AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 3, Supplement 1, 1987Mary Ann Liebert, Inc., Publishers

Prospects for Development of a Vaccine AgainstHTLV-III-Related Disorders

THOMAS J. MATTHEWS,1 H. KIM LYERLY,1 KENT J. WEINHOLD,1A. J. LANGLOIS,1 JAMES RUSCHE,2 SCOTT D. PUTNEY,2

ROBERT C. GALLO,3 and DANI P. BOLOGNESI1'Duke University Medical Center, Durham, NC

2Repligen Corporation, Cambridge, MA 'Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD

INTRODUCTION

With the etiology and diagnosis of AIDS largely resolved, the chal-lenges for the future clearly lie in its prevention and treatment. Given thealarming projections for the spread of this epidemic within the world popula-tion over the next 5 years (1), development of vaccines, antiviral strategiesand immune restorative approaches are imperative. To achieve success in any ofthese, a better understanding of the virus, the course of infection and theprocess of disease progression is required. Remarkable advances have been madein defining the genomic structure of the virus as well as the function of itsunique genetic elements. Many of the viral genes have been cloned and ex-pressed both in prokaryotic and eukaryotic vectors. The availability of largequantities of these materials in pure form paves the way for both production ofvaccines and design of anti-viral agents. Indeed, the life cycle processes ofthis retrovirus as well as understanding its mechanism of pathogenesis are wellwithin our grasp.

This discussion will be limited to the possibility of developing avaccine against the human retroviruses associated with AIDS. However, exclu-sion of all the necessary public health and educational measures should notdetract from their critical value toward stemming the tide of infection withthis agent in the human population. Likewise, the use of immunoprophylaxiswith anti-viral immune elements or chemoprophylaxis mediated by anti-viral com-pounds could be very effective means of reducing virus load and preventing theonset of disease if applied sufficiently early after exposure. Indeed, it isthe combined role of all approaches to prevention which will be necessary tomake a significant impact on this disease (1).

RETROVIRUS ANTIGENS WHICH ELICIT IMMUNITY TO INFECTION

The viral component which is responsible for the salient immunobiolo-gical features of retroviruses is its major exterior glycoprotein (gp) (forreview see 2). First, the gp is required for infection and mediates attachmentof the virus to the host cell surface. It is also the viral component most

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gp120 9P41

PE3 PB1 PENV9

itr-an-o-«—i-oîxziï-l^d

H * variable regions™ |-1 100 amlno acids

|] = conserved regions

Fig. 1. Location of proteins PB1, PE3, and PENV9 in relation to gpl20 andgp41. The envelope is synthesized as a precursor, gpl60, that is cleaved togive gpl20 and gp41. The secretion signal sequence is hatched. The locationof the variable and conserved regions are shown as darkened or open boxes,respectively.

directly involved in the phenomenon of interference as it relates to attachmentof virus receptors at the cell surface. Finally, the gp specifies the patternof neutralization by anti-viral antibodies. Consistent with these propertiesis its strategic location on the outer surface of the virion (3), as is illus-trated in Figure 1.

Another antigen in the envelope of the virion is a hydrophobic trans-membrane protein (tmp) which non-covalently anchors the gp to the particle (4).The tmp can either contain or be devoid of carbohydrate, but in every case thedegree of glycosylation is considerably less than that of the exterior gp. Thetmp can also be a target for neutralizing antibodies, but this is generallyweak unless complement is present (4).

The viral gp and tmp are also present in infected cells, concentratedat the sites of virus budding but also distributed on other areas of the cellsurface (5). As such, the envelope components represent a cellular target forimmune attack (6).

Animals infected with retroviruses usually respond with easily detect-able humoral immunity against the virus envelope components. For the most part,these are selective for the infecting agent, i.e., they are type specific (7).This is one of the three immunogenic domains of the external gp. The othersrepresent determinants which are common to all viruses of a given species(group) or extend to those in widely different species (interspecies) (2). Itis only in rare cases that animals respond to these latter domains undernatural conditions; with the exception of antibodies to the tmp which are di-rected predominantly to highly conserved regions of the molecule (8,9). On theother hand, one can immunize animals with virus or purified envelope componentsand obtain antibody responses which are much broader in their reactivity thannatural antibodies (2). This is particularly the case when heterologous spe-cies are employed. Thus, potent neutralizing as well as cytotoxic antibodiescan be raised artifically against gp which display strong group and interspe-cies specific reactivity.

VACCINATION AGAINST RETROVIRUS INFECTIONS

As noted above, immunization with gp elicits strong neutralizing anti-bodies as well as antibodies which are cytotoxic for infected cells. Mice im-munized with purified gp can indeed resist substantial challenges of infectious

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leukemogenic virus (10). The immune response associated with protection exhi-bits both type and group specific reactivity (10, 11).

