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Can an AIDS Vaccine Be Developed?
Can an AIDS Vaccine Be Developed? Jay A. Levy
T
HE HUMAN IMMUNODEFICIENCY virus (mv) , the etiologic agent of the acquired immunodeficiency syndrome (AIDS) is a member of the lentivirus subfamily of human retroviruses. 1 Similar viruses found in animals (sheep, goats, horses, cows, cats, monkeys) readily infect cells of the hematopoietic system, particularly macrophages, and cause immunologic and neurologic disease. 2 Thus far, no effective control of this infection in animal species has been achieved, but efforts at developing a vaccine against these infectious agents have been pursued in only a limited manner. Much can be learned from the study of these animal systems, and should be encouraged. Nevertheless, it is clear that the tasks of treatment and prevention of a lentivirus infection will be extremely difficult. They will require a basic knowledge of the biology of the virus and the immune responses necessary to control and eliminate infection. Work has begun towards developing a vaccine against mv, but the results thus far only reflect the difficulties involved. FACTORS INFLUENCING THE DEVELOPMENT OF AN ANTI-HIV VACCINE
Several features of HIV infection should be appreciated when considering approaches at developing an antiviral vaccine (Table 1). They can pose major problems in obtaining an effective HIV vaccine. The Virus-Infected Cell
Epidemiological data, obtained soon after AIDS was recognized in 1981, indicated that the causative agent was spread through contact with blood (transfusions, intravenous [IV] drug use), intimate sexual contact (infected genital secretions), and From the Department of Medicine, Cancer Research Institute, University of California, School of Medicine, San Francisco. Supported by Grant Nos. AI-24499, POI-AI-24286from the National Institutes of Health and the California State Universitywide Task Force on AIDS. Address reprint requests to Jay A. Levy, MD, Department of Medicine, Cancer Research Institute, University of California, School of Medicine, San Francisco, CA. © 1988 by Grune & Stratton, Inc. 0887-7963/88/0204-0009$03.00/0 264
events occurring during gestation or delivery (ie, passage of virus from mother to child). 3 Results in our laboratory as well as others have indicated a very low level of infectious-free virus in most body fluids. Aside from cerebrospinal fluid that can contain an estimated 100 to 10,000 infectious particles (IP) per milliliter, other body fluids contain considerably less. 4 ,5 Only serum or plasma and, in some cases, seminal fluid have levels that are readily detected by cell culture procedures (l0-50 IP/mL). Other body fluids contain 10- to 100-fold less virus, and would thus not be a major source of contagion. In contrast, genital fluids as well as blood can carry considerable amounts of mV-infected cells that serve as a source for HIV transmission. 5 The variations in virus transmission among sexual partners suggests that the relative number of infected cells in seminal or cervicaV vaginal fluids influence the transmission rate. In our laboratory, Dr Masatoshi Tateno demonstrated up to 5% of cells in some seminal fluids are infected. 5 During anal/genital contact, these cells in the seminal fluid would be the most likely carriers of the infection to the bowel mucosa that recent evidence suggests can be directly infected with the virus. 6 Alternatively, small lesions in the mucosal lining of the gastrointestinal (01) tract can permit entrance of infected cells into the circulation. This information is consistent with epidemiologic data indicating the receptive partner (both men and women) in anal/genital contact has the greatest risk for mv infection.? In vaginal intercourse, the infection could occur most readily when the cervical os is open during menses or shortly thereafter; infected cells could then find their way via the cervical os into the endometrium where the resident macrophages and lympJ;lOcytes can spread mv in the host. 5 These observations emphasize the role of the infected cell as a source of infection during sexual contact. Transmission by blood and the maternal route may also result primarily via infected cells. Vaccines must therefore elicit cytotoxic responses to eliminate these sources of infection. Virus Integration
An important feature of the infected cell is the integration of the viral genome into the cellular
Transfusion Medicine Reviews, Vol 2, No 4 (December), 1988; PP 264-271
CAN AN AIDS VACCINE BE DEVELOPED?
Table 1. Features of HIV Infection That Affect Vaccine Development 1. 2.
3. 4. 5. 6. 7.
The infected cell is a major source of virus transmission. Virus infection involves integration of the viral genome into the chromosome of the infected cell. This cell becomes a reservoir for persistent virus production, The infected cell can transfer the virus by cell-to-cell contact. The infected cells can remain "latent" and express very few viral antigens. Several independent serotypes and subtypes of HIV can be identified. HIV infection occurs at specific sites in the host (eg, the rectum), Portions of HIV proteins resemble normal cellular proteins.
chromosome. The cell does not die quickly and can continue producing progeny virus for days, up to months. 4 Macrophages that are not as susceptible as T cells to the cytopathic effects of HIV are particularly effective reservoirs for this transfer of the virus in the host, as well as to other individuals. 4 ,5 Thus, as noted above, elimination of the virus requires killing of its infected cell. Cell-to-Cell Contact
Whereas virus transmission can take place by direct infection, in many and perhaps most cases, cell-to-cell contact is involved. The HIV envelope gp41 protein appears to mediate this intracellular contact via fusion. 4 ,8 This mechanism for virus spread would not be prevented by anti-HIV neutralizing antibodies. A cytotoxic response is the best means of control. Latency
In some individuals, the infected cell may remain in a latent state in which very little viral protein or RNA is made. Thus, without sufficient expression of viral antigens, the cell would not be readily recognized by the immune system and could enter the individual unchallenged. Encountering host cells, these infected cells could be activated to produce virus progeny and transmit the infection. 4 HIV Serotypes
Another noteworthy feature of HIV is its various serotypes; many may need to be incorporated into a vaccine to give protection against all HIV. Chim-
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panzees inoculated with one strain of HIV can be superinfected by another. 9 Most likely humans can also be infected by more than one HIV strain. In addition, a new subtype of HIV, HIV-2,lO has been recognized in West Africa and is spreading throughout the world. Some individuals have evidence of infection by both types of HIV. 4, 11 Clearly, infection by one virus may not prevent infection by another. HIV-2 is associated with AIDS, but differs considerably in its envelope region from HIV-l. It may presage other related human lentiviruses that must be controlled by an anti-HIV vaccine. Local Immunity If indeed a major source of HIV transmission is via the rectal mucosa, a strong immune response at this site would be required to ward off the initial infection. Similarly, in women, local immunity in the vagina and endometrium would be necessary to prevent transmission of HIV, particularly by the infected cell. Whether this response can be elicited by conventional vaccine approaches must be evaluated.
Autoimmunity
One other potential complication of HIV vaccines is the possibility that antibodies against the viral proteins will cross-react with normal cellular proteins. Autoimmunity has been described in individuals with HIV infection, but in most cases, these autoimmune syndromes appear to result from a polyclonal activation of B cells and hypergammaglobulinemia that commonly occurs with viral infections. 4 Because several regions of HIV resemble portions of normal cellular proteins 4 (Table 2), the induction of a reaction against these cellular counterparts might occur following vaccination. For this reason, the present trials in human volunteers using the gp160 envelope protein made in the baculovirus system (insect cells) should be very informative. Immunization of 48 volunteers has thus far taken place with no reported side effects. Nevertheless, the possible induction of autoimmunity or other compromising events to the immune system must be considered, particularly if antibodies to interleukin-2 (IL-2) or interferon are elicited. HUMORAL IMMUNE RESPONSES TO HIV
Standard procedures for preventing virus infections such as measles, mumps, polio, and chicken
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JAY A. LEVY Table 2. Regions of HIV that Cross-react with Cellular Proteins Shared Region
IL-2 'Y- 1FN
",-1 Thymosin Neuroleukin (phosphohexose isomerase) Peptide T HLA antigen HLA antigens
Location*
LTR, env LTR gag (p17)
env env env virion surfacet
Abbreviations: IFN. interferon; IL-2. interleukin-2. * Denotes region of HIV that has potential cross-reactivity with a normal cellular protein as deduced from sequence analysis. 4 t Incorporated into the viral envelope from the cell surface.
pox, have been based on the ability of the vaccination program to induce humoral immune responses. These lead to production of neutralizing antibodies that inactivate the incoming free viral agent. The above discussion suggests that this type of immunity would give only limited protection against HIV, because the virus-infected cells would not necessarily be killed. Nevertheless, the initial studies with HIV were directed at understanding if neutralization of the virus can be observed with sera from infected individuals. These studies frrst indicated very low titers of neutralizing activity in infected individuals. However, with recent improvements in these assays, substantial levels have been found in many infected individuals including those suffering from severe disease. 4,12 This latter observation has led to the conclusion that these antibodies, produced following infection, are probably not helpful in protecting against progression of disease. Work by Dr Cheng-Mayer et al have shown that different mv isolates can be distinguished by their sensitivity to neutralization;12 some are easily neutralized by certain sera, others are not. In particular, viruses from Africa appear to be neutralized only by individuals coming from that continent, and similar observations have been made from studies of Haitian isolates. This neutralization appears to be mediated primarily by an interaction of antibody with the viral envelope, gp120, that can be highly variable. 4,13 Thus, it seems certain that if neutralization is an important part of antiviral approaches, polyvalent vaccines using envelope proteins from a variety of HIV will be needed for effective protection.
