ICV were also protected from a FIV Petaluma
challenge exposure. In addition, these cats were partially protected from challenge with 75
IDS0 FIV Bangston (subtype B), a distinctly heterologous isolate, given eight months after
the initial challenge without any intervening booster.
In conclusion, vaccine protocols employing recombinant ALVAC-based FIV vaccines
alone or in combination with conventional inactivated FI V-infected cell vaccine can prevent
establishment of FIV infection in cats. This immunity may even protect or delay infection
with FIV isolates of other subtypes than those used to generate the vaccine. It remains
unclear as to what constituted protective immunity in the protected animals. The obtained
data suggest a role for cell-mediated responses. However, a role for FIV specific humoral
responses including viral neutralizing antibody responses can not be excluded.
xi

CHAPTER I
LENTI VIRUSES AND VACCINE DEVELOPMENT
Introduction
Lentiviruses comprise a group of viruses known to cause life-long chronic
infections in a number of species (Table 1.1). The most prominent member of this group is
the human immunodeficiency virus (HIV), the causative agent of an acquired
immunodeficiency syndrome (AIDS) in man. HIV was first isolated from young male
homosexual patients in 1983 (Barre-Sinousi et al. 1983; Gallo et al. 1984). These patients
presented with a high incidence of a pneumonia caused by Pneumocystis carinii thus far
only known to cause disease in immuno-compromised people and a rare cancer, Kaposi
sarcoma (Ammann et al. 1983; Gyorkey et al. 1984; Selik et al. 1984; Weissler 1990).
More important, it was noticed that the numbers of circulating lymphocytes, in particular
those of the CD4+ T-helper phenotype, were severely reduced in these patients, a
phenomenon which is now considered one of the main hallmarks of HIV infection in man
(Huet et al. 1990). Many years prior to the discovery of HIV, a number of viruses, much
later classified as lentiviruses, had been described as pathogens in animals. This included
the equine infectious anemia virus (EIAV) which was identified in 1904 as the causative
agent of a disease in horses characterized by recurrent episodes of fever and hemolytic
anemia (Vallee and Carre 1904). This also included the maedi-visna virus (MW) which
was first isolated from sheep in Iceland that presented with severe chronic pneumonia
(maedi), wasting and paralysis (visna) (Gislason 1947; Narayan et al. 1977). Based on the
long incubation period and the fact this virus could manifest its effects over a long period,
1

2
MW was named a lenti- (slow) virus (Sigurdsson 1954). This virus has since become
the prototype of the lentivirus genus. In 1974, a virus similar to MW was identified in
goats, the caprine arthritis encephalitis virus (CAEV). CAEV infection presents as chronic
inflammation of the joints in adult goats and progressive encephalopathy in younger goats
(Clements et al. 1980; Cork 1974).
Thus, several animal lentiviruses had been long known before HIV. However, it
was the discovery of HIV that resulted in an increased interest in these viruses and led to
the search and isolation of lentiviruses in other species. For example, a number of
lentiviruses were isolated from monkeys. These viruses have collectively been named the
simian immunodeficiency viruses (SIV) and are specified by the particular monkey species
they have been isolated from (Table 1.1) (Huet et al. 1990; Kanki et al. 1985; Ohta et al.
1988; Tjusimotoetal. 1988; Hirsch etal. 1993; Peeters et al. 1992; Fultz et al. 1986). In
contrast to HIV infection of man, SIV infection of natural host African monkey species, is
relatively nonpathogemc. However, the exception to this is SIV infection in Asian
macaques (Daniel et al 1985; Benvisti et al. 1986). Infected Asian macaques develop an
acquired immunodeficiency syndrome similar to that of HIV infected individuals
(Murphey-Corb et al. 1986). Interestingly, most of the nonpathogenic SIV strains have
been isolated from monkeys in the wild. SIV^, however, has only been isolated from
macaques held in captivity and has never been isolated from this species in its natural
habitat. Since these monkeys are of Asian origin whereas other SIV infected monkeys are
of African origin it has been suggested that this virus was transmitted to macaques during
captivity from an African monkey species, most likely the mangebeys (Murphey-Corb et al.
1986).
The second human lentivirus, now known as HIV-2, was first isolated in 1986
(Clavel et al. 1986). HIV-2 is predominantly found in West-African prostitutes and also
causes AIDS although milder in its pathogenesis as marked by a longer incubation period
and a lower rate of transmission (Marlink et al. 1994). Much like SIV infection of Asian

3
macaques, the emergence of HIV in humans is thought to be caused by cross-species
transmission. This is supported by the fact that HIV-2 genetically closely resembles the
SIV_ and SlV^-isolates, and HIV-1 more closely resembles the SIVcpz-isolate (Hirsch
and Johnson 1994; Franchini etal. 1987).
Table 1.1 The Lenti viruses
Virus
subtypes
species
Clinical signs of disease
EIAV
horse
anaemia
fever
weight loss
Maedi-visna
sheep
encephalomyelitis
wasting
pneumonia
CAEV
goats
arthritis
encephalomyeli tis
wasting
BIV
cows
lymphadenopathy
lymphocytosis
wasting
FIV
cats
immunodeficiency
opportunistic infections
neurological disorders
HIV
HIV-1
human
immunodeficiency
lymphadenopathy
neurological syndrome
opportunistic infections
HIV-2
human
i mmunodeficiency
1 y mphadenopathy
opportunistic infections
SIV
SIV
mac
SIV
smm
SlV^m
macaques
sooty mangabey
African green monkey
immunodeficiency
neurological disease
SIVmnd
mandrill
no obvious clinical signs
SIVsyk
SIV^
Sykes monkey
chimpanzee
of disease

4
In addition to the primate lentiviruses, a virus similar in morphology and genetic
composition, the bovine immunodeficiency virus (BIV), was isolated from cows in 1985.
Although, the pathogenesis of BIV is poorly defined, infection in calves has been
associated with lymphocytosis and lymphadenopathy (Gonda et al. 1987, 1994).
The feline homologue of HIV, feline immunodeficiency virus (FIV) was first
isolated in 1986 from a cattery in California (Pedersen et al. 1987). In this cattery, several
cats presented with a loss in immune function after the introduction of a sentinel cat. The
loss of immune function could not be linked to feline leukemia virus (FeLV), another
member of the Retroviridae, already known to cause immunosupression in cats (Jarret et al.
1964). This led to the discovery of a novel retrovirus that differed from FeLV and more
closely resembled HIV in morphology and the Mg2+- rather than Mn2+-dependence of its
reverse transcriptase (Yamamoto et al. 1988a). Subsequent genetic analysis demonstrated
that this virus belonged to the lentivirus family.
The seven known members of the lentivirus family as listed above are commonly
divided into two groups based on differences in cell tropism and disease manifestation.
Those affecting the ungulate species EIAV, MW, and CAEV are predominantly
macrophage-tropic and cause immune-mediated diseases that target specific organs. Those
affecting primates, HIV and SI V, are tropic for lymphocytes and macrophages and cause a
major loss of immune function that results in an increased susceptibility to opportunistic
pathogens. FIV resembles the primate viruses in cell tropism and disease manifestation but
is genetically more closely related to the nonprimate lentiviruses (EIAV and MVV)
(Olmsted et al. 1989b).
Common to all lentiviruses is the long incubation period, the ability to affect
multiple organs, and most importantly the persistence in the face of host-immune
responses. The ability to escape from the host immunity is in part explained by a high
mutation rate of the lentiviral genome resulting in continuous antigenic variation (Rigby et

5
al. 1993). As such, lentiviruses present a major challenge to the development of
therapeutic strategies and vaccines.
The Feline Immunodeficiency Virus
The FIV virion, like other members of the Retrovindae, is enveloped and
approximately 100-125nm in diameter (Pedersen et al. 1987). A electron micrograph of a
mature lentivirion is depicted in Figure 1.1. The FIV core, composed of the capsid protein
(CA, p24), has the physical appearance of a cone, typical of all lentiviruses. The core
encloses the viral genome which consists of two identical single stranded RNA molecules
of approximately 9kb (Elder et al. 1993). Associated with the viral genome is a small
nucleocapsid protein (NC, p8), which is thought to play a role in viral assembly and
disassembly (Elder et al. 1993; Aldovini and Young 1990). Also packaged within the
virion are the enzymes essential for replication, the reverse transcriptase (RT), the integrase
(IN) and the deoxyundine triphosphatase (dUTPase; DU)(Elder et al. 1993). The RT is
responsible for transcription of the viral RNA genome into DNA. The integrase functions
as an endonuclease which cuts the cellular genome to allow integration of the provirus.
The dUTPase is unique to the nonprimate viruses and its function in the FIV life cycle still
remains unknown. However, it is speculated that the dUTPase is required for replication
of FIV in nondividing macrophages or resting T-lymphocytes (Miyazawa et al. 1993).
Additionally, it has been observed that lack of functional FIV dUTPase results in an
increased mutation frequency of FIV propagated in macrophages (Lemer et al. 1995).
Surrounding the viral core is a matrix protein layer (MA, pl5) which is closely associated
with the viral envelope. The viral envelope is derived from the cellular membrane by
budding of the viral particles and consists of lipids, inserted viral proteins and cellular
proteins (Gelderblom et al. 1987). The virally derived envelope glycoprotein (Env) is
composed of two subunits; the transmembrane protein (TM, gp40) and the surface protein

6
(SU, gpl20). The TM protein protrudes through the viral membrane and noncovalently
anchors the outer SU protein, which appears as a knob-like structure on the viral surface.
Figure 1.1 Electron micrograph of FI V x70,000.
FI V genomic organization and regulation of gene expression
Lentiviral genomes are among the smallest of the known viruses and are more
complex in their genomic organization than other members of the retrovirus family. The
FIV genome is a positive stranded polyadenylated RNA of approximately 9kb. It contains
three major open reading frames (ORFs) encoding the structural and enzymatic proteins
(Env,Gag and Pol) necessary for the viral life cycle (Olmsted et al. 1989a, 1989b;

7
Miyazawa 1993). These genes are organized in the order of 5-gag-pol-env-3, typical of
all replication-competent retroviruses. In addition, lentiviruses contain several small open
reading frames (ORFs) that encode for auxiliary proteins. At least 4 ORFs have been
identified for FIV which may encode for regulatory proteins similar to those described for
the primate lentiviruses (Miyazawa et al. 1993; Olmsted et al. 1989b). Flanking the
proviral genome are long terminal repeat (LTR) regions which are crucial for integration of
the proviral DNA into the cellular genome. These regions also contain enhancer and
promoter elements which are required for efficient transcription of the retroviral genome, as
well as, a polyadenylation signal sequence (Phillips et al. 1992; Talbott et al. 1989).
Transcription of the integrated proviral DNA genome initiates at the 5LTR to
generate full length viral mRNA transcripts (Miyazawa et al. 1993). Initially, these
transcripts will undergo multiple splicing and give rise to small mRNAs that encode for
regulatory proteins such as Tat and Rev. The Tat protein facilitates gene expression from
the 5LTR. Tat activity is essential for replication of all primate lentiviruses. In contrast,
Tat is not essential for replication of FIV in T-lymphoblast cells (Sparger et al. 1992;
Miyazawa et al. 1993). The Rev protein, encoded by all members of the lentiviridae, is
responsible for shifting viral gene expression from early regulatory proteins to that of
structural and enzymatic proteins. It accomplishes this by binding to the Rev-responsive
element (RRE) in the env coding region of single spliced and unspliced full length mRNAs.
In doing so, it promotes the stability and transport of incompletely spliced mRNAs from
the nucleus to the cytoplasm (Phillips et al. 1992; Cochrane et al. 1990; Hammarskjold et
al. 1989; Stephens et al. 1992). The single spliced messages give rise to the Env precursor
protein which is cleaved into the transmembrane glycoprotein (TM) and the outer surface
glycoprotein (SU) by cellular proteases (Stephens etal. 1991; Talbott etal. 1989).
The unspliced full length mRNA serves as both a template for the Gag and Gag-Pol
proteins and as genome that is packaged into the viral core. Regular translation of full
length message gives rise to the Gag precursor protein which is cleaved into the mature

8
capsid (CA), matrix (MA) and nucleocore (NC) proteins by the viral protease encoded
within thepol gene (Elder et al. 1993). Expression of the Gag-Pol precursor protein from
the unspliced full length mRNA is accomplished by a ribosomal frameshift that prevents
termination of translation at the gag stop codon (Morikawa and Bishop 1992). The
efficiency of this shift is about 5%, so that the production of Gag protein is 20 fold higher
than that of Gag-Pol polyprotein. The Gag-Pol precursor protein is proteolytically
processed into the viral protease (PR), the reverse transcriptase (RT), the deoxyundine
triphosphatase (DU) and the integrase (IN) proteins (Elder et al. 1993).
FIV replication
The first step in the replication of FIV is the attachment of the virus to the cell
receptor. For HIV, the major receptor has been identified as the CD4 molecule, present on
T-helper lymphocytes (Dagleish et al. 1984; Klatzman et al. 1984). In addition, the CD26
molecule and the recently described fusin and CKR-5 molecules have been reported to play
a role in HIV attachment and fusion (Callebaut et al. 1993; Feng et al. 1996; Alkhatib
1996). The fusin is thought to act as a coreceptor for T cell line-tropic HIV strains whereas
the CKR-5 is thought to actas a coreceptor for macrophage-tropic HIV strains (Feng et al.
1996; Alkhatib 1996). The target receptor(s) for FIV, however, is still unknown.
Nonlymphoid feline cell lines transfected with cDNA encoding the feline CD4 (fCD4)
protein failed to support productive infection indicating that the fCD4 alone is not sufficient
(Nonmine et al. 1993). Others have proposed a putative role for a receptor homologous to
the human CD9 molecule that is expressed on both haematopoietic and nonhaematopoietic
cells (Willetet al. 1994; Hosie et al. 1993; Boucheix and Beiot 1988). Anti-CD9 antibodies
effectively block replication of FIV infection on lymphoid cells and ectopic expression of
CD9 on feline lymphoma cells causes an enhancement of viral infection with cell culture

9
adapted FIV strains. However, ectopic expression of CD9 on these cells does not seem to
render these cells more susceptible infection with primary FIV isolates (Willetet al. 1996).
Following attachment, the FIV enters the cell either by receptor mediated
endocytosis or by fusion of the viral envelope with the cellular membrane. Upon entry the
viral genome is released into the cellular cytoplasm. Subsequent, the viral RNA genome is
transcribed into double-stranded proviral DNA by the viral reverse transcriptase packaged
within the virion. Similar to HIV, initiation of first strand cDNA synthesis is primed by
cellular tRNAlys (Olmsted etal. 1989b; Talbott etal. 1989). Integration of the provirus into
the host cellular genome is facilitated by the viral Integrase and occurs at random sites. The
integrated provirus can stay quiescent or give rise to progeny viral particles. During
productive infection, the proviral DNA is transcribed into viral mRNAs by cellular RNA
polymerase II. These mRNAs are translated into the Gag precursor (p55), the Gag-Pol
precursor (pl60) and the Env precursor proteins (gpl60). The Gag precursor is further
processed by proteolysis to give rise to the capsid (CA), matrix (MA), and nucleocapsid
(NC) proteins. Proteolysis is mediated by the viral protease which also facilitates its own
cleavage (Elder et al. 1993). Similar to HIV, the FIV matrix protein is myristolated.
Myristolation is the attachment of a C14 fatty acid and is required for proper targeting of the
MA protein to the cellular membrane (Elder et al. 1993). The Env precursor protein is
cleaved into the mature SU and TM proteins by cellular proteases and further processed by
glycosylation. For FIV, 22 possible N-linked glycosylation sites have been identified; 18
in the SU and 4 in the TM protein (Stephens et al. 1991; Elder et al. 1993). The final step
in the FIV replication cycle involves the assembly of the virions and their release from the
cell by budding.

10
FIV cell tropism
FIV has a broad cell tropism and infects cells of both lymphoid and
monocyte/macrophage origins. In contrast to HIV, which is thought to primarily replicate
in CD4+ T-lymphocytes and not in CD8+ T-lymphocytes, FIV productively infects both
CD4+ and CD8+ T-lymphocytes (Brown et al. 1991). Additionally, FIV has been shown to
replicate in B-cells, thus further supporting the view that the feline CD4 receptor is not the
primary cell receptor for FIV, as it is for HIV (English et al. 1993; Norimine et al. 1993).
The macrophage/monocyte cell types supporting FIV replication include peritoneal
macrophages, Kupffer cells in the liver, microglial cells, astrocytes, and endothelial cells in
the central nervous system (Steffan et al. 1994; Dow et al. 1990; Martin et al. 1995;
Brunner and Pederson 1989). Furthermore, a number of FIV isolates have been shown to
infect cells of nonlymphoid origin including Crandell feline kidney cells (CrFK) and feline
tongue cells (Fc3Tg) (Yamamoto et al. 1988a).
FIV epidemiology and pathogenesis
FIV has been isolated from cats worldwide. The virus infects domestic cats (Felis
catus) and is species specific (Yamamoto et al. 1988b, 1989). FIV-related viruses have
been isolated from several wild felids including the African lion (Panthera leo), and the
Pallas cat (Felis tnanul) (Barr et al. 1989, 1995; Poli et al. 1995; Brown et al. 1994).
Furthermore, serologic surveys in African and Asian lions revealed that the serum of the
majority of these animals reacted positively with FIV (Brown et al. 1994).
The prevalence of FIV varies throughout the world. In North America the average
incidence is estimated at 1.4% in healthy animals and 7.4% in diseased cats (Yamamoto et
al. 1989; Shelton et al. 1990). The incidence of infections is the highest in free roaming,
outdoor male cats. Since FIV is shed in the saliva, the major route of transmission is most

11
likely due to biting between male cats as part of territorial behavior (Yamamotoet al. 1989).
In addition to saliva, FIV can be recovered from blood, serum, plasma, and cerebrospinal
fluid of infected cats (Pedersen et al. 1987; Dow et al. 1990). Horizontal transmission
through contact alone appears to be inefficient (Pedersen et al. 1987; Yamamoto et al.
1988b). Vertical transmission in tero or postpartum via the milk has been reported and
was found to occur most frequently in queens that became viremic during pregnancy
(Callanan et al. 1991; Wasmoen et al. 1992; ONeil et al. 1996). Although high rate of
perinatal transmission has been reported for queens that had been infected with a highly
pathogenic FIV strain 4 to 30 months prior to conception (Ueland and Nesse 1992; ONeil
et al. 1996).
Similar to HIV, five clinical stages can be defined for FIV infection in cats. The
acute viremic phase, 2 to 4 weeks after infection, is characterized by fever, neutropenia,
and generalized lymphadenopathy. These symptoms vary in duration and severity between
individual cats and are mostly recognized in experimentally infected cats but rarely in
naturally infected cats (Yamamoto et al. 1988b, 1989). Once a full immune response is
established and most of the virus is cleared from the plasma, a period of months to years
follows with no obvious clinical signs of disease, defined as the asymptomatic phase.
However, during this period changes in lymphocyte counts, such as a decrease in CD4+
lymphocytes and CD4/CD8 ratios, takes place (Ackley et al. 1990). This period is
followed by a phase equivalent to that of AIDS related complex (ARC) in men. Cats with
ARC often present with chronic illness such as stomatitis/gingivitis, lower urinary tract
infections, skin disorders and diarrhea (Yamamotoet al. 1989). Finally, cats may develop
a stage similar to that of AIDS in men, characterized by severe lymphoid depletion, weight
loss and opportunistic infections. Opportunistic pathogens reported in cats suffering from
AIDS include toxoplasmosis, cryptococcoses, candidiasis, mycobacteriosis, feline calici-
and herpes virus (Lapin et al. 1989; Knowles et al. 1989; Ishida and Tomoda 1989). At
this stage, CD4+ T-cell counts have dropped dramatically and CD4:CD8 ratios are inversed

12
(Ackley et al. 1990). Other immunologic abnormalities reported include hypergamma
globulinemia and reduced T-cell responses to T-independent antigens (Ackley et al. 1990;
Tortenetal. 1991). The occurrence of neurological abnormalities as seen in AIDS patients
has only been reported in a small percentage of cats infected with neurotropic isolates of
FIV (Podelletal. 1993; Dow etal. 1990).
FIV as an animal model for HIV
With the emergence of the HIV pandemic and an estimated 12 million infected
people, animal models to study antiviral drugs and vaccine strategies have become very
important. The search for an appropriate animal model for HIV-1 infection in man has,
however, been complicated by the host specificity of HI V-l.
To date, HIV-1 has only been shown to infect three other species; pig-tailed
macaques, gibbons, and chimpanzees (Agey et al. 1992; Fultz et al. 1986). These animals
become viremic and mount specific antibody responses similar to HIV infected humans.
However, infection does not result in depletion of CD4+ T-lymphocytes,
immunosuppression, or opportunistic infections. Furthermore, chimpanzees are an
endangered species, therefore limiting the availability of these animals for research
purposes. Consequently, the infection of macaques with SIV has become the most
prevalent animal model used. Both the SIVniac- and SIVsmm- isolates have been shown to
cause an AIDS-like disease in rhesus-, cynomolgus-, and stumptail macaques (Murphey-
Corb etal. 1986). Initial infection results in viremiaand is characterized by fever, diarrhea
and lymphadenopathy. Like HIV-1 in humans, monkeys in the end-stages of disease
present with opportunistic infections and show decreased CD4+ T-lymphocyte counts and
inversed CD4:CD8 ratios. Additional promising models include the infection of baboons
and pig-tailed macaques with specific isolates of HIV-2 (Barnett et al. 1994; Novembre et
al. 1994). A large percentage of these monkeys become persistently infected and show

13
depletion of CD4+ T-lymphocytes and susceptibility to opportunistic pathogens. In
addition, several alternative models have been developed with some success. For example,
transient HIV infection of SCID mice reconstituted with human lymphocytes or peripheral
blood mononuclear cells (PBMC) and the infection of rabbits with HIV-1 (Reina et al.
1993; Mosier et al. 1991; Namikawa et al. 1988). These models, however, are highly
artificial and the relevance to HIV pathogenesis in humans should be interpreted with
caution.
The infection of FIV in domestic cats offers several advantages over the models
discussed above. First, FIV is a natural pathogen of cats and the pathogenesis closely
resembles that of HIV in man. Obvious advantages of the feline model include the
availability and costs which allow the use of larger study groups. Especially relevant to
vaccine development is the availability of a wide variety of FIV subtypes which is lacking
in the SIV model. FIV isolates have been grouped into 4 subtypes (A-D) versus 7 for HIV
(Sodora et al. 1994; Kakinuma et al. 1995; Rigby et al. 1993). This grouping is primarily
based on antigenic diversity in the Env and Gag proteins. Hence, the FIV model provides
a means to evaluate the protective efficacy of vaccine strategies against multiple subtypes
and as such has implications to the development of multiple subtype HIV vaccines.
Further, FIV like HIV replication is sensitive to antiviral drugs such as AZT and protease
inhibitors (North etal. 1989, 1990). Thus, FIV infection in cats also provides a model to
assess the efficacy of these and other newly developed drugs.
Vaccine development
Vaccine development initiated with the work of Dr. Edward Jenner in 1798. He
observed that milkmaids which had recovered from cowpox did not contract the more
virulent smallpox. Based on this observation he postulated that smallpox infection of man
could be prevented by prior exposure to cowpox. He successfully proved this hypothesis