While monomeric gp is capable of inducing protective immunity, rela-tively large quantities of purified antigen were required to accomplish thisreproducibly (10). In other animal systems, notably the cat, similar attemptswere not successful. On the other hand, the use of gp linked to the tmp so asto form multimeric aggregates (Figure 1) resulted in a much more effective im-munogen both in the mouse (11) and cat (12). It is likely that similar struc-tures are present in supernatants of FeLV-infected cells following serum starva-tion (13). This is the basis for a vaccine against feline leukemia virus whichhas been licensed for use in cats.

Recently, from work by Morein and colleagues (14), the capture ofvirus envelope components (gp and tmp) by glycoside lattices through hydropho-bic interaction with tmp generates a multimeric matrix structure (ISCOM; immunestimulating complex) which is an unusually powerful immunogen. When comparedto monomeric gp, an ISCOM preparation containing an equal amount of gp elicitsat least a ten-fold increase in the protective end point (15). It may be thatthese complex structures are more easily recognized, presented, taken up andprocessed by macrophages for antigen presentation to the immune system. In arecent report (16), Hunsmann and his colleagues could show that similar resultscould be obtained, even in the absence of tmp, provided that appropriate aggre-gates of gp could be formed.

SPECIAL CONSIDERATION FOR A VACCINE AGAINST AIDS

One can sum up the prospects for developing a vaccine as being diffi-cult but achievable. A successful vaccine will have to overcome significantobstacles imposed by HIV and other AIDS-associated retrovi ruses. The first isthe well-recognized feature of extensive genomic diversity, particularly in theenvelope gene (17), which is highly reminiscent of the situation with lenti-viruses (18). An effective vaccine should contain all possible neutralizationdeterminants either in the form of a single universal epitope or as an appro-priate cocktail of individual epitopes that span the divergence of the virusnature. At this point in time, it is not clear how either or both of thesealternatives will be pursued.

The second major obstacle is equally foreboding. This is based on thedistinct possibility that the disease is transmitted not only by free virusbut also via infected cells. This situation, coupled with two unique featuresof the virus, 1) its ability to exist in latent form and 2) the capacity ofinfected cells expressing gpl20 to fuse with uninfected cells, generates mecha-nisms to disseminate the virus in covert form even in the face of an immune re-

response. One significant hope to overcome these problems is for the vaccineto be able to induce specific cell-mediated immunity. To accomplish this, notonly will the appropriate viral antigens or epitopes need to be identified butalso their optimal mode of presentation to the immune system. In addition, or

perhaps even in place of cellular immunity, the generation of antibodies ableto mediate ADCC (antibody dependent cell cytotoxicity) would be of considerablebenefit.

PROGRESS IN CHARACTERIZATION OF POTENTIAL IMMUNOGENS FOR VACCINE STRATEGIES

The structural components associated with the envelope of retroviruseshave thus far received the most attention with regard to vaccine strategies.This derives not only from their strategic location on the surface of thevirion and the infected cell (2), but because they are responsible for thesalient immunobiological properties of the virus such as host tropism, inter-ference and neutralization (3). The envelope glycoproteins associated with HIVare derived from a precursor, gpl60, that is proteolytically cleaved to generatethe external envelope glycoprotein gpl20 and the transmembrane envelope glyco-protein gp42 (Figure 2). Several lines of evidence support the notion that a

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subunit vaccine based on gpl20 may be possible. 1) Antibodies that neutralizeHIV infection have been found in sera of people with AIDS, ARC (AIDS-relatedcomplex), as well as asymptomatic individuals infected with the virus (19,20)and such antibodies bind the affinity resins bearing purified gpl20 (21); 2)gpl20 elicits production of neutralizing antibodies (22), as does a large seg-ment of gpl20 produced in mammalian cells (23); and 3) direct binding of gpl20to the T4 receptor has been demonstrated, and monoclonal antibodies recognizingcertain epitopes of the virus receptor prevent viral infection (24,25). Recentstudies have also shown that monoclonal antibodies that neutralize HIV are spe-cific for a subregion of gpl20 (M. Robert-Guroff, personal communication).

The protein backbone of gpl20 represents approximately half of its ap-parent molecular weight on SDS gels (26) with the remainder consisting of car-bohydrate. This extensive degree of glycosylation might be expected to play arole in the determination and accessibility of both binding and neutralizingepitopes. We recently undertook studies to determine the extent of carbohy-drate involvement in both the induction of neutralizing antibodies to the virusand in binding of the envelope to CD4 as evidenced by inhibition of HTLV-III-mediated cell fusion. To achieve this, antibodies raised against native gpl20,gpl20 from which the carbohydrate had been enzymatically removed, and selectedrecombinant fragments of gpl20 expressed in E. col i were compared in variousimmunologie and biologic assays (27).