A portion of the viral transmembrane protein, gp41, is also exposed on the outside surface of the virus 4 and antibodies to this envelope protein have shown some neutralizing activity as well. 14 This result is promising, because regions of gp41 are shared by more mv isolates than gp120. However, because all HIV appear to use a similar mechanism for attachment to the cellular receptor (the CD4 molecule),4 antibodies against this attachment site (presumed present on gp120) would appear to be the most conserved among all mv. Evidence indicates that mv-2 also uses the CD4 protein for infection of T cells. However, the particular viral epitope involved has not yet been identified. Nevertheless, recent data suggest that other cellular receptors or means of virus activity may be involved, because cells lacking the CD4 antigen can be infected by HIV. 4,15 Most recently, observations have indicated a property of mv infection that was not expected. Some antibodies to the virus may actually help it enter macrophages and T cells. 15a This presumed opsonization of virus-antibody complexes leading to infection poses an important problem for vaccine approaches: The induction of non-neutralizing antibodies might facilitate the infection rather than prevent it. One humoral immune response that could be of benefit in eliminating the virus-infected cell is antibody-directed cellular cytotoxicity (ADCC). This assay has indicated that anti-envelope antibodies (both gp120 and gp41) are involved in the killing of infected cells by the host's natural killer (NK) or T cells. 16 These antibodies do not appear to decrease substantially during development of AIDS, and thus progression of disease must result from a reduction in the number or function of these effector cells in the host. ADCC could be extremely important soon after infection, because this response could immediately kill incoming i!rl'ected cells and prevent establishment of the infection. CELLULAR IMMUNE RESPONSES TO HIV
Studies of cell-mediated .immune responses in mY-infected individuals have been limited. Recent data have indicated that cytotoxic activity is associated with NK cells or T cells from infected individuals. 17 ,18 These observations suggest that vaccination programs might elicit a strong cellular immune response against the infected cell. Studies have also identified T helper cells with specific
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CAN AN AIDS VACCINE BE DEVELOPED?
responses to selective epitopes on the viral envelope. 19 These appear to be different from those recognized by antibodies. Thus, it appears that the humoral and cellular immune responses are directed against certain components of HIV and the specificity of each may differ. Both types of epitopes may need to be incorporated into a vaccine. Then, a reaction against not only the virus but also, most importantly, the infected cell can be elicited. The combination of ADCC and a strong cellular immune response would seem at present to be the most effective way of preventing HIV transmission by infected cells. Neutralizing antibodies with good cross-reactivity with a variety of different HIV would be the major mechanism for preventing infection by free virus. APPROACHES CONSIDERED FOR HIV VACCINES
Inactivated Virus
Inactivated virus vaccines have been a major approach for preventing infections with other human viruses such as polio, smallpox, and yellow fever (Table 3). A formalin-inactivated type D simian retrovirus vaccine has shown promise in prevention of SAIDS in monkeys. 20 However, this method has not been considered seriously for HIV because of the potential danger of a virus surviving the inactivation procedure. Such an event did occur during development of some of the inactivated human viral vaccines. 21 Moreover, studies with paramyxoviruses, that are also enveloped viruses like retroviruses, have shown other complications because the inactivated virions used did not elicit immune responses to all the viral proteins. With respiratory syncytial virus (RSV) , for instance, vaccination with inactivated virus gave rise to severe disease in some immunized infants subsequently infected by RSV. 21 The reason for this observation is not certain, but the data suggest that
vaccination against inactivated virus elicits antibodies to many of the RSV proteins, but not to the protein responsible for fusion of the virus with the cell surface membrane. Thus, virus entry by fusion and spread by cell-to-cell contact can occur, despite the presence of antiviral antibodies. The infected cells that then become established in the host express virus and viral antigens that are attacked by the immune system of the host. These immune reactions with antibody-antigen complexes lead to strong inflammatory responses (eg, Arthus reaction) and more cytopathology (including pneumonias) than would normally have occurred during a natural RSV infection. Therefore, any inactivated virus must be effective in eliciting an immune response against all the viral proteins mediating infection, and the inactivation procedure must be completely effective. Non-Pathogenic Variants
Some researchers have suggested that manipulating HIV so that it no longer has its reverse transcriptase, protease, or its transactivating (tat) proteins could produce a non-infectious agent with the three-dimensional shape of a total virion. These approaches have not been considered seriously by investigators because of the potential that these modified viruses could still interact with genes (perhaps endogenous viral) in the host and emerge as fully infectious pathogenic virions. Using naturally occurring non-pathogenic strains as live vaccines is also an approach not considered seriously. With other human viruses, attenuated strains have made excellent vaccines (eg, polio, measles), but in rare cases, a virulent wild type virus has emerged. 21 Nevertheless, combining inactivation with the use of non-infectious variants may be a feasible approach for an HIV vaccine, since both the three-dimensional structure and the safety of the vaccine (even if some virions were not inactivated) might be better assured.
Table 3. Approaches to HIV Vaccines 1. 2. 3.
4. 5. 6.
