|HIV Vaccine Development: From Basic Research to Human Trials (UNAIDS, 1996, 33 p.)|
Reference: AIDS 1996, 10 (suppl A): S123-S132.
Jossparza, William L. Heyward and Saladin Osmanov
From the Joint United Nations Program on HIV/AIDS
Request for reprints to: Dr. Jossparza, UNAIDS, 20 Avenue
CH-1211, Geneva 27, Switzerland.
There is general agreement that a safe, effective and available HIV preventive vaccine is urgently needed to bring the HIV/AIDS epidemic under control [1, 2]. However, little agreement exists on what should be the characteristics of such a vaccine(s), the type of immune responses these vaccines must induce, and when and how to proceed to Phase III trials to evaluate vaccine protective efficacy in humans [3 - 13]. Some proponents feel that further basic research is needed and maintain that efficacy trials should not be initiated until we have a better understanding of the immune correlates of protection against HIV infection in humans, and new candidate vaccines are developed based on such information . Others argue that some of the existing candidate vaccines based on envelope recombinant proteins have been shown to induce protective immunity in chimpanzees, are safe and immunogenic in Phase I/II trials in humans, and are reasonable candidate vaccines available now for Phase III efficacy trials [9, 13 - 17]. In this article we propose that a parallel development process - additional basic research of different vaccine concepts in close interaction with vaccine efficacy trials - may represent the most rational approach to accelerate the process of HIV vaccine development.
The need for further basic research
AIDS may be different from most other vaccine-preventable diseases in that HIV infection may persist or AIDS may develop in spite of a broad immune response by the host. Therefore, the principles used for the development of other viral vaccines may not work in the case of HIV/AIDS, emphasizing the need for more basic research to understand better the mechanisms of protective immunity to HIV infection in humans [5, 10].
Immunological correlates of protection
The major conceptual problem for HIV vaccine development is the lack of information on immune responses known to correlate with protection against HIV infection or disease. In this regard, there has been controversy about whether a vaccine must induce humoral and/or cell-mediated immunity for effective protection and about the potential importance of inducing mucosal immunity.
One possible research approach to identify the immunological correlates of protection is to study situations in which specific host responses might be involved in controlling HIV infection. However, it is important to keep in mind that the immune responses needed to prevent the initial replication of HIV after exposure, might not necessarily be the same as those needed to contain, control, and potentially resolve the infection once it has been established in an individual. It is conceivable that even a highly efficient barrier immunity could allow some early, limited virus replication which in turn will boost certain qualities of the preexisting immune responses, conferring protection against the establishment of chronic infection, or against development of disease.
One situation in which immune responses may be effective in controlling HIV replication is the decline in virus load following the initial high-level viraemia which is characteristic of primary infections. This decline coincides with the development of HIV-1 specific CD8+ cytotoxic T lymphocytes (CTLs), before the appearance of neutralizing antibodies, suggesting a role for cell-mediated immunity in the initial control of HIV infection [18 - 22]. Another approach has been pursued by studying HIV-infected long-term non-progressors [23 - 30]. In fact, low levels of viraemia appear to be the most significant prognostic marker for prolonged survival in HIV-infected persons [28, 31, 32], a situation which has also been observed in the SIV/macaque model [33 - 35]. Correlates of protection might also be identified by studying persons who remain apparently uninfected even after frequent and/or extensive exposure to the virus, either perinatally or sexually . In these cases, it has been hypothesized that such resistance could be due to genetic factors, alloimmunization to cellular antigens, or naturally acquired cell-mediated immunity as a result of exposure to subinfectious doses of the virus .
Results from the above studies are providing initial evidence for the importance of CTLs in controlling HIV replication in infected persons. However, more cautious views have been expressed about the possible role of CTLs in preventing or clearing HIV infection [37 - 41]. Likewise, data about the role of neutralizing antibodies in preventing disease progression in humans is also inconclusive. Anti-HIV antibodies administered passively, or actively induced by candidate vaccines, have been shown to protect experimental animals from HIV infection, indicating that neutralizing antibodies may also play a role in protection against HIV infection [42 - 44].
Therefore, at this stage of vaccine development, it might be wise to assume that both arms of the immune system may play a role in protective immunity against HIV infection and disease, and that preventive vaccines may need to induce both types of immune responses .
Antigenic significance of HIV genetic variability
Vaccine-induced protection may be affected by the antigenic variability resulting from the genetic differences among envelope genes. Phylogenetic analysis of the nucleotide sequences of the env and gag genes of numerous HIV-1 virus strains has resulted in their classification within a major M-group, which is subdivided into at least nine genetic subtypes (A-I), and a genetically distant outlier O-group, with different subtypes unevenly distributed in different parts of the world [45 - 47]. This genetic variability is generally perceived as a potential major obstacle to the development of broadly effective HIV vaccines. However, at the present time it is not clear what is the significance of genetic variability in relation to the antigenic characteristics of HIV-1 [46, 47].
The principal neutralization domain in gp120 is located within one of the five hypervariable regions of the molecule, known as the V3 loop, which induces the production of strain-specific neutralizing antibodies. In addition, a number of conformational epitopes are also present in gp120, and the neutralizing antibodies which they induce are more broadly reactive, some of them being capable of preventing the binding of gp120 to the CD4 receptor molecule . Recent studies with human monoclonal antibodies suggest that most gp120 conformational epitopes involved in CD4-binding and virus neutralization are conserved among strains belonging to different genetic subtypes [48, 49]. In addition, cross-neutralization assays of HIV-1 primary isolates from different genetic subtypes, using autologous and heterologous plasma in in vitro assays using peripheral blood mononuclear cells (PBMC), have failed to define any pattern of genetic subtype-specific neutralization [unpublished data from the WHO Network for HIV Isolation and Characterization [50, 51]. Moreover, cross-neutralization has also been reported between group-M and group-O viruses . Such cross-neutralizing antibodies are probably directed to the conformational epitopes of gp120.