14
by demonstrating that a boy, injected with material from a cowpox pustula, failed to
develop disease upon exposure to smallpox. This technique became known as vaccination
(Jenner 1798).
Though the underlying mechanisms were not known at the time, it is now
understood that success of vaccination lies in the ability of the immune system to generate
long lasting immunity. This immunity is mediated by memory B and T lymphocytes which
are capable of rapid anamnestic responses upon exposure to foreign antigens. Responses
mediated by B cells include the production of specific antibodies that could prevent the
entry of pathogens into host cells by interfering with microbial attachment or fusion upon
attachment to host cells. These antibodies, in respect to viral pathogens, have been defined
as viral neutralizing (VN) antibodies. Further, antibodies may directly destroy microbes by
complement mediated lysis or promote phagocytosis by macrophages and natural killer
cells through opsonization. T-lymphocyte responses include those mediated by T-helper
cells and T-cytotoxic lymphocytes (CTLs). T-helper cells are of the CD4+ phenotype and
recognize exogenously produced antigens presented in the context of MHC-class II found
predominantly on B-lymphocytes and macrophages. Upon recognition these cells produce
interleukins to facilitate the activation of macrophages and maturation of B cells into
antibody producing plasma cells. Cytotoxic T-lymphocytes of the CD8+ phenotype
function by the direct destruction of infected cells displaying foreign antigens in association
with MHC-class I molecules. Class I MHC molecules are found on the majority of cells
and present endogenously synthesized antigens. As such CTL responses are especially
critical to the clearance of intracellular bacteria and virally infected cells. Together, humoral
and cell mediated responses are capable of preventing the invasion of pathogens.
Several different types of vaccines have been developed. Most commonly used
vaccines against viral pathogens are composed of live attenuated viruses, inactivated whole
virus, or inactivated virus infected cells. The majority of these vaccines induce VN
antibody responses and some, in particular attenuated live viruses, also induce cell-

15
mediated responses including CTL reponses. More recent developments in the vaccine
field involve the use of viruses, bacteria or naked DNA as vaccine vectors. These vectors
are genetically engineered to carry and express foreign genes encoding immunogens of
pathogens. Upon inoculation of these vectors into the host, the inserted immunogen is
expressed and presented to the host immune system. In fact, viral antigens encoded within
these vectors are presented to the host immune system in a manner simulating natural
infection. Furthermore, vector based vaccines are thought to be more effective in eliciting
CTL responses than conventional inactivated vaccines. As such, vaccine strategies
employing vector based vaccines may be especially useful against viral and intracellular
bacterial pathogens.
To date, success of viral vaccines has been limited to a confined group of viruses.
These viruses display constant antigenic specificity and consist of a single or limited
number of serotypes. Furthermore, spontaneous recovery has usually been observed
shortly after natural infection with these viruses. Lentiviruses, including FIV, do not fall
into this category. Lentiviruses are subject to continuous antigenic variation and consists of
many serotypes. For FIV a total of 4 subtypes have been defined based on genetic
differences in the env and gag coding regions (Sodora et al. 1994; Kakinuma et al. 1995;
Rigby et al. 1993). Spontaneous recovery upon infection with lentiviruses has not been
reported. Moreover, these viruses integrate into the host cellular genome and can stay
latent without the expression of viral proteins. Latently infected cells serve as a reservoir
and fail to be recognized by the immune system, allowing the virus to persist. Thus, the
development of effective vaccines against lentiviruses faces additional challenges.
FIV vaccine development
An optimal FIV vaccine should induce long-lasting protective immunity. This
immunity should be effective against a wide range of FIV strains within as well as across

16
subtypes (A-D). In addition, this vaccine should induce protective immunity against cell-
free and cell-associated virus and against various routes of infection. Since FIV is
predominantly transmitted through biting, protection should be directed in particular against
this route of exposure.
In order to properly evaluate the immunogenicity and protective efficacy of FIV
vaccines, several factors should be taken into consideration. One factor is the method used
to assess the induction of VN antibody responses as these are often used as a parameter for
the effectiveness of viral vaccines. The ability of antibodies to neutralize lentiviral infection
in vitro may however differ with the specificity of the target cell line used in the assay.
For example, it has been found that vaccine induced antibodies capable of neutralizing FIV
infection on a feline Crandell kidney cell line (CrFK) failed to neutralize FIV infection on
feline thymocytes (Siebelink et al. 1993).
Likewise, the assessment of protective efficacy is influenced by several factors. As
for viruses in general, these include the route of infection and the dose of challenge
inoculum. In most FIV vaccine trials, the challenge inoculum is given either intravenously
or intraperitoneally. Experimental infection through these routes is obtained more readily
than through mucosal exposure, e.g. requires less viral particles. Furthermore, the origin
of the vaccine virus and challenge virus inoculum play an important role in the outcome of
vaccine efficacy. Lentiviruses are enveloped and incorporate cellular antigens into the viral
envelope when budding from the host cell. These host derived proteins present in the
lentiviral envelope may play a role in protective efficacy of inactivated virus and infected
cell vaccines, in particular. For example, monkeys immunized with uninfected human cells
were shown to be protected from challenge with SIV grown on the identical human cell
line. Protected monkeys had significantly higher levels of antibodies directed to host-cell
major histocompatibility complex (MHC) antigens than monkeys that were not protected.
In fact, protection against SIV has been obtained with vaccines composed of MHC
molecules solely (Chan et al. 1992; Stott 1991; Langlois et al. 1992). Likewise, protection

17
against feline leukemia virus (FeLV), another feline retrovirus, has been afforded in the
presence of antibody responses directed against cat-cell antigens (Lee et al. 1982). Thus,
the cellular origin of the vaccine virus and the challenge virus should be taken into
consideration when evaluating protective efficacy.
In the majority of FIV vaccine trials, the challenge inoculum virus is produced from
FIV strains adapted to cell cultures in the laboratory. In vitro cell culturing, however, can
lead to changes in virulence, cell tropism, and sensitivity to VN antibodies. To overcome
this, FIV isolated freshly from infected cats have been used. A relatively new approach is
the use of molecularly cloned FIV. The viral genome is incorporated into a plasmid and as
such used to infect animals.
FIV vaccine trials
Early FIV vaccine trials utilizing inactivated whole virus failed to demonstrate
protective immunity (Table 1.2 and Table 1.3). These trials included the immunization of
specific pathogen free (SPF) cats with inactivated FIV UK8 purified from T cells and
incorporated into immune stimulating complexes (ISCOMs) (Morein et al. 1984; Hosie
1994). Similarly, an inactivated virus vaccine produced from Crandell feline kidney cells
(CrFK) infected with the FIV UT113 isolate and adjuvanted with aluminum hydroxide-oil,
failed to protect cats against low dose homologous challenge (Hosie 1994) (Table 1.2).
Initial trials involving inactivated FI V-infected cell vaccines were also unsuccessful.
Cats immumzedwith inactivated FIV UK8 infected T-cells or helper T-cells (Q201) became
readily infected upon a homologous challenge with 20IDS0 (Hosie 1994, Hosie et al. 1992)
(Table 1.3). Comparable results were obtained with a vaccine consisting of inactivated
CrFK cells infected with the FIV UT113 isolate (Verschoor et al. 1995). In contrast,
partial protection (3 out of 5) was observed in cats immunized with a cell vaccine consisting
of FIVUT113-infected thymocytes. However, one of three control cats immunized with

18
uninfected thymocytes alone also remained virus-negative (Verschoor et al. 1994). Thus,
immune responses against cellular antigens and not viral antigens may have been
responsible for the observed protection in this study.
Other unsuccessful FIV vaccine trials include those based on FIV subunit proteins
and synthetic peptides corresponding to FIV epitopes (Table 1.4). Vaccines composed of
nonglycosylated FIV Env produced in Escherichiacoli and glycosylated Env produced in a
baculovirus system failed to protect cats against low dose challenge (Lutz et al. 1995).
Similar results were obtained with vaccines consisting of bacterial produced Env fragments
fused to galactokinase or glutathione-S-transferase (Verschoor et al. 1996). Also
unsuccessful were vaccines composed of synthetic peptides corresponding to the V3 region
of the FIV surface envelope protein (SU) (Lombardi et al. 1994). This region resembles
the V3 loop of the HIV-1 Env surface protein which is thought to contain the principal
neutralizing determinant (PND) of HIV (Pancino et al. 1994). Following immunization, all
cats developed V3-specific antibodies however no protection against low dose challenge
was observed (Lombardi et al. 1994). Interestingly, immunized animals showed
enhancement of infection compared to controls as indicated by a higher virus load in the
peripheral blood. Enhancement of infection as a result of immunization was also observed
in cats immunized with a FIV envelope produced by recombinant vaccinia virus (Siebelink
et al. 1995). In addition, subunit vaccines consisting of recombinant Gag protein p24 or
native purified p24, lacked prophylactic efficacy despite the presence of high anti-p24
antibody titers (Hosie et al. 1992).
FIV vaccine trials using recombinant vector based vaccines have also been
unsuccessful (Table 1.4). This included a trial in which the efficacy of a replicative
defective adenovirus engineered to express the env gene of FIV was evaluated. After
immunization, Env-specific antibody responses could not be detected and all cats became
infected upon challenge (Gonin et al. 1995). Likewise, vaccine protocols involving

Table 1.2 Conventional inactivated whole virus FI V vaccine trials
Type of Immunization
Cellular Origin
(Vaccination Route)
Vaccine Virus'
(FIV subtype)
Vaccine Dose
(mg)
Vaccination
Protocol(wks)
Type of
Adjuvantb
Challenge Inoculum
Cellular Origin& Dose (IDS0)
Route & Strain
Protection
rate
(Hosie et al. 1992)
Whole-virus
feline T-cell(s.c.)
UK-8 (A)
10c
0,5,18
iscom
PBMC (i.p.)
20UK-8
0/5 (0%)
Unvaccinated
(-)
(-)
(-)
(-)
(-)
PBMC (i.p.)
20UK-8
1/4(25%)
(V erschoor,unpublished)
Whole virus
CrFK(s.c.)
UT113(A)
100
0,6
AluOH-oil
Thymocytes(s.c.)
10UT113
0/5 (0%)
(Yamamoto et al. 1991b)
Whole-virus
FL-4 (s.c.)
PET (A)
200
0,2,46
CFA/IFA
FeTl (i.p.)
10PET
3/3(100%)
Whole-virus
FL-4 /Fetd(s.c.)
PET (A)
200/107
0,2,4,6
CFA/IFA
FeTl (i.p.)
10PET
2/3 (67%)
Adjuvant alone
(-)
(-)
(-)
0,2,46
CFA/IFA
FeTl (i.p.)
10PET
0/3 (0%)
(Yamamoto et al.1993)
Whole-vims
FL-4 (s.c.)
PET (A)
250
0,2,5
A-MDP
FeTl (i.p.)
10PET
13/15(87%)
Adjuvant alone
(-)
(-)
(-)
0,2,5
A-MDP
FeTl (i.p.)
10PET
0/10 (0%)
Whole-viras(boost)'
FL-4 (s.c.)
PET (A)
250
38e
A-MDP
FeTl (i.p.)
10DDC(A)
13/13(100%)
Adjuvant alone
(-)
(-)
(-)
38e
A-MDP
FeTl (i.p.)
10DIX
0/5 (0%)
(Hosie et al. 1995)
Pelleted-virus
FL-4 (s.c.)
PET (A)
250
0,2,4,7,10,17
T-MDP
FeTl (i.p.)
10PET
5/6 (83%)
Control
(-)
(-)
(-)
(-)
(-)
FeTl (i.p.)
10PET
0/6 (0%)
Pelleted-virus
FL-4 (s.c.)
PET (A)
250
0,2,4,7,10,17
T-MDP
Q201(i.p.)
5UK-8
0/5 (0%)
Control
(-)
(-)
(-)
(-)
(-)
Q201(i.p.)
5UK-8
0/5 (0%)
Gradient-purified vims
FL-4 (s.c.)
PET (A)
250
0,3,6
T-MDP
FeTl (i.p.)
10PET
5/5 (100%)
Control
(-)
(-)
(-)
(-)
(-)
FeTl (i.p.)
10PET
0/5 (0%)
Gradient-purified vims
FL-4 (s.c.)
PET (A)
250
0,3,6
T-MDP
Q201 (i.p.)
10PET
4/5 (80%)
Adjuvant alone
(-)
(-)
(-)
0,3,6
T-MDP
Q201(i.p.)
10PET
0/5 (0%)
Gradient-purified vims
FL-4 (s.c.)
PET (A)
250
0,3,6
T-MDP
Q201 (i.p.)
10UK-8
1/5 (20%)
Adjuvant alone
(-)
(-)
(-)
(-)
T-MDP
Q201 (i.p.)
10UK-8
0/5 (0%)
(Johnson et al.1995)
Whole-vims
FL-4 (s.c.)
PET (A)
250
0,2,46,8
T-MDP
FeTl (nasal)
10PET
3/5 (60%)
Adjuvant alone
(-)
(-)
(-)
0,2,46,8
T-MDP
FeTl (nasal)
10PET
0/5 (0%)
Whole-vims
FL-4 (s.c.)
PET (A)
250
0,2,4,6
T-MDP
FeTl (i.p.)
10SHI (D)
0/2 (0%)
Adjuvant alone
(-)
(-)
(-)
0,2,4,6
T-MDP
FeTl (i.p.)
10SHI
0/7 (0%)
a UK-8, United Kingdom-8; PET, Petaluma; DIX, Dixon; SHI, Shizuoka.
b isom, immune stimulating complex;; A-MDP, adenyl-muramyldipeptide; CFA, complete Freunds adjuvant; IFA, incomplete Freunds; T-MDP, threonyl-muramyldipeptide.
c Vaccine dose was lOug of P17 and P24
d Vaccine consisted of 200mg /dose of inactivated whole-virus mixed with lxlO7 cells/dose of uninfected FeT-1 cells
e Vaccinated cats protected from FIVpet challenge were boosted 38 wks after the first immunization and challenged 3 weeks after the boost with FlVdix strain.

Table 1.3 Conventional inactivated FI V-infected cell vaccine trials
Type of Immunization
Cellular Origin
(Vaccination Route)
V accine Virus1
(FIV subtype)
Vaccine Dose
(mg/PFU)
Vaccination
Protocol(wks)
Type of
Adjuvant6
Challenee Inoculum
Cellular Origin& Dose (ID50)
Route & Strain
Protection
rate
(Hosie et al. 1992)
Infected cell
feline T-cell(s.c.)
UK-8 (A)
2x10*
0,3,6,9,12,15
QuilA
PBMC (i.p.)
20 UK-8
0/5 (0%)
Uninfected cell
(-)(s.c.)
(-)
2x1o6
0,3,6,9,12,15
QuilA
PBMC (i.p.)
20 UK-8
1/5 (20%)
(Hosie,unpublished)
Infected cell
Q201(s.c.)
UK-8 (A)
lxlO7
0,3,6
QuilA
Q201 (i.p.)
20 UK-8
0/4 (0%)
Adjuvant alone
(-)(s.c.)
(-)
(-)
0,3,6
QuilA
Q201 (i.p.)
20 UK-8
0/4 (0%)
Control
(-Xs.c.)
(-)
(-)
0,3,6
(-)
Q201 (i.p.)
20 UK-8
1/4 (25%)
(Verschoor et al. 1995)
Infected cell
CrFK(i.m.)
UT-113 (A)
2.5x107
0,3,6
alu/MDP
PBMC (i.p.)
10 UT-113
0/5 (0%)
Uninfected cell
CrFK(i.m)
(-)
2.5x107
0,3,6
alu/MDP
PBMC (i.p.)
10 UT-113
0/3 (0%)
Infected cell
Thymocyte s(i.m.)
UT-113 (A)
1.5xl07
0,3,6
alu/MDP
PBMC (i.p.)
10 UT-113
2/5 (40%)
Uninfected cell
Thymocytesfi.m.)
(-)
1.5xl07
0,3,6
alu/MDP
PBMC (i.p.)
10 UT-113
1/3 (33%)
Control
(-)
(-)
(-)
(-)
(-)
PBMC (i.p.)
10 UT-113
0/2 (0%)
(Yamamoto et al. 1991b)
Infected cell
Fetl (s.c.)
PET (A)
lxlO7
0,2,4,6,8,16
T-MDP
PBMC (i.p.)
10 PET
4/5 (80%)
Infected cell
FL4(s.c.)
PET (A)
lxlO7
0,2,4,6,8,16
T-MDP
PBMC (i.p.)
10 PET
2/4(50%)
Uninfected FeTl
(-)(s.c.)
(-)
lxlO7
0,2,4,6,8,16
T-MDP
PBMC (i.p.)
10 PET
0/5 (0%)
Adjuvant alone
(-)(s.c.)
(-)
(-)
0,2,4,6,8,16
T-MDP
PBMC (i.p.)
10 PET
0/5 (0%)
(Yamamoto et al. 1993)
Infected cell
FL4 (s.c.)
PET (A)
2.5x107
0,2,5
A-MDP
Fetl (i.p.)
10 PET
15/15(100%)
Adjuvant alone
(-Xs.c.)
(-)
(-) ,
0,2,5
A-MDP
Fetl (i.p.)
10 PET
0/10 (0%)
Uninfected Fetl
FL4 (s.c.)
PET (A)
2.5x107
38
A-MDP
Fetl (i.p.)
10DIX
14/15 (93%)
Adjuvant alone
(-Xs.c.)
(-)
(-)
38
A-MDP
Fetl (i.p.)
10DIX
0/5 (0%)
(Matteucci et al. 1995)
Infected cell
MBM(s.c.)
M-2 (B)
3x107
0,3,6,9,12,15
IFA
plasma (i.p.)
10 M-2
5/6 (83%)
Uninfected cell
MBM(s.c.)
(-)
3xl07
0,3,6,9,12,15
IFA
plasma (i.p.)
10 M-2
0/3 (0%)
Control
(-Xs.c.)
(-)
(-)
0,3,6,9,12,15
(-)
plasma (i.p.)
10 M-2
0/6 (0%)
(Johnson et al. 1994)
Infected cell
Fl-4(s.c.)
PET (A)
2.5x107
0,2,4,6,8
A-MDP
Fetl (nasal)
10 PET
2/5 (40%)
Infected cell
H-4(s.c.)
PET (A)
2.5x107
0,2,4,6
A-MDP
Fetl (i.p.)
20 SHI
1/8(12%)
a UK-8, United Kingdom 8; UT113, Utrecht 113; PET, Petaluma; M-2, Milan-2; SHI, Shizuoka.
b T-MDP, threonyl-muramyldipeptide; A-MDP, adenyl-muramyldipeptide; IFA, incomplete Freunds adjuvant; QuilA, saponin.

Table 1.4 Subunit and vector-based FIV vaccine trials
Type of Immunization
Cellular Origin
(Vaccination Route)
Vaccine Virus"
(FIV subtype)
Vaccine Dose
(mg/PFU)
Vaccination
Protocol) wks)
Type of
Adjuvant1*
Challenge Inoculum
Cellular Origin& Dose (ID50)
Route & Strain
Protection
rate
(Hosie, et al. 1992)
p24
E. coli(s.c.)
UK-8 (A)
50
0,3,5,7
iscom
PBMC (i.p.)
20 UK-8
0/4 (0%)
(Lutz et al. 1995)
gplOO(denatured)
Insect cell (i.m.)c
Z2 (A)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
0/5 (25%)
gplOO(native)
Insect cell (i.m.)
Z2 (A)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (20%)
gplOOfhighly purified)
Insect cell (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (20%)
gplOO(native )
Insect cell (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
0/5 (0%)
gplOO(denatured)
E..Coli (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (25%)
Control(Ovalbumin)
(-)
(-)
100
0,2,4,8
(-)
PBMC (i.p)
20 Z2
2/7 (29%)
(Osterhaus et al. 1996)
Env(cleavage)
NA(s.c.)
AM 19 (A)
100
0,4,10
iscom
PBMC (i.m)
20AM19
0/6 (0%)
Env(no cleavage)6
NA(s.c.)
AM 19 (A)
100
0,4,10
iscom
PBMC (i.m.)
20AM19
0/6 (0%)
Env(no cleavage)6
NA(s.c.)
AM 19 (A)
100
0,4,10
QuilA
PBMC (i.m.)
20AM19
0/6 (0%)
Env-b Gal
NA(s.c.)
AM19 (A)
100
0,4,10
QuilA
PBMC (i.m.)
20AM19
0/6 (0%)
Control(PBS)
(-XS.C.)
(-)
(-)
0,4,10
(-)
PBMC (i.m.)
20AM19
0/6 (0%)
(Flynn et al. 1995)
V3-peptide
synthetic (s.c.)
UK-8 (A)
100
0,3,6
QuilA/AlOH
PBMC (i.p.)
10 UK-8
0/15(0%)
Adjuvant alone
(-) (s.c.)
(-)
100
0,3,6
QuilA/AlOH
PBMC (i.p.)
10 UK-8
0/5 (0%)
(Lombardi, 1994)
V3-peptide
synthetic(s.c.)
PET(A)
500
0,2,4,6,8
CFA
F14(i.v.)
20-PET
0/3 (0%)
Control
(-)
(-)
(-)
(-)
(-)
F14(i.v.)
20-PET
0/3 (0%)
(Verschoor et al. 1996)
V3-fusion protein F
E..Coli(s.c.)
UT113 (A)
100
0,4,6,8,10
AlOH
NA(s.c.)
10-20UT113
0/5 (0%)
V3-fusion protein I
E..Coli(s.c.)
UT113
100
0,6,10
QuilA
NA(s.c.)
10-20UT113
0/5 (0%)
Control(PBS)
(-)(s.c.)
(-)
(-)
0,6,10
(-)
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-Env
(-)oronasal/s.c.
UT113
10SPFU
0
(-)
Boost V3-peptide
E. Coli (i.m.)
UT113
100
4,8
AlOH
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-Env
(-)oronasal/s.c.
UT113
105PFU
0
(-)
Boost V3 peptide
E.Coli (i.m.)
UT113
100
4,8
QuilA
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-b Gal.
(-)oronasal/s.c.
(-)
10SPFU
0
(-)
Boost PBS
(-)(s.c.)
(-)
(-)
4,8
(-)
NA(s.c.)
10-20UT113
0/5 (0%)
(Gonin et al.1995)
Adenovirus-env
(-)(i.m.)
Wo (A)
11.8-9.2PFU
0,4,30
ISA206
NA(NA)
20 Wo
0/4 (0%)
Adeno-psuedorabies(control)
(Kim.)
(-)
11.8-9.2PFU
0,4,30
ISA708
NA(NA)
20 Wo
0/4 (0%)
a UK-8, United Kingdom; Z2, Zurich; BANG, Bangston; AM19, Amsterdam 19; PET, Petaluma,
b Recombinant vaccinia virus expressed
c Baculovirus expression
d iscom, immune stimulating complex; A10H/QS21, aluminumhydroxide and non-toxic fraction from Quillaja saponaria; ISA206, water/oil adjuvant;ISA708, water/oil adjuvant,
e V3-fusion protein was composed of the FIV V3 region fused to galactokinase.
f Deletion of the cleavage site between the envelope surface(SU) and transmembrane protein(TM).