Inactivated virus (natural or engineered) Non-pathogenic variants (natural or engineered) Subunit vaccine (natural or engineered) Envelope glycoprotein, gp120 Envelope-transmembrane glycoprotein, gp41 Gag protein Viral proteins in infectious recombinant virus (eg, vaccinia) Sequence-derived peptides of HIV Anti-idiotypes of neutralizing antibodies
Viral Proteins-Natural or Genetically Engineered
This approach is aimed at eliminating all nucleic acids from the vaccination material and using proteins that are required for protective anti-HIV responses. These proteins would be either purified naturally (limited in part by the feasibility and expense of growing large amounts of virus) or produced by genetically engineered procedures em-
268
ploying bacteria, yeast, insect, or mammalian cells. The latter approach would be the most appropriate, because normal glycosylation of the viral proteins (eg, the envelope) would occur, and thus elicit a more effective immune response. The problems inherent in this approach involve determining which proteins are important and producing these proteins in sufficient amounts. Moreover, because the proteins removed from the structure of the virion would not have the threedimensional shape of the virus, an appropriate response of the immune system might not take place. For this latter reason, some investigators are considering the incorporation of viral proteins into liposomes (or lipid vesicles) that would simulate a virion structure. Viral Proteins In Infectious Recombinant Viruses
Another mechanism for presenting HIV proteins to the host is to incorporate their genes into the genome of other infectious viruses such as vaccinia (smallpox) and baculoviruses. In this way, immunization can be performed with a live virus that will express the mv proteins in infected cells and elicit an immune response to these virus proteins in a manner similar to natural infection. The virus vector may also help stimulate this immune response. This approach has induced neutralizing antibodies and cell-mediated responses when vaccinia-HIV recombinant viruses were used. Nevertheless, these vaccines (using gp 120 or gp 160) have not protected immunized animals from virus challenge. 34 Synthetic Peptides
An alternative approach to using proteins produced via viral genes is to synthesize the peptides coded for by the known viral sequences. This approach completely dismiss the use of viral genes, and in principal would be safe from any possible contamination. Nevertheless, these peptides without the correct glycosylation and three-dimensional form could be potentially less effective in inducing an effective immune response to HIV. Thus far, they have been useful in evaluating the natural immune response in infected individuals. Anti-Idiotype Vaccine
A relatively new approach to vaccines initially uses an antibody that neutralizes the virus as the immunogen. Because this antibody interacts with
JAY A. LEVY
the epitope on the virus that appears required for infection, the antibody to this antibody (the antiidiotype) should be a mirror image of the virus epitope itself. The subsequent use of this anti-idiotype in vaccination procedures should then elicit an immune response against the virus. This approach with HIV would avoid any contact with viral peptides or infectious virus, and is considered the safest approach for a vaccine. It has been successful in eliciting protective neutralizing antibodies to hepatitis B virus 22 and has been used in preliminary studies to make antibodies to the mv envelope protein. 23 Nevertheless, this method for mv must produce not only neutralizing antibodies but also cellular immune responses. This latter parameter thus far has not been measured. Moreover, adverse effects may result from this approach, particularly if the anti-idiotype antibody attacks, in the immunized individual, the viral receptor on normal cells. 21 PRELIMINARY STUDIES AIMED AT DEVELOPMENT OF AN ANTI-HIV VACCINE
The initial attempts to evaluate the feasibility of an anti-HIV vaccine concentrated, as expected, on the outside envelope region. Studies in a variety of animal species used either purified gp120 from virus, synthetic peptides, genetically engineered cells, or recombinant forms of this protein. 24-36 These latter products were made by direct transfection of viral genes into Escherichia coli, yeast, or mammalian cells (Chinese hamster ovary) or by using vectors such as vaccinia or baculovirus. All these experiments successfully produced antigp120 and gp41 antibodies (Table 4). Virus neutralization by some sera from immunized animals was also achieved, but at very low titer « 1 to 20), and in nearly all cases was type-specific. Only the virus whose envelope protein was used for the immunization was recognized by the animal sera. However, one recent study inrabbits using synthetic peptides from the second conserved region of gp120 gave promising results on neutralizing antibodies and cross-reactivity. 31 Conceivably, with different adjuvants, these immune responses could be increased. Nevertheless, these early studies do suggest that polyvalent vaccines will need to be used to induce a response to all HIV isolates. Very few of these immunization studies evaluated T cell responses, which, as noted above, are an important part of the defense mechanism
CAN AN AIDS VACCINE BE DEVELOPED?
269
Table 4. Research Aimed at Developing an Anti·HIV Vaccine Immunogen
Animal
Response
Investigators
Mouse Mouse
v-env (gp160) v-env5 v-env2
anti-gp120 anti-gp41 anti-gp120
Chakrabarti et al 24 Hu et al 25
Guinea pig, Rabbit Goat, Horse, Rhesus monkey Goat
gp120, (CHO cell) gp 120 (cellular)
anti-gp120 (neutraliz) (neutraliz)
Lasky et al 26 Robey et al 27
gp120, (E coli)
Putney et al 28
Rabbits Mice Rabbit Goat Rabbit
synthetic peptide (gp120)
anti-gp120 anti-gp41 (neutraliz) anti-gp120 (neutraliz)
Kennedy et al 29
gp120, gp120r (cellular, E coli) synthetic peptide (gp120)
anti-gp120 (neutraliz)
Krohn et al 30 Ho et al 31
Goat Macaque
v-env5
Chimpanzee
v-env 5 (gp160)
Chimpanzee Human
gp120 (virus) v-env, (gp160)
anti-gp120 (cross-neutraliz) neutraliz anti-gp41 anti-gp120 T cell response anti-env gp41 (no neutraliz) T cell response no protection neutraliz neutraliz T cell response
synthetic peptide (gp120)
Palker et al 32 Zarling et al 33 Hu et al 34 Zarling et al 35 Arthur et al 36 Zagury et al 39
Abbreviations: v, vaccinia virus vector; env, envelope; neutraliz, neutralizing antibodies; r, recombinant protein.
against the virus and infected cells. Some experiments with chimpanzees using gp120 or a vaccinia-recombinant gp160 (v-env-5) have shown the induction of both anti-envelope antibodies and T cell responses. 34- 36 Nevertheless, these studies have been limited and none of the immunized chimpanzees had high levels of neutralizing antibodies nor were protected from subsequent challenge. 34 Thus far, immunization procedures have not given the type or extent of immune response in these animals as has direct virus infection. 37 Some results have suggested that antibodies to the viral p17 (that presumably cross-reacts with a thymosin) can neutralize HIV. 38 This observation probably reflects the presence of the viral protein just below the virus outer membrane where an external portion could be present. 4 Experimental studies to evaluate a vaccine using this human gag protein are planned. Finally, Zagury et al 39 have immunized seronegative human volunteers with a vaccinia-recombinant HIV envelope virus. These studies produced neutralizing antibodies at a titer of 1 to 40 against the HTLV-IIIB strain of HIV represented in the vaccine. A much lower level of neutralization was found to the RF strain of HIV from Haiti. Some cellular immune responses were
also noted to the HTLV-IIIB strain, and to a lesser extent against the RF strain. Further boosters are planned to determine if these approaches can give substantial amounts of neutralizing antibodies and strong cellular immune responses. These studies represent the only human trials of a vaccine that ,have yielded any results. The studies cited above with purified gp 160 produced in insect cells are now in progress. CONCLUSIONS
As discussed in the introduction, a retrovirus, and particularly one from the lentivirus subfamily, poses substantial problems in the development of a vaccine. While the approaches described above are being considered by laboratories throughout the world, the chance of success with anyone of them seems limited because of the properties of HIV infection (Table 1). No successful prototype lentivirus vaccine has yet been developed. The trials thus far with purified envelope proteins from the virus, synthetic peptides of the envelope of the virus, and recombinant viruses (eg, vaccinia) carrying the envelope viral gene have led to the development of anti-envelope antibodies, but with
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JAY A. LEVY Table 5. Ideal Properties of an Anti-HIV Vaccine
1. 2. 3. 4. 5. 6. 7.
Elicits neutralizing antibodies that react with all HIV strains and subtypes. Induces cytotoxic responses against virus-infected cells. Induces immune responses that recognize latently infected cells. Does not induce antibodies that enhance HIV infection. Induces local immune response at all sites of HIV entry in the host. Vaccine is safe, showing no toxic effects. The effect of the vaccination procedure is long-lasting.
low neutralizing activity in the immunized animals. Most importantly, no protection from challenge by HIV was observed. 34 Moreover, studies of cell-mediated immune responses that are the most important in virus infection have been limited. Because the immunization procedures were not successful against free virus inoculation, prevention of infection after a challenge with infected cells would be even less likely to occur. Thus, the possibility of an anti-HIV vaccine in the near future seems remote. The problems that are involved in the development of a vaccine against HIV are considerable, and the approaches being considered must address these issues successfully (Table 5). Whether an effective vaccine will be developed depends on the capability of an immunization strategy employing an appropriate adjuvant to elicit a strong response of both humoral and cellular origin. All HIV serotypes and subtypes must be recognized as well as the infected and latently infected cell. Antiviral antibodies must inactivate and not potentiate the infection. Moreover, the response must be rapid and present at all sites of virus entry. In this way,
a virus-infected cell entering the host by any route can immediately be recognized and destroyed before it is able to fuse with a host cell or release progeny that is infectious. And, this vaccine must be completely safe, nontoxic, and long-lasting. The latter feature is particularly important if the vaccine is to be effective in remote areas where administering frequent boosters would be difficult. The recent reports with malaria vaccines offer some hope,40,41 but the results are very preliminary. The use of several proteins linked by polymerization gave effective prevention in some individuals. 41 A similar approach employing several viral proteins presented in a highly immunogenic manner may also be effective with HIV. The malarial parasite, like HIV, establishes infection of host cells within minutes after entry, and has antigenic heterogeneity. However, with malaria, only the free infectious agent and not an infected cell generally needs to be considered. Thus, cytotoxic responses may be more important in HIV prevention. Similarities to HIV can be drawn as well from the difficulties encountered in making vaccines to the herpesviruses, particularly EpsteinBarr virus (EBV) and cytomegalovirus (CMV). These agents can also be passed by the infected cells, and have different serotypes. Prevention and control, as with HIV, depend on an active cellular immune system. Progress has been substantial in understanding the features of HIV infection and the potential immunologic responses to the virus. Hopefully, continuing studies will bring to light a direction that will eventually lead to a safe and effective vaccine. Nevertheless, without a new and creative approach to this problem, the solution will certainly take time.