These studies indicate that genetic subtypes of HIV-1 do not represent classical virus neutralization subtypes, although earlier studies suggested that subtypes B and E viruses from Thailand may belong to two different neutralization immunotypes . However, it is not possible to rule out the existence of neutralization immunotypes, although they may not necessarily correspond to env genetic subtypes. Nevertheless, these results are very encouraging, because they indicate extensive antigenic conservation among HIV-1 strains from different subtypes, and if neutralizing antibodies are important for vaccine-induced protection, then development of a broadly effective vaccine may be possible.
Biological variability of HIV
The genetic variability of HIV-1 strains has also been correlated with in vitro growth characteristics of the virus, some of which may be relevant for vaccine development. Some strains, referred to as slow/low, replicate slowly and to low titers in PBMC cultures, do not replicate in T-cell lines and are not syncytia-inducing (NSI). In contrast, other strains, referred to as rapid/high, are able to efficiently replicate not only in PBMC but also in T-cell lines, and to induce syncytia (SI) [54 - 56]. Most viruses isolated from recently infected persons exhibit the slow/low-NSI phenotype, whereas viruses isolated from patients with more advanced disease are of the rapid/high-SI phenotype. These findings suggest that HIV is usually transmitted as a NSI virus and later evolves to SI forms with disease progression [57 - 59]. HIV strains have also been distinguished according to their in vitro passage history. Viruses which have only been passed very few times in PBMC, and have never been grown in T-cell lines, are known as primary, clinical or field isolates. On the other hand, laboratory strains are those which have been adapted to grow in T-cell lines. In general, laboratory strains have the SI phenotype, whereas primary isolates can be either SI or NSI viruses.
Of potential relevance to vaccine development is the observation that primary and laboratory isolates of HIV differ in their susceptibility to neutralizing antibodies. Laboratory strains can be neutralized by sera from infected individuals and by antibodies induced by experimental vaccines in animals or humans. On the other hand, primary isolates are only marginally neutralized by sera from infected persons and are resistant to neutralization by sera from immunized volunteers [3, 60 - 62]. This differential neutralization is probably due to a different configuration of the envelope glycoprotein in primary and laboratory-adapted strains [63 - 70]. More specifically, the principal neutralization domain of gp120 (the V3 loop) seems to be relatively inaccessible among NSI clinical isolates, whereas it is more accessible among laboratory strains which have been adapted to continuous culture in established T cell lines. Natural HIV infection results in the development of neutralizing antibodies directed against both the V3 loop and the conformational epitopes of gp120. On the other hand, existing candidate vaccines based on monomeric gp120 or gp160, induce neutralizing antibody responses mainly directed against the V3 loop . This has led to speculation that the currently available candidate vaccines, which are based on laboratory strains of HIV, might not be able to induce protective immunity against naturally circulating viruses [3, 8]. It has been proposed that effective vaccines must induce neutralizing antibodies against conformational epitopes present on oligomeric gp120 molecules, which are expected to be more efficient in neutralizing primary NSI isolates, and therefore new experimental vaccines should be developed based on selected primary isolates. Contradicting this hypothesis are animal experiments to be discussed later, which indicate that protection against HIV infection could be achieved even in the absence of neutralizing antibodies against the challenge virus.
Animal protection experiments with experimental vaccines
The two animal models which have been extensively used for vaccine development are Simian Immunodeficiency Virus (SIV) infection in macaque monkeys and HIV-1 infection in chimpanzees [2, 71]. Protection experiments with HIV-2 infection in macaques are also being conducted. Experimental HIV-1 vaccines based on subunit recombinant envelope proteins produced in mammalian cells have reproducibly protected chimpanzees against HIV-1 infection. This is in contrast to experimental SIV vaccines, which have been notoriously poor in protecting macaques against SIV infection. This dichotomy in the ability of candidate vaccines to induce protection in these two primate models should be kept in mind when interpreting the relevance of these experiments for humans.
The first successful protection experiments in the SIV/macaque model used whole inactivated virus vaccines, although later studies indicated that the protection was largely due to immune responses against cellular proteins incorporated into both the immunizing and challenge virus, rather than to virus-specific immune responses [72 - 74]. Subsequent studies, using inactivated or subunit SIV vaccines (mostly envelope glycoproteins) have been less successful, conferring only partial protection against SIV infection, although in many cases they suppressed virus burden after challenge and slowed progression to disease [2, 34, 75 - 78].