Immunization with a recombinant segmentof gpl20 (PB-1) or with deglycosylated gpl20 envebodies that neutralize HIV infection in vitro whito those obtained using purified native gpl20 as

fragments, including some extending into the gp41in this regard. Cross-absorption and direct antilocalize the dominant neutralizing epitopes in PBthe same epitopes elicit antibodies which prevent(Figure 3). Studies now in progress are aimed atrespective biological activities associated withof the envelope.

at the carboxy-terminal halflope protein produced anti-ch were surprisingly superiorimmunogen (Table 1). Otherregion, were not effective

gen competition studies indeed-1 and document further thatvirus-mediated cell-fusionfurther definition of the

PB-1 as well as other regions

However, in all cases the antibodies produced were specific for theisolate from which the immunogen was derived and did not neutralize divergent

gpi20START

>

StCM

I— gpl20

gpl20/gp41JUNCTION

gp41END

CO

S*»

gp4i _JFig. 2.

A SUBREGION OF GPl20 IS RESPONSIBLE FOR INDUCTION OF

ANTIBODIES THAT NEUTRALIZE VIRUS INFECTIVITY AND BLOCK

CELL FUSION.

A BACTERIAL RECOMBINANT FRAGMENT (PB1) REPRESENTING THIS

REGION CAN INDUCE HIGH TITERS OF NEUTRALIZING AND FUSION

BLOCKING ANTIBODIES,

3. The representative fragment can itself block the

neutralizing and cell fusion inhibitory activities of

various antibodies raised to larger components of the

env gene, including gpl60 and gp120.

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Experiment 1GP120 -» CHIMP

WAIT FOR- DEVELOPMENT OF

IMMUNE RESPONSES

CHALLENGE WITHHOMOLOGOUS VIRUS

Experiment 2

GP120 - - CHIMPIBID

EXP.1CHALLENGE WITHDIVERGENT VIRUS

Experiment 3

GP120-

IBIDEXP.1

CHALLENGE WITHVIRUS INFECTED

CHIMP CELLS

PARALLEL STUDIES SHOULD FOCUS ON:

1) Are other viral antigens necessary (or desirable) to achieve protective response?

2) Approaches to maximize the immunogenicity of viral subunits: role of adjuvants

3) Role of cell mediated immunity in protection

4) Impact of routes of Immunization and challenge

5) Role of passively administered antibodies in protection

6) Assessment of immunotoxicity of viral subunit immunogens

Fig. 3. HYPOTHETICAL SCHEME FOR EVALUATING CANDIDATE VACCINE ANTIGENS

HIV viruses (21) (Table 1). This indicates that a variable region of gpl20 isa dominant neutralizing epitope and while conserved regions elicit strong im-mune responses, the resultant antibodies do not prevent virus infectivity.These findings suggest that "cocktails" of divergent virus envelope componentsmay be required for effective vaccination strategies.

A likely target of opportuniof biologic significance is the bindisurface marker. Recent studies demonpresence of carbohydrate, suggestingdeterminant may be responsible (28).bility that linear sequences are invoantibodies or specific peptides couldsuch site was recently described (L

ty which would represent a conserved reging site on gpl20 for the CD4 lymphocytestrate that binding to CD4 requires thethat a complex rather than a simple linea

However, this does not exclude the possilved in the binding and that appropriatespecifically block this interaction. On

Laskey, personal communication).ANTI-VIRAL IMMUNE RESPONSES WHICH ARE CYTOTOXIC

Various investigative groups are attempting to define both humoral ancell-mediated cytotoxic responses against HTLV-III infected cells. Recentstudies using gpl20 as an immunogen in various animal species, including sub-human primates, indicate that the molecule can induce both T-cell-mediatedimmunity (29) and complement-dependent cytotoxic antibodies (30). Similaractivities were detected in chimpanzees infected with HTLV-IIIß (30) or witha recombinant vaccinia virus containing the virus envelope gene (31). Althougno complement-fixing cytotoxic antibodies could be demonstrated in humans infeted with HIV, virus-specific cytotoxic T cells have recently been identified(B. Walker; Nature, in press).

Our recent studies also demonstrate the presence of antibodies inpatients which mediate ADCC against virus-infected targets (32). This activit.was directed against a specific viral antigen, the envelope gpl20. Further-more, using normal CEM cells whose CD4 surface antigens were saturated with

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Table 1

HIV VARIANT SPECIFIC NEUTRALIZATION

BY ANTI-ENVELOPE SERA

H9 Infectivity* Cell Fusion Blocking**

IIlB MN RF IIIb MN RF

PBI-IIIb 1000 0 0 60 0 0

PBI-RF 0 N.T. 20 0 0 10

rl60-IIlB 1000 0 0 60 0 0

gpl20-IIIB 100 0 0 10 0 0

ARC sera 300 200 250 10 10 10

* Titer is the dilution at which HIV infectivity is 50% of preimmune sera.