REFERENCES 1. Levy JA, Kaminsky LS, Morrow WJW, et al: Infection by the retrovirus associated with the acquired immunodeficiency syndrome. Ann Intern Med 103:694-699, 1985 2. Haase AT: Pathogenesis of lentivirus infections. Nature 322:130-136, 1986 3. CurranJW, Jaffe HW, Hardy AM, et al: Epidemiology of HIV infection and AIDS in the United States. Science 239:610-616, 1988 4. Levy J: The human immunodeficiency yirus (HIV): Detection and pathogenesis, in Levy J (ed): AIDS: Pathogenesis and Treatment, New York, Dekker, (in press) 5. Levy JA: The transmission of AIDS: The case of the infected cell. JAMA 259:3037-3038, 1988
6. Nelson JA, Wiley CA, Reynolds-Kohler C, et al: Human immunodeficiency virus detected in bowel epithelium of patients with gastrointestinal symptoms. Lancet 1:259-262, 1988 7. Winkelstein W Jr, Lyman DM, Padian N, et al: Sexual practices and risk of infection by the human immunodeficiency virus: The San Francisco men's health study. JAmMed Assoc 257:321-325, 1987 8. Sodroski J, Goh WC, Rosen C, et al: Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. Nature 322:470-474,1986 9. Fultz PN, Srinivasan A, Greene CR, et al: Superinfection of a chimpanzee with a second strain of human immunodeficiency virus. J ViroI61:4026-4029, 1987
CAN AN AIDS VACCINE BE DEVELOPED?
10. Clavel F, Guetard D, Brun-Vezinet F, et al: Isolation of a new human retrovirus from West African patients with AIDS. Science 233:343-346, 1986 11. Denis D, Barin F, Gershy-Damet G, et al: Prevalence of human T lymphotropic retroviruses type III (HIV) and type IV in Ivory Coast. Lancet 1:408-411, 1987 12. Cheng-Mayer C, Homsy I, Evans LA, Levy IA: Identification of HIV subtypes with distinct patterns of sensitivity to serum neutralization. Proc Nat! Acad Sci (USA) 85:2815-2819, 1988 13. Starcich BR, Hahn BH, Shaw GM, et al: Identification and characterization of conserved and variable regions in the envelope gene of HTLV-III/LAV, the retrovirus of AIDS. Cell 45:637-648, 1986 14. Chanh TC, Dreesman GR, Kanda P, et al: Induction of anti-HIV neutralizing antibodies by synthetic peptides. EMBO 15:3065-3071, 1986 15. Cheng-Mayer C, Rutka JT, Rosenblum ML, et al: The human immunodeficiency virus (HIV) can productively infect cultured human glial cells. Proc Nat! Acad Sci (USA) 84:3526-3530, 1987 15a. Homsy J, Tateno M, Levy I: Antibody-dependent enhancement of HIV infection. Lancet 1:1285-1286, 1988 16. Evans LA, Thomson-Honnebier G, Steimer K, et al: Antibody-dependent cellular toxicity (ADCC) is directed against envelope proteins of the human immunodeficiency virus. (submitted for publication) 17. Walker B, Chakrabarti S, Moss B, et al: HIV-specific cytotoxic T lymphocytes in lung disorders. Nature 328: 345-348, 1987 18. Plata F, Autran B, Martins LP, et al: AIDS virus-specific cytotoxic RT lymphocytes in lung disorders. Nature 328:348-351, 1987 19. Walker CM, Steimer KS, Rosenthal KL, et al: Identifi- . cation of HIV envelope type-specific T helper cells in an HIV-infected individual. (submitted for publication) 20. MarxPA, Pedersen NC, Lerche NW, et al: Prevention of simian acquired immune deficiency syndrome with a formalin-inactivated type D retrovirus vaccine. I Virol 60:431-435, 1986 21. Hilleman MR: Conclusions: In pursuit of an AIDS virus vaccine, in Levy I (ed): AIDS: Pathogenesis and Treatment, New York, Dekker, (in press) 22. Kennedy RC, Eichberg JW, Lanford RE, Dreesman GR: Anti-idiotypic antibody vaccine for type B viral hepatitis in chimpanzees. Science 232:220-223, 1986 23. Zhou E-M, Chanh TC, Dreesman GR, et al: Immune response to human immunodeficiency virus. In vivo administration of anti-idiotype induces an anti-gp 160 response specific for a synthetic peptide. I Immunol 139:2950-2956, 1987 24. Chakrabarti S, Robert-Guroff M, Wong-Staal F, et al: Expression of the HTLV-III envelope gene by a recombinant vaccinia virus. Nature 320:535-537, 1986 25. Hu SoL, Kosowski SG, Dalrymple 1M: Expression of AIDS virus envelope gene in recombinant vaccinia viruses. Nature 320:537-540, 1986
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26. Lasky LA, Groopman IE, Fennie CW, et al: Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein. Science 233:209-212, 1986 27. Robey WG, Arthur LO, Matthews TJ, et al: Prospect for prevention of human immunodeficiency virus infection: Purified 120-kDa envelope glycoprotein induces neutralizing antibody. Proc Natl Acad Sci (USA) 83:7023-7027, 1986 28. Putney SD, Matthews TJ, Robey WG, et al: HTLV-III! LAV-neutralizing antibodies to an E coli-produced fragment of the virus envelope. Science 234: 1392-1395, 1986 29. Kennedy RC, Dreesman GR, Chanh TC, et al: Use of a resin-bound synthetic peptide for identifying a neutralizing antigenic determinant associated with the human immunodeficiency virus envelope. J BioI Chern 262:5769-5774, 1987 30. Krohn K, Robey WG, Putney S, et al: Specific cellular immune response and neutralizing antibodies in goats immunized with native or recombinant envelope proteins derived from human T-lymphotropic virus type IIIB and in human immunodeficiency virus-infected men. Proc Nat! Acad Sci (USA) 84:4994-4998, 1987 31. Ho DD, Kaplan IC, Rackauskas IE, Gurney ME: Second conserved domain of gp 120 is important for HIV infectivity and antibody neutralization. Science 239: 1021-1023, 1988 32. Palker RI, Clark ME, Langlois AI, et al: Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides. Proc Natl Acad Sci (USA) 85:1932-1936, 1988 33. Zarling 1M, Morton WM, Moran PA, et al: T-cell responses to human AIDS virus in macaques immunized with recombinant vaccinia viruses. Nature 323:344-346, 1986 34. Hu SoL, Fultz PN', McClure HM, et al: Effect of immunization with a vaccinia-HIV env recombinant on HIV infection of chimpanzee. Nature 328:721-723, 1987 35. Zarling 1M, Eichberg JW, Moran PA, et al: Proliferative and cytotoxic T cells to AIDS virus glycoproteins in chimpanzees immunized with a recombinant vaccinia virus expressing AIDS virus envelope glycoproteins. I Immunol 139:988-990, 1987 36. Arthur LO, Pyle SW, Nara PL, et al: Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp 120) subunit vaccine. Fmc Nat! Acad Sci (USA) 84:8583-8587, 1987 37. Morrow WJW, Homsy I, Eichberg IW, et al: Immunosuppressive therapy did not induce disease in baboons, rhesus monkeys, and chimpanzees inoculated with the human immunodeficiency virus (HIV) (submitted for publication) 38. Sarin PS, Sun DK, ThomtonAH, et al: Neutralization of HTLV-III/LAV replication by antiserum to thymosin alpha-I. Science 232:1135-1137, 1986 39. Zagury D, Leonard R, Fouchard M, et al: Immunization against AIDS in humans. Nature 326:249-250, 1987 40. Miller LH, Howard RI, Carter R, et al: Research toward malaria vaccines. Science 234:1349-1356, 1986 41. Patarroyo ME, Amador R, Clavijo P, et al: A synthetic vaccine protects humans against challenge with asexual blood stages of Plasmodiumfalciparum malaria. Nature 332: 158-161, 1988
T
HE HUMAN IMMUNODEFICIENCY virus (mv) , the etiologic agent of the acquired immunodeficiency syndrome (AIDS) is a member of the lentivirus subfamily of human retroviruses. 1 Similar viruses found in animals (sheep, goats, horses, cows, cats, monkeys) readily infect cells of the hematopoietic system, particularly macrophages, and cause immunologic and neurologic disease. 2 Thus far, no effective control of this infection in animal species has been achieved, but efforts at developing a vaccine against these infectious agents have been pursued in only a limited manner. Much can be learned from the study of these animal systems, and should be encouraged. Nevertheless, it is clear that the tasks of treatment and prevention of a lentivirus infection will be extremely difficult. They will require a basic knowledge of the biology of the virus and the immune responses necessary to control and eliminate infection. Work has begun towards developing a vaccine against mv, but the results thus far only reflect the difficulties involved. FACTORS INFLUENCING THE DEVELOPMENT OF AN ANTI-HIV VACCINE
Several features of HIV infection should be appreciated when considering approaches at developing an antiviral vaccine (Table 1). They can pose major problems in obtaining an effective HIV vaccine. The Virus-Infected Cell
Epidemiological data, obtained soon after AIDS was recognized in 1981, indicated that the causative agent was spread through contact with blood (transfusions, intravenous [IV] drug use), intimate sexual contact (infected genital secretions), and From the Department of Medicine, Cancer Research Institute, University of California, School of Medicine, San Francisco. Supported by Grant Nos. AI-24499, POI-AI-24286from the National Institutes of Health and the California State Universitywide Task Force on AIDS. Address reprint requests to Jay A. Levy, MD, Department of Medicine, Cancer Research Institute, University of California, School of Medicine, San Francisco, CA. © 1988 by Grune & Stratton, Inc. 0887-7963/88/0204-0009$03.00/0 264