Since subunit non-replicating immunogens are generally not efficient inducers of CD8+ CTLs, much work has been invested in the development of live-vector vaccines, especially poxvirus vectors. In this regard, it has been disappointing that vaccination of rhesus monkeys with a recombinant vaccinia vector expressing SIV envelope and core antigens was not more effective than subunit envelope proteins in protecting against the pathogenic SIVmac251 strain . It was earlier reported that envelope (gp160)-based vaccines, when used in a recombinant vaccinia virus priming and subunit protein boosting regime, protected macaques against infection by a cloned homologous SIVmne, but not against uncloned virus . More recent studies indicate that addition of gag and pol antigens to the immunization regime strengthen and broaden the immune response, achieving protection against uncloned SIVmne [81,82]. Since immunization with gag and pol antigens alone was not sufficient to induce any protection in the immunized animals, it was concluded that the inclusion of multiple antigens, inducing both humoral and cell-mediated immunity, is required for more effective protection. In contrast to the above described vaccine approaches, nef deleted attenuated SIV has been reported to induce solid protection against virus challenge . Although the mechanisms of protection in these animals are not well understood, this approach opens possibilities for the development of a new generation of live-attenuated vaccines . Interestingly, immunization of rhesus macaques with attenuated macrophage-tropic SIV was able to confer protection against challenge with a heterologous SIV, and the protective responses were associated with the appearance of cross-reactive neutralizing antibodies . However, the administration of a live-attenuated retrovirus to an uninfected person may have an appreciable degree of risk, necessitating further research to understand the potential risks and benefits before decisions could be made regarding the initiation of human trials [84, 86 - 90].
Although immunized macaques are difficult to protect against SIV challenges, macaques have been protected against HIV-2 infection using an HIV-2/ISCOM vaccine . Likewise, macaques have been protected against HIV-2 infection by different immunization regimes which included priming with recombinant poxvirus vectors (attenuated vaccinia or canarypox expressing the HIV-2 gag, pol and env genes) and boosting with baculovirus derived rgp160 in alum, although clear correlates of protective immunity were not identified . Intriguingly, a similar immunization approach, priming with attenuated vaccinia or canarypox HIV-1 recombinants and boosting with HIV-1 protein subunits, protected some immunized macaques against an HIV-2 challenge . This cross-protection is in agreement with the recent results suggesting that natural HIV-2 infection may confer protection against HIV-1 , indicating a greater potential for in vivo cross-protection among HIV viruses than what has been inferred from laboratory studies.
The only animal model where vaccine efficacy against HIV-1 infection can be tested is the chimpanzee. In this animal model, envelope subunit recombinant HIV-1 vaccines (rgp120 or rgp160) produced in mammalian cell systems, have reproducibly protected against intravenous cell-free homologous challenges . More importantly, chimpanzees immunized with envelope proteins derived from the MN strain, have been protected against infection with the SF2 strain, a heterologous subtype B virus, whose envelope amino acid sequence is approximately 18% different from that of the immunizing virus [15, 95 - 96]. The SF2 strain used in cross-challenge experiments was a primary isolate grown only in PBMC. In agreement with previous observations, sera from the immunized chimpanzees neutralized laboratory strains of HIV-1, but failed to neutralize in vitro the SF2 challenge virus. Nevertheless, the immunized chimpanzees were protected against the challenge with the SF2 virus. This observation should bring some caution in the interpretation of in vitro neutralization tests as predictors of potential vaccine efficacy against clinical isolates.
Experiments in chimpanzees have been very instructive in other ways. Data from different laboratories, using different immunization schedules, suggest that protection against HIV infection seems to correlate with the level of neutralizing antibodies against the immunizing virus [96,97]. Sterilizing immunity has been achieved when the level of neutralizing antibodies is high. However, with lower levels of neutralizing antibodies, partial protection was obtained with delayed and decreased viraemia after challenge, which in some cases became undetectable after a few weeks of follow-up . Thus, even in the absence of sterilizing immunity, vaccination modified the outcome of HIV infection, suppressing virus infection and reducing virus load. In fact, minimal replication of the infecting virus could boost the pre-existing immunity in the vaccinated animals, perhaps resulting in effective and long-lasting protection. In contrast to the protective immunity obtained with envelope subunit vaccines, probably mediated by antibodies, preliminary experiments using canarypox virus expressing gp160-MN proteins aimed at inducing CTLs, have failed to induce protective immunity in chimpanzees , which could be possibly explained by the low dose of vaccine used (Girard M, Personal communication, 1995).
The chimpanzee experiments described above indicate that intra-clade B heterologous protection may be obtained with subunit recombinant envelope vaccines. However, the question remains as to the ability of candidate vaccines to induce inter-clade heterologous protection. Preliminary experiments have suggested that chimpanzees chronically infected with a clade B virus appeared to be resistant to superinfection by a heterologous clade E virus when inoculated in the uterine cervix, but not when inoculated intravenously . However, replication of the superinfecting clade E virus was restricted and possibly cleared after six weeks of infection, a result which could be interpreted as non-sterilizing cross-clade protection . Results from cross-protection experiments in chimpanzees immunized against one HIV-1 subtype and challenged with another subtypes are eagerly awaited.
In addition, other novel vaccine approaches are being tested in animal models. One of the most exciting is the use of naked DNA, which may have many of the advantages of live attenuated vaccines, without many of its disadvantages.
Phase I/II safety and immunogenicity trials
Since 1987, more than 21 HIV-1 preventive candidate vaccines have been tested in Phase I trials to assess their safety and immunogenicity. Although the immune correlates of protection against HIV infection are not known, the first generation of candidate vaccines were aimed at inducing neutralizing antibodies, and most of them have been based on the subunit recombinant envelope concept (rgp120 or rgp160 produced in expression systems using baculovirus, yeast, or mammalian cells) or on synthetic V3 peptides. Newer generations of candidate vaccines, aimed at inducing both neutralizing antibodies and CTLs, are based on live recombinant vectors, especially using poxvirus vectors (vaccinia or canarypox), followed by boosting with subunit recombinant envelope vaccines or with synthetic V3 peptides. The initial candidate vaccines were mostly based on the LAI strain of HIV-1, although newer generations are based on SF2 and MN strains, which are more representative of subtype B viruses.