22
pruning with a feline herpes virus engineered to express the FlVenv gene followed by
booster immunizations with bacterial Env-fusion proteins failed to induce protection against
low dose challenge (Verschoor et al. 1996).
Successful FIV vaccine protocols include the use of inactivated cells infected with
the FIV Petaluma isolate (FIVPet; subtype A) or inactivated cell free FIVPct (Yamamoto et al.
1991b, 1993). These vaccines were produced from either feline lymphoid cells
productively infected with FIVPet (FL-4) or an IL-2 dependent feline lymphoid cell line
(FeT) infected with FIVPet (Yamamoto et al. 1991a). Using these vaccines, a protection
rate of 70%-90% has been observed against low dose experimental challenge with
homologous HVPet and slightly heterologous FIV Dixon (FIVDU; subtype A) (less than 9%
divergence in the env coding region) (Yamamoto et al. 1991b, 1993; Hosie et al. 1995).
Further, these vaccines afforded protection against FIV challenge inoculum virus
propagated on different cell lines including FeT 1, FL-4, and allogeneic PBMC. Protection
was achieved against intraperitoneal challenge and oral-nasal challenge in a small number of
animals tested (Yamamotoetal. 1991b, 1993; Johnson etal. 1994). These same vaccines,
however, failed to induce protection against a high challenge dose of 5x1o4 ID50 with the
homologous FIVPet isolate. Furthermore, these vaccines failed to induce protection against
experimental challenge with a moderate heterologous FIV UK8 isolate (subtype-A) and a
distinctly heterologous FIV Shizouka (FIVshi; subtype D) isolate. The Env amino acid
sequences of these isolates differ from the FIVPet Env sequence by 11% and 21%,
respectively. In addition, immunization schemes employing a similar vaccine produced
from MBM lymphoid cells infected with the Italian isolate FIV M2 have been shown to
induce protective immunity against a homologous plasma derived virus inoculum
(Mattuecci et al. 1996).
In summary, conventional inactivated vaccines are capable of inducing protective
immunity against low dose homologous FIV challenge and slightly heterologous FIV
challenge. Similar vaccine approaches have also been successful in other animal models

23
such as SIV in macaques and HIV in chimpanzees (Desrosiers et al. 1989; Fultz et al.
1992; Murphey-Corb et al. 1989). FIV subunit vaccines consisting of either Env or Gag
proteins as well as recombinant vector based vaccines expressing Env did not elicit
protective immunity against low dose homologous challenge. In contrast, protective
immunity has been obtained against homologous SIV challenge in macaques immunized
with Env subunit vaccines and against homologous and heterologous HIV challenge in
chimpanzees immunized with Env subunit vaccines (Hu et al. 1992; Girard et al. 1995).
Mechanisms of protection
Protection obtained with conventional inactivated whole virus and infected cell
vaccines produced with the FIVPet isolate, correlated most with high levels of envelope-
specific and VN antibodies (Yamamotoet al. 1991b, 1993). Protection obtained with these
vaccines did not correlate with anti-MHC antibodies as could be concluded from the lack of
protection in cats that were immunized with u ninfec ted cel Is alone (Yamamoto et al. 1991b,
1993) (Table 1.3). Similar vaccines produced with FIV isolates grown in feline T cells,
thymocytes and CrFK cells failed to induce protective immunity (Verschoor et al. 1995;
Hosie etal. 1992; Hosie 1994) (Table 1.3). However, these vaccines elicited lower levels
of envelope-specific antibodies and VN antibodies. In fact, only the FIV vaccines
produced from infected feline T-cell lines FL-4 and FeTl cells, induced VN antibody titers
similar to those observed in infected cats (Tozzinm et al. 1992). This could be due to the
larger quantities of envelope protein produced by FL-4 and FeTl cell lines and the fact that
the envelope protein present on FL-4 and FeTl cells is well preserved following
purification. Additional contributing factors may have been the choice of inactivating
agent. Inactivation of successful vaccines was accomplished by paraformaldehyde which
is thought to be more effective in maintaining antigenicity of immunogens when compared
to other inactivating agents i.e. fl-propriolactone (Allison and Byars 1991; Warren et al.

24
1986). Furthermore, the type of adjuvant may have affected the protective efficacy, as it
influences the proportion and intensity of humoral vs. cell-mediated responses upon
immunization (Byars and Allison 1987).
Additional support for a role of Env-specific and VN antibody responses in the
protection obtained with the FTVPet FL-4/FcT 1 vaccines comes from passive immunization
studies. In these studies, cats were passively immunized with pooled sera from cats
immunized with inactivated FI VPet infected T-cells (FL-4) or sera from cats experimentally
infected with FIVPet (Hohdatsu et al. 1993). Control cats received either phosphate
buffered saline (PBS) or pooled sera from cats immumzed with inactivated uninfected FeTl
cells (related to FL-4 cell line) or uninfected 3201 cells (allogeneic feline T-cell line). Upon
low dose homologous challenge with 5 IDS0, all control cats became infected whereas 3 out
of 3 cats passively immunized with FlVPl;t (FL-4) vaccine sera and 4 out of 4 cats
immunized with FIV infected cat sera did not. Protected cats showed VN antibody titers
averaging between 100-200 whereas uninfected cell or PBS control sera had no VN
antibody titers. These findings resemble those reported for SIV. Cynomolgus monkeys
passively immunized with sera from SI V^ infected and HIV-2 vaccinated monkeys were
protected in the presence of high titer antiviral antibodies against homologous challenge at
10 to 100 IDS0 (Putkonen et al. 1991). In contrast, passive immunization studies in
macaques immunized with SIV^ indicated that the levels of anti-cellular and not anti-viral
antibodies correlated mostly with protection (Rosenthal et al. 1992).
Findings from studies evaluating protective immunity in kittens bom to queens
vaccinated with FIVPet FL-4/FeTl vaccines also imply a role for VN responses (Pu et al.
1995). In these studies, kittens received colostrum/milk from either vaccinated or sham
vaccinated queens and were challenged shortly after birth with low dose homologous
challenge of 5 IDS0. It was found that only those kittens born to and nursed by vaccinated
queens were protected. Furthermore, protected kittens showed high levels of VN titers
(500-5000) at five days postparturition. The role of transplancental maternal antibody

25
reponses was evaluated in kittens bom to vaccinated queens and nursed by sham
immunized queens. These kittens became infected and showed significantly lower titers of
VN (10-100) postparturition as compared to littermate controls receiving colostrum from
vaccinated queens. Nevertheless, these kittens showed lower levels of viremia as
compared to littermate controls which were bom to and nursed by unvaccinated queens (Pu
et al. 1995). As such, these data suggest that VN maternal antibodies transferred via
colostrum/milk or placenta play a role in preventing establishment of FIV infection.
Supporting this are additional studies on kittens born to queens infected for various lengths
of time. It was found that kittens bom to queens infected for more than 7 months were
protected in the presence of high VN titers in the colostrum. In contrast, kittens bom to
short term (< 2mo.) infected queens became infected in the presence of low VN titers in
colostrum (Pu et al. 1995).
Opposing a role for Env specific and VN responses in protection against FIV
infection are findings from a number of subunit vaccines trials. Vaccines composed of the
FIV envelope or envelope fragments alone failed to induce protective immunity. This
included nonglycosylated, glycosylated, native, and denatured whole FIV Env and Env
fragment vaccines in combination with different adjuvants (Lutz et al. 1995; Verschoor et
al. 1996; Lombardi et al. 1994). The majority of these vaccines, however, effectively
induced Env-specific antibody responses as well as VN antibody responses. Therefore, it
could be suggested that the tertiary structure of the envelope protein is crucial as it affects
the presentation of the Env to the immune system or that other epitopes besides Env are
required to obtain protective immunity. A subunit vaccines composed of Gag proteins
alone, however, failed to induce protection despite the induction of Gag specific humoral
responses (Hosie et al. 1992). Interestingly, several of the envelope subunit vaccines
caused enhancement of infection upon challenge (Hosie et al. 1992; Siebelink et al. 1995;
Osterhaus et al. 1996). Similar enhancement of viremia has been observed in horses
immunized with a recombinant envelope vaccine against equine infectious anemia virus

26
(EIAV), another len ti virus (Wang et al. 1994). This phenomenon has also been observed
with other nonlentiviral vaccines in particular macrophage and monocyte-tropic viruses
(Halstead and ORourke 1977). It is thought to be mediated by viral specific antibodies
elicited upon immunization that can facilitate viral transport to susceptible host cells by
binding of the Fc portion of antibodies to the surface of macrophages.
The role of VN antibodies as a factor in preventing FIV infection is not clear.
Although, a majority of cats immunized with FlVPet FL-4/FeTl vaccines showed high
levels of VN antibody responses at challenge, some cats were protected in their absence.
In addition vaccine trials employing inactivated MBM cells infected with the FIV M2
isolate, a vaccine similar to the FlVPet FL-4/FeTl vaccines, induced protective immunity, in
the absence of detectable VN antibody titers (Mattuecci et al. 1996).
In summary, protection against low dose experimental challenge with FIV may in
part be mediated by antiviral humoral responses. However, cell-mediated responses such
as CTL or underlying immune effector activities i.e. chemokines may also play a role. In
fact, recent studies demonstrated the induction of FIV specific CTL responses directed
against Env and Gag epitopes in cats immunized with the FIVPet (Fl-4) inactivated infected
cell vaccine (Flynn et al. 1995a). The importance of these responses in protective
immunity however remains to be established. Induction of FIV specific CTL responses
was also observ ed in cats immunized with a synthetic peptide vaccine corresponding to the
FIV V3 region. These cats, however, became infected upon challenge even though V3-
specific CTL responses were detected at the time of challenge (Flynn et al. 1994, 1995b).
Recombinant poxvirus-based vaccines
Members of the poxvirus family comprise a group of large viruses that infect a
number of species. Poxviruses are enveloped and contain a single double stranded DNA
genome of 130 to 300kbp. These viruses encode their own enzymes required for viral

27
DNA replication and mRNA synthesis, and are unique in the fact that they replicate within
the cytoplasmic compartment of infected cells (Moss 1990).
The use of poxviruses as vaccine vectors was preceded by advances in the field of
molecular biology that allowed the manipulation of viruses in such away that foreign genes
could be inserted and expressed (Piccini et al. 1987; Perkus et al. 1989). Poxviruses have
since become candidate vaccine-vectors for a wide variety of pathogens (Perkus et al.
1995). In comparison to other candidate vector-viruses, poxviruses are exceptionally well-
suited due to their physical stability, low production costs, and ease of administration.
Furthermore, poxviruses have large genomes that allow the insertion of multiple genes.
The most widely used member of the poxvirus family is the vaccinia virus,
prototype of the Orthopoxviruses (Esposito 1991). This virus has been engineered to
express antigens of bacterial and viral pathogens and shown to induce protective immunity
in vivo (Perkus et al. 1995). Immune-responses induced upon inoculation with vaccinia
based vaccines include both humoral responses and cell mediated responses directed
against the inserted foreign antigens. There are however some concerns about the safety of
vaccinia when used in a large population. As vaccinia exhibits a broad host-range there is a
potential risk of spread to the general environment. Moreover, vaccinia has been shown to
cause disseminated infection in immuno-compromised people (Fulgitini et al. 1968).
For these reasons, the development of poxviruses as vector vaccines has been
extended to attenuated poxviruses and poxviruses with a more restricted host-range. One
such example is the NY VAC vector. This vector was derived from the Copenhagen
vaccinia strain by the selective deletion of 18 open reading frames, encoding genes
involved in host-specificity and virulence (Tartaglia et al. 1992). These deletions resulted
in a virus which replication is highly impaired on cell lines from several species including
human cells. Furthermore, this virus lacks virulence in immuno-compromised animal
models. The MVA vaccinia strain is another example of an attenuated vector strain used in
vaccine development. This strain was derived by extensive passaging of the Ankara

28
vaccinia strain on primary chick embryo-fibroblast. As a result, the MVA virus is severely
attenuated and lacks replication on nonavian cell lines (Sutter et al. 1994).
An alternative to the use of attenuated vaccinia viruses is the use of poxviruses that
exhibit species specificity (Baxby and Paoletti 1992). These include the suipoxviruses,
capri poxviruses and avipoxviruses. Avipoxviruses only productively infect cells of avian
origin and were originally developed as vaccine vectors for the poultry industry.
Unexpectedly, it was found that nonavian cells inoculated with avipoxvectors expressed the
inserted antigen despite the absence of vector replication (Taylor et al 1992b). Moreover, it
was found that these vectors when administered to nonavian species were capable of
eliciting protective immunity. It is now understood that these viruses undergo abortive
infection in nonavian cells resulting in expression of early gene and inserted gene products.
Two avipoxviruses, the fowlpoxvirus and canary poxvirus, have been developed as
vaccine vectors (Plotkin et al. 1995). ALVAC represents a vector derived from an
attenuated canarypoxvirus strain originally used to immunize canaries. The protective
potential of recombinant ALVAC vectors has been tested against several viral pathogens
including rabies, measles, Japanese encephalitis virus (JEV), cytomegalovirus (CMV), and
equine influenza virus (EIV) (Table 1.5) (Cadoz et al. 1992; Konishi et al. 1994; Taylor et
al. 1992a, 1992b; Gonczol et al. 1995). The majority of these ALVAC-based vaccines
were shown to elicit antigen-specific antibody responses, including VN antibody responses
specific for the inserted antigens. More important, cell-mediated immunity was also
induced in individuals immunized with these vaccines. In one study, lymphocytes from
human volunteers immunized with an ALVAC-rabies recombinant had proliferative
responses to the rabies antigens, demonstrating the induction of antigen specific T-helper
cells (Cadoz et al. 1992; Taylor et al. 1991). This same recombinant vaccine was used to
evaluate the ability of ALVAC-based vaccines to elicit memory immune responses in dogs.

Table 1.5 Immunogenicity and prophylactic efficacy of ALVAC-based vaccines
Pathogen
genus
Test
species
Humoral
responses
Cell-mediated
responses
Protection
Rabies3
Rhabdovindae
mice
+
ND
+
dog
+
ND
+
cats
+
ND
+
squirrel monkeys
+
ND
ND
rhesus macaques
+
ND
ND
chimpanzees
+
ND
ND
humans
+
ND
ND
Cytomegalovirusb
Herpesviridae
mice
+
+
ND
guinea pigs
+
+
ND
Canine distemper virusc
Paramyxoviridae
dogs
+
ND
+
Japanese encephalitis virus'1
Flaviviridae
mice
+
ND
+
3 Cadoz et al. 1992; Taylor et al. 1991.
b Gonczol et al. 1995.
c Taylor et al. 1992a, 1992b.
d Konishi et al. 1994.
ND=not determined

30
It was found that dogs could be protected from a rabies challenge given 36 weeks after a
single ALVAC-rabies immunization (Cadoz et al. 1992).
The efficacy of ALVAC-based vaccines has also been tested against a number of
retroviral pathogens (Table 1.6). Immunization with ALVAC recombinants expressing the
Envelope and Gag proteins of the feline leukemia virus (FeLV) protected cats against
experimental infection with FeLV (Tartagliaet al. 1993). Protection was afforded in the
absence of detectable VN antibody responses. Interestingly, protected animals developed
FeLV-specific VN antibody titers at 9-12 weeks postchallenge whereas control animals
failed to develop VN antibody titers. The data presented in this study should be interpreted
with some caution since no analysis of FeLV by PCR was performed on tissues of the
protected animals (Tartagliaet al. 1993). Thus, immunization may not have resulted in
sterilizing immunity but resulted in a reduced viral load explaining the development of VN
titers at 9-12 weeks postchallenge. Further, the evaluation of cell-mediated responses was
not included in this study.
ALVAC-based vaccines have protective efficacy against human T cell
leukemia/lymphoma virus type I (HTLV-1), the causative agent of adultT cell leukemia and
tropical spastic paraparesis in humans (Barre-Sinoussi et al. 1983). In the rabbit model
used, it was found that two inoculations with ALVAC-based vaccines encoding the
HTLV-1 envelope protein (ALVAC-gp65), protected rabbits against challenge infection
with HTLV-1 infected cells. Protective immunity was afforded in the absence of HTLV-1
VN antibody responses. Again, this study lacked the evaluation of cell-mediated reponses.
Interestingly, rabbits boosted with baculovirus expressed envelope protein (gp65) in
addition to ALVAC-gp65 immunizations failed to be protected (Franchini et al. 1995b).
The rhesus macaque model was used to assess the prophylactic efficacy of
ALVAC-based vaccines against HIV-2 challenge (Franchini et al. 1995a). It should be
kept in mind that rhesus macaques can be infected with HIV-2 but do not develop an AIDS
like syndrome. Rhesus macaques were given two immunizations with ALVAC

31
recombinants expressing HIV-2 Env and Gag/Prot followed by two immunizations with
recombinant Env (gpl60) and a final ALVAC-HIV-2 Env, Gag/Prot boost. Upon
challenge with 100ID50 HIV-2, both macaques were free of viremiaand tested negative for
the virus by virus isolation and virus specific PCR. Prior to challenge, HIV-2-specific
CTL responses were detected. In addition, one of the macaques had a transient VN
antibody titer prior to challenge. After challenge both animals developed significant VN
antibody titers as such resembling findings in the ALVAC-FeLV trials (Franchini et al.
1995a; Tartagliaetal. 1993). The macaques were given a second HIV-2 challenge of 100
IDS0 at 7 months post the primary challenge without any intervening boosters. At this time,
both macaques became infected despite the presence of VN antibody titers at the time of
challenge. Cytotoxic T-lymphocyte responses were not measured at the time of the second
challenge, however, both animals were positive for HIV-2 specific CTL activity at one
month following the second challenge.
To evaluate the strain specificity of the immunity generated upon immunization with
ALVAC-based vaccines, rhesus macaques were immunized with an ALVAC-recombinant
expressing Env, Gag/Prot of the HIV-1,^ isolate and challenged with a distinctly
heterologous HIV-2 isolate (> 40% difference) (Abimiku et al. 1995). Two rhesus
macaques received a combination of primary ALVAC-HIV-1^. immunization and
subsequent subunit boosts with either p24 and gpl60 or a tandem V3-peptide. Both
animals developed virus-specific CTL and VN antibodies after immunization. Upon
challenge, the animal boosted with the V3-peptide was considered partially protected, based
on the low VN titers and the absence of virus by virus isolation and PCR at six months
postchallenge. The other animal exhibited a delay by two months and had lower VN
antibody titers as compared to control animals. All control animals became positive at 1
month after challenge and remained virus positive throughout the study. Perhaps the most
interesting observation made in this study was that the VN antibody responses generated
upon immunization did not cross react with the HIV-2 isolate used in the challenge

32
inoculum. Based on this it was speculated that cell mediated responses attributed to the
partial protection observed in these animals. However, the assessment of cross-reactive
CTL responses to the HIV-2 isolate was not included in this study.
In addition to the macaque model, the chimpanzee model has been used to assess
the prophylactic efficacy of ALVAC-based vaccine protocols against HIV infection. In one
study, the efficacy of an ALVAC recombinant expressing both the HIV-1^ Env and Gag
(ALVAC-HIVj^, gpl20TM, Gag/Prot) was evaluated against cell-associated HIV-1IIIB(LAI)
challenge in two chimpanzees. Each animals received a total of five immumzations and
were challenged one month after the final immunization. HIV-specific antibody responses
including VN antibodies were detected after the 4th and 5th immunization in both animals.
Upon challenge, one animal remained virus negative and the other, including a naive
control animal, became infected. Interestingly, the protected animal had a higher VN
antibody titer at the time of challenge than the nonprotected animal (Van der Ryst et al.
1996). Importantly, these same vaccine protocols failed to induce protective immunity
against challenge with the heterologous HI V-DH12 isolate (personal communication).
Further, the vaccine efficacy of ALVAC-HIV^ gpl20TM, Gag/Prot recombinants
was evaluated against mucosal challenge in chimpanzees. One group consisting of two
animals was immunized by the intra-muscular (i.m.), cervico-vaginal, and rectal route
simultaneously. Another group was immumzed by the i.m., oral and nasal route and one
animal was immunized by the i.m. route. All animals were challenged intra-cervically with
2500 TCIDS0 of HIV-1lvi passaged in chimpanzees. All vaccinated animals were free from
virus infection whereas the nonvaccinated control animals were infected. HIV-specific
antibody responses, including VN antibody titers, were low or undetectable at the time of
challenge, thus implying that mucosal protection occurred through mechanisms other than
VN antibody responses (Girard et al. 1996).
Another study reported on the efficacy of ALVAC-based vaccine strategies against
heterologous HIV challenge in chimpanzees. Chimpanzees were immunized with an

33
ALVAC recombinant vaccine encoding Env of the HIV-1^. strain and boosted with
recombinant envelope glycoproteins (gpl60) of the HIV-1N1N and HIV-1^, both classified
as subtype B strains. The animals were challenge intravenously with HIV-1SF2 which is
also classified as a subtype B virus but differs significantly from the HIV-1^ strains.
Upon challenge, virus was isolated from both vaccinated animals. However, in
comparison to the infected control animals the vaccinated ammals had a lower viral load
(Girard etal. 1995)
In summary, ALVAC-based recombinants were effective against lentiviral infection
in several animal models. However, it is not clear as to what constituted vaccine
protection. Some of these studies suggest a role for VN antibody responses. On the other
hand, other studies showed protection in the absence of detectable VN antibody responses.
Conflicting results may also stem from the fact that only a limited number of ammals was
used in these trials. Furthermore, the assessment of immune responses, in particular cell-
mediated responses, was frequently omitted in these trials. Nevertheless, the data obtained
from these animal trials are promising and studies on the safety and immunogemcity of
these vectors in human volunteers have been initiated. Thus far, no adverse effects have
been reported in human subjects immunized with an ALVAC-recombinant expressing HIV
Env (gpl60), ALVAC-HIVMNgpl60, followed by a boost with recombinant gpl60
(Clements et al. 1996, Lawrence et al. 1996). Further, it was found that immumzation
with this ALVAC recombinant alone failed to induce VN antibody responses. However,
VN antibodies were detected in most subjects after the rgp 160N1N/IjU boost. Env-specific T-
cell proliferative responses were detected in a small percentage of subjects after ALVAC
immunizations and in all subjects following rgpl60N1NiI AI boosts. The presence of HIV
Env-specific CTL activity was detected in some of the subjects, even without the subunit
boost (Pialoux et al. 1995). In a similar study, immune responses elicited with ALVAC-
HlV^.gplO alone was compared to those elicited by ALVAC-HIVN1Ngp 160 priming
followed by a HIV-1 rgpl20SF2 boost. Boosting with envelope protein significantly

34
enhanced VN antibody responses, ADCC, and CTL responses as compared to
immunization with either ALVAC- HlV^gplO or rgpl20SF2 alone (Clements et al. 1996).
In a follow-up study, volunteers received immunizations with an ALVAC
recombinant expressing both HIV-1 Env and Gag and a boost with recombinant Env
(Pialoux et al. 1995; Clements et al. 1996). The immunogenicity resembled that of the
previous studies (Lawrence et al. 1996). Interestingly, Gag-specific CTL responses were
detected in the majority of subjects whereas Env-specific CTL-responses were only
detected in a small percentage of the subjects. Together, these studies demonstrate that
immunization schemes involving ALVAC-based HIV vaccines, in combination with whole
protein boosts, are safe and can elicit both humoral and cell-mediated responses specific for
the inserted immunogens.

Table 1.4 Subunit and vector-based FIV vaccine trials
Type of Immunization
Cellular Origin
(Vaccination Route)
Vaccine Virus
(FIV subtype)
Vaccine Dose
(mg/PFU)
V accination
Protocol(wks)
Type of
Adjuvant11
Challenge Inoculum
Cellular Origin& Dose (IDS0)
Route & Strain
Protection
rate
(Hosie, et al. 1992)
p24
E. coli(s.c.)
UK-8 (A)
50
0,3,5,7
iscom
PBMC (i.p.)
20 UK-8
0/4 (0%)
(Lutz et al. 1995)
gplOO(denatured)
Insect cell (i.m.)c
Z2 (A)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
0/5 (25%)
gplOO(native)
Insect cell (i.m.)
Z2 (A)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (20%)
gplOO(highly purified)
Insect cell (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (20%)
gplOO(native )
Insect cell (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
0/5 (0%)
gplOO(denatured)
E..Coli (i.m.)
BANG (B)
100
0,2,4,8
A10H/QS21
PBMC (i.p.)
20 Z2
1/5 (25%)
Control(Ovalbumin)
(-)
(-)
100
0,2,4,8
(-)
PBMC (i.p)
20 Z2
2/7 (29%)
(Osterhaus et al. 1996)
Env(cleavage)
NA(s.c.)
AM19 (A)
100
0,4,10
iscom
PBMC (i.m)
20 AM19
0/6 (0%)
Env(no cleavage)
NA(sc)
AM19 (A)
100
0,4,10
iscom
PBMC (i.m.)
20 AM19
0/6 (0%)
Env(no cleavage)
NA(s.c.)
AM 19 (A)
100
0,4,10
QuilA
PBMC (i.m.)
20AM19
0/6 (0%)
Env-b Gal
NA(s.c.)
AM 19 (A)
100
0,4,10
QuilA
PBMC (i.m.)
20 AM19
0/6 (0%)
Control(PBS)
(-)(s.c.)
(-)
(-)
0,4,10
(-)
PBMC (i.m.)
20AM19
0/6 (0%)
(Flynn etal. 1995)
V3-peptide
synthetic (s.c.)
UK-8 (A)
100
0,3,6
QuilA/AlOH
PBMC (i.p.)
10 UK-8
0/15(0%)
Adjuvant alone
(-) (s.c.)
(-)
100
0,3,6
QuilA/AlOH
PBMC (i.p.)
10 UK-8
0/5 (0%)
(Lombardi, 1994)
V3-peptide
synthetic(s.c.)
PET(A)
500
0,2,4,6,8
CFA
F14 20-PET
0/3 (0%)
Control
(-)
(-)
(-)
(-)
(-)
F14 20-PET
0/3 (0%)
(Verschoor et al. 1996)
V3-fusion protein Ie
E..Coli(s.c.)
UT113 (A)
100
0,4,6,8,10
AlOH
NA(s.c.)
10-20UT113
0/5 (0%)
V3-fusion protein I
E..Coli(s.c.)
UT113
100
0,6,10
QuilA
NA(s.c.)
10-20UT113
0/5 (0%)
Control(PBS)
(-)(s.c.)
(-)
(-)
0,6,10
(-)
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-Env
(-)oronasal/s.c.
UT113
10s PFU
0
(-)
Boost V3-peptide
E. Coli (i.m.)
UT113
100
4,8
AlOH
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-Env
(-)oronasal/s.c.
UT113
105PFU
0
(-)
Boost V3 peptide
E.Coli (i.m.)
UT113
100
4,8
QuilA
NA(s.c.)
10-20UT113
0/5 (0%)
Feline Herpes-b Gal.
(-)oronasal/s.c.
(-)
105PFU
0
(-)
Boost PBS
(-)(s.c.)
(-)
(-)
4,8
(-)
NA(s.c.)
10-20UT113
0/5 (0%)
(Gonin et al. 1995)
Adenovirus-env
(-)(i.m.)
Wo (A)
11.8-9.2PFU
0,4,30
ISA206
NA(NA)
20 Wo
0/4 (0%)
Adeno-psuedorabies(control)
(-)(i.m.)
(-)
11.8-9.2PFU
0,4,30
ISA708
NA(NA)
20 Wo
0/4 (0%)
a UK-8, United Kingdom; Z2, Zurich; BANG, Bangston; AM19, Amsterdam 19; PET, Petaluma.
b Recombinant vaccinia virus expressed
c Baculovirus expression
d iscom, immune stimulating complex; A10H/QS21, aluminumhydroxide and non-toxic fraction from Quillaja saponaria; ISA206, water/oil adjuvant;ISA708, water/oil adjuvant,
e V3-fusion protein was composed of the FIV V3 region fused to galactokinase.
f Deletion of the cleavage site between the envelope surface(SU) and transmembrane protein(TM).