events occurring during gestation or delivery (ie, passage of virus from mother to child). 3 Results in our laboratory as well as others have indicated a very low level of infectious-free virus in most body fluids. Aside from cerebrospinal fluid that can contain an estimated 100 to 10,000 infectious particles (IP) per milliliter, other body fluids contain considerably less. 4 ,5 Only serum or plasma and, in some cases, seminal fluid have levels that are readily detected by cell culture procedures (l0-50 IP/mL). Other body fluids contain 10- to 100-fold less virus, and would thus not be a major source of contagion. In contrast, genital fluids as well as blood can carry considerable amounts of mV-infected cells that serve as a source for HIV transmission. 5 The variations in virus transmission among sexual partners suggests that the relative number of infected cells in seminal or cervicaV vaginal fluids influence the transmission rate. In our laboratory, Dr Masatoshi Tateno demonstrated up to 5% of cells in some seminal fluids are infected. 5 During anal/genital contact, these cells in the seminal fluid would be the most likely carriers of the infection to the bowel mucosa that recent evidence suggests can be directly infected with the virus. 6 Alternatively, small lesions in the mucosal lining of the gastrointestinal (01) tract can permit entrance of infected cells into the circulation. This information is consistent with epidemiologic data indicating the receptive partner (both men and women) in anal/genital contact has the greatest risk for mv infection.? In vaginal intercourse, the infection could occur most readily when the cervical os is open during menses or shortly thereafter; infected cells could then find their way via the cervical os into the endometrium where the resident macrophages and lympJ;lOcytes can spread mv in the host. 5 These observations emphasize the role of the infected cell as a source of infection during sexual contact. Transmission by blood and the maternal route may also result primarily via infected cells. Vaccines must therefore elicit cytotoxic responses to eliminate these sources of infection. Virus Integration
An important feature of the infected cell is the integration of the viral genome into the cellular
Transfusion Medicine Reviews, Vol 2, No 4 (December), 1988; PP 264-271
CAN AN AIDS VACCINE BE DEVELOPED?
Table 1. Features of HIV Infection That Affect Vaccine Development 1. 2.
3. 4. 5. 6. 7.
The infected cell is a major source of virus transmission. Virus infection involves integration of the viral genome into the chromosome of the infected cell. This cell becomes a reservoir for persistent virus production, The infected cell can transfer the virus by cell-to-cell contact. The infected cells can remain "latent" and express very few viral antigens. Several independent serotypes and subtypes of HIV can be identified. HIV infection occurs at specific sites in the host (eg, the rectum), Portions of HIV proteins resemble normal cellular proteins.
chromosome. The cell does not die quickly and can continue producing progeny virus for days, up to months. 4 Macrophages that are not as susceptible as T cells to the cytopathic effects of HIV are particularly effective reservoirs for this transfer of the virus in the host, as well as to other individuals. 4 ,5 Thus, as noted above, elimination of the virus requires killing of its infected cell. Cell-to-Cell Contact
Whereas virus transmission can take place by direct infection, in many and perhaps most cases, cell-to-cell contact is involved. The HIV envelope gp41 protein appears to mediate this intracellular contact via fusion. 4 ,8 This mechanism for virus spread would not be prevented by anti-HIV neutralizing antibodies. A cytotoxic response is the best means of control. Latency
In some individuals, the infected cell may remain in a latent state in which very little viral protein or RNA is made. Thus, without sufficient expression of viral antigens, the cell would not be readily recognized by the immune system and could enter the individual unchallenged. Encountering host cells, these infected cells could be activated to produce virus progeny and transmit the infection. 4 HIV Serotypes
Another noteworthy feature of HIV is its various serotypes; many may need to be incorporated into a vaccine to give protection against all HIV. Chim-
265
panzees inoculated with one strain of HIV can be superinfected by another. 9 Most likely humans can also be infected by more than one HIV strain. In addition, a new subtype of HIV, HIV-2,lO has been recognized in West Africa and is spreading throughout the world. Some individuals have evidence of infection by both types of HIV. 4, 11 Clearly, infection by one virus may not prevent infection by another. HIV-2 is associated with AIDS, but differs considerably in its envelope region from HIV-l. It may presage other related human lentiviruses that must be controlled by an anti-HIV vaccine. Local Immunity If indeed a major source of HIV transmission is via the rectal mucosa, a strong immune response at this site would be required to ward off the initial infection. Similarly, in women, local immunity in the vagina and endometrium would be necessary to prevent transmission of HIV, particularly by the infected cell. Whether this response can be elicited by conventional vaccine approaches must be evaluated.
Autoimmunity
One other potential complication of HIV vaccines is the possibility that antibodies against the viral proteins will cross-react with normal cellular proteins. Autoimmunity has been described in individuals with HIV infection, but in most cases, these autoimmune syndromes appear to result from a polyclonal activation of B cells and hypergammaglobulinemia that commonly occurs with viral infections. 4 Because several regions of HIV resemble portions of normal cellular proteins 4 (Table 2), the induction of a reaction against these cellular counterparts might occur following vaccination. For this reason, the present trials in human volunteers using the gp160 envelope protein made in the baculovirus system (insect cells) should be very informative. Immunization of 48 volunteers has thus far taken place with no reported side effects. Nevertheless, the possible induction of autoimmunity or other compromising events to the immune system must be considered, particularly if antibodies to interleukin-2 (IL-2) or interferon are elicited. HUMORAL IMMUNE RESPONSES TO HIV
Standard procedures for preventing virus infections such as measles, mumps, polio, and chicken
266
JAY A. LEVY Table 2. Regions of HIV that Cross-react with Cellular Proteins Shared Region
IL-2 'Y- 1FN
",-1 Thymosin Neuroleukin (phosphohexose isomerase) Peptide T HLA antigen HLA antigens
Location*
LTR, env LTR gag (p17)
env env env virion surfacet
Abbreviations: IFN. interferon; IL-2. interleukin-2. * Denotes region of HIV that has potential cross-reactivity with a normal cellular protein as deduced from sequence analysis. 4 t Incorporated into the viral envelope from the cell surface.
pox, have been based on the ability of the vaccination program to induce humoral immune responses. These lead to production of neutralizing antibodies that inactivate the incoming free viral agent. The above discussion suggests that this type of immunity would give only limited protection against HIV, because the virus-infected cells would not necessarily be killed. Nevertheless, the initial studies with HIV were directed at understanding if neutralization of the virus can be observed with sera from infected individuals. These studies frrst indicated very low titers of neutralizing activity in infected individuals. However, with recent improvements in these assays, substantial levels have been found in many infected individuals including those suffering from severe disease. 4,12 This latter observation has led to the conclusion that these antibodies, produced following infection, are probably not helpful in protecting against progression of disease. Work by Dr Cheng-Mayer et al have shown that different mv isolates can be distinguished by their sensitivity to neutralization;12 some are easily neutralized by certain sera, others are not. In particular, viruses from Africa appear to be neutralized only by individuals coming from that continent, and similar observations have been made from studies of Haitian isolates. This neutralization appears to be mediated primarily by an interaction of antibody with the viral envelope, gp120, that can be highly variable. 4,13 Thus, it seems certain that if neutralization is an important part of antiviral approaches, polyvalent vaccines using envelope proteins from a variety of HIV will be needed for effective protection.