Results from earlier trials have been extensively reviewed elsewhere [2, 99 - 101]. In summary, candidate vaccines have been found to be safe and well tolerated in doses that are capable of inducing HIV-1-specific immune responses. These immune responses include binding antibodies (measured by gp120- or gp160-enzyme-linked immunosorbent assay, Western Blot, or V3 peptide binding assays), as well as functional antibodies which have the ability to neutralize virus infectivity, to mediate cell cytotoxicity, to block binding of gp120 to the CD4 molecule, and to inhibit fusion of HIV infected cells.
Subunit recombinant envelope vaccines produced in mammalian cells induce higher levels of neutralizing antibodies than those produced in baculovirus or yeast systems. It appears that rgp120 molecules are better inducers of neutralizing antibodies than rgp160. These antibodies are able to neutralize laboratory adapted strains of HIV-1, but not primary isolates, and in a dose-related fashion can also neutralize heterologous strains from the same HIV-1 subtype. All vaccines have been found to induce T lymphocyte memory as detected by HIV-1 specific lymphoproliferative responses. Likewise the subunit envelope vaccines are capable of inducing CD4+ CTLs in some immunized volunteers, although only immunization with recombinant live poxvirus vectors reproducibly resulted in CD8+ CTLs in approximately 20% of vaccinees .
More recent results have been published from Phase I clinical trials of several subunit recombinant envelope vaccines produced in mammalian cells: rgp120MN produced in Chinese hamster ovary (CHO) cells, using alum as adjuvant (Genentech, Inc, South San Francisco, California) ; rgp120SF2 produced in CHO cells and combined with a novel adjuvant, MF59 (Biocine, Emeryville, California) [15, 104]; and rgp160LAI produced in monkey kidney (Vero) cells, using alum and deoxycholate as adjuvant (Immuno AG, Vienna, Austria) [105 - 107]. In general, at least two injections of 50-300 µg of the immunogen was required to induce neutralizing antibodies against the homologous strain in the majority of volunteers. After three or more doses, most volunteers also developed neutralizing antibodies against other B subtype strains. Current research is exploring different dose schedules, and the initial data suggest that schedules with a rest period before the final booster immunization are superior to those with closely spaced immunizations [15, 107, 108]. Two of the candidate vaccines described above (rgp120MN and rgp120SF2) are presently being evaluated in a Phase II trial in the US among 300 healthy volunteers, to compare safety and immune responses in a population where an efficacy trial could be conducted in the future .
The use of live recombinant vector vaccines, especially using poxvirus vectors, is another vaccine concept which is being studied. In most cases they are combined with subunit recombinant envelope products, aiming at inducing both neutralizing antibodies and CD8+ CTLs. Early clinical trials indicated that priming with live recombinant vaccinia vector encoding gp160LAI (Oncogen/Bristol-Myers Squibb, Seattle, Washington) resulted in low levels of antibodies, although a booster immunization with a subunit rgp160LAI envelope product (MicroGeneSys, Meriden, Connecticut) resulted in increased antibody levels, which in some subjects also cross-reacted with heterologous subtype B viruses [109 - 111].
Due to the risk of adverse effects associated with administration of live vaccinia virus, especially to immunosuppressed persons, the use of other vectors is being explored, such as canarypox virus (ALVAC, Virogenetics, Troy, New York), an avian poxvirus which undergoes only an abortive replication cycle in mammalian species . Initial results indicate that administration of a nonreplicating canarypox vector expressing the HIV-1MN env gene (Pasteur Meux Serums et Vaccins, Marnes-la-Coquete, France) resulted in induction of high levels of env-specific CD8+ CTLs in 2 of 12 volunteers and did not induce antibody response even after two injections . On the other hand, when the canarypox priming was followed by a booster dose with rgp160MN/LAI (Transgene, Strasbourg, France), homologous neutralizing antibodies were detected in the majority of the volunteers, of whom almost half also had env-specific CTLs . To expand the humoral and cellular immune responses, a Phase I trial is presently being conducted in which priming with a canarypox vector expressing gp120MN/gag/protease is followed by a booster dose with a p24E-V3MN peptide .
Another vaccine approach being tested in Phase I trials is based on the use of synthetic V3 peptides. Data from ongoing trials with oligomeric V3-MAPS (multiple antigen-presentation systems) (United Biomedical, Inc., Hauppauge, New York) using HIV-1MN peptides, or a mixture of 15 peptides representing 5 different HIV-1 subtypes, have not yet been published . A V3MN loop synthetic peptide conjugated to purified protein derivative (V3-PPD) (Swiss Serum and Vaccine Institute, Berne, Switzerland) was capable of inducing neutralizing antibodies against the laboratory HIV-1MN strain, but not against other clade viruses .