CHAPTER II
IMMUNOGENICITY AND PROTECTIVE EFFICACY EVALUATION OF
CANARYPOXVIRUS (ALVAC)-BASED FIV VACCINES AGAINST HOMOLOGOUS
FIV CHALLENGE
Introduction
The feline immunodeficiency virus (FIV) is the causative agent of an immuno
deficiency syndrome in cats (Pedersen et al. 1987). The immunological and pathological
changes observed in FIV infected cats closely resemble those observed in humans infected
with HIV the causative agent of AIDS (Yamamoto et al. 1988b; Ackley et al. 1990). Based
on these similarities FIV infection in cats has become a valuable model for the evaluation of
vaccine and prophylactic strategies. Similar to HIV, it is still unknown what constitutes
protective immunity against FIV. However, vaccine protection against experimental FIV
infection in cats has been achieved with conventional vaccines such as inactivated whole-
virus and inactivated FI V-infected cell vaccines (Yamamoto et al. 1991, 1993; Hosie et al.
1995; Verschoor et al. 1995). This vaccine approach has also been successful against
lentiviral infection in other animal models, such as SIV in macaques and HIV in
chimpanzees (Murphey-Corb et al. 1986; Fultz et al. 1992). The use of such vaccine
approach in humans, however, may not be feasible because of safety issues, such as
incomplete inactivation of vaccine virus which could potentially lead to infection of
immunized subjects.
An alternative to the use of conventional vaccines, is the use of viral vectors based
vaccines which can be engineered to encode specific components of these viruses. One
36

37
vector evaluated in current trials of HIV vaccines, is the canarypoxvirus vector, ALVAC
(Baxby et al. 1992; Perkus et al. 1995).Canarypoxvirus (ALVAC)-based vaccines are
considered safe due to their restricted host-range and their inability to undergo full-
replication in cells of non-avian origin (Baxby et al. 1992). The efficacy of ALVAC-based
vaccines against lentiviruses has been tested in several animal models. Rhesus macaques
immunized with ALVAC-recombinants expressing Env and Gag of HIV-2 were protected
from infection with homologous HIV-2 (Franchini et al. 1995a). Additionally, ALVAC-
HIV-1 vaccines have been proven effective against experimental infection with homologous
HIV-1 in a small number of chimpanzees tested (Girard et al. 1995, 1996; Van der Ryst et
al. 1996). Although, the number of animals in these trials was small, these findings
suggest that ALVAC-based vaccines can induce protective immunity against lentiviral
infection.
Hence, the main objective of this study was to assess the immunogenicity and
protective efficacy of recombinant canarypoxvirus (ALVAC)-vectored FIV vaccines alone
or in combination with conventional inactivated FIV-infected cell vaccines. Four distinct
ALVAC-FIV recombinants were tested. This included recombinants that encoded the FIV
Env (ALVAC-env), the FIV Gag and Prot (ALVAC-gag/prot) or both the FIV Env, Gag
and Prot (ALVAC-e/iv,gag/prot). Also included was a recombinant that encoded the FIV
Gag and a modified FIV Env from which a putative immunosuppressive region had been
deleted (ALVAC-977MG).
This study was conducted to address the following specific aims:
I. In vitro evaluation of ALVAC-FIV recombinant expression.
Determine if ALVAC-FIV recombinants are able to infect non-avian, non-permissive, feline
cells and properly express the inserted FIV gene constructs in these cells.

38
II. In vivo evaluation of ALVAC-FIV recombinant vaccines.
a) Identify the immune responses, both humoral and cell-mediated, elicited in cats after
immunization with ALVAC-FIV recombinants alone or after priming with ALVAC-FIV
recombinants followed by boosting with inactivated FI V-infected cell vaccine (ICV).
b) Assess the protective efficacy of immunization protocols employing ALVAC-ewv,
ALVAC-gag/prot, ALVAC-97TMG and ALVAC-env,gag/prot against experimental
infection with the homologous subtype FIV Petaluma (FIVPet) isolate in cats.
c) Assess the efficacy of a combination prime-boost protocol consisting of ALVAC-
env,gag/prot priming followed by inactivated FI V-infected cell boost, against experimental
infection with the homologous subtype FlVPet isolate in cats.
Materials and Methods
Construction of ALVAC-recombinants
ALVAC is a canarypoxvirus vector derived from a canarypoxvirus vaccine strain
used to immunize canaries, Kanapox (Rhone-Merieux, Lyon, France). The ALVAC-FIV
recombinants were generated using standard procedures similar to those used by
Virogenetics to generate ALVAC-based FeLV recombinants (Piccini et al. 1987; Tartaglia et
al. 1993). Briefly, the coding region of the FIVViUefranche isolate (subtype A) env, gag and
prot were amplified by polymerase chain reaction (PCR) and fused in a precise ATG to
ATG fashion with vaccinia early/late promotors FI6 or I3L, respectively ( Figure 2.1). The
protease gene was included in the constructs to ensure proper proteolytic cleavage of the
Gag precursor protein into the mature matrix protein (MA, pl5), capsid protein (CA, p24)

39
and nucleocapsid protein (NC, p7). The promotor-FIV gene constructs were then cloned
into the pC6L donor plasmid that contained ALVAC C6 flanking regions to enable insertion
of the F1V promotor-gene constructs into the ALVAC non-essential C6 genetic locus.
Prior to insertion into the donor plasmid, TsNT sequence elements were removed from the
FIV coding sequences without altering the amino acid sequence. TsNT motifs are
recognized by the poxvirus transcriptional apparatus as termination signals at early times
postinfection and their retention is known to result in diminished expression of foreign
gene products (Yuen and Moss 1987; Earl et al. 1990). The donor plasmid was then
transfected into ALVAC-infected chicken embryo fibroblast cells to generate full ALVAC-
recombinants by homologous recombination. Recombinants were subjected to several
rounds of plaque purification and analyzed by nucleotide sequence analysis to ensure
proper insertion.
ALVAC-FTV recombinants generated for this trial included ALVAC-chv containing
the whole FIV env coding region, ALVAC-gag/prot containing the FIV gag and prot
coding regions and ALVAC-env, gag/prot containing the FIV env, gag and prot coding
regions. The ALVAC-97TMG recombinant contained the FIV gag and prot gene and a
modified env coding region, of which a 714bp fragment encoding a putative
immunosupressive element, had been deleted (Figure 2.1).
In vitro Expression of the FIV Env and Gag
The ability of ALVAC-FIV recombinants to infect non-permissive non-avian cells
was evaluated on CrFK cells, a feline fibroblastic kidney cell line. Petn dishes seeded with
a monolayer of CrFK cells were inoculated with ALVAC-FIV recombinants or ALVAC
vector alone at a multiplicity of infection (m.o.i.) of 10 for 1 h at 37C in a humidified
atmosphere containing 5% COz. The ALVAC inoculum was removed and cells were
incubated with fresh medium for an additional 48 h at 37C. At 48 h, cells were removed

A.
H6 aTC

env 2571 bp (857 aa)
TAA

B.
13 L ATC gag/prot 1 71 Obp (570 aa)
/
TAA
r
c.
H6 ATC env 2571 bp
/'
TAA I3L ATC gag/prot 171 Obp TAA
{'///////A (
D.
TAA
t
gag/prot 1 71 Obp
GTA I 3 L H 6 ATC
Aen v 1857bp (61 9 aa) TAA
A
is
1
ALVAC-env
A LV AC-gag/prot
ALVAC -env,gag/prot
ALVAC-97TMG
Figure 2.1 Schematic representation of ALVAC constructs

41
by scraping and washed three times in phosphate-buffered saline (PBS). DNA was
extracted from the cells by resuspending in lysis buffer consisting of 0.021 M Tris (pH
7.5), 0.029 MEDTA (pH 8.0), 0.1 MNaCl, 1% SDS and proteinase K (10 mg/ml). Cells
in lysis buffer were incubated at 56C for 3-4 h followed by a 20 min incubation at 95C to
inactivate Proteinase K activity. The presence of FIV-specific DNA encoded by the
ALVAC-recombinants in the obtained DNA samples was determined by PCR using FIV
env- and gag-specific primers [FIV env specific primers 5-GAAATGTATAATATTGCTGG- 3
and 5 -GAATTGATTITGATTACATCC- 3; 5 -GGTAGGAGAGATTCTACA- 3 and 5-
CTGCATTCTTGCTGGTGC- 3 FIV gag specific primers]. PCR reactions were carried out in
a final volume of 50 pi containing: 1.0 pg DNA, 50 mM KC1, 10 mM Tris-HCl (pH 8.3),
2.5 mM MgCl2, 0.2 mM of each dNTP (dATP, dCTP, dTTP and dGTP), 20 pmol of each
primer, and 2.5 units of Taq DNA polymerase. The reaction mixtures were incubated for 5
min at 94C and cycled 30 times through 1 min at 94C, 1 min at 55C, 1.5 min at 72C,
followed by 10 min incubation at 72C. Finally, the PCR reaction products were separated
by electrophoresis on a 1% agarose gel and visualized by staining with ethidium bromide.
Detection of FIV-specific mRNA transcripts by RT-PCR
The expression of messenger RNA corresponding to the FIV genes encoded within
the ALVAC-recombinants was evaluated in non-permissive CrFK cells. Monolayers of
CrFK cells were inoculated with ALVAC-FIV recombinants or ALVAC vector alone as
described above. At 48 h postinoculation, messenger RNA was isolated using the Micro-
Fast track mRNA isolation kit (Invitrogen, San Diego, CA). The isolated mRNA was then
reverse transcribed into cDNA. Briefly, 1 pg of isolated mRNA was mixed with 7 pi
DEPC-treated H20 and 2 pi pdN6 and incubated at 65C for 5 min. Subsequently, the
samples were mixed with 4 pi 5X buffer, 1.3 pi DTT, 0.7 pi RNase inhibitor (40 U/pl), 1
pi dNTPs (10 mMdATP, dGTP, dTTP, dGTP), 2.0 pi acetylated BSA and 2 pi M-MLV

42
reverse transcriptase (Superscript RT 200 U/pl, Rnase H, Gibco BRL, Gaithersburg, MD)
and incubated at 42C for 1 h, followed by 10 min at 95C. The generated cDNA served as
a template for PCR reactions using env and gag specific primers. PCR was performed as
described above.
Indirect immunofluorescence
Protein expression of the ALVAC encoded FI V env and gag genes was analyzed by
indirect immunofluorescence on CrFK cells inoculated with ALVAC-FIV recombinants.
CrFK were seeded at a density of 5x10s cells per 35mm2 dish on sterile glass coverslips
and infected at a m.o.i. of 10 with ALVAC-FIV recombinants or the ALVAC vector alone.
Indirect immunofluorescence was performed at 48 h postinoculation. Cells were fixed in
4% paraformaldehyde for 10 min, washed in PBS, and permeabilized in PBS containing
0.2% Triton X-100. Cells were then incubated with pooled FIV-positive serum for 30
min, washed, and incubated with FITC-labeled anti-cat IgG. All serum and antibody
dilutions were made in PBS containing 3% bovine serum albumin (BSA). Finally, cells
were washed and counterstained with Evans Blue (0.5% in PBS) for 10 min, observed
under a microscope and photographed.
ALVAC vaccine production and titering
Both ALVAC vector and ALVAC-FIV recombinants were amplified on permissive
primary chicken embryo fibroblast (CEF) and titered in vitro by measuring the number of
plaque forming units (PFU). Vaccines were produced from clarified lysates of infected
CEF cells in serum free medium and aliquoted at IX108 PFU per dose.

43
Inactivated-cell vaccine preparation and titering
The inactivated FIV-infected cell vaccine (ICV) was produced from an IL-2
independent feline lymphoid cell line (FL-4) chronically infected with the FIV Petaluma
isolate (subtype A). This cell line was cloned from an IL-2 dependent feline T-cell line
(FeTl) infected with FIVPet and stains positive for CD8,CD4 and PanT surface markers and
negative for IgM heavy and light chains (Yamamoto et al. 1991a). The ICV vaccine was
generated by inactivation of FL-4 cells with 1.25% paraformaldehyde for 24 h, followed
by extensive dialysis against PBS. A single vaccine dose consisted of 2.5xl07 fixed
infected cells mixed with 250pg SAF/muramyl dipeptide (MDP) (Chiron Corporation).
Animals
A total of 36 specific pathogen free (SPF) cats (Felis catus, domestic short hair), 12
weeks of age, were purchased from Liberty Research Inc. (Waverly, NY). The animals
were housed at the Infectious Disease complex of Animal Resource Services and cared for
in accordance with the policies set by the Environmental Health and Safety division
(EH&S) and the Animal Care Committee of the University of Florida. All cats received a
combination vaccine against feline herpes virus, calicivirus and panleukopeniavirus (Fel-O-
Vax, Ford Dodge laboratories, Mason City, IA). Animals were not vaccinated against
feline leukemia virus (FeLV). Prior to immunization, all animals tested negative for
Toxoplasma gondii, FeLV and FIV by immunoblot analysis.
Grouping and Immunization Protocol
Cats were divided into 7 groups, with equal numbers of males and females in each
group (Table 2.1). Littermates were evenly spread over all groups. Cats were immunized

44
Table 2.1 Grouping and Immunization
Group
Cat ID#
sex
Vaccine(Number of Immunizations)
QH4
F
PY1
M
Group A
QOl
F
ALVAC-env (3X)
QC1
M
QU1
M
QL2
F
QQl
M
QA5
F
Group B
QU2
F
ALVAC-gag/prot (3X)
QX3
M
QI1
M
QL3
F
QH5
F
PY3
M
Group C
QS4
F
ALVAC-env,gag/prot (3X)
QC3
M
QG3
F
QE2
M
QQ2
M
PY5
F
Group D
Q02
F
ALVAC-97TMG (3X)
QX4
M
QI2
M
QL4
F
QH2
M
PY2
M
Group E
QA4
F
ALVAC (3X)
QC4
M
QG5
F
QE3
F
QH3
M
Group F
PY4
M
ALVAC-env,gag/prot (2X)
QA6
F
ICV(IX)
QC5
F
Group G
QG4
F
ALVAC (2X)
QE4
M
&ICV(1X)
F=Female M=Male

45
a total of three times at monthly intervals. The ALVAC vaccine was administered
intramuscularly at lxlO8 PFU/cat. The inactivated FlV-infected cell vaccine (ICV) was
mixed with 250 fig SAF/MDP adjuvant and was administered subcutaneously.
Challenge
The challenge inoculum consisted of cell-free culture fluid from PBMC infected
with FI VPet, previously titered in vivo in SPF cats. The challenge inoculum of 50 IDS0 was
given intraperitoneally (i.p.) four weeks after the final immunization.
FIV immunoblot assay
Sucrose gradient purified FlVPet from chronically infected FL-4 lymphoid cells was
separated by a 10% S DS pol yacryl am i de gel (SDS-PAGE). Proteins were transferred to
nitrocellulose sheets (pore diameter of 0.45 pm) by wet blotting. After transfer, the sheets
were blocked for 1-2 h at 37C in gelatin buffer (PBS containing 3% gelatin and 0.02%
sodium azide) and cut into strips. Serum samples of immunized and non-immunized cats
were diluted at 1:100 in Buffer 3 (0.05 Tris at pH7.4 containing 0.15 M sodium chloride,
0.001 M EDTA, 0.05 % Tween-20, and 1 % BSA) and incubated with immunoblot strips
for 4 h at 37C. The reactions were stopped with ddiHzO and strips were washed 3 times
with ELISA buffer (see ELISA protocol). The strips were incubated with biotinylated anti
cat IgG (Southern Biotechnology) for 1 h at 37C followed by three washes with ELISA
buffer. Strips were then incubated with streptavidin conjugated to horseradish peroxidase
for 1 h at 37C. The reactions were stopped and washed 3 times with ELISA buffer.
Finally, strips were incubated with fresh substrate solution (0.1 M Tris at pH7.4 containing
0.05% diamino benzidine, 400 mg/ml of NiCl2 and 0.01% H202). Upon appearance of
visible bands, reactions were stopped with an excess of ddiH20.

46
The titer of FIV-specific antibodies, if detected, was determined by testing 10-fold
serial dilutions of serum samples (1:100 to 1:1000000) as described above and defined as
the reciprocal of the highest dilution (in 10Log) at which FIV-specific bands could be
visualized.
ALVAC immunoblot assay
ALVAC immunoblots were generated similar to that described for FTV
immunoblots, using ALVAC derived from clarified lysates of ALVAC-infected primary
chicken embryo fibroblasts. Serum samples obtained from ALVAC immunized animals
and control non-immunized animals were tested at a serum dilution of 1:100 in Buffer 3.
Reaction were carried out as described (see FI V immunoblot assay).
Enzyme-linked immunosorbent assays (ELISA)
Synthetic peptides corresponding to both conserved and variable regions in the
FIVPet envelope surface(SU) and transmembrane(TM) protein were coated on 96 well
Immunolon microtiter plates at 250 ng/well with bicarbonate buffer (pH 9.6).[ V3-1
(SKWEEAKVKFHCQRTQSQPGS), V3-2 (GSWFRAISSWKQRNRWEWRDF), V3-3
(DFESKKVKISLQCNSTKNLFA) and TM (QLEGCNQNQFFCKI)]. The plates were
washed with Buffer 3 immediately prior to use and blocked with 5% dry non-fat milk in
H20. Serum samples were diluted 1:200 in Buffer 3 containing 5% newbom-calf serum
and incubated in the coated wells for 30 min at 37C, washed 3 times with ELISA wash
solution (0.05 % Tween-20 in 0.15M sodium chloride), and incubated with biotinylated
anti-cat IgG (Vector laboratories, Burlingame, CA) for 30 mm at37C. Subsequently, the
wells were washed three times and incubated with streptavidin conjugated to horse radish
peroxidase (Vector laboratories, Burlingame, CA), washed 3 times with ELISA wash

47
solution, followed by incubation with ELISA substrate solution (0.005 %
tetramethylbenzidine and 0.015 % H202 in 0.96 % citrate solution). The reactions were
stopped with 0.1 M sulfuric acid upon establishment of visible reaction color. The plates
were read in a ELISA reader at 414 nm.
Assessment of viral neutralizing (VN) antibodies
The presence of FIV specific VN antibodies was evaluated using a standard assay
(Yamamotoet al. 1991). Serum samples obtained preimmunization, postimmunization and
after challenge were diluted at various concentrations (1:5 to 1:100) and incubated at 56C
for 30 minutes to inactivate complement. The diluted sera were then incubated with 100
TCIDS0 (tissue culture cell infective doses of HVpJ for 45 min at 37C in a 24-well
microtiter plate. Subsequently, peripheral blood mononuclear cells (PBMC) were added to
this mixture at lxlO6 cells/well. After three days of culturing, cells were washed to remove
residual virus from the culture and resuspended in fresh culture media (see RT media).
Virus infection was monitored by Mg2+ dependent reverse transcriptase (RT) activity (see
RT assay) in culture fluid harvested on Day 6, 9, 12, 15 and 18 of culturing. The VN
antibodies titers were defined as the reciprocal of the highest final dilution which gave
>50% reduction in reverse transcriptase activity as compared to the reverse transcriptase
activity detected in fluids from control cell cultures that contained SPF serum and virus.
Assessment of FIV-specific proliferative T-cell responses
FIV-specific proliferative responses were evaluated using a 3[H]-thymidine
incorporation assay (Yamamoto etal. 1991). Freshly isolated PBMC were cultured in 96-
well microtiter plates in a final volume of 200 pi in RPMI1640 media supplemented with
5% heat inactivated fetal calf serum, lOmM HEPES buffer, 50 mg/ml gentamycin, 5X10 s

48
M 2-mercaptoethanol at a final concentration of lxlO6 cells/ml (2x10scells/well). Triplicate
cultures were stimulated with inactivated FI V (5 pg/well) and incubated at 37C for 4 days
in a humidified atmosphere containing 5% C02. On Day 4, cells were pulsed with 1 pCi
3 [H]-thymidine (Amersham, Indianapolis, IN) per well for 18 h. Cells were harvested
onto filter paper using a cell harvester. The discs were air-dned and 3[H]-thymidine
incorporation was assessed by liquid scintillation counting. Results of triplicate samples
were expressed as the stimulation index (S.I.), calculated as the mean incorporation in the
presence of inactivated FI V divided by the mean incorporation in the absence of inactivated
FIV.
Assessment of cytotoxic T-lymphocyte(CTL) responses
PBMC were tested for their ability to lyse autologous lymphoblastoid cells infected
with FIVPet. Freshly isolated PBMC were cultured at 2xl06 cells/ml in RPMI1640 medium
containing 10% FBS and stimulated with Con A (5 mg/ml) for three days. On Day 3, 3.6 x
106 Con A lymphoblasts were removed, infected with FIVPet and cultured for 5 days. The
remaining cells (effector cells) were maintained in RPMI 1640 medium supplemented with
10% FBS and IL-2 (100 U/ml) for 5 days. After5 days, l-5xl06 of the FIV-infected cells
were inactivated by UV treatment and added as antigen-presenting cells(APC) to the
effector cells at a ratio of 1:15 (APC:effector cells). Effector cells and FIV-infected APC
cells were cocultured for an additional 5-7 days in no IL-2 medium. Effector cells were
assayed for cytolytic activity against autologous FIV-infected target cells by a standard slCr
release assay (Song et al. 1992). Target cells were labeled with slCr for 2 h at 37C and
washed three times prior to use in the assay. Effector and slCr -labeled target cells were
then mixed at effector to target ratios ranging from 50:1 to 10:1 and incubated for 4 h at
37C without IL-2. After 4 h, 100 pi of supernatant was removed from each well and
slCr-specific activity was measured in a y-counter. Results are shown as the percentage of

49
specific cytotoxicity for triplicate assays. Maximum release was obtained by repeated
freeze-thawing of labeled target cells. Spontaneous release was obtained from slCr labeled
target cell cultures in the absence of effector cells. The percentage of FIV-specific release
was calculated as 100 x (mean cpm test release mean spontaneous release)/(mean cpm
maximum release mean cpm spontaneous release). The spontaneous release did not
exceed 20% of the maximum release. Specific lysis values equal or greater than 10% were
considered positive for CTL activity.
Assessment of natural killer cell activity
The level of natural killer (NK) cell activity was determined using a 4 h slCr release
assay similar to that described for the CTL assay. Various target cell types were used
including FeT-J (feline lymphoid cell line), FL-4 (feline lymphoid cell line chronically
infected with FlVPet), non-autologous PBMC, and autologous PBMC. Target cells were
labeled with slCr for 2 h at 37C and washed three times prior to use in the assay. Freshly
isolated PBMC effector cells were cocultured at effector to target cell ratios ranging
between 100:1 to 10:1 for 4 h at 37C in a humidified atmosphere containing 5% C02.
After 4 h, 100 pi of supernatant was removed from each well and slCr-specific activity was
measured in a y-counter. Results are shown as the percentage of cytotoxicity for tnplicate
assays. Maximum release was obtained by repeated freeze-thawing of labeled target cells.
Spontaneous release was obtained from slCr labeled target cell cultures in the absence of
effector cells. The percentage of NK activity was calculated as 100 x (mean cpm test
release mean cpm spontaneous release)/ (mean cpm maximum release mean cpm
spontaneous release). The spontaneous release did not exceed 20% of the maximum
release.