A portion of the viral transmembrane protein, gp41, is also exposed on the outside surface of the virus 4 and antibodies to this envelope protein have shown some neutralizing activity as well. 14 This result is promising, because regions of gp41 are shared by more mv isolates than gp120. However, because all HIV appear to use a similar mechanism for attachment to the cellular receptor (the CD4 molecule),4 antibodies against this attachment site (presumed present on gp120) would appear to be the most conserved among all mv. Evidence indicates that mv-2 also uses the CD4 protein for infection of T cells. However, the particular viral epitope involved has not yet been identified. Nevertheless, recent data suggest that other cellular receptors or means of virus activity may be involved, because cells lacking the CD4 antigen can be infected by HIV. 4,15 Most recently, observations have indicated a property of mv infection that was not expected. Some antibodies to the virus may actually help it enter macrophages and T cells. 15a This presumed opsonization of virus-antibody complexes leading to infection poses an important problem for vaccine approaches: The induction of non-neutralizing antibodies might facilitate the infection rather than prevent it. One humoral immune response that could be of benefit in eliminating the virus-infected cell is antibody-directed cellular cytotoxicity (ADCC). This assay has indicated that anti-envelope antibodies (both gp120 and gp41) are involved in the killing of infected cells by the host's natural killer (NK) or T cells. 16 These antibodies do not appear to decrease substantially during development of AIDS, and thus progression of disease must result from a reduction in the number or function of these effector cells in the host. ADCC could be extremely important soon after infection, because this response could immediately kill incoming i!rl'ected cells and prevent establishment of the infection. CELLULAR IMMUNE RESPONSES TO HIV
Studies of cell-mediated .immune responses in mY-infected individuals have been limited. Recent data have indicated that cytotoxic activity is associated with NK cells or T cells from infected individuals. 17 ,18 These observations suggest that vaccination programs might elicit a strong cellular immune response against the infected cell. Studies have also identified T helper cells with specific
267
CAN AN AIDS VACCINE BE DEVELOPED?
responses to selective epitopes on the viral envelope. 19 These appear to be different from those recognized by antibodies. Thus, it appears that the humoral and cellular immune responses are directed against certain components of HIV and the specificity of each may differ. Both types of epitopes may need to be incorporated into a vaccine. Then, a reaction against not only the virus but also, most importantly, the infected cell can be elicited. The combination of ADCC and a strong cellular immune response would seem at present to be the most effective way of preventing HIV transmission by infected cells. Neutralizing antibodies with good cross-reactivity with a variety of different HIV would be the major mechanism for preventing infection by free virus. APPROACHES CONSIDERED FOR HIV VACCINES
Inactivated Virus
Inactivated virus vaccines have been a major approach for preventing infections with other human viruses such as polio, smallpox, and yellow fever (Table 3). A formalin-inactivated type D simian retrovirus vaccine has shown promise in prevention of SAIDS in monkeys. 20 However, this method has not been considered seriously for HIV because of the potential danger of a virus surviving the inactivation procedure. Such an event did occur during development of some of the inactivated human viral vaccines. 21 Moreover, studies with paramyxoviruses, that are also enveloped viruses like retroviruses, have shown other complications because the inactivated virions used did not elicit immune responses to all the viral proteins. With respiratory syncytial virus (RSV) , for instance, vaccination with inactivated virus gave rise to severe disease in some immunized infants subsequently infected by RSV. 21 The reason for this observation is not certain, but the data suggest that
vaccination against inactivated virus elicits antibodies to many of the RSV proteins, but not to the protein responsible for fusion of the virus with the cell surface membrane. Thus, virus entry by fusion and spread by cell-to-cell contact can occur, despite the presence of antiviral antibodies. The infected cells that then become established in the host express virus and viral antigens that are attacked by the immune system of the host. These immune reactions with antibody-antigen complexes lead to strong inflammatory responses (eg, Arthus reaction) and more cytopathology (including pneumonias) than would normally have occurred during a natural RSV infection. Therefore, any inactivated virus must be effective in eliciting an immune response against all the viral proteins mediating infection, and the inactivation procedure must be completely effective. Non-Pathogenic Variants
Some researchers have suggested that manipulating HIV so that it no longer has its reverse transcriptase, protease, or its transactivating (tat) proteins could produce a non-infectious agent with the three-dimensional shape of a total virion. These approaches have not been considered seriously by investigators because of the potential that these modified viruses could still interact with genes (perhaps endogenous viral) in the host and emerge as fully infectious pathogenic virions. Using naturally occurring non-pathogenic strains as live vaccines is also an approach not considered seriously. With other human viruses, attenuated strains have made excellent vaccines (eg, polio, measles), but in rare cases, a virulent wild type virus has emerged. 21 Nevertheless, combining inactivation with the use of non-infectious variants may be a feasible approach for an HIV vaccine, since both the three-dimensional structure and the safety of the vaccine (even if some virions were not inactivated) might be better assured.
Table 3. Approaches to HIV Vaccines 1. 2. 3.
4. 5. 6.