Whereas most of the early preclinical investigations and early Phase I trials have been conducted in industrialized countries, repeat Phase I/II trials are now being conducted in developing countries including Brazil, China and Thailand. These repeat trials are important in order to evaluate the safety and immunogenicity of candidate vaccines in other populations and settings where endemic diseases, genetic factors, and nutritional conditions may differ, and to strengthen research infrastructures in preparation for Phase III trials. Two Phase I trials with the oligomeric V3-MAPS synthetic peptide candidate vaccine (Unite Biomedical Inc.) have been recently conducted in China and Thailand among healthy HIV-negative volunteers, and a similar trial is currently underway in Brazil. A Phase I/II trial with the rgp120MN/alum candidate vaccine (Genentech) is currently underway in Bangkok, Thailand, among HIV-negative injecting drug users and another Phase I trial with the rgp120SF2/MF59 candidate vaccine (Biocine) has recently begun in Thailand among healthy HIV-negative volunteers .
Phase I/II trials are providing important information on the safety and immunogenicity of candidate vaccines. However, these trials are not designed to provide information on the protective efficacy of these candidate vaccines. Since 1987, 1706 healthy volunteers have participated in Phase I/II trials of different candidate vaccines sponsored by the US National Institutes of Health, and they have been followed for more than 2.400 person-years . During these trials, 4 of 345 placebo recipients and 16 of 1361 vaccine recipients have become infected with HIV-1 due to continuous high-risk behaviour [101, 119; Clements ML, Personal communication, 1995]. One third of the infected vaccinees had only received one or two immunizations prior to exposure, and the others had received three or more immunizations. In one reported case, infection occurred after the full course of immunization and after neutralizing antibodies had developed, suggesting that the immunological response induced by the vaccine did not protect the volunteer from HIV-1 infection . These cases of breakthrough infections are being intensively investigated to try to understand the reasons for possible vaccine failure, or the possibility that pre-existing anti-HIV-1 immunity might alter the course of HIV-1 infection. However, thus far there is no evidence of enhancing antibodies in sera from vaccinees prior to infection, nor that the course of HIV-1 infection in vaccinees is different from that of unvaccinated controls.
When conducting trials in populations at high risk, infections among vaccinees should be expected since immunizations are given over a period of several months, and since no vaccine is 100% effective. For this reason, conclusions about vaccine efficacy should not be drawn from Phase I/II trials, which are solely designed to study the safety and immunogenicity of the candidate vaccines. Properly designed large-scale Phase III trials, involving thousands of volunteers at risk of infection, are required to appropriately address the question of vaccine protective efficacy.
Phase III efficacy trials
Although no country at this time has decided to proceed with a Phase III trial to determine the efficacy of candidate vaccines, several developed and developing countries are now actively preparing populations and research infrastructures for such trials [121 - 124]. These preparations include characterization of potential trial populations and the establishment of cohorts of HIV-negative volunteers for the determination of HIV incidence and their willingness to participate in trials ; social-behavioural studies to ensure proper education, counselling, and informed consent [126 - 128]; virological studies to characterize incident HIV-1 strains in trial populations [46, 47, 129, 130]; and the development of guidelines to ensure the highest scientific and ethical standards in the conduct of trials , with the appropriate participation of the community.
Phase III trials will require extensive international collaboration and coordination, and it is likely that developing countries will play a major role in these trials. This is appropriate since some of the highest HIV incidence rates are found in developing countries, and conducting trials among these populations would reduce the sample size and duration of the trial. Moreover, since more than 90% of all HIV infections are occurring in developing countries, they would eventually benefit the most from an HIV vaccine. Multiple efficacy trials, conducted in parallel or sequentially, in different international sites may be necessary because of the worldwide genetic variability of HIV-1, the presence of various co-factors and routes of transmission in different geographic areas, and the necessity to test and compare different vaccine concepts.
In view of the many scientific uncertainties described previously, decisions to initiate Phase III trials will be difficult [13, 17, 132, 133]. However, these decisions must be based not only on an analysis of the scientific data (animal protection experiments, safety and immunogenicity in Phase I/II trials), but also on important public health considerations (severity of the epidemic), as well the feasibility of conducting large-scale trials, including the ability to recruit and follow a large number of volunteers, and the political and community support to ensure successful implementation of the trial.
Phase III efficacy trials will be an enormous scientific and social challenge. They will be expensive and time-consuming. Some have raised the issue that potential vaccine efficacy trial side effects may occur, such as false expectations by the community that an effective vaccine will soon be available, which could interfere with other prevention strategies; that serious rare adverse events such as antibody-dependent enhancement of infectivity may occur when large numbers of volunteers are vaccinated; and that a poorly designed or conducted trial, or even a trial with a no efficacy result, may create an atmosphere of pessimism or rejection of further efforts in HIV vaccine development.
Thus, the decision to initiate a Phase III trial should be based on a careful risk/benefit analysis. Testing available candidate vaccine(s) must be weighed against the severity of the epidemic and the consequences of waiting for more data or better candidate vaccines in the future.
Discussions on strategies of how to develop HIV vaccines are often hampered by the confrontation of two truisms: One states that the more information we obtain from basic research, the better off are we to develop more effective HIV vaccines. The other argues that laboratory research alone will never be a substitute for large-scale clinical trials to obtain definitive information on vaccine efficacy. A passionate and uncompromising defense of either position will not help those people who have to take the practical decisions, nor will this effectively promote HIV vaccine development in general. Thus, a sensible strategy is to accept the uncertainties of proceeding with efficacy trials of available products which have met previously defined minimal requirements , and at the same time continue basic research to obtain additional information on the nature of protective immune responses in humans, some of which would likely be derived from the efficacy trials themselves.