50
Viral reverse transcriptase (RT) assay
Freshly isolated PBMC were either stimulated with ConA (5mg/ml) or co-cultured
and cultured for 4 weeks in RPMI1640 medium containing 5% heat inactivated FCS,
lOmMHEPES buffer, 50 mg/ml gentamycin, 5X10 M 2-mercaptoethanol and 100 U/ml
human recombinant IL-2, or cocultured with ConA lymphoblasts from SPF cats. Culture
supernatants were collected every 3-4 days and assayed for the presence of viral reverse
transcriptase (RT) activity. The virus was pelleted from the supernatants by ultra
centrifugation (1 h at 17,000rpm). The virus pellet was then incubated with an RT cocktail
containing 100 mM Tris (pH8.3), 150 mM KC1, 10 mM MgCl2, 4 mM diethiothreitol
(DTT), 0.6 Units of Poly (rA), oligo(dT), and 60 pCi of 3[H]TTP per ml. After incubation
at 37C for lh, the cDNA was spotted onto filter paper discs that had been prewashed with
0.1 M sodium pyrophosphate. Discs were washed in the following sequence: twice in
10% cold trichloroacetic acid (TCA), once in 5% TCA, once in 5% TCA containing 0.5 %
SDS, and once in ethanol. The filter discs were air-dried and placed in scintillation vials
with 3 ml scintillation fluid. 3[H]-TTP incorporation was measured in using a liquid
scintillation counter. Supernatants were considered positive for RT activity if cpm in test
samples were equivalent or higher than 3 times the cpm of the negative control sample
(supernatants from SPF control cats).
Detection of proviral DNA by polymerase chain reaction (PCR)
Proviral DNA (latent infection) was monitored by env specific PCR on DNA
extracted from PBMC, bone marrow (BM) cells, and lymph node (LN) cells after culturing
for 4 weeks with FIV-free ConA lymphoblasts. BM cells were obtained from 1-2 ml of
aspirates taken from the femur. LN cells were obtained from the popliteal lymph nodes.

51
DNA was extracted as described. In the PCR reaction, the following FIV env specific
primer sets were used: 5-GAAATGTATAATATTGCTGG-3 and 5-
GAATTGATTTTGATTACATCC-3. The PCR reactions were carried out in 50 pi
reaction mixtures containing 1.0 pg genomic DNA, 50 mM KC1, 10 mM Tris-HCl (pH
8.3), 2.5 mM MgCl2, 0.2 mM of each dNTP (dATP, dCTP, dTTP and dGTP), 20 pmol of
each primer, and 2.5 units of Taq DNA polymerase. The reaction mixture was incubated
for 5 min at 94C and cycled 30 times through 94C for 1 min, 55C for 1 min, 72C for
1.5 min, followed by 10 mm at 72C. The specificity of the PCR-amplified 455bp product
was verified by nucleotide sequence analysis.
DNA sequencing
DNA sequencing was performed using the Amplicycle sequencing kit (Perkin
Elmer, Norwalk, CT). Primers (approximately 10 pM) were labeled at the 5-end with 20
pCi y-32P ATP (6000Ci/mmol) and 20 U Polynucleotide kinase in a final reaction volume
of 6.2 pi for 10 min at 37C. The reactions were terminated by incubation at 90C for 5
min. For the sequencing reactions, four separate reaction mixture were prepared
containing: 1 pi labeled primer, 4 pi 10X reaction buffer and approximately 10-50 fmol of
PCR template and 2 pi of either G, A, T, or C termination mix. Reaction mixtures were
then incubated at 95C for 15 seconds, cycled 25 times at 95C for 1 min, 68C for 1 min,
followed by 7 mm at 78C. Upon cycling, 4 pi of formamide stop solution containing
0.05% bromophenol blue and xylene cyanol were added. The reactions were analyzed on a
6% polyacry lamide gel containing 7 M urea and IX TBE.

52
In vivo assessment of viral-status
Three SPF kittens (#DH2, #DH5, #DE4), 12 weeks of age, were transfused
intravenously with a total of 1.1 x 108 cells obtained 8 months after the FIVPet challenge
from either ALVAC-gag/prot immunized cats (#QQ1 and #QX3) or FIV-mfected ALVAC-
control cat (#PY2). Cells consisted of 3 x 107 of PBMC isolated by ficoll hypaque density
centrifugation, 7 x 107 BM cells and lxlO7 LN cells. Prior to transfusion, these cells were
washed in sterile PBS and resuspended in 2 ml PBS. Cat #DH2 received cells from
infected control cat #PY2, cat #DH5 received cells from ALVA C-gag/prot immumzed cat
#QQ1 and cat #DE4 received cells from ALVAC-gag/prot immunized cat #QX3.
General parameters
Throughout the trial, all cats were momtored for hematological changes (complete
blood count, differential leukocyte count and total protein count) and abnormal clinical
manifestations (diarrhea, vomiting, lymphadenopathy, weight loss, elevated rectal
temperature and neurological signs).
CD4/CD8 ratios
CD4/CD8 ratios were determined by indirect immunofluorescence staining and flow
cytometry. Briefly, 5X10S PBMC isolated by ficoll hypaque density centrifugation, were
washed in FACS buffer (PBS containing 0.325% sodium azide and 2.5% BSA) and
incubated with feline CD4 or CD8 specific monclonal antibodies at 37C for 1 h.
Subsequently, cells were washed in FACS buffer and incubated with secondary antibody,
FITC labeled goat F(ab)2 anti-mouse IgG (H+L) (Southern Biotechnology Associates,
Inc.) for 1 h at 37C. Finally, cells were washed and analyzed by flow cytometry on a

53
Becton Dickinson FACSSORT. Monoclonal antibodies to feline CD4 and CD8 were
kindly provided by N. Gengozian, University of Tennessee.
T-cell mitogen proliferative responses
T-cell mitogen proliferative responses were measured by 3[H]-thymidine uptake
assays (Ackley et al. 1990). Freshly isolated PBMC were resuspended at 2x10s cells/ml
and stimulated with either Con A (5 mg/ml) or SEA (1 mg/ml). Cells were cultured for 48
h and then pulsed with lpCi3[H]-thymidine/well. After 18 h, cells were harvested using a
cell harvester onto filter paper discs. The filter discs were air-dried and 3[H]-thymidine
incorporation was assessed by liquid scintillation counting. The results were expressed as
the stimulation index (S.I.), calculated as incorporation (mean cpm of triplicate samples) in
the presence of mitogen divided by incorporation (mean cpm of triplicate samples) in the
absence of mitogen.
Statistical analysis
The statistical significance of the data was evaluated by a Fishers exact test, which
is a modification of the chi square test. This test should be used when comparing two sets
of discontinuous, quantal (all or none) data. The analysis was set up as follows:
Vaccinated
Unvaccinated
Infected
A
B
Uninfected
C
D

54
The P (Probability) for a one-tailed test was calculated as (A+B)!(C+D)!(A+C)!(B+D)! /
N!A!B!C!D! combined with the P value of stronger combinations. The obtained P value
tells if the groups differ significantly and the degree of significance. In this study, a P-
value equal or less than 0.05 was considered significant.
Results
In vitro assessment
The ability of ALVAC-recombinants to infect non-permissive feline cells was
demonstrated by PCR analysis on DNA extracted from feline CrFK cells inoculated with
ALVAC-'/zv and ALVAC-gag/prot recombinants (Figure 2.2 and 2.3). The obtained PCR
products corresponded in size to PCR-products obtained with DNA extracted from FL-4
cells, a lymphoid cell line chronically infected with FIVPrt. The correct nucleotide sequence
was verified by DNA sequencing (data not shown). No FI V-specific PCR products could
be detected in CrFK cells infected with ALVAC vector alone or FeT-J, a FIV-negative
feline lymphoid cell line.
Similarly, the expression of messenger RNA (mRNA) specific for FIWenv and gag
products was demonstrated in CrFK cells inoculated with the ALVAC-env and ALVAC-
gag/prot recombinants (Figure 2.4). RT-PCR on mRNA extracted from CrFK cells
infected with ALVAC-env revealed a 450bp band and from those infected with ALVAC-
gag/prot revealed a 700bp band consistent with the bands obtained from the FIV-infected
FL-4 cell line. No RT-PCR products could be detected in cells infected with ALVAC
vector alone and FeT-J control cells (FIV negative feline lymphoid cell line). In addition,
no PCR products were obtained PCR reactions in which mRNA extracts were used as
template, indicating that the obtained RT-PCR products were not the result of DNA
contamination of mRNA extracts (data not shown).

55
Cell
lane
PI4 Crfk FetJ
FIV+ Alvac- FI V-
Ctrl Env Gag
Ml 2 3 4 5
X + + "
450 bp
Figure 2.2 FIV-env specific PCR on CrFK inoculated with ALVAC-recombmants.
Photograph of PCR products following electrophoresis on a 1.0 % agarose gel in Tris
acetate-buffer(40mMTris-acetate, 1 mM EDTA, pH 7.6). LaneM, \ DNA EcoRI/Hindlll
marker (lpg/lane). Lane 1, positive control, FIV-env specific PCR on DNA extracted
from FL4 (FIV+) cells. Lane 2, FIV-Env specific PCR on CrFK cells inoculated with
ALVAC-control. Lane 3, FI\-env specific PCR on CrFK cells infected with ALVAC-env.
Lane 4, FIV-cnv specific PCR on CrFK cells infected with ALVAC-gag/prot. Lane 5,
negative control, FIV-Env specific PCR on Fet-J (FlV)cells.

56
Cell
FetJ
Crfk
FI4
FI V-
Alvac-
FIV+
Ctrl Env Gag
lane
M
1
2 3 4
5
X

- +
+
Figure 2.3 FIV-gag specific PCR on CrFK inoculated with ALVAC-recombinants.
Photograph of PCR products following electrophoresis on a 1.0 % agarose gel in Tris
acetate-buffer (40mM Tris-acetate, 1 mMEDTA,pH 7.6). Lane M, marker EcoRl/Hindlll
cutDNA (1 pg). Lane 1, negative control, FI V-gag specific PCR on DNA extracted from
Fet-J (FIV ) cells. Lane 2, FI V-gag specific PCR on CrFK cells inoculated with ALVAC-
control. Lane 3, FIV-gag specific PCR on CrFK cells infected with ALVAC-env. Lane 4,
FTV-gag specific PCR on CrFK cells infected with ALVAC-gag/prot.. Lane 5, positive
control, FIV-gag specific PCR on F14 (FIV+)cells.

57
Cell
primers
lane M
FI4 CRFK
FIV+ Alvac-
Ctrl. Env Gag
E G E G E G
1 2 3 4 5 6
Figure 2.4 RT-PCR on mRNA extracted from CrFK inoculated with ALVAC-
recombinants. Photograph of PCR products following electrophoresis on a 1.0 % agarose
gel in Tris acetate-buffer (40mM Tris-acetate, 1 mM EDTA, pH 7.6). Lane M, X DNA
EcoRUHindUI marker (lpg/lane). RT-PCR on mRNA extracted from F1V+ FL-4 cells
shows specific amplification of FlV-env (Lane 1) and FIV-gag (Lane 2). RT-PCR on
mRNA extracted from CrFK cells infected with ALVAC-control show no amplification of
FlV-env (Lane 3) or FIV-gag (Lane 4). Lane 5, FlV-^nv specific RT-PCR on mRNA
extracted from CrFK cells infected with ALVAC-cnv. Lane 6, FI V-gag specific RT-PCR
on mRNA from CrFK cells infected with ALVAC-gag/prot.

Figure 2.5 Indirect immunofluorescence analysis on permeabilized CrFK cells infected
with ALVAC-recombinants at a m.o.i. of 10. Expression of FIV Env glycoprotein and
Gag proteins was detected using pooled serum from FIV-mfected cats as the primary
antibody and fluorescein isothiocyanate-conjugated mouse anti-cat IgG as the secondary
antibody. Panel A, control uninfected CrFK cells. Panel B, control CrFK cells infected
with ALVAC-control. Panel C, CrFK cells infected with ALVAC-e/iv. Panel D, CrFK
cells infected with ALVAC-gag/prot. Panel E, CrFK cells infected with ALVAC-
env,gag/prot. Panel F, CrFK cells infected with ALVAC-97TMG.

59

60
Further, the expression of the ALVAC encoded FIV Env as a membrane associated
protein and of the FIV-Gag core protein as intracellular protein was demonstrated by
indirect immunofluorescence on CrFK cells inoculated with the ALVAC-FIV recombinants
(Figure 2.5). At 48 h postinfection, cells infected with ALVAC-env showed fluorescence
predominantly at the surface (panel C) whereas cells infected with ALVAC-gag/prot,
ALVAC-env,gag/prot and ALVAC-97TMG showed strong fluorescence in the cytoplasm
(see Figure 2.5, panel D-F). Control cells, uninfected CrFK and CrFK infected with
ALVAC vector alone, did not show immunofluorescence (panel A and B).
In vivo assessment
A total of 36 SPF cats were used to evaluate the prophylactic efficacy of
immunizations protocols employing ALVAC-FIV recombinants alone or in combination
with ICV. Cats were divided into 7 groups (A-G) and inoculated three times at monthly
intervals with either ALVAC-env (n=6), ALV AC-gag/prot (n=6), ALVA C-env,gag/prot
(n=6), ALVAC-97TMG (n=6) or ALVAC vector alone (n=6). Cats in group F and G
were immunized twice with ALVA C-env, gag/prot (n=3) or ALVAC (n=3), respectively,
followed by a boost with inactivated FIV-infected cell vaccine (see Table 2.1). All cats
were challenged 4 weeks after the final immunization with 50 IDS0 FIVPet. The FIVPet
isolate is a subtype A virus, and differs 3% in the Env and 1% in the Gag protein coding
region from the FIVvi]]efranche isolate (subtype A) used to generate the ALVAC recombinants.
General immunologic parameters
No adverse effects and no significant changes in blood chemistry (CBC, HgB,
PCV, TPP) were noticed in any of the cats upon immunization with ALVAC-FIV
recombinants or after boosting with inactivated FIV-infected cell vaccine (data not shown).

Figure 2.6 Representative FACS analysis of binding of anti-fCD4 and anti-fCD8
monoclonal antibodies to PBMC. Expression of fCD-4 and fCD-8 was detected by using
anti-fCD4 and anti-fCD8 antibodies as the primary antibody and fluorescein isothiocyanate-
conjugated goat anti-mouse IgG as the secondary antibody. Panel A, scattered dot-plot of
PBMC isolated from cat#QG3. The depicted histograms respresents cells gated under gate
1 (predominantly lymphocytes) and gate 2 (negative control, cells of the macrophage and
monocyte lineages). Panel B, histogram of CD4 and CD8 staining of cells in gate 1. Panel
C, histogram of CD4 and CD8 staining of cells in gate 2.

62
FL-HEIGHT
FL-HEIGHT

63
Table 2.2 CD4/CD8 ratios before and after immunization and challenge
Cat ID#
Immunizations
Before After
-4 -1
months
post-challenge
+3
+5
+6
+7
+8
+ 10
+ 12
QH4
4.7
4.4
3.2
3.0
3.3f
PY1
4.3
4.2
2.4
2.4
2.5f
QOl
3.9
2.6
3.5f
QC1
3.3
3.3
2.9
2.5
l.SF
QU1
ND
3.8
3.6
3.3
2.5f
QL2
3.6
3.3
2.2
1.9
1.7f
QH5
6.7
4.2
3.8
2.6
2.5F
PY3
5.1
4.9
4.3
3.3
2.6F
QS4
6.0
4.8
2.6
2.3
2.3F
QC3
3.2
2.6
3.6
2.1
2.0F
QG3
5.0
4.0
3.6
3.2
2.4f
QE2
3.1
ND
3.0
2.0f
QQl
4.9
2.7
2.1
1.9
1.6
2.3
QA5
4.3
2.2
2.6
2.5
2.9
2.5
QU2
4.0
2.6
2.4
2.0
2.3
2.9
QX3
2.0
2.5
2.0
1.8
1.8
2.3
QI1
6.1
3.9
3.9
3.3
3.3
3.9
QL3
4.9
2.0
2.6
1.9
2.0
2.2
QQ2
2.7
3.4
2.7
2.0
2.2f
PY5
5.1
3.8
2.5
2.1
l.^
Q02
3.3
4.0
2.4f
QX4
1.3
ND
1.5
1.1
1.0F
QI2
4.9
2.5
2.1
2.0
l.SF
QL4
4.4
4.1
3.8
2.3
2.5f
QH2
3.9
3.5
4.6f
PY2
3.3
2.9
3.5
2.7
3.8
2.5
QA4
2.9
2.6
2.4
1.7
1.8f
QC4
4.2
3.1
2.7
2.4
1.6f
QG5
4.0
2.5
2.7f
QE3
2.1
2.3
2.7
1.7
2.0f
QH3
5.5
4.1
4.0
3.4
2.0
4.3
PY4
2.8
4.0
3.1
2.6
2.6
2.4
QA6
4.3
3.6
2.9
2.5
2.1
2.7
QC5
2.8
2.0
2.1F
QG4
5.1
3.5
2.9f
QE4
2.6
3.1
1.8
2.0
1.6
2.5
F=Final testing of CD4/CD8 ratio before animal was euthanized.

Table 2.3 Proliferation responses to T-cell mitogens (ConA and SEA).
Stimulation Index
Vaccine
Cat ID#
Number of immunizations
Pre- IX
Immunization
3X
Post-
Challenge3
ConA
SEA
ConA
SEA
ConA
SEA
ConA
SEA
ALVAC-ewv
QH4
2.8
26.7
7.9
17.4
22.5
24.9
2.5
3.1
PY1
4.3
29.0
6.6
12.6
4.0
4.0
3.2
3.1
A L V A C-gag/>prot
QA5
7.5
16.9
32.1
77.4
30.3
14.4
22.7
23.2
QI1
50
93.8
2.2
7.5
25.8
28.2
18.3
17.8
ALVAC-env,
QS4
6
29
3.1
6.7
35.0
35.2
37.1
36.7
gag/prot
QG3
2.9
6.9
1.0
2.3
38.1
35.0
30.7
22.7
ALVAC-97TMG
QQ2
2.7
9.2
1.0
2.0
16.8
18.0
17.2
12.5
QL4
78.4
99.0
9.6
20.3
29.4
27.2
18.8
17.2
ALVAC-env,
PY4
13.4
26.8
4.0
15.9
20.5
19.7
63.2
36.7
gag/prot &ICV
ALVAC
QH2
4.2
7.2
2.4
5.5
9.5
9
28.7
26.2
QA4
46
65
5.5
9.3
44.3
52.8
15.2
17.3
ALVAC
QG4
4
8
12.5
23.5
7.4
7.4
25.4
23.5
&ICV
3 weeks post challenge

65
CD4/CD8 ratios were monitored prior to immunization, postimmunization and after
challenge, a representation of a typical FACS analysis is depicted in Figure 2.6. Prior to
immunization, most cats displayed CD4/CD8 ratios considered normal for three month old
kittens (average ratio 4.0 + 1.2) except for cat #QX3 which displayed a ratio of 1.3 which
is considered low. A littermate of this cat (#QX4) also displayed a relatively low ratio,
suggesting that genetic factors attributed to this. After immunization, most cats showed an
average CD4/CD8 ratio of 3.3 + 0.8. The observed decline in ratio is expected with the
increase in age. No inversion (<1) in CD4/CD8 ratios were noticed after challenge (Table
2.2). This was as expected since the FIVPct at the challenge dose used generally does not
cause inversion of CD4/CD8 ratio until 1.5-2 years postchallenge.
To determine if ALVAC vaccinations influenced lymphocyte function, we evaluated
lymphocyte proliferation upon exposure to concanavalin A (ConA) and staphylococcal
enterotoxin A (SEA) at various times in selected cats. Following immunizations and
challenge no abnormalities in T-cell proliferative responses were detected in any of the cats
tested (Table 2.3).
Humoral responses
The generation of ALVAC-specific antibodies was evaluated before and after
immunization in serum samples taken from cat #QS4 immunized with ALVAC-
env,gag/prot and cat #PY2 immunized with ALVAC vector alone by immunoblotting.
ALVAC specific humoral responses were detected in both cats upon a single immunization
and additional immunizations resulted in increased titers of these antibodies (Figure 2.7).
Serum samples obtained from SPF and FIV infected control cats tested negative.
Next, we tested if the immunization schemes used were able to generate FIV-
specific antibody responses. Immumzation with ALVAC-FIV recombinants alone (#QC3)
failed to induce detectable FI V-specific antibody responses even after three immunizations

Figure 2.7 ALVAC-specific immunoblot. Serum samples taken from ALVAC-control immunized cat #PY2 and ALVAC-env,gag/prot
immunized cat #QS4 prior to immunization (pre-) and after the first, second and third immunization and 17 weeks postchallenge (17pc)
were diluted 1:100 in Buffer 3 and incubated with ALVAC-specific westemblot strips. Negative controls: pooled sera from FIV-infected
cats and pooled sera from FIV-negative SPF cats.

Immunoblot ALVAC
Cat # : PY2
Vaccine; Alvac-control
pre 1st 2nd 3rd 17pc
83 kDa
18 kDa
serum dilution: 1:1 00
Alvac-env,gag/prot

Figure 2.8 FIV-specific immunoblot. Serum samples from ALVAC-env,gag/prot immunized cat #QC3, ALVAC-env,gag/prot
combined with ICV immunized cat #QA6 and ALVAC combined with ICV immunized cat #QC5, obtained before and after
immunizations, were diluted 1 to 100 in Buffer 3 and incubated with FIV-specific westemblot strips. The positive control was incubated
with pooled serum from FI V-infected cats. The negative control was incubated with serum from a SPF cat.

Immunoblot FIV
Cat # QC3
Vaccine Alvac-Env,Gag/pro(3X)
QA6
Alvac-Env,Gag/pro(2X)
ICV(IX)
pre 1 st 2nd 3rd
pre 1st 2nd 3rd
QC5
Alvac-control(2X)
ICV(IX)
pre 1st 2nd 3rd

70
Table 2.4 FI V-specific antibody titers before and after immunizations and challenge.
FTV-specific antibody titer2
Cat ID# Vaccine Boost pre- post Mo.postchallenge
immunizations 2 8
QA6
ALVAC-env,gag/prot
ICV(IX)
<2
5-6
4-5
4-5
QH3
ALVAC-env,gag/prot
ICV(IX)
<2
5
3-4
4
PY4
ALVAC-env,gag/prot
ICV(IX)
<2
5
4-5
4-5
QG4
ALVAC(2X)
ICV(IX)
<2
4-5
5
ND
QC5
ALVAC(2X)
ICV(IX)
<2
3-4
5-6
ND
QE4
ALVAC(2X)
ICV(IX)
<2
3-4
5-6
5-6
QS4
ALVAC-env,gag/prot (3X)
<2
<2
5-6
ND
QC3
ALVAC-env,gag/prot (3X)
<2
<2
<2
ND
PY3
ALVAC-env,gag/prot (3X)
<2
<2
<2
ND
PY2
ALVAC(3X)
<2
<2
5-6
5-6
2 FI V-specific titers expressed as the reciprocal of the highest dilution (in 10log) at which
FIV specific bands could be detected by immunoblotting.
ND= not determined
(Figure 2.8). In contrast, all cats boosted with ICV (group F and G) developed detectable
FIV-specific antibody responses. Interestingly, cats primed with ALVAC-env,gag/prot
(#QA6) developed approximately 10-fold higher antibody titers than those primed with
ALVAC vector alone (#QC5) (Figure 2.8) upon the ICV boost (see Table 2.4)
Selected cats of each group were also tested for the presence of antibody responses
to peptides corresponding to the V3 region of the FIV surface envelope glycoprotein by
ELISA. This region is thought to be equivalent to the V3 region of HIV which contains the
principal neutralizing domain (Pancino et al. 1994). None of the immunized cats exhibited
significant levels of antibody titers to the three V3 peptides tested even after three
immunizations (data not shown).