Inactivated virus (natural or engineered) Non-pathogenic variants (natural or engineered) Subunit vaccine (natural or engineered) Envelope glycoprotein, gp120 Envelope-transmembrane glycoprotein, gp41 Gag protein Viral proteins in infectious recombinant virus (eg, vaccinia) Sequence-derived peptides of HIV Anti-idiotypes of neutralizing antibodies
Viral Proteins-Natural or Genetically Engineered
This approach is aimed at eliminating all nucleic acids from the vaccination material and using proteins that are required for protective anti-HIV responses. These proteins would be either purified naturally (limited in part by the feasibility and expense of growing large amounts of virus) or produced by genetically engineered procedures em-
268
ploying bacteria, yeast, insect, or mammalian cells. The latter approach would be the most appropriate, because normal glycosylation of the viral proteins (eg, the envelope) would occur, and thus elicit a more effective immune response. The problems inherent in this approach involve determining which proteins are important and producing these proteins in sufficient amounts. Moreover, because the proteins removed from the structure of the virion would not have the threedimensional shape of the virus, an appropriate response of the immune system might not take place. For this latter reason, some investigators are considering the incorporation of viral proteins into liposomes (or lipid vesicles) that would simulate a virion structure. Viral Proteins In Infectious Recombinant Viruses
Another mechanism for presenting HIV proteins to the host is to incorporate their genes into the genome of other infectious viruses such as vaccinia (smallpox) and baculoviruses. In this way, immunization can be performed with a live virus that will express the mv proteins in infected cells and elicit an immune response to these virus proteins in a manner similar to natural infection. The virus vector may also help stimulate this immune response. This approach has induced neutralizing antibodies and cell-mediated responses when vaccinia-HIV recombinant viruses were used. Nevertheless, these vaccines (using gp 120 or gp 160) have not protected immunized animals from virus challenge. 34 Synthetic Peptides
An alternative approach to using proteins produced via viral genes is to synthesize the peptides coded for by the known viral sequences. This approach completely dismiss the use of viral genes, and in principal would be safe from any possible contamination. Nevertheless, these peptides without the correct glycosylation and three-dimensional form could be potentially less effective in inducing an effective immune response to HIV. Thus far, they have been useful in evaluating the natural immune response in infected individuals. Anti-Idiotype Vaccine
A relatively new approach to vaccines initially uses an antibody that neutralizes the virus as the immunogen. Because this antibody interacts with
JAY A. LEVY
the epitope on the virus that appears required for infection, the antibody to this antibody (the antiidiotype) should be a mirror image of the virus epitope itself. The subsequent use of this anti-idiotype in vaccination procedures should then elicit an immune response against the virus. This approach with HIV would avoid any contact with viral peptides or infectious virus, and is considered the safest approach for a vaccine. It has been successful in eliciting protective neutralizing antibodies to hepatitis B virus 22 and has been used in preliminary studies to make antibodies to the mv envelope protein. 23 Nevertheless, this method for mv must produce not only neutralizing antibodies but also cellular immune responses. This latter parameter thus far has not been measured. Moreover, adverse effects may result from this approach, particularly if the anti-idiotype antibody attacks, in the immunized individual, the viral receptor on normal cells. 21 PRELIMINARY STUDIES AIMED AT DEVELOPMENT OF AN ANTI-HIV VACCINE
The initial attempts to evaluate the feasibility of an anti-HIV vaccine concentrated, as expected, on the outside envelope region. Studies in a variety of animal species used either purified gp120 from virus, synthetic peptides, genetically engineered cells, or recombinant forms of this protein. 24-36 These latter products were made by direct transfection of viral genes into Escherichia coli, yeast, or mammalian cells (Chinese hamster ovary) or by using vectors such as vaccinia or baculovirus. All these experiments successfully produced antigp120 and gp41 antibodies (Table 4). Virus neutralization by some sera from immunized animals was also achieved, but at very low titer « 1 to 20), and in nearly all cases was type-specific. Only the virus whose envelope protein was used for the immunization was recognized by the animal sera. However, one recent study inrabbits using synthetic peptides from the second conserved region of gp120 gave promising results on neutralizing antibodies and cross-reactivity. 31 Conceivably, with different adjuvants, these immune responses could be increased. Nevertheless, these early studies do suggest that polyvalent vaccines will need to be used to induce a response to all HIV isolates. Very few of these immunization studies evaluated T cell responses, which, as noted above, are an important part of the defense mechanism
CAN AN AIDS VACCINE BE DEVELOPED?
269
Table 4. Research Aimed at Developing an Anti·HIV Vaccine Immunogen
Animal
Response
Investigators
Mouse Mouse
v-env (gp160) v-env5 v-env2
anti-gp120 anti-gp41 anti-gp120
Chakrabarti et al 24 Hu et al 25
Guinea pig, Rabbit Goat, Horse, Rhesus monkey Goat
gp120, (CHO cell) gp 120 (cellular)
anti-gp120 (neutraliz) (neutraliz)
Lasky et al 26 Robey et al 27
gp120, (E coli)
Putney et al 28
Rabbits Mice Rabbit Goat Rabbit
synthetic peptide (gp120)
anti-gp120 anti-gp41 (neutraliz) anti-gp120 (neutraliz)
Kennedy et al 29
gp120, gp120r (cellular, E coli) synthetic peptide (gp120)
anti-gp120 (neutraliz)
Krohn et al 30 Ho et al 31
Goat Macaque
v-env5
Chimpanzee
v-env 5 (gp160)
Chimpanzee Human
gp120 (virus) v-env, (gp160)
anti-gp120 (cross-neutraliz) neutraliz anti-gp41 anti-gp120 T cell response anti-env gp41 (no neutraliz) T cell response no protection neutraliz neutraliz T cell response
synthetic peptide (gp120)
Palker et al 32 Zarling et al 33 Hu et al 34 Zarling et al 35 Arthur et al 36 Zagury et al 39
Abbreviations: v, vaccinia virus vector; env, envelope; neutraliz, neutralizing antibodies; r, recombinant protein.
against the virus and infected cells. Some experiments with chimpanzees using gp120 or a vaccinia-recombinant gp160 (v-env-5) have shown the induction of both anti-envelope antibodies and T cell responses. 34- 36 Nevertheless, these studies have been limited and none of the immunized chimpanzees had high levels of neutralizing antibodies nor were protected from subsequent challenge. 34 Thus far, immunization procedures have not given the type or extent of immune response in these animals as has direct virus infection. 37 Some results have suggested that antibodies to the viral p17 (that presumably cross-reacts with a thymosin) can neutralize HIV. 38 This observation probably reflects the presence of the viral protein just below the virus outer membrane where an external portion could be present. 4 Experimental studies to evaluate a vaccine using this human gag protein are planned. Finally, Zagury et al 39 have immunized seronegative human volunteers with a vaccinia-recombinant HIV envelope virus. These studies produced neutralizing antibodies at a titer of 1 to 40 against the HTLV-IIIB strain of HIV represented in the vaccine. A much lower level of neutralization was found to the RF strain of HIV from Haiti. Some cellular immune responses were
also noted to the HTLV-IIIB strain, and to a lesser extent against the RF strain. Further boosters are planned to determine if these approaches can give substantial amounts of neutralizing antibodies and strong cellular immune responses. These studies represent the only human trials of a vaccine that ,have yielded any results. The studies cited above with purified gp 160 produced in insect cells are now in progress. CONCLUSIONS
As discussed in the introduction, a retrovirus, and particularly one from the lentivirus subfamily, poses substantial problems in the development of a vaccine. While the approaches described above are being considered by laboratories throughout the world, the chance of success with anyone of them seems limited because of the properties of HIV infection (Table 1). No successful prototype lentivirus vaccine has yet been developed. The trials thus far with purified envelope proteins from the virus, synthetic peptides of the envelope of the virus, and recombinant viruses (eg, vaccinia) carrying the envelope viral gene have led to the development of anti-envelope antibodies, but with
270
JAY A. LEVY Table 5. Ideal Properties of an Anti-HIV Vaccine
1. 2. 3. 4. 5. 6. 7.
Elicits neutralizing antibodies that react with all HIV strains and subtypes. Induces cytotoxic responses against virus-infected cells. Induces immune responses that recognize latently infected cells. Does not induce antibodies that enhance HIV infection. Induces local immune response at all sites of HIV entry in the host. Vaccine is safe, showing no toxic effects. The effect of the vaccination procedure is long-lasting.
low neutralizing activity in the immunized animals. Most importantly, no protection from challenge by HIV was observed. 34 Moreover, studies of cell-mediated immune responses that are the most important in virus infection have been limited. Because the immunization procedures were not successful against free virus inoculation, prevention of infection after a challenge with infected cells would be even less likely to occur. Thus, the possibility of an anti-HIV vaccine in the near future seems remote. The problems that are involved in the development of a vaccine against HIV are considerable, and the approaches being considered must address these issues successfully (Table 5). Whether an effective vaccine will be developed depends on the capability of an immunization strategy employing an appropriate adjuvant to elicit a strong response of both humoral and cellular origin. All HIV serotypes and subtypes must be recognized as well as the infected and latently infected cell. Antiviral antibodies must inactivate and not potentiate the infection. Moreover, the response must be rapid and present at all sites of virus entry. In this way,
a virus-infected cell entering the host by any route can immediately be recognized and destroyed before it is able to fuse with a host cell or release progeny that is infectious. And, this vaccine must be completely safe, nontoxic, and long-lasting. The latter feature is particularly important if the vaccine is to be effective in remote areas where administering frequent boosters would be difficult. The recent reports with malaria vaccines offer some hope,40,41 but the results are very preliminary. The use of several proteins linked by polymerization gave effective prevention in some individuals. 41 A similar approach employing several viral proteins presented in a highly immunogenic manner may also be effective with HIV. The malarial parasite, like HIV, establishes infection of host cells within minutes after entry, and has antigenic heterogeneity. However, with malaria, only the free infectious agent and not an infected cell generally needs to be considered. Thus, cytotoxic responses may be more important in HIV prevention. Similarities to HIV can be drawn as well from the difficulties encountered in making vaccines to the herpesviruses, particularly EpsteinBarr virus (EBV) and cytomegalovirus (CMV). These agents can also be passed by the infected cells, and have different serotypes. Prevention and control, as with HIV, depend on an active cellular immune system. Progress has been substantial in understanding the features of HIV infection and the potential immunologic responses to the virus. Hopefully, continuing studies will bring to light a direction that will eventually lead to a safe and effective vaccine. Nevertheless, without a new and creative approach to this problem, the solution will certainly take time.