From a practical point of view, it would be important to address three questions: (1) What type of additional information is necessary to proceed to efficacy trials with the greatest likelihood of success? (2) How realistic are the expectations that relevant information will be obtained from additional laboratory, animal protection or natural history studies in the absence of efficacy trials? (3) From the candidate vaccines which have entered Phase I/II trials, are there products which meet minimal conditions to proceed to Phase III trials?
Answering the above questions is not easy. Since natural immune responses to HIV are complex (including both humoral and cellular responses) and obviously not very efficient, focusing laboratory research on limited aspects of the human immune response to HIV infection and disease may lead in false directions. Likewise, great uncertainties remain concerning the relevance of animal models as predictors of vaccine efficacy in humans.
Several candidate vaccines, based on different concepts, are at different stages in the HIV vaccine development pipeline. Candidate vaccines based on the subunit recombinant envelope concept and produced in mammalian cells, have been shown to protect chimpanzees from HIV-1 infection, and to be safe and reasonably immunogenic in humans, inducing neutralizing antibodies. A second generation of candidate vaccines, which are based on live vectors expressing the envelope and other HIV-1 genes, and which are capable of inducing CTLs, are beginning to be evaluated in human trials. Newer generations of candidate vaccines now being mostly explored in animal experiments are using combinations of subunit recombinant proteins or live-vectored vaccines with other immunogens (such as synthetic peptides or pseudovirions), or are based on more novel approaches, including nucleic acid immunization and perhaps whole-inactivated or live-attenuated vaccines.
With our present state of knowledge, it is not possible for laboratory assays to accurately predict which vaccine concept, or concepts, will induce protection against HIV infection in humans. Unless major advances are made in our understanding of the nature of protective immune responses to HIV-1 in humans, that information will only be obtained through the conduct of Phase III field efficacy trials. However, in view of the rate of progression of the HIV pandemic, especially in developing countries, it would not be ethical to wait in the hope that such advances will occur soon, thus postponing trials with candidate vaccines. In fact these trials, conducted in parallel or sequentially, may represent our best chance to enhance our basic knowledge of the nature of protective immune responses to HIV infection.
Thus, in order to avoid the unacceptable alternative of perpetual uncertainty, or to delay the development of a much needed vaccine, there is no other choice but to effectively integrate further basic research with the initiation of large-scale field efficacy trials in the process of HIV vaccine development. These Phase III trials will present unique opportunities to: (1) establish if different vaccine concepts can induce protection in humans; (2) validate the primate models presently being used in HIV vaccine research; (3) obtain information on immune correlates of vaccine-induced protection; (4) explore the significance of viral genetic variability in relation to vaccine-induced protection; (5) evaluate different end-points for vaccine efficacy (prevention of infection, establishment of chronic infection, or disease); and (6) generate additional data on vaccine safety.
The development of an HIV vaccine will be a long and difficult process. Multiple efficacy trials and case-control studies will ultimately be required before a safe, effective and affordable vaccine is available for widespread public health use. With more than 6000 new infections occurring every day worldwide, there is urgency to proceed.
We thank Drs Peter Piot and Stefano Bertozzi, from UNAIDS, and Drs Ronald Montelaro and Erling Norrby, editors of the Immunology and Vaccines section of this volume, for valuable comments.
1. Merson MH: Slowing the spread of HIV: agenda for the 1990s. Science 1993, 260:1266 - 1268.
2. Esparza J, Osmanov S, Heyward WL: HIV preventive vaccines: progress to date. Drugs 1995, 50:792 - 804.
3. Bolognesi DP: The dilemma of developing and testing AIDS vaccines. In Global Challenge of AIDS. Edited by Shiokawa Y, Nakamura T. Tokyo: Kodansha/Karger; 1995:147 - 157.
4. Hilleman M: Vaccinology, Immunology, and comparative pathogenesis of measles in the quest for a preventative against AIDS. AIDS Res Hum Retroviruses 1994, 10:3 - 12.
5. Levy JA: HIV research: a need to focus on the right target. Lancet 1995, 345:1619 - 1621.
6. Levy J-P: The problem of a vaccine against AIDS (in French). Medicine/Science (Paris) 1995, 11:407 - 419.
7. Montelaro RC, Bolognesi DP: Vaccines against retroviruses. In The Retroviridae, Vol 4. Edited by Levy JA. New York: Plenum Publishing Corporation; 1995.
8. Moore JP: Back to primary school. Nature 1995, 376: 115.
9. Moore J, Anderson R: The WHO and why of HIV vaccine trials. Nature 1994, 372: 313 - 314.
10. Paul WE: Reexamining AIDS research priorities. Science 1995, 267:633 - 636.
11. Schwartz DH: Potential pitfalls on the road to an effective HIV vaccine. Immunology Today 1994, 15:54 - 57.
12. Osborn JE: The rocky road to an AIDS vaccine. J Acquir Immune Defic Syndr Human Retrovirol 1995, 9:26 - 29.
13. World Health Organization: Scientific and public health rationale for HIV vaccine efficacy trials. AIDS 1995, 9:WHO1 - WHO4.
14. Mascola JR, McNeil JG, Burke DS: AIDS vaccines: are we ready for human efficacy trials? JAMA 1994, 272:488 - 489.
15. Duliege A-M, Sinangil F, Walker C, Dekker C, Steimer K: Status and future of Biocine® HIV-1 rgp120SF2 subunit vaccines: the experience of the Biocine Company. In Retroviruses of Human AIDS and Related Animal Diseases, 9e Colloque des Cent Gardes. Edited by Girard M, Valette L. Marnes-La-Coquette, Paris: Fondation Marcel Meux; 1994:301 - 307.