71
Viral neutralizing antibody responses were measured in selected cats before and
after immunization. The neutralization assay was performed using ConA lymphoblast as
FIV-susceptible cells and FIVPet propagated on a feline lymphoid cell line (FL-4) as the
virus inoculum. VN antibody responses were absent before immunization and after
immunization in all cats tested including those boosted with ICY (Table 2.5).
Table 2.5 Viral neutralizing antibody titers after immunization and challenge
VN titer1*
Vaccine
pre- post- Months post-
Cat ID# immunizations challenge
3
12
ALVAC-cnv
QU1
<5
<5
<5
NT
PY1
<5
<5
>100
NT
ALV AC-gag/prot
QX3
<5
<5
<5
<5
QQi
<5
<5
<5
<5
QIl
<5
<5
<5
<5
QL3
<5
<5
<5
<5
ALV AC-env, gag/prot
QS4
<5
<5
>100
NT
PY3
<5
<5
<5
NT
ALVA C-env, gag/prot
QH3
<5
<5
5-20
<5
&ICV
QA6
<5
<5
5-20
<5
PY4
<5
<5
5-20
5-20
ALVAC
QC4
<5
<5
>100
NT
PY2
<5
<5
>100
>100
QA4
<5
<5
<5
NT
QE3
<5
<5
<5
NT
ALVAC
QG4
<5
<5
>100
NT
&ICV
QC5
<5
<5
>100
NT
QE4
<5
<5
>100
>100
a VN Titer expressed as the reciprocal of the highest final dilution which gave > 50%
reduction in reverse transcriptase activity as compared to reverse transcriptase activity
observed in control cultures which contained SPF serum. NT= not tested.

72
T-helper Lymphoproliferative Responses
Selected cats from each group were tested after the second and third immunization
and challenge for lymphocyte proliferation in response to inactivated FIV (Table 2.6). The
inactivated FIV preparation used in this assay, included significant amounts of Gag
allowing the detection of proliferative responses to both FIV Env and Gag. After two
immunizations, no significant levels of FIV-specific lymphoproliferative responses were
detected in any of the tested cats. After the third immunization lymphoproliferative
responses were detected in two cats. However, the observed levels were low as compared
to those detected in cats immunized multiple times with ICV vaccines (S.I. 4-6). This
included cat #QC3 immunized with ALVAC-env,gag/prot and cat #PY4 immumzed with
ALVAC-env,gag/prot and boosted with ICV. Upon challenge, lymphoproliferative
responses were absent in all of the tested cats even in cats that became viremic except for
cat #QX4 which showed low levels of FIV-specific proliferative responses (Table 2.6).
Cytotoxic T-cell responses
Since viral-vector based vaccines are thought in general to be effective in eliciting
CTL responses, FIV-specific CTL responses were measured after each immunization in the
peripheral blood of selected cats. PBMC isolated were cultured in the presence of FIV
antigen-presenting cells and assayed for their ability to lyse autologous PBMCs infected
with FI VPet. FIV-specific CTL activity was detected in one of two cats tested after a single
immunization with ALVAC-env,gag/prot (see Table 2.7). After the second and third
immunization, CTL activity was detected in some cats of each group immunized with
ALVAC-FIV recombinants alone and those immunized with ALVAC-env,gag/prot and
boosted with ICV. No major variance between the different immunization schemes, with
respect to intensity of CTL activity or percentage of cats displaying CTL activity within

73
Table 2.6 T-helper responses to FIV upon immunization and challenge
Stimulation Index
Vaccine
Cat ID#
Number
oflmmunizations
Post-
IX
3X
challenge
ALVAC -env
QL2
1.1
1.2
ND
QH4
ND
ND
1.0
QC1
ND
ND
0.7
ALV AC-gag/prot
QX3
1.0
1.3
1.4
ALV AC-env, gag/prot
QG3
0.9
1.6
ND
QC3
ND
2.6
1.3
PY3
ND
1.6
ND
QH5
ND
ND
0.7
ALV A C- 9 7TMG
QI2
1.1
1.7
ND
QX4
ND
ND
2.1
ALVAC
QC4
1.2
ND
ND
QE3
ND
1.1
ND
QH2
ND
ND
1.3
ALVAC-env,
PY4
ND
2.4
ND
gag/prot &ICV
QH3
ND
ND
0.7
ALVAC
QC5
0.7
1.3
0.7
&ICV
QG4
ND
1.0
ND
a 4 weeks post-challenge
ND= not determined.

74
Table 2.7 FI V specific CTL activity in peripheral blood after immunizations
% specific 51Cr release3
Vaccine Cat ID# Number of Immunizations
IX
2X
3X
QH4
ND
3.9
9.5
ALVAC-cnv
QC1
ND
ND
22.9
PY1
ND
ND
3.7
QQl
ND
ND
22
ALV AC-gag/prot
QA5
ND
ND
4
QI1
ND
ND
8
QC3
0.1
ND
18.4
ALV AC-env, gag!prot
PY3
25
9.9
36.7
QS4
ND
22
7
QH5
ND
ND
1
QQ2
ND
25.6
19
ALVA C-97TMG
PY5
ND
ND
12.5
QI2
ND
ND
9.0
QH3
ND
0.4
55.2
ALV AC-env, gag/prot
QA6
ND
ND
27.3
&ICV
PY4
ND
ND
8.0
ALVAC&ICV
QG4
ND
ND
10
QA4
ND
ND
7
ALVAC
QH2
ND
1.3
ND
PY2
ND
ND
0.0
QC4
ND
ND
10
a Percentage specific release as observed at an average effector to target cell ratio
of 1 to 20-30.
ND= not determined

75
each group, was observed. Further, cats primed with ALVAC-env,gag/prot and boosted
with ICV displayed similar levels of CTL activity as detected in those immunized with
ALVAC-env, gag/prot alone. Control cats immunized with ALVAC vector alone or
immunized with ALVAC (2X) and boosted with ICV failed to demonstrate significant
levels of CTL activity (< 10%).
The detected CTL activity was found to be MHC restricted as effector cells were
only capable of lysing autologous FIV-infected target cells and failed to lyse non-
autologous FIV-infected target cells (data not shown), thus implying that the detected
activity was due to CTL, as opposed to NK cell activity. Further, no lysis was observed
using uninfected autologous target cells (data not shown).
Table 2.8 NK activity postchallenge
Vaccine
Cat ID#
% specific slCr release
Target cell
autologous
PBMC
heterologous
PBMC
FL-4
Fet-J
ALVAC-env
QC1
0
0
10.1
20.7
ALV AC-gag/prot
QC3
ND
0
12.5
18.8
ALVA C-env,
QX3
0
0
8.7
22.8
gag/prot
ALVAC-97TMG
QX4
ND
0
10.7
16.9
ALVAC
QC5
ND
0
5.5
17.8
&ICV
ALVAC
QC4
ND
12.2
6.5
8.2
ND= not determined

76
NK cell activity
NK cells are the principal effector cells in clearance of viral infections early in the
course of infection. For this reason, we evaluated levels of NK activity in selected cats at
3.5 weeks postinfection (Table 2.8). NK activity was measured using various target cells
including autologous PBMC as negative controls. All cats displayed normal levels of NK
activity and no significant differences were observed between the infected and non-infected
ALVAC-immunized cats.
Protective efficacy
The presence or absence of FIV following challenge was measured at monthly
intervals using several methods. This included the assessment of viral reverse transcriptase
(RT) activity and FIV-specific PCR analysis of cultured PBMC. These assays were also
used to test LN and BM cells for the presence of FIV, as these organs function as major
reservoirs for the virus. In addition, FIV infection was determined by comparing the level
of FIV-specific antibody responses in the serum before and after FIV challenge. FIV-
specific immunoblotting (WB) was performed as well as ELISA using a peptide
corresponding to the transmembrane (TM) region of the FIV Env. Sera from FIVPet
infected cats have shown to react strongly to this peptide. Further, VN antibody responses
were measured in selected cats before and after FIV challenge. In general, the induction
and persistent elevation of FIV-specific antibody responses and VN antibodies are
indicative of an active viral infection.
All control cats (n=3) immunized with ALVAC vector alone and boosted with ICV
became viremic as assessed by RT for infectious virus and FIV-specific PCR for proviral
DNA in peripheral blood and tissue samples (BM and LN cells) (Table 2.9b and Table
2.10). Further, these cats developed high titer VN (>100) responses indicative of active

Table 2.9a Virus isolation (RT and PCR) on PBMC and immunoblot analysis.
Vaccine
Immunizations
pre- post-
Cat ID# -3 -1 +1
WB RT PCR WB RT PCR WB RT PCR
Months post-challenge
+2 +3 +4 +8 +12
WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR
QH4
PY1 ___ ___ + + + + + + + + + + +
ALVAC-env QOl - + + + + + + + +
QC1
QU1
QL2 ___ ___ + + + + + + + + + + +
QQl
QA5
ALVAC-gag/prot QU2
QX3
QI1
QL3
QH5
PY3
ALVAC-e/iv, QS4
gag/prot QC3
QG3
QE2
- + + + + + + + + + + +
+ -- + + + +- +
-4
-4
WB= western blot (FIV-specific), RT= reverse transcriptase, PCR using FIV-specific primers.

Table 2.9b Virus isolation (RT and PCR) on PBMC and immunoblot analysis.
Immunization
pre- post-
Months post-challenge
Vaccine
Cat ID#
-3 -1
+1
+2 +3 +4
+8
+ 12
WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR WB RT PCR
QQ2
PY5
ALVAC-97TMG Q02
QX4
QI2
QL4
+ + + + + + + +
+ + + + + + + + +--
+ + + + + + + + + +
QH2
- -
- + +
+
+
+
+
+
+
PY2
- -
- -
+
+
+
+
+
+
+
+
+
+ + +
ALVAC
QA4
- -
- -
-
-
-
-
-
-
-
-
-
QC4
- -
- -
+
+
+
+
+
+
+
+
+
QG5
QE3

- + +
+
+
+
+
+
+
ALVAC-env,
QH3
+ -
+ -
+
+
+
+ -
gag/prot &ICV
PY4
+ -
+ -
+
-
-
+
-
-
+
-
-
+ -
QA6
+ -
+ -
+
-
-
+
-
-
+
-
-
+ -
ALVAC
QC5
+ -
+ + +
+
+
+
+
+
+
+
+
+
&ICV
QG4
+ -
+ + +
+
+
+
+
+
+
+
+
+
QE4
+ -
+ -
+
+
+
+
+
+
+
+
+
+ + +
WB= western blot (FIV-specific), RT= reverse transcriptase, PCR using FIV-specific primers.

79
Table 2.9 Virus isolation on PBMC and tissue samples and WB and ELISA data
Vaccine Cat#
Weeks
p .c.
WB/ELISA
PBMC
RT PCR
Tissue
BM LN
RT PCR RT PCR
THY
RT PCR
QH4
27
-
-
-
+
-
+
_
+
_
PYl
24
+
+
-
-
-
-
-
+
-
Alvac-env
Q01
10
+
+
+
+
+
+
+
+
ND
QC1
28
-
-
-
-
-
-
-
-
ND
QUl
28
ND
QL2
24
+
+
-
-
+
+
+
+
- -
QQ1
29
ND
QA5
29
-
-
-
-
-
-
-
-
ND
Alvac-gragr/prot
QU2
29
ND
QX3
29
ND
QI1
29
-
-
-
-
-
-
-
-
ND
QL3
29
-
-
-
-
-
-
-
-
ND
QH5
28
PY3
27
-
-
-
-
-
-
-
-
ND
Alvac-env,
QS4
24
+
+
-
-
-
+
+
+
-
gag,prot
QC3
28
-
-
-
-
-
-
-
-
- -
QG3
28
-
-
-
-
-
-
-
-
- -
QE2
28
+
+
-
-
-
+
-
+
- +
QQ2
27
_
_
_
_
_
_
+
PY5
28
Alvac-97TMG
Q02
10
+
+
+
+
+
+
+
+
ND
QX4
25
+
+ /-
-
-
-
-
-
-
- -
QI2
25
+
-
-
-
-
-
-
-
-
QL4
27
-
-
-
-
-
-
-
-
- -
QH2
10
+
+ /-
+
+
+
+
+
+
ND
PY2
39
+
-
+
+
-
+
-
-
ND
Alvac-control
QA4
28
- -
QC4
26
+
+
+
+
+
+
+
+
+ +
QG5
10
+
+
-
+
-
+
+
+
ND
QE3
28
-
-
-
-
-
-
-
-
- -
Alvac-env,
QH3
36
+
_
_
ND
gag/prot&ICV
PY4
36
+
-
-
-
-
-
-
-
ND
QA6
36
+
-
-
-
-
-
-
-
ND
Alvac-control
QC5
10
+
+
+
+
+
+
ND
&ICV
QG4
10
+
+
-
+
-
+
+
+
ND
QE4
39
+
+
-
-
+
+
ND
ND
WB=westem blot, PBMC= peripheral blood mononuclear cells, BM= bone marrow, LN=lymph node,
Thy=thymus, ND= not determined.

80
viral infection (Table 2.5). In contrast, only 4 of 6 control cats immunized with ALVAC
vector alone became viremic as determined by RT, PCR and development of FIV-specific
antibodies including VN antibodies. The two other cats (#QA4 and #QE3) in this group
tested negative consistently by RT and PCR analysis in peripheral blood samples taken 1,
2, 3, 4 and 7 mo after challenge. In addition, BM, LN and thymus tissues taken from
these two cats 7 mo after challenge were negative for virus by RT and PCR analysis (Table
2.9b). These cats also failed to develop FIV-specific antibody responses as assessed by
immunoblot (WB) and ELISA and lacked detectable levels of VN antibody responses,
further supporting lack of infection in these cats.
In the group immumzed with ALVAC-e/iv two of six cats tested negative for virus
by RT, and PCR analysis at all time-points postchallenge (Table 2.9a). In addition, these
cats failed to develop detectable levels of FIV-specific and VN antibody responses (Table
2.5). Another cat (#QH4) in this group also tested negative by all these criteria up to 6 mo
postchallenge but tested positive for virus by PCR analysis in PBMC, BM and LN cells
taken 7 mo postchallenge (Table 2.10). The three remaining cats in this group developed
viremia upon challenge at a rate similar to that observed in the ALVAC control cats that
became infected. Partial protection was also observed in the groups immunized with
ALVAC-env,gag/prot and ALVAC-97TMG. Four out of 6 cats immunized with ALVAC-
env,gag/prot and 3 out of 6 cats immunized with ALVAC-97TMG resisted infection
following challenge. The remaining cats in these groups tested positive for virus by all
criteria except for cat #QQ2 which tested positive by PCR analysis only in lymph node
tissue taken 7 months after challenge (Table 2.9b and Table 2.10).
In contrast, full protection was observed in the group immunized with ALVAC-
gag/prot All six cats tested negative by RT and PCR in peripheral blood and lymphoid
tissues. Further, these cats lacked detectable levels of FIV-specific antibodies and VN
antibodies up to one year postchallenge ( Table 2.5). Similarly, cats primed with ALVAC-
env,gag/prot and boosted with ICV tested negative by RT and PCR analysis of peripheral

81
blood and lymphoid tissues throughout the vaccine trial (Table 2.9a and 2.10). FIV-
specific humoral responses elicited by vaccination remained following challenge but slowly
declined thereafter. Interestingly, serum samples obtained three months postchallenge
contained low VN antibody titers (5-20) whereas no VN titers were detected in the sera
taken from these cats after the third immunization. At 8 mo postchallenge, VN antibody
titers persisted at low level in cat #PY4 and could no longer be detected in cat #QA6 and
#QH3. The low titer of VN antibodies detected in this group as compared to those detected
in infected control cats and the observed delay suggest that these responses were the result
of anamnestic responses to the challenge inoculum rather than active viral infection.
In vivo transfer study
As an additional means of analyzing the viral status of ALVAC-gag/prot
immunized cats after challenge, two naive SPF kittens were transfused with PBMC, BM
and LN cells isolated from cat #QX3 and #QQ1 at 8 months postchallenge. An additional
SPF kitten, which served as a positive control, was transfused with cells from FI V-infected
control cat #PY2. Following challenge, kittens were monitored for viral infection at
monthly intervals by methods analogous to those described previously. Kitten #DH2,
which was transfused with cells from FTVPet infected cat #PY2, became readily infected as
shown by RT, PCR (data not shown), and immunoblotting (Figure 2.9). In contrast,
kittens #DFL5 and #DE4 which were transfused with cells from ALM AC-gag/pro t
immunized cats, were negative for virus by RT and PCR up to 5 months after the
transfusion in peripheral blood, LN and BM tissues (data not shown). Further, these cats
failed to develop FIV-specific humoral responses as determined by immunoblotting (Figure
2.9).

Figure 2.9 FIV-specific immunoblot. Serum samples obtained before (pre) and 16 weeks (16pc) after transfusion from kittens #DH5 and
#DE4, transfused with cells from ALVAC-gag/prot immunized cats, and cat #DH2 transfused with cells from ALVAC-control
immunized cat, were diluted 1 to 100 in Buffer 3 and incubated with FIV-specific westemblot strips. The positive control was incubated
with pooled serum from FI V-infected cats. The negative control was incubated with serum from a SPF cat.

Cat #
DH2
cell
inoculum
PY2
pre 16pc
p24
DH5
DE4
QQ1
QX3 Controls
pre 16pc
pre 1 6pc +
oo
CO

84
Statistical analysis
The Fishers exact test was used to analyze the statistical significance of the
protective efficacy data (Table 2.11). The infectivity rate of the ALVAC-FIV recombinant
immunized groups was compared to that of the ALVAC (n=6) control group alone or to
that of the ALVAC control group combined with the ALVAC/ICV (n=3) immunized control
group (total n=9). The infectivity rate of AJJVAC-env.gag/protlCV immunized cats was
compared to either the ALVAC/ICV-immunized control group (n=3) or to both the
ALVAC-immunized and ALVAC/ICV-immunized control groups (n=9). Based on this
test, immunization schemes employing ALVAC-gag/prot and ALVAC-env,gag/prot
together with ICV, showed significant protection as indicated by a P (Probability) value
equal or less than 0.05 (Table 2.11). Protection observed by the other immunization
schemes was not significant (P value > 0.05).
Table 2.11 Statistical analysis
Viral status
vaccine control P value significant
group group
Vaccine +/- +/- (single-tailed)
ALVAC-env
3/3
4/2
0.5
no
3/3
7/2
0.28
no
ALVA C-gag/prot
0/6
4/2
0.0303
yes
0/6
7/2
0.00914
yes
ALVAC-env, gag/pro t
2/4
4/2
0.28
no
2/4
7/2
0.118
no
ALVAC-97TMG
3/3
4/2
0.5
no
3/3
7/2
0.28
no
ALVAC-env, gag/prot
0/3
3/0
0.05
yes
& ICV
0/3
7/2
0.00914
yes

85
Discussion
The aim of this study was to evaluate the immunogenicity and protective efficacy of
ALVAC-based FIV vaccines alone or in combination with ICV against experimental FIV
challenge in cats. ALVAC recombinants tested in this study included ALVAC constructs
encoding the FIV Env, the FIV Gag or both the FIV Env and Gag. Additionally, a
recombinant was tested which encoded both the FIV Gag and a modified Env in which a
putative immunosupressive region had been deleted.
We demonstrated that these recombinants were able to effectively infect non-
permissive feline cells and express the inserted FIV genes. Upon inoculation in cats,
ALVAC-specific humoral responses were readily detected. In contrast, FIV-specific
humoral responses were detected only in cats that received a booster immunization with
ICV. VN antibody titers were undetectable in all cats pnor to challenge, even in those
boosted with ICV. These observations are consistent with those of ALVAC-based HIV
vaccine candidates in macaques, chimpanzees and humans in which humoral responses
were weak or undetectable unless the animals received booster immunizations with subunit
proteins (Franchini etal. 1995a; Abimikuetal. 1995; Piaoloux et al. 1995; Clements et al.
1996). Also, the induction of VN antibodies required boosting with HIV Env or peptides
corresponding to the V3 region. Humoral responses including VN responses have been
detected in chimpanzees immunized with ALVAC-HI V-l recombinants alone (Van der Ryst
et al. 1996; Girard et al. 1996). However, these responses were detected after a minimum
of four immunizations only. Other FIV vaccine candidates composed of viral vectors
including an attenuated adenovirus and a herpesvirus engineered to express the FIV Env,
also failed to elicit FIV-specific humoral responses (Gonin et al. 1995; Verschoor et al.
1996).
The induction of humoral responses after the ICV boost is consistent with previous
studies in our laboratory in which ICV vaccines were found to elicit FIV-specific humoral

86
responses but failed to induce VN antibody responses after a single immunization.
Interestingly, cats immunized with ALVAC-env,gag/prot showed approximately a 10-fold
higher immunoblot antibody titer than cats immumzed with ALVAC vector alone after the
ICV boost. Therefore it is possible that ALVAC-env,gag/prot induced FIV-specific T-
helper responses resulting in a more efficient generation of FIV-specific humoral responses
after exposure to ICV. This has been reported in other studies in which chimpanzees were
immunized with an ALVAC recombinant encoding the HIV Env and boosted with
recombinant Env. Chimpanzees immunized with ALVAC-Env produced antibodies
following a single recombinant Env immunization, whereas chimpanzees immunized with
recombinant Env alone failed to developed such antibodies after a single immunization
(Girard etal. 1995). Similar priming of T-helper responses has also been shown in human
volunteers immunized with ALVAC-HIV candidate vaccines (Piaoloux et al. 1995;
Clements et al. 1996)(Tartaglia, personal communication)
The effectiveness of ALVAC-based vaccines in priming CTL responses as has been
reported previously was further confirmed by findings in this study (Cox et al. 1993).
Detectable levels of FIV-specific CTL responses were detected even after a single
immunization. Although, the phenotype of the effector cells was not determined it was
found that the effector cells reacted in a MHC-restricted manner. This finding excludes NK
cells as the effector cell and implies a role for CD8+ T-lymphocytes known to react in a
MHC class I restricted manner. However, we can not exclude a role for CD4+ CTL
responses since the target cells (autologous PBMC) used in the assay could have presented
FIV antigens in the context of both MHC class I and II. In an attempt to identify the F1V
epitope recognized by these effector cells, CTL assays were performed using autologous
target cells infected with vaccinia recombinants expressing FIV Env or Gag. These
experiments did not demonstrate presence of CTL activity (data not shown), possibly due
to technical difficulties of the CTL assay itself. The ability of ALVAC-based vaccines to
prime CTL responses has been reported in several studies. Mice immunized with an