REFERENCES 1. Levy JA, Kaminsky LS, Morrow WJW, et al: Infection by the retrovirus associated with the acquired immunodeficiency syndrome. Ann Intern Med 103:694-699, 1985 2. Haase AT: Pathogenesis of lentivirus infections. Nature 322:130-136, 1986 3. CurranJW, Jaffe HW, Hardy AM, et al: Epidemiology of HIV infection and AIDS in the United States. Science 239:610-616, 1988 4. Levy J: The human immunodeficiency yirus (HIV): Detection and pathogenesis, in Levy J (ed): AIDS: Pathogenesis and Treatment, New York, Dekker, (in press) 5. Levy JA: The transmission of AIDS: The case of the infected cell. JAMA 259:3037-3038, 1988
6. Nelson JA, Wiley CA, Reynolds-Kohler C, et al: Human immunodeficiency virus detected in bowel epithelium of patients with gastrointestinal symptoms. Lancet 1:259-262, 1988 7. Winkelstein W Jr, Lyman DM, Padian N, et al: Sexual practices and risk of infection by the human immunodeficiency virus: The San Francisco men's health study. JAmMed Assoc 257:321-325, 1987 8. Sodroski J, Goh WC, Rosen C, et al: Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. Nature 322:470-474,1986 9. Fultz PN, Srinivasan A, Greene CR, et al: Superinfection of a chimpanzee with a second strain of human immunodeficiency virus. J ViroI61:4026-4029, 1987
CAN AN AIDS VACCINE BE DEVELOPED?
10. Clavel F, Guetard D, Brun-Vezinet F, et al: Isolation of a new human retrovirus from West African patients with AIDS. Science 233:343-346, 1986 11. Denis D, Barin F, Gershy-Damet G, et al: Prevalence of human T lymphotropic retroviruses type III (HIV) and type IV in Ivory Coast. Lancet 1:408-411, 1987 12. Cheng-Mayer C, Homsy I, Evans LA, Levy IA: Identification of HIV subtypes with distinct patterns of sensitivity to serum neutralization. Proc Nat! Acad Sci (USA) 85:2815-2819, 1988 13. Starcich BR, Hahn BH, Shaw GM, et al: Identification and characterization of conserved and variable regions in the envelope gene of HTLV-III/LAV, the retrovirus of AIDS. Cell 45:637-648, 1986 14. Chanh TC, Dreesman GR, Kanda P, et al: Induction of anti-HIV neutralizing antibodies by synthetic peptides. EMBO 15:3065-3071, 1986 15. Cheng-Mayer C, Rutka JT, Rosenblum ML, et al: The human immunodeficiency virus (HIV) can productively infect cultured human glial cells. Proc Nat! Acad Sci (USA) 84:3526-3530, 1987 15a. Homsy J, Tateno M, Levy I: Antibody-dependent enhancement of HIV infection. Lancet 1:1285-1286, 1988 16. Evans LA, Thomson-Honnebier G, Steimer K, et al: Antibody-dependent cellular toxicity (ADCC) is directed against envelope proteins of the human immunodeficiency virus. (submitted for publication) 17. Walker B, Chakrabarti S, Moss B, et al: HIV-specific cytotoxic T lymphocytes in lung disorders. Nature 328: 345-348, 1987 18. Plata F, Autran B, Martins LP, et al: AIDS virus-specific cytotoxic RT lymphocytes in lung disorders. Nature 328:348-351, 1987 19. Walker CM, Steimer KS, Rosenthal KL, et al: Identifi- . cation of HIV envelope type-specific T helper cells in an HIV-infected individual. (submitted for publication) 20. MarxPA, Pedersen NC, Lerche NW, et al: Prevention of simian acquired immune deficiency syndrome with a formalin-inactivated type D retrovirus vaccine. I Virol 60:431-435, 1986 21. Hilleman MR: Conclusions: In pursuit of an AIDS virus vaccine, in Levy I (ed): AIDS: Pathogenesis and Treatment, New York, Dekker, (in press) 22. Kennedy RC, Eichberg JW, Lanford RE, Dreesman GR: Anti-idiotypic antibody vaccine for type B viral hepatitis in chimpanzees. Science 232:220-223, 1986 23. Zhou E-M, Chanh TC, Dreesman GR, et al: Immune response to human immunodeficiency virus. In vivo administration of anti-idiotype induces an anti-gp 160 response specific for a synthetic peptide. I Immunol 139:2950-2956, 1987 24. Chakrabarti S, Robert-Guroff M, Wong-Staal F, et al: Expression of the HTLV-III envelope gene by a recombinant vaccinia virus. Nature 320:535-537, 1986 25. Hu SoL, Kosowski SG, Dalrymple 1M: Expression of AIDS virus envelope gene in recombinant vaccinia viruses. Nature 320:537-540, 1986
271
26. Lasky LA, Groopman IE, Fennie CW, et al: Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein. Science 233:209-212, 1986 27. Robey WG, Arthur LO, Matthews TJ, et al: Prospect for prevention of human immunodeficiency virus infection: Purified 120-kDa envelope glycoprotein induces neutralizing antibody. Proc Natl Acad Sci (USA) 83:7023-7027, 1986 28. Putney SD, Matthews TJ, Robey WG, et al: HTLV-III! LAV-neutralizing antibodies to an E coli-produced fragment of the virus envelope. Science 234: 1392-1395, 1986 29. Kennedy RC, Dreesman GR, Chanh TC, et al: Use of a resin-bound synthetic peptide for identifying a neutralizing antigenic determinant associated with the human immunodeficiency virus envelope. J BioI Chern 262:5769-5774, 1987 30. Krohn K, Robey WG, Putney S, et al: Specific cellular immune response and neutralizing antibodies in goats immunized with native or recombinant envelope proteins derived from human T-lymphotropic virus type IIIB and in human immunodeficiency virus-infected men. Proc Nat! Acad Sci (USA) 84:4994-4998, 1987 31. Ho DD, Kaplan IC, Rackauskas IE, Gurney ME: Second conserved domain of gp 120 is important for HIV infectivity and antibody neutralization. Science 239: 1021-1023, 1988 32. Palker RI, Clark ME, Langlois AI, et al: Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides. Proc Natl Acad Sci (USA) 85:1932-1936, 1988 33. Zarling 1M, Morton WM, Moran PA, et al: T-cell responses to human AIDS virus in macaques immunized with recombinant vaccinia viruses. Nature 323:344-346, 1986 34. Hu SoL, Fultz PN', McClure HM, et al: Effect of immunization with a vaccinia-HIV env recombinant on HIV infection of chimpanzee. Nature 328:721-723, 1987 35. Zarling 1M, Eichberg JW, Moran PA, et al: Proliferative and cytotoxic T cells to AIDS virus glycoproteins in chimpanzees immunized with a recombinant vaccinia virus expressing AIDS virus envelope glycoproteins. I Immunol 139:988-990, 1987 36. Arthur LO, Pyle SW, Nara PL, et al: Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp 120) subunit vaccine. Fmc Nat! Acad Sci (USA) 84:8583-8587, 1987 37. Morrow WJW, Homsy I, Eichberg IW, et al: Immunosuppressive therapy did not induce disease in baboons, rhesus monkeys, and chimpanzees inoculated with the human immunodeficiency virus (HIV) (submitted for publication) 38. Sarin PS, Sun DK, ThomtonAH, et al: Neutralization of HTLV-III/LAV replication by antiserum to thymosin alpha-I. Science 232:1135-1137, 1986 39. Zagury D, Leonard R, Fouchard M, et al: Immunization against AIDS in humans. Nature 326:249-250, 1987 40. Miller LH, Howard RI, Carter R, et al: Research toward malaria vaccines. Science 234:1349-1356, 1986 41. Patarroyo ME, Amador R, Clavijo P, et al: A synthetic vaccine protects humans against challenge with asexual blood stages of Plasmodiumfalciparum malaria. Nature 332: 158-161, 1988