16. Francis DP: Laboratory empiricism, clinical design, and social value: the rough road toward vaccine development. In Vaccine Design: The subunit and Adjuvant Approach. Edited by Powell MF, Newman MJ. New York: Plenum Press; 1995:135 - 139.
17. Plotkin SA, Duliege A-M, Esparza J, et al.: Round table: should the candidate HIV vaccines be tested for efficacy in phase III trials? In Retroviruses of Human AIDS and Related Animal Diseases, 9e Colloque des Cent Gardes. Edited by Girard M, Valette L. Marnes-La-Coquette, Paris: Fondation Marcel Meux; 1994:321 - 333.
18. Pantaleo G, Graziosi C, Fauci AS: The immunopathogenesis of human immunodeficiency virus infection. N Engl J Med 1993, 328:327 - 335.
19. Koup RA, Ho DD: Shutting down HIV. Nature 1994, 370:416.
20. Koup RA, Safrit JT, Cao Y, et al.: Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol 1994, 68:4650 - 4655.
21. Pantaleo G, Demarest JF, Soudeyns H, et al.: Major expansion of CD8+ T cells with a predominant Vusage during the primary immune response to HIV. Nature 1994, 370:463 - 467.
22. Moore JP, Cao Y, Ho DD, Koup RA: Development of the anti-gp120 antibody response during seroconversion to human immunodeficiency virus type 1. J Virol 1994, 68:5142 - 5155.
23. Easterbrook PJ: Non-progression in HIV infection. AIDS 1994, 8:1179 - 1182.
24. Buchbinder SP, Katz MH, Hessol NA, OMalley PM, Holmberg SD: Long-term HIV-1 infection without immunological progression. AIDS 1994, 8:1123 - 1128.
25. Cao Y, Quin L, Zhang L, Safrit J, Ho DD: Virologic and immunologic characterization of long-term survivors of human immunodeficiency virus type 1 infection. N Engl J Med 1995, 332:201 - 208.
26. Wong MT, Warren RQ, Anderson SA, et al.: Longitudinal analysis of the humoral response to human immunodeficiency virus type 1 (HIV-1) gp160 epitopes in rapidly progressing and nonprogressing HIV-1-infected subjects. J Infect Dis 1993, 168:1523 - 1527.
27. Huang Y, Zhang L, Ho DD: Characterization of nef sequences in long-term survivors of human immunodeficiency virus type 1 infection. J Virol 1995, 69:93 - 100.
28. Pantaleo G, Menzo S, Vaccarezza M, et al.: Studies in subjects with long-term nonprogressive human immunodeficiency virus infection. N Engl J Med 1995, 332:209 - 216.
29. Kirchhoff F, Greenough TC, Brettler DB, Sullivan JL, Desrosiers RC: Absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N Engl J Med 1995, 332:228 - 232.
30. Michael NL, Chang G, DArcy LA, et al.: Defective accessory genes in a human immunodeficiency virus type 1-infected long-term survivor lacking recoverable virus. J Virol 1995, 69:4228 - 4236.
31. Baltimore D: Lessons from people with nonprogressive HIV infection. N Engl J Med 1995, 332:259 - 260.
32. Mellors JW, Kingsley LA, Rinaldo CR, et al.: Quantitation of HIV-1 RNA in plasma predicts outcome after seroconversion. Ann Int Med 1995, 122:573 - 579.
33. Chakrabarti L, Cummont M-C, Montagnier L, Hurtrel B: Variable course of primary simian immunodeficiency virus infection in lymph nodes: relation to disease progression. J Virol 1994, 68:6634 - 6642.
34. Hirsch VM, Goldstein S, Hynes NA, et al.: Prolonged clinical latency and survival of macaques given a whole inactivated simian immunodeficiency virus vaccine. J Infect Dis 1994, 170:51 - 59.
35. Hirsch VM, Dapolito G, Johnson PR, et al.: Induction of AIDS by simian immunodeficiency virus from an african green monkey: species-specific variation in pathogenicity correlates with the extent of in vivo replication. J Virol 1995, 69:955 - 967.
36. Rowe P: Resistance to HIV infection. Lancet 1993, 341:624.
37. Bevan MJ, Braciale TJ: Why cant cytotoxic T cells handle HIV? Proc Natl Acad Sci USA 1995, 92:5765 - 5767.
38. Ferbas J, Kaplan AH, Hausner MA, et al.: Virus burden in long-term survivors of human immunodeficiency virus (HIV) infection is a determinant of anti-HIV CD8+ lymphocyte activity. J Infect Dis 1995, 172:329 - 339.
39. Moss PAH, Rowland-Jones SL, Frodsham PM, et al.: Persistent high frequency of human immunodeficiency virus-specific cytotoxic T cells in peripheral blood of infected donors. Proc Natl Acad Sci USA 1995, 92:5773 - 5777.
40. Koenig S, Conley AJ, Brewah YA, et al.: Transfer of HIV-1-specific cytotoxic T lymphocytes to an AIDS patient leads to selection for mutant HIV variants and subsequent disease progression. Nature Medicine 1995, 1:330 - 336.
41. Mathieson BJ: CTL to HIV-1: surrogates or sirens. Nature Medicine 1995, 1:304 - 305.
42. Emini EA, Schleif WA, Numberg JM, et al.: Prevention of HIV-1 infection in chimpanzees by gp120 V3 domain-specific monoclonal antibody. Nature 1992, 355:728 - 730.