** Titer is the highest dilution at which no multinucleated cells form.

exogenously added gpl20, virus-specific cytotoxic lymphocytes were found invirus-exposed individuals (33). The significance of these reactivities, partic-ularly in relation to the stage of infection and disease progression, will beof interest to study and relevant to the vaccine issues discussed here.

An important aspect with regard to generation of cell-mediated immuni-ty is the identity of the virus gene product(s), which are critical in thisregard. Based on studies in other virus models (34), it is likely that notonly the envelope but also the internal components of the virus may be required.This suggests that unique sequences may be responsible for recognition byT cells during antigen presentation. Two of these have recently been mapped todistinct regions of gpl20 with one residing in the PB-1 region (35). Neverthe-theless, while the neutralizing and certain T-cell epitopes reside on the virusenvelope, a complex vaccine including the internal antigens may be needed toelicit the full immune spectrum necessary for protection against infection.

ANIMAL MODELS FOR PRE-CLINICAL VACCINE TESTING

An additional challenge toward developing a successful vaccine is anappropriate animal model where efficacy and safety can be determined. To datethe chimpanzee is the only species which can be infected with the human viruses(36,37). Unfortunately, the chimp does not develop the full blown syndrome ofAIDS, but a few immunological abnormalities are noted. While imperfect, it isadequate for testing the power of a vaccine preparation to resist challenge byeither free virus and/or virus-infected cells. At the present time, some pro-mising immunogens are available which, in other animal species, have raisedneutralizing antibodies. These are represented by native gpl20 in purifiedform (38,39), a recombinant gpl20 expressed in mammalian cells (40) and recom-binant envelope products produced in bacteria (27) and yeast (41). The majordrawbacks for vaccine testing of the chimp model are the scarcity of theanimals, their expense and difficulties in handling and the assignation of thechimp as an endangered species. Efforts to find a smaller and more plentiful

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animal that can be infected with the human isolates would thus represent amajor breakthrough in this regard. In this context the close resemblance ofSTLV-III with HIV indicates that the further development of the experimentalmodels in rhesus macaques and African green monkeys will add a great deal toour knowledge in the meantime. Valuable lessons concerning the nature of im-munity required to protect against infection can also be gained by exploitingother animal virus models, particularly those of the ruminant lentiviruses butnot excluding either the feline or murine systems which have been so instruc-tive thus far.

Independent of which model is used for testing, the issues of genomicdiversity and mode of transmission will have to be seriously considered.Hence, the model must be amenable to challenge with different substrains ofHIV including those which exhibit the greatest degree of diversity. In addi-tion, inclusion of infected cells in the challenge will have to be addressed.Once the viral elements to be included in a vaccine regimen have been defined,issues such as mode of presentation, routes of inoculation, appropriate adju-vants (Figure 4) and other principles which have been alluded to with theanimal retrovirus vaccines (see above) will have to be addressed. This shouldbe followed by intense immunologie monitoring of the inoculated animals withparticular attention to both neutralizing antibodies and cell-mediated immuni-ty (Figure 4). Otherwise stated, the science of vaccinology will have to becarefully applied in order to obtain an optimal protective response.

However, because any animal model for HIV infecton and disease islikely to be imperfect, testing of primary vaccine candidates directly in manmust proceed to determine safety, immunogenicity and efficacy.CONCLUDING REMARKS

In summmary, while the AIDS epidemic may eventually prove to be one ofthe most severe devastations by an infectious agent to affect mankind, it re-mains fortunate that it has occurred during a period when the biomédical com-

munity has the opportunity to deal with it. One must find considerable comfortin the vast knowledge that exists about retroviruses in general and their humancounterparts in particular and expect that effective counter-measures againstthese agents will be developed. In the like manner, our rapidly expandingunderstanding of how the immune system functions will no doubt lead to specificapproaches to restore the damage the virus has perpetrated. In both areas,modern biotechnology is likely to provide the methodology and capability ofdealing with this problem on a world scale.

As one progresses down the avenues to control this disease, valuableprinciples will be gained which will apply to other human diseases associatedwith retroviruses. Because some of these represent malignant neoplasms (42),the opportunity to control a cancer by direct attack on the etiological agentclearly presents itself. The possibility that other widespread diseases suchas autoimmune and degenerative syndromes might also involve this group ofagents (43) further highlights the valuable byproducts of the intense studiesin HIV and AIDS.

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Address reprint requests to:Dani Bolognesi, Ph.D.

P.O. Box 2926Department of Surgery

Duke University Medical CenterDurham, NC 27710

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