87
ALVAC-recombinant encoding the HIV-1 Env were shown to elicit CTL responses,
including memory T-cell responses (Cox et al. 1993). The effector cells in these studies
were characterized as CD8+ T-lymphocytes. In addition, specific CTL responses mediated
by CD8+ T-lymphocytes were detected in 30% of the human volunteers immunized with an
ALVAC recombinant encoding the HIV-1 Env (Pialoux et al. 1995; Egan et al. 1995).
Interestingly, volunteers immunized with an ALVAC recombinant encoding both the HIV-1
Env and Gag, mounted CTL responses specific for Gag more often than for Env
(Lawrence et al. 1996). We were unable to distinguish if this was the case in our study.
In summary, ALVAC-based FIV vaccines were found to differ from inactivated
FIV-infected cell vaccines in their ability to elicit cell-mediated and humoral responses.
This can be explained, in part, by a difference in the processing and presentation of
ALVAC encoded immunogens. Immunogens encoded within ALVAC, require de novo
expression within host cells to be presented to the host immune system. In this study,
ALVAC immunizations were given intramuscularly and therefore it is likely that the
majority of ALVAC infected muscle cells. These cells are capable of presenting
immunogens in association with MHC class I and as such are expected to stimulate
primarily CTL responses. Additionally, part of the ALVAC inoculum may have been taken
up by macrophages or infected cells of the monocyte lineage, such as dendritic cells. These
cells are capable of presenting antigens in association with both MHC class I and class II
molecules and could therefore have stimulated the generation of both CTL and T-helper cell
responses. This is supported by the low level of proliferative responses detected in some
of the cats immunized with ALVAC-FIV recombinants alone. The direct stimulation of B-
cell responses by ALV AC-encoded immunogens would require the expression and release
of the immunogens from ALV AC-infected host cells. This process may have occurred at
low level only, since ALVAC-FIV recombinants failed to induce detectable levels of
humoral responses. Further, the nature of the immunogen itself may have played a role
since ALVAC-recombinants expressing epitopes of viral pathogens other than retroviruses

88
have been shown to effectively elicit humoral responses including VN antibodies to the
inserted immunogens (Taylor et al. 1992a, 1992b, 1991).
The protective efficacy ALVAC-based FIV vaccines was evaluated against 50 IDS0
FIVPet (subtype A). This isolate differs only slightly (3% in Env and 1% in the Gag amino-
acid coding region) from the FIVviU(. franche isolate (subtype A) used to generate the ALVAC
recombinants and is identical to the vaccine virus. After challenge, complete protection was
obtained in all cats immunized with the ALVAC-gag/prot recombinant. The protection
observed in this group correlated significantly with immunization as determined by the
Fishers exact test. In addition, virus was not detected in two naive SPF kittens transfused
with cells obtained eight months postchallenge from ALVA C-gag/prot immunized cats
whereas a kitten transfused with cells from an infected control cat became virus positive.
Full protection was also observed in cats immunized with ALVAC-env,gag/prot and
boosted with ICV. No significant protection was observed in cats immunized with
ALVAC-env, ALVAC-env,gag/prot, and ALVAC-9777VG as determined by the Fishers
exact test (R>0.05). Important to emphasize at this point is the fact that we failed to obtain
100% infectivity in the control group immunized with ALVAC vector alone. Two out of
six cats in this group tested virus negative by all entena throughout the duration of the
study. The infective dose (IDS0) of the virus challenge inoculum was titrated in cats
obtained from a vendor different than the one used in this study. Therefore the true titer of
the challenge inoculum may have been less than 50 IDS0 explaining the lack of 100%
infectivity in the control group. Further, lack of full infectivity could have been due to a
general elevation of immune function by the ALVAC vector itself, although, no indication
for such an elevation has been reported in any of the vaccine studies conducted with
ALVAC. The inability to obtain full infectivity of control animals has also been observed
in other FIV vaccine trials as well as SIV and HIV vaccine trials. In general, this is
attributed to differences in genetic background which causes some animals to be more
resistant to infection than others. For example, the HLA and HLA related genes have been

89
implemented in influencing the intensity and specificity of host immune-responses (Haynes
et al. 1996). Also of some relevance to this issue are recent studies in which individuals
with a homozygous defect in the CKR-5 loci encoding a coreceptor for HIV, were less
likely found to become infected with HIV than individuals without this defect (Liu et al.
1996). Similar genetic based mechanism(s), yet to be determined, may be behind the
observed variance in susceptibility to FIV infection in cats.
What constituted protective immunity in the ALVAC-gag/prot immunized cats is not
clear. At the time of challenge and after challenge, the sera of these cats tested negative for
Gag-specific and VN antibodies. Therefore, the presence of these antibodies did not
appear to be crucial to the protection observed in this group. Moreover, vaccine trials in
which cats were immunized with Gag proteins lacked protective efficacy despite the
presence of Gag-specific antibodies. In fact, in these studies immumzed cats showed
enhanced infection. Thus, even if these responses would have been present they may not
have played a role in the observed protection. However, we can not exclude a role for
antibodies directed against Gag epitopes other than those tested for in the immunoblot and
VN assays. Recent studies have demonstrated that HIV Gag proteins are displayed on the
surface of infected cells (Ikatu et al. 1989; Shang et al. 1991). Similarly, cells infected
with ALV AC-gag/prot may have expressed Gag on the host-cell surface and resulted in the
induction of Gag-specific antibody dependent cell-mediated cytotoxic responses (ADCC).
ADCC responses have been detected in HIV-infected individuals but these responses were
predominantly directed against Env epitopes and attempts to demonstrate Gag-specific
ADCC have failed thus far (Koup et al. 1988; OToole and Lowdell 1990). Protection in
the ALVAC-gag/prot, may have been accomplished through Gag specific CTL responses.
The induction of Gag-specific CTL responses has been reported in infected cats as well as
cats immumzed with ICV (Song et al. 1992; Flynn et al. 1995a). The importance of these
responses in terms of protection, however, is still unknown. A vaccine based on a
synthetic peptide containing epitopes of both the FIV Env and Gag was shown to

90
effectively induce Gag-specific CTL responses but failed to protect cats. No efficacy
studies have been undertaken in monkeys to assess protection of ALVAC recombinants
encoding Gag alone. Protocols in these models often involve priming and boosting with
multiple antigenic determinants, making it difficult to resolve which epitope(s) are crucial
for protection. However, there have been reports suggesting the importance of Gag
epitopes in vaccine trials against other retroviruses. Vaccine efficacy of a herpes
recombinant vector expressing FeLV Env was significantly enhanced with the inclusion of
the Gag (Wardley et al. 1992). Furthermore, mice immunized with a vaccinia recombinant
expressing the Gag of the Friends murine leukemia retrovirus (F-MuLV) were protected
against disease upon exposure to F-MuLV (Miyazawa et al. 1992).
Env-expressing ALVAC recombinants in our study lacked protective efficacy,
similar to adenovirus and herpesvirus vectored vaccines encoding FIV Env (Gonin et al.
1995; Verschoor et al. 1996). Similar to ALVAC-chv, these vector vaccines failed to
induce Env-specific humoral responses including VN antibodies. The evaluation of cell-
mediated responses was not included in these studies. In our study, ALVAC-e/zv
immunized cats were shown to elicit FIV-specific CTL responses. However, the presence
of these responses did not correlate with protection. Chimpanzees immunized with an
ALV AC-recombinant encoding the HIV-1 Env and boosted with recombinant Env (gpl60)
also failed to be protected. Lack of protection in these studies correlated with low VN
antibody titers, since, chimpanzees immunized simultaneously with recombinant Env
(gpl60) and boosted with V3 peptide were protected in the presence of high VN antibody
titers (Girard et al. 1995). Further, vaccine trials employing ICV and inactivated whole
FIV in cats indicate that Env-specific humoral responses, including VN antibodies may be
responsible for the observed protection (Yamamoto etal. 1991, 1993). Thus, the inability
to generate such responses by the ALVAC-cnv vaccine could explain the lack of protective
efficacy. Additionally, differences in the processing pathway, as discussed above, may

91
have resulted in immune responses against Env epitopes other than those generated upon
immunization with ICV and inactivated whole virus.
The lack of protective immunity in the group immunized with ALVAC-env,gag/prot
is somewhat surprising. It is possible that the inclusion of Env interfered with efficient
presentation of Gag and as a result failed to properly prime the immune system. Immune
responses directed against certain Env epitopes have been reported to cause enhancement of
infection and as such could have negated the protective efficacy (Hosie et al. 1992;
Siebelinket al. 1995; Osterhaus et al. 1996). This enhancement, however, was thought to
be mediated by VN antibodies. In our study, VN antibodies were not detected prior to
challenge. Further, virus isolation data did not show differences in the viral load among
infected cats in the ALVAC-env group and infected cats in the control group immunized
with ALVAC vector alone. Alternatively, a putative immunosupressive region in the
transmembrane portion of Env could have interfered with the development of protective
immune responses. However, cats immunized with the AUVAC-97TMG, a recombinant in
which this region had been removed were readily infected at a same ratio as the ALVAC-
env,gag/prot -immunized cats, implying that this region did not play a role. However, other
immunosupressive regions located elsewhere than in the Env transmembrane region could
have attributed to the reduction in immunogemcity of ALVAC-env,gag/prot recombinants.
Similar to our findings, ALVAC recombinants expressing both the Env and Gag of HIV-2
failed to protect cynomolgus monkeys from HIV-2 challenge (Biberfeld et al. 1994). The
challenge in this trial, however, was given by the mucosal route. Opposing our findings are
studies in the chimpanzee model. Chimpanzees immunized with ALVAC recombinants
expressing HIV-1 Env and Gag were protected from homologous HIV challenge (Van der
Ryst et al. 1996; Girard et al. 1996). These animals, however, exhibited HIV-1-specific
humoral responses including VN antibodies prior to challenge. Therefore, it would be of
interest to determine if a higher dose of ALVAC-env,gag/prot or an increased number of
immunizations could elicit such antibody responses in cats and enhance protective efficacy.

92
Interestingly, cats immunized with ALVAC-env,gag/prot but boosted with ICV
were protected. The difference between this group and the ALVAC-env,gag/prot
immunized group was the presence of FIV-specific humoral responses (antibodies detected
by FIV immunoblot analysis) at the time of challenge. Control cats immunized with
ALVAC vector alone and boosted once with ICV became infected despite the presence of
FIV-specific antibody responses. The antibody titers in these control cats, however, were
10-fold lower than those detected in the ALV AC-env,gag/prot-pnmed cats. Based on this
it could be speculated that priming with ALVAC-env,gag/prot enhanced the
immunogenicity of ICV in a such a manner that protective immunity could be elicited after a
single ICV immunization rather than after the three to four immunizations that are usually
required to obtain protection. Interestingly, the protected cats in this group displayed low
VN antibody titers shortly after challenge. These titers (5-20) differed significantly from
those detected in infected control cats (>100). Therefore, VN antibody production may
have been the result of anamnestic responses to the challenge inoculum. This is supported
by the fact that VN antibodies in two of three cats could no longer be detected at 8 mo post
challenge. In the case of active infection, an increase rather than a decrease in VN antibody
titers, is expected. The low level VN titer detected at 8 mo postchallenge in cat #PY4
presented a dilemma as this may have been the result of low level viral infection
undetectable by RT and PCR analysis. Alternatively, this may have been the result of
strong immunity that persisted which is more likely since this cat tested persistently
negative by all other criteria up to one year postchallenge. Moreover, this cat was not able
to resist supennfection with another FIV isolate (see chapter III) whereas two infected
control cats resisted superinfection with this same FIV isolate. The generation of VN
responses upon challenge in this group resembles observations in macaques which had
been immunized with ALVAC recombinants encoding HIV-2 Env and Gag (Franchim et al.
1995a). After challenge, protected animals tested negative for virus but developed
significant levels of viral neutralizing antibodies. Furthermore, the protective efficacy

93
obtained with ALVAC recombinants, in combination with protein based booster
immunizations is similar to that observed in other animal models. Cynomolgus monkeys
immunized with ALVAC-env,gag/prot (HIV-2) alone failed to be protected whereas those
immunized with ALVAC-env,gag/prot and boosted with recombinant Env were partially
protected from HIV-2 challenge (Biberfeld et al. 1994).
In summary, protective immunity can be obtained with ALVAC-based FIV vaccines
encoding Gag or ALVAC-based FIV vaccines encoding Env and Gag in combination with
ICV. This is the first study to report the induction of protective immunity against
experimental FIV challenge in cats utilizing a viral vector-based vaccine. Protection
obtained with ALVAC-based vaccines encoding Gag may have occurred via cell-mediated
responses including CTL and ADCC, or other mechanisms. Protection in ALVAC-
env,gag/prot and ICV immunized cats may have been mediated by these same mechanisms.
Additionally, humoral responses including low but significant VN antibody titers may have
contributed to the protection observed in this group.

CHAPTER III
EFFICACY EVALUATION OF CANARYPOXVIRUS (ALVAC)- BASED FIV
VACCINE COMBINED WITH INACTIVATED FI V-CELL VACCINE AGAINST
HETEROLOGOUS FIV CHALLENGE IN CATS
Introduction
Based on genetic variation in the Env and Gag coding regions, HIV isolates
obtained worldwide have been classified into several subtypes or clades (Cheingsong-
Popov etal. 1994; Brodineetal. 1995). Optimally, a vaccine against HIV should induce
immune responses that can cross react with a wide variety of these HIV subtypes. Thus
far, HIV vaccine trials have concentrated mainly on a single subtype, subtype B, as this
represents the predominant type found in Europe and the United States. However, the
emergence of HIV isolates other than subtype B is increasing significantly. Furthermore,
90% of the reported HIV cases are in the developing countries where the HIV epidemic
encompasses multiple subtypes. These countries in particular would benefit from the
development of broad spectrum vaccines since drug treatments, even if they would
become available, would be too expensive.
Similar to HIV, FIV strains have been classified into different subtypes (A-D),
based on aa sequence differences in Env and to a lesser extent in the Gag (Sodora et al.
1994; Kakinuma et al. 1995; Rigby et al. 1993) (Figure 3.1). As such, the FIV model
provides a means to assess the protective efficacy of vaccine strategies against multiple
HIV subtypes. Vaccine protection against homologous and slightly heterologous FIV
strains (within one subtype) has been achieved with inactivated whole virus and
inactivated FIV-infected cell vaccines (ICV) (Yamamoto et al. 1991, 1993; Verschoor et
al. 1995; Johnson et al. 1994; Hosie et al. 1995). These same vaccines,
94

95
however, failed to induce protective immunity against distinctly heterologous FV strains
from heterologous subtypes (Yamamoto et al. 1993; Johnson et al. 1994; Hosie et al.
1995). Thus, a modified or different vaccine approach is required to induce immune
responses that will cross-react with a wide variety of FIV subtypes.
Cross-protective immunity has been obtained against heterologous HIV-1
infection in chimpanzees using vaccines composed of whole Env or Env fragments from
multiple isolates (Girard et al. 1995). However, the use of viral vector-based vaccines
may overcome the need for the inclusion of antigenic determinants from multiple isolates.
Viral vector-based vaccines have been shown to effectively prime cell-mediated responses
whereas conventional vaccines have been shown to prime predominantly humoral
responses. The epitopes recognized by cell-mediated responses may be directed against
epitopes that are more conserved among different isolates and as such provide protection
against a wider variety of HIV isolates. In fact, preliminary findings from vaccine trials
in which macaques were immunized with a canarypoxvirus (ALVAC) vectored HIV-1
vaccines suggest that this may be the case (Abimiku et al. 1995). After immunization
these macaques were partially protected from infection with a distinctly heterologous
HIV-2 isolate.
In the previous chapter, we demonstrated that cats immunized with ALVAC-
recombinants encoding FIV Env and Gag {ALV AC-env, gag/prot) and boosted with a
conventional inactivated FIV-infected cell vaccine (ICV), resisted infection w'ith FIV
Petaluma (FIVPet), a subtype A isolate. This isolate was closely related to the isolate used
to generate the ALVAC-FIV recombinant (FIVviUe franche, subtype A) and identical to the
FIV isolate used to generate the ICV vaccine. In this study, we evaluated if these
ALV AC-env, gag/prot/lCV immunized cats could be protected from a second challenge
with a distinctly heterologous FIV isolate, FIV Bangston (FIVBang). The FIVBang isolate is
classified as a subtype B virus and differs from the HVPet isolate (subtype A) by 21% in
the Env and 2.4% in the Gag amino-acid sequence.

Figure 3.1. Phylogenetic relationship between FIV-isolates comparing envelope sequences. The four subtypes (A-D) are grouped with
circles.
5% diversity

97
Materials and Methods
Animals and grouping
Eight cats were used in this study including three ALV AC-env,gag/prot/\CV
immunized cats (#PY4, #QH3, #QA6) and two FIVPet infected control cats (#PY2 and
#QE4) (see Chapter II, Materials and Methods). Also included were three age matched
SPF cats (#EJ2, #DH3, #GU5), purchased from Liberty Research Inc., which received
no immunizations prior to the FI VBang challenge.
Challenge inoculum
The challenge inoculum consisted of cell-free culture fluid from PBMC infected
with FIVBang previously titered in SPF cats. The challenge inoculum of 75 IDS0 was given
i.p. 8 months after the initial FI VPet challenge.
Viral status monitoring
Viral infection was monitored by RT activity and FIV-specific PCR and by
evaluating the level of FIV-specific humoral responses, including viral neutralizing (VN)
antibody responses to both the FIVPet and FIVBang isolate, as described previous (see
Chapter II, Materials and Methods).
DNA sequencing
Nucleotide sequencing of the amplified PCR products was performed as described
previously using FIV specific primers: FIVPct (5-TAGTAGTTATAGTGGTACTA-3) and
FIVBang (5-GGGACTACTAGCAATGGAATA-3) (see Chapter II, Materials and Methods).

98
Results
In this study, ALVAC-env,gag/prot/lCV immunized cats, which were previously
shown to resist challenge with FIVPet a homologous subtype strain (subtype A), were
given a second challenge with 75 IDS0 FIVBang, a subtype B virus. Additionally, two
FlVPet-infected control cats were challenged with FIVBang to determine if active infection
could prevent superinfection with the FIVBang isolate. As presented in Table 3.1, all
nonimmunized/noninfected control cats became readily viremic after FI VBang challenge. In
contrast, ALV AC-env, gag!protHCV immunized cat #QA6 remained virus negative as
determined by virus isolation (RT) and PCR in peripheral blood up to four months
postchallenge. Cats #PY4 and #QH3 remained virus negative as determined by virus
isolation (RT) in peripheral blood but tested positive by PCR at three months and four
months postchallenge, respectively. Nucleotide sequence analysis of the amplified PCR
product from PBMC revealed HVBang-specific sequences only (data not shown). The two
FIVPct -infected control cats (#QE4 and #PY2) tested positive by RT and PCR before and
after the FlVBang challenge. However, nucleotide sequence analysis of the FlV-specific
PCR products obtained from these cats only verified FlVPet-specific sequences up to 4
months after the FIVBang challenge (data not shown).
Prior to challenge, FIV-specific antibodies, as determined by immunoblot, were
present in all ALVAC-env,gag/prot/lCV immunized cats. Similarly, sera from FIVPet -
infected control cats (#PY2 and #QE4) contained FIV-specific antibodies which were
slightly higher in titer than those detected in the ALV AC-env,gag/prot/\CV immunized
group (Table3.1). As expected, sera from the naive control cats (#EJ2, #DH3, #GU5)
did not contain detectable levels of FIV-specific antibodies before the FIVBang challenge
(Table 3.1). Viral neutralizing antibodies to either FIVPet or FIVBang were absent in all
naive control cats and in two of three ALV AC-env,gag/protflCV-imm\m7£d cats (#QA6,
#QH3). In contrast, sera of the FlVPet-infected control cats (#PY2 and #QE4) had high

Table 3.1 Immune parameters and viral status before and after FIVBang challenge.
virus status
Cat# Vaccine pre-2nd-challengea post-2nd-challengeb RT/PCR
WB VNPet VNBang WB VNPel VnBang (wks p.c.)
2 6 10 14 18
QA6
ALVAC-env,gag/prot & ICV
+(4-5)
<5
<5
+
5-20
<5
-/-
-/-
-/-
-/-
-/-
PY4
ALVAC-env,gag/prot & ICV
+(4-5)
5-20
5-20
+
5-20
<5
-/-
-/-
-/-
+7+
-/+
QH3
ALVAC-env,gag/prot & ICV
+(4)
<5
<5
+
5-20
<5
-/-
-/-
-/-
-/-
-/+
EJ2
_
_
ND
<5
+
ND
<5
-/-
+/+
+/+
+/+
NT
GU5
-
-
ND
<5
+
ND
<5
-/-
+/+
+/+
+/+
NT
DH3
-
-
ND
<5
+
ND
>100
-/-
+/+
+/+
+/+
+/+
PY2
ALVAC
+(5-6)
>100
<5
+
>100
<5
+/+
-/-
+/+
+/+
-/+
QE4
ALVAC&ICV
+(5-6)
>100
<5
+
>100
<5
+/+
-/+ +/+
+/+
+/+
a serum samples taken at the day of challenge.
b serum samples taken 3-4 months post-2nd-challenge.
c weakly positive by RT (2X background cpm)
ND= not determined
NT= not tested, animal euthanized.

100
VN antibody titers (>100). More importantly, these responses were specific for FlVPet
and did not cross-neutralize FIVBang in vitro. Interestingly, serum taken before the FI VBang
challenge from cat #PY4 contained low level VN antibodies(<20) which neutralized both
FlVPet and FIVBang in vitro.
After challenge, all naive control cats developed FIV-specific antibodies as
determined by immunoblotting. In contrast, FlVBang-specific VN antibodies were detected
in only one of the three cats (#DH3). Interestingly, all three cats immunized with
ALV AC-env,gag/protHCV developed low level of VN antibodies to FIVPet similar to that
observed after the initial FI VPet challenge but failed to develop VN antibodies specific for
FIVBang. Similarly, FIVPet-infected control cats failed to develop FIVBang-specific VN
antibodies after challenge whereas FIVPet-specific antibodies persisted at high titers
(>100).
Discussion
In this study, cats immunized with both ALV AC -env,gag/prot and ICV were
partially protected from heterologous FIV subtype challenge given eight months after an
initial homologous FIV challenge. These data should be interpreted with some caution as
they reflect only a 4 month period postchallenge. Nevertheless, immunized cats showed a
delay in infection, as all control cats became viremic within 6 weeks postchallenge
whereas two of the immunized cats became positive based on PCR analysis only at 14
and 18 weeks postchallenge, respectively. Furthermore, throughout the study these cats
were negative for virus isolation, demonstrating a significant reduction in viral load.
The exact immune-mechamsm(s) responsible for the delay in infection or the
reduction in viral load in the ALV AC-env,gag/prot/lCV immunized cats is unclear. It is
possible that FIV-specific humoral responses present prior to the FIVBang challenge played
a role. Both the ALV AC-env, gag!protHCV immunized cats and FIVPet-infected control

101
cats had high titer of FIV-specific humoral responses prior to challenge whereas the naive
control cats lacked such responses. On the other hand, ICV vaccines capable of eliciting
high titers of FIV-specific antibody responses were effective against homologous and
slightly heterologous challenges but not against distinctly heterologous FIV isolates
(Yamamotoet al. 1991, 1993; Johnson et al. 1994). It is possible that the presentation of
both exogenous and endogenous FIV antigens in the ALVAC-env,gag/protHCV
immunized cats resulted in the generation of higher titers of antibody responses or
antibodies to a wide variety of FIV epitopes including those shared by FIVPet and FIVBang
isolates. Similarly, active FlVPet infection in the infected control cats could have
broadened humoral responses, resulting in resistance to superinfection. This is consistent
with results from a previous study in which a long-term FIVBang-infected cat was
protected from FIVPet infection in the presence of high titer FIV-specific antibody
responses. Likewise, cross-protection by infection with HIV-2 in high risk woman has
been found to correlated with resistance to infection with HIV-1 which is genetically
highly divergent from HIV-2 (Kanki et al. 1995). Additionally, monkeys infected with
an attenuated macrophage tropic SIV strain were resistant to superinfection with a highly
virulent SIV strain displaying 16% difference in the Env amino-acid sequence (Clements
et al. 1995). In these studies, the length of infection and the concomitant broadening of
humoral immune responses positively correlated with protection. Moreover, naive
monkeys passively immumzed with serum from these long-term infected monkeys were
partially protected from subsequent heterologous challenge. The broadening of humoral
responses in these monkeys, however, also included the broadening of VN antibody
responses capable of cross-reacting with the challenge inoculum virus. In contrast,
chimpanzees immunized with recombinant Env and V3 peptides corresponding to two
HIV-1 isolates, were protected from heterologous subtype challenge in the presence of
VN antibodies that neutralized vaccine strains but not the challenge strain (Girard et al.
1995).

102
In our studies, cross-reactive VN antibody responses may not have played a key
role in vaccine protection. Poor to the FIVBang challenge, VN antibody titers were
detected in both FIVPct-infected control cats and in the ALV AC-env,gag/prot/lCV-
immunized cat #PY4. Except for the low VN antibody titer detected in cat #PY4, these
VN antibody responses were specific for FIVPet and did not cross-react with FIVBang
Surprisingly, cat #PY4 was the first one of the ALV AC-env,gag/protllCV immunized
group to become positive by PCR analysis, suggesting that the presence of FIVBang-
specific VN antibodies prior to challenge was not beneficial. Again, this resembles the
findings from supennfection studies in which FIVBang-infected cats which developed
FIVBang-specific antibody responses were susceptible to superinfection FlVPel whereas
those that lacked FTVBang-specific VN antibodies resisted superinfection (Okada et al.
1994). Furthermore, cats #QA6 and #QH3 showed a delay in infection (i.e. partial
protection) without detectable VN antibodies at the time of challenge, suggesting that
these antibodies were not responsible for this delay. However, the low titers of VN
antibodies detected alter challenge, similar to those observed after the initial FIVPet
challenge may have contributed, although the mechanisms behind this remains to be
identified.
In addition to humoral immunity, FIV-specific CTL activity may have contributed
to the delay in infection and/or reduction in viral load. In the previous chapter, the
presence of FlVPet-specific CTL activity was reported in the ALVAC-env,gag/prot /ICV
immunized cats. Unfortunately, cross reactivity to FIVBang was not evaluated due to
limited amount of PBMC that could be harvested from these cats. However, the
generation of cross-reactive CTL responses against FIVBang has been detected in cats long
term infected with the FlVPet (unpublished data). Thus, ALV AC-env, gag/prot and ICV
immunizations could have elicited similar cross-reactive CTL responses. These
responses may have been directed against Gag epitopes in particular, since the amino acid
sequence difference between the FIVPet and FIVBang Gag protein is significantly less

103
(approx. 2.4 %) than the difference displayed at the Env (approx. 21 %). These findings
resemble those of ALVAC-HIV-1 trials in macaques (Abimiku et al. 1995). Macaques
immunized with ALVAC-HIV-1 recombinants and boosted with subumt proteins were
partially protected and had a delay in infection after challenge with distinctly heterologous
HIV-2. Prior to challenge these macaques exhibited HIV-1 specific cell-mediated
responses and VN responses that effectively prevented HIV-1 infection but failed to
prevent HIV-2 infection in vitro.
In summary, prime/boost protocols involving priming with ALVAC-HV
recombinants followed by boosting with inactivated FIV-infected cells vaccines can elicit
partial protection or delay in infection of distinctly heterologous FIV isolates from
heterologous subtypes. The exact immune-correlates of protection are unclear. Although,
the findings in our study suggest a role for both cell mediated and humoral responses.

CHAPTER IV
SUMMARY AND FUTURE STUDIES
Synopsis
Comparative studies on the efficacy and immunogemcity of various ALVAC-based
FIV vaccines alone or in combination with inactivated FIV-infected cell vaccines support
the following conclusions:
(1) ALVAC-based FIV vaccines are ineffective in priming B-cell responses but
effective in priming cytotoxic T-cell responses and low level T-helper responses specific
for the inserted FIV antigens.
(2) A booster immunization with ICV following immunization with ALVAC-based
FIV vaccines enhances immunogemcity as determined by induction of detectable FIV-
specific humoral responses.
(3) ALVAC-based FIV vaccines encoding the FIV Gag and Prot can induce
protective immunity against experimental challenge with slightly heterologous FIV isolates.
(4) ALVAC-based FIV vaccines combined with ICV can induce protective
immunity against a slightly heterologous FIV isolate and delay infection with distinctly
heterologous FIV isolates of other subtypes.
The data obtained in this study were generated from a relatively small study group.
Future studies, should include larger numbers of animals to add to the statistical
significance of the obtained data. Further, the duration of the ALVAC-FIV-induced
protective immunity and the efficacy against various routes of challenge, in particular the
104

105
mucosal route, should be evaluated. In addition, the protective efficacy should be
examined against FIV field isolates, higher challenge doses and other types of challenge
inocula, such as cell-associated and plasma-derived virus. Moreover, studies should be
undertaken to further assess the protective efficacy against FIV strains of heterologous
subtypes. It will be particularly interesting to evaluate if cats immunized with ALVAC-
gag/prot alone can resist heterologous subtype challenge since sequence variability in the
gag gene is significantly lower between different FIV isolates when compared to the
variability observed in the env gene. Therefore, protective immune responses directed
against Gag epitopes may cross-react with a wider range of FIV isolates. If such protection
can not be achieved, ALVAC recombinants encoding Gag of multiple FIV isolates could be
tested for their ability to elicit broad-range protective immunity.
Furthermore, it would be of interest to evaluate immunization protocols involving
priming with ALVAC-gag/prot in combination with inactivated FIV-infected cell vaccine.
This may be a more optimal combination than ALVAC-env,gag/prot in combination with
ICV as ALVAC-env,gag/prot alone lacked protective efficacy.
Although we were able to demonstrate protective immunity upon immumzation with
ALVAC-based FIV vaccines, the mechamsm(s) involved in the observed protection
remains unclear. This important issue needs to be addressed in future studies. To further
delineate the role of cell mediated and humoral responses naive cats could be passively
immunized with serum or cells from ALVAC-FIV immunized cats and subsequently tested
for their ability to resist experimental FIV infection. Further, efforts should be made to
improve the current methods used to assess the induction of cell-mediated responses in
particular CTL responses. At present, these assays require large numbers of cells and
prolonged in vitro culturing which may result in inaccurate representation of what actually
occurs in vivo. Since our study implies a role for Gag in protection the next step would be

106
to evaluate which of the Gag proteins, CA, NC or MA are required to induce protective
immunity. Particular attention should be paid to the Gag pl7 protein as vaccine studies
involving HIV-1 pl7 Gag protein have demonstrated both the induction of CTL, T-help
and VN antibody responses. Moreover, SCID mice transfused with PBMC from human
volunteers immunized with a synthetic pl7 were reported to be protected from HIV-1
challenge whereas control mice transfused with cells from nonimmunized subjects became
infected (Goldstein et al. 1993).
Overall, these studies should add to our understanding of the interactions between
lentiviral pathogens and the host immune system and aid in the development of safe and
effective vaccines against FIV as well as HIV. Additionally, these studies may aid in the
development of vaccines against other pathogens that display continuous antigenic variation
and pathogens for which current vaccine approaches have failed.

107
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BIOGRAPHICAL SKETCH
Marinka Tellier was born in the summer of 1968 and grew up on the islands
surrounded by the salty waters of the Noordzee and the Schelde in the Netherlands. At
the age of 4 she was first sent to school by her parents not knowing that this episode would
continue for the next 24 years of her life. In the first eight she visited the Prinsenhoven
School in Middelburg. She then went to the Christelijke Scholengemeenschap Walcheren
from which she graduated in 1986. She continued her education at the College of
Pharmacy at the State University of Utrecht in the Netherlands. After enjoying student life
for a period of five years she graduated with a M.S. degree in pharmacy in 1991.
In the summer of 1993, she started her graduate work under the supervision of Dr.
Yamamoto with no experience in the feline field. In fact, at the time she did not even
realize there was such a thing as feline immunodeficiency virus. In the last three years she
has become impressed by this little creature that encompasses a little more than 9000
basepairs. Now, a little wiser and a little more confused, she will go on and keep on
wondering if our curiosity will do to us what it did to the cat.
121




41
by scraping and washed three times in phosphate-buffered saline (PBS). DNA was
extracted from the cells by resuspending in lysis buffer consisting of 0.021 M Tris (pH
7.5), 0.029 MEDTA (pH 8.0), 0.1 MNaCl, 1% SDS and proteinase K (10 mg/ml). Cells
in lysis buffer were incubated at 56C for 3-4 h followed by a 20 min incubation at 95C to
inactivate Proteinase K activity. The presence of FIV-specific DNA encoded by the
ALVAC-recombinants in the obtained DNA samples was determined by PCR using FIV
env- and gag-specific primers [FIV env specific primers 5-GAAATGTATAATATTGCTGG- 3
and 5 -GAATTGATTITGATTACATCC- 3; 5 -GGTAGGAGAGATTCTACA- 3 and 5-
CTGCATTCTTGCTGGTGC- 3 FIV gag specific primers]. PCR reactions were carried out in
a final volume of 50 pi containing: 1.0 pg DNA, 50 mM KC1, 10 mM Tris-HCl (pH 8.3),
2.5 mM MgCl2, 0.2 mM of each dNTP (dATP, dCTP, dTTP and dGTP), 20 pmol of each
primer, and 2.5 units of Taq DNA polymerase. The reaction mixtures were incubated for 5
min at 94C and cycled 30 times through 1 min at 94C, 1 min at 55C, 1.5 min at 72C,
followed by 10 min incubation at 72C. Finally, the PCR reaction products were separated
by electrophoresis on a 1% agarose gel and visualized by staining with ethidium bromide.
Detection of FIV-specific mRNA transcripts by RT-PCR
The expression of messenger RNA corresponding to the FIV genes encoded within
the ALVAC-recombinants was evaluated in non-permissive CrFK cells. Monolayers of
CrFK cells were inoculated with ALVAC-FIV recombinants or ALVAC vector alone as
described above. At 48 h postinoculation, messenger RNA was isolated using the Micro-
Fast track mRNA isolation kit (Invitrogen, San Diego, CA). The isolated mRNA was then
reverse transcribed into cDNA. Briefly, 1 pg of isolated mRNA was mixed with 7 pi
DEPC-treated H20 and 2 pi pdN6 and incubated at 65C for 5 min. Subsequently, the
samples were mixed with 4 pi 5X buffer, 1.3 pi DTT, 0.7 pi RNase inhibitor (40 U/pl), 1
pi dNTPs (10 mMdATP, dGTP, dTTP, dGTP), 2.0 pi acetylated BSA and 2 pi M-MLV


27
DNA replication and mRNA synthesis, and are unique in the fact that they replicate within
the cytoplasmic compartment of infected cells (Moss 1990).
The use of poxviruses as vaccine vectors was preceded by advances in the field of
molecular biology that allowed the manipulation of viruses in such away that foreign genes
could be inserted and expressed (Piccini et al. 1987; Perkus et al. 1989). Poxviruses have
since become candidate vaccine-vectors for a wide variety of pathogens (Perkus et al.
1995). In comparison to other candidate vector-viruses, poxviruses are exceptionally well-
suited due to their physical stability, low production costs, and ease of administration.
Furthermore, poxviruses have large genomes that allow the insertion of multiple genes.
The most widely used member of the poxvirus family is the vaccinia virus,
prototype of the Orthopoxviruses (Esposito 1991). This virus has been engineered to
express antigens of bacterial and viral pathogens and shown to induce protective immunity
in vivo (Perkus et al. 1995). Immune-responses induced upon inoculation with vaccinia
based vaccines include both humoral responses and cell mediated responses directed
against the inserted foreign antigens. There are however some concerns about the safety of
vaccinia when used in a large population. As vaccinia exhibits a broad host-range there is a
potential risk of spread to the general environment. Moreover, vaccinia has been shown to
cause disseminated infection in immuno-compromised people (Fulgitini et al. 1968).
For these reasons, the development of poxviruses as vector vaccines has been
extended to attenuated poxviruses and poxviruses with a more restricted host-range. One
such example is the NY VAC vector. This vector was derived from the Copenhagen
vaccinia strain by the selective deletion of 18 open reading frames, encoding genes
involved in host-specificity and virulence (Tartaglia et al. 1992). These deletions resulted
in a virus which replication is highly impaired on cell lines from several species including
human cells. Furthermore, this virus lacks virulence in immuno-compromised animal
models. The MVA vaccinia strain is another example of an attenuated vector strain used in
vaccine development. This strain was derived by extensive passaging of the Ankara


Figure 2.7 ALVAC-specific immunoblot. Serum samples taken from ALVAC-control immunized cat #PY2 and ALVAC-env,gag/prot
immunized cat #QS4 prior to immunization (pre-) and after the first, second and third immunization and 17 weeks postchallenge (17pc)
were diluted 1:100 in Buffer 3 and incubated with ALVAC-specific westemblot strips. Negative controls: pooled sera from FIV-infected
cats and pooled sera from FIV-negative SPF cats.


115
Morikawa, S. and Bishop, D.H.L. 1992. Identification and analysis of the gag-pol
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comparison of inhibitor sensitivities of reverse transcriptases from feline and human
immunodeficiency virus. Antimicrob. Agents Chemother. 34, 1505-1507.
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model for reverse transcriptase-targeted chemotherapy for acquired immunodeficiency
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Nakai, M., Honjo, S., andHayami, M. 1988. Isolation of simian immunodeficiency
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Retroviruses 10, 1739-1746.


81
blood and lymphoid tissues throughout the vaccine trial (Table 2.9a and 2.10). FIV-
specific humoral responses elicited by vaccination remained following challenge but slowly
declined thereafter. Interestingly, serum samples obtained three months postchallenge
contained low VN antibody titers (5-20) whereas no VN titers were detected in the sera
taken from these cats after the third immunization. At 8 mo postchallenge, VN antibody
titers persisted at low level in cat #PY4 and could no longer be detected in cat #QA6 and
#QH3. The low titer of VN antibodies detected in this group as compared to those detected
in infected control cats and the observed delay suggest that these responses were the result
of anamnestic responses to the challenge inoculum rather than active viral infection.
In vivo transfer study
As an additional means of analyzing the viral status of ALVAC-gag/prot
immunized cats after challenge, two naive SPF kittens were transfused with PBMC, BM
and LN cells isolated from cat #QX3 and #QQ1 at 8 months postchallenge. An additional
SPF kitten, which served as a positive control, was transfused with cells from FI V-infected
control cat #PY2. Following challenge, kittens were monitored for viral infection at
monthly intervals by methods analogous to those described previously. Kitten #DH2,
which was transfused with cells from FTVPet infected cat #PY2, became readily infected as
shown by RT, PCR (data not shown), and immunoblotting (Figure 2.9). In contrast,
kittens #DFL5 and #DE4 which were transfused with cells from ALM AC-gag/pro t
immunized cats, were negative for virus by RT and PCR up to 5 months after the
transfusion in peripheral blood, LN and BM tissues (data not shown). Further, these cats
failed to develop FIV-specific humoral responses as determined by immunoblotting (Figure
2.9).


42
reverse transcriptase (Superscript RT 200 U/pl, Rnase H, Gibco BRL, Gaithersburg, MD)
and incubated at 42C for 1 h, followed by 10 min at 95C. The generated cDNA served as
a template for PCR reactions using env and gag specific primers. PCR was performed as
described above.
Indirect immunofluorescence
Protein expression of the ALVAC encoded FI V env and gag genes was analyzed by
indirect immunofluorescence on CrFK cells inoculated with ALVAC-FIV recombinants.
CrFK were seeded at a density of 5x10s cells per 35mm2 dish on sterile glass coverslips
and infected at a m.o.i. of 10 with ALVAC-FIV recombinants or the ALVAC vector alone.
Indirect immunofluorescence was performed at 48 h postinoculation. Cells were fixed in
4% paraformaldehyde for 10 min, washed in PBS, and permeabilized in PBS containing
0.2% Triton X-100. Cells were then incubated with pooled FIV-positive serum for 30
min, washed, and incubated with FITC-labeled anti-cat IgG. All serum and antibody
dilutions were made in PBS containing 3% bovine serum albumin (BSA). Finally, cells
were washed and counterstained with Evans Blue (0.5% in PBS) for 10 min, observed
under a microscope and photographed.
ALVAC vaccine production and titering
Both ALVAC vector and ALVAC-FIV recombinants were amplified on permissive
primary chicken embryo fibroblast (CEF) and titered in vitro by measuring the number of
plaque forming units (PFU). Vaccines were produced from clarified lysates of infected
CEF cells in serum free medium and aliquoted at IX108 PFU per dose.


57
Cell
primers
lane M
FI4 CRFK
FIV+ Alvac-
Ctrl. Env Gag
E G E G E G
1 2 3 4 5 6
Figure 2.4 RT-PCR on mRNA extracted from CrFK inoculated with ALVAC-
recombinants. Photograph of PCR products following electrophoresis on a 1.0 % agarose
gel in Tris acetate-buffer (40mM Tris-acetate, 1 mM EDTA, pH 7.6). Lane M, X DNA
EcoRUHindUI marker (lpg/lane). RT-PCR on mRNA extracted from F1V+ FL-4 cells
shows specific amplification of FlV-env (Lane 1) and FIV-gag (Lane 2). RT-PCR on
mRNA extracted from CrFK cells infected with ALVAC-control show no amplification of
FlV-env (Lane 3) or FIV-gag (Lane 4). Lane 5, FlV-^nv specific RT-PCR on mRNA
extracted from CrFK cells infected with ALVAC-cnv. Lane 6, FI V-gag specific RT-PCR
on mRNA from CrFK cells infected with ALVAC-gag/prot.


72
T-helper Lymphoproliferative Responses
Selected cats from each group were tested after the second and third immunization
and challenge for lymphocyte proliferation in response to inactivated FIV (Table 2.6). The
inactivated FIV preparation used in this assay, included significant amounts of Gag
allowing the detection of proliferative responses to both FIV Env and Gag. After two
immunizations, no significant levels of FIV-specific lymphoproliferative responses were
detected in any of the tested cats. After the third immunization lymphoproliferative
responses were detected in two cats. However, the observed levels were low as compared
to those detected in cats immunized multiple times with ICV vaccines (S.I. 4-6). This
included cat #QC3 immunized with ALVAC-env,gag/prot and cat #PY4 immumzed with
ALVAC-env,gag/prot and boosted with ICV. Upon challenge, lymphoproliferative
responses were absent in all of the tested cats even in cats that became viremic except for
cat #QX4 which showed low levels of FIV-specific proliferative responses (Table 2.6).
Cytotoxic T-cell responses
Since viral-vector based vaccines are thought in general to be effective in eliciting
CTL responses, FIV-specific CTL responses were measured after each immunization in the
peripheral blood of selected cats. PBMC isolated were cultured in the presence of FIV
antigen-presenting cells and assayed for their ability to lyse autologous PBMCs infected
with FI VPet. FIV-specific CTL activity was detected in one of two cats tested after a single
immunization with ALVAC-env,gag/prot (see Table 2.7). After the second and third
immunization, CTL activity was detected in some cats of each group immunized with
ALVAC-FIV recombinants alone and those immunized with ALVAC-env,gag/prot and
boosted with ICV. No major variance between the different immunization schemes, with
respect to intensity of CTL activity or percentage of cats displaying CTL activity within


Immunoblot FIV
Cat # QC3
Vaccine Alvac-Env,Gag/pro(3X)
QA6
Alvac-Env,Gag/pro(2X)
ICV(IX)
pre 1 st 2nd 3rd
pre 1st 2nd 3rd
QC5
Alvac-control(2X)
ICV(IX)
pre 1st 2nd 3rd


A.
H6 aTC

env 2571 bp (857 aa)
TAA

B.
13 L ATC gag/prot 1 71 Obp (570 aa)
/
TAA
r
c.
H6 ATC env 2571 bp
/'
TAA I3L ATC gag/prot 171 Obp TAA
{'///////A (
D.
TAA
t
gag/prot 1 71 Obp
GTA I 3 L H 6 ATC
Aen v 1857bp (61 9 aa) TAA
A
is
1
ALVAC-env
A LV AC-gag/prot
ALVAC -env,gag/prot
ALVAC-97TMG
Figure 2.1 Schematic representation of ALVAC constructs


4
In addition to the primate lentiviruses, a virus similar in morphology and genetic
composition, the bovine immunodeficiency virus (BIV), was isolated from cows in 1985.
Although, the pathogenesis of BIV is poorly defined, infection in calves has been
associated with lymphocytosis and lymphadenopathy (Gonda et al. 1987, 1994).
The feline homologue of HIV, feline immunodeficiency virus (FIV) was first
isolated in 1986 from a cattery in California (Pedersen et al. 1987). In this cattery, several
cats presented with a loss in immune function after the introduction of a sentinel cat. The
loss of immune function could not be linked to feline leukemia virus (FeLV), another
member of the Retroviridae, already known to cause immunosupression in cats (Jarret et al.
1964). This led to the discovery of a novel retrovirus that differed from FeLV and more
closely resembled HIV in morphology and the Mg2+- rather than Mn2+-dependence of its
reverse transcriptase (Yamamoto et al. 1988a). Subsequent genetic analysis demonstrated
that this virus belonged to the lentivirus family.
The seven known members of the lentivirus family as listed above are commonly
divided into two groups based on differences in cell tropism and disease manifestation.
Those affecting the ungulate species EIAV, MW, and CAEV are predominantly
macrophage-tropic and cause immune-mediated diseases that target specific organs. Those
affecting primates, HIV and SI V, are tropic for lymphocytes and macrophages and cause a
major loss of immune function that results in an increased susceptibility to opportunistic
pathogens. FIV resembles the primate viruses in cell tropism and disease manifestation but
is genetically more closely related to the nonprimate lentiviruses (EIAV and MVV)
(Olmsted et al. 1989b).
Common to all lentiviruses is the long incubation period, the ability to affect
multiple organs, and most importantly the persistence in the face of host-immune
responses. The ability to escape from the host immunity is in part explained by a high
mutation rate of the lentiviral genome resulting in continuous antigenic variation (Rigby et


22
pruning with a feline herpes virus engineered to express the FlVenv gene followed by
booster immunizations with bacterial Env-fusion proteins failed to induce protection against
low dose challenge (Verschoor et al. 1996).
Successful FIV vaccine protocols include the use of inactivated cells infected with
the FIV Petaluma isolate (FIVPet; subtype A) or inactivated cell free FIVPct (Yamamoto et al.
1991b, 1993). These vaccines were produced from either feline lymphoid cells
productively infected with FIVPet (FL-4) or an IL-2 dependent feline lymphoid cell line
(FeT) infected with FIVPet (Yamamoto et al. 1991a). Using these vaccines, a protection
rate of 70%-90% has been observed against low dose experimental challenge with
homologous HVPet and slightly heterologous FIV Dixon (FIVDU; subtype A) (less than 9%
divergence in the env coding region) (Yamamoto et al. 1991b, 1993; Hosie et al. 1995).
Further, these vaccines afforded protection against FIV challenge inoculum virus
propagated on different cell lines including FeT 1, FL-4, and allogeneic PBMC. Protection
was achieved against intraperitoneal challenge and oral-nasal challenge in a small number of
animals tested (Yamamotoetal. 1991b, 1993; Johnson etal. 1994). These same vaccines,
however, failed to induce protection against a high challenge dose of 5x1o4 ID50 with the
homologous FIVPet isolate. Furthermore, these vaccines failed to induce protection against
experimental challenge with a moderate heterologous FIV UK8 isolate (subtype-A) and a
distinctly heterologous FIV Shizouka (FIVshi; subtype D) isolate. The Env amino acid
sequences of these isolates differ from the FIVPet Env sequence by 11% and 21%,
respectively. In addition, immunization schemes employing a similar vaccine produced
from MBM lymphoid cells infected with the Italian isolate FIV M2 have been shown to
induce protective immunity against a homologous plasma derived virus inoculum
(Mattuecci et al. 1996).
In summary, conventional inactivated vaccines are capable of inducing protective
immunity against low dose homologous FIV challenge and slightly heterologous FIV
challenge. Similar vaccine approaches have also been successful in other animal models


Table 1.5 Immunogenicity and prophylactic efficacy of ALVAC-based vaccines
Pathogen
genus
Test
species
Humoral
responses
Cell-mediated
responses
Protection
Rabies3
Rhabdovindae
mice
+
ND
+
dog
+
ND
+
cats
+
ND
+
squirrel monkeys
+
ND
ND
rhesus macaques
+
ND
ND
chimpanzees
+
ND
ND
humans
+
ND
ND
Cytomegalovirusb
Herpesviridae
mice
+
+
ND
guinea pigs
+
+
ND
Canine distemper virusc
Paramyxoviridae
dogs
+
ND
+
Japanese encephalitis virus'1
Flaviviridae
mice
+
ND
+
3 Cadoz et al. 1992; Taylor et al. 1991.
b Gonczol et al. 1995.
c Taylor et al. 1992a, 1992b.
d Konishi et al. 1994.
ND=not determined


Ill
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