43. Prince AM, Reesink H, Pascal D, et al.: Prevention of HIV infection by passive immunization with HIV immunoglobulin. AIDS Res Hum Retroviruses 1991, 7:971 - 973.
44. Putkonen P, Thornstensson B, Ghavamzadeh L, et al.: Prevention of HIV-2 and SIVsm infection by passive immunization in cynomolgus monkeys. Nature 1991, 352:436 - 438.
45. Myers G, Korber BTM, Smith RF, Berzofsky JA, Pavlakis GN: Human Retroviruses and AIDS. Los Alamos, NM: Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, 1993.
46. Osmanov S, Heyward WL, Esparza J: The World Health Organization network for HIV isolation and characterization: summary of a pilot study. AIDS Res Hum Retroviruses 1994, 10:1325 - 1326.
47. WHO Network for HIV Isolation and Characterization: HIV type 1 variation in World Health Organization-sponsored vaccine evaluation sites: genetic screening, sequence analysis, and preliminary biological characterization of selected viral strains. AIDS Res Hum Retroviruses 1994, 10: 1327 - 1343.
48. Moore JP, McCutchan FE, Poon S-W, Mascola J, Liu J, Cao Y, Ho DD: Exploration of antigenic variation in gp120 from clades A through F of human immunodeficiency virus type 1 by using monoclonal antibodies. J Virol 1994, 68:8350 - 8364.
49. Trkola A, Pomales AP, Yuan H, et al.: Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG. J Virol 1995, 69:6609 - 6617.
50. Moore JP, Cao Y, Qin L, Korber B, Ho DD: Inter- and intraclade neutralization of human immunodeficiency virus type 1: Genetic clades do not correspond to neutralization serotypes but partially correspond to gp120 antigenic serotypes. J Virol 1996, 70:427-444.
51. Kostrikis LG, Cao Y, Ngai H, Moore JP, Ho DD: Quantitative analysis of serum neutralization of human immunodeficiency virus type 1 from subtypes A, B, C, D, E, F, and I: Lack of direct correlation between neutralization serotypes and genetic subtypes and evidence for prevalent serum-dependent infectivity enhancement. J Virol 1996, 70:445-458.
52. Nkengansong JN, Peeters M, Ndumbe P, et al.: Cross-neutralizing antibodies to HIV-1ANT70 and HIV-1IIIB in sera of African and Belgian HIV-1-infected individuals. AIDS 1994, 8:1089 - 1096.
53. Mascola JR, Louwagie J, McCutchan FE, et al.: Two antigenically distinct subtypes of human immunodeficiency virus type 1: viral genotype predicts neutralization serotype. J Infect Dis 1994, 169:48 - 54.
54. j Morfeldt-Manson L, Albert J, Biberfeld G, Karlsson A, Lindman K, Feny: Replicative capacity of human immunodeficiency virus from patients with varying severity of HIV infection. Lancet 1986, ii:660 - 662.
55. Tersmette M, De Goede REY, Winkel I, et al: Differential syncytium-inducing capacity of human immunodeficiency virus isolates: Frequent detection of syncytium-inducing isolates in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. J Virol 1988, 62:2026 - 2032.
56. Feny, Morfeldt-Manson L, Chiodi F, et al: Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates. J Virol 1988, 62:4414 - 4419.
57. De Wolf F, Hogervorst E, Goudsmit J, et al.: Syncytium-inducing and non-syncytium inducing capacity of human immunodeficiency virus type 1 subtypes other than B: phenotypic and genotypic characteristics. AIDS Res Hum Retroviruses 1994, 10:1387 - 1400.
58. Rubsamen-Waigmann H, Von Briesen H, Holmes H, et al.: Standard conditions of virus isolation reveal biological variability if HIV type 1 in different regions of the world. AIDS Res Hum Retroviruses 1994, 10:1401 - 1408.
59. Zhu T, Mo H, Wang N, et al.: Genotypic and phenotypic characterization of HIV-1 in patients with primary infection. Science 1993, 261:1179 - 1181.
60. Bolognesi D: Humoral immune responses to primary HIV isolates: implications for vaccine development. In Retroviruses of Human AIDS and Related Animal Diseases, 9e Colloque des Cent Gardes. Edited by Girard M, Valette L. Marnes-La-Coquette, Paris: Fondation Marcel Meux; 1994:285 - 291.
61. Hanson CV: Measuring vaccine-induced HIV neutralization: report of a workshop. AIDS Res Hum Retroviruses 1994, 10:645 - 648.
62. Matthews TJ: Dilemma of neutralization resistance of HIV-1 field isolates and vaccine development. AIDS Res Hum Retroviruses 1994, 10:631 - 632.
63. Bou-Habib DC, Roderiquez G, Oravecz T, Berman PW, Lusso P, Norcross MA: Cryptic nature of envelope V3 region epitopes protects primary monocytotropic human immunodeficiency virus type 1 from antibody neutralization. J Virol 1994, 68:6006 - 6013.
64. Broder CC, Earl PL, Long D, Abedon ST, Moss B, Doms RW: Antigenic implications of human immunodeficiency virus type 1 envelope quaternary structure: oligomer-specific and -sensitive monoclonal antibodies. Proc Natl Acad Sci USA 1994, 91:11699 - 11703.
65. Earl PL, Broder CC, Long D, et al.: Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities. J Virol 1994, 68:3015 - 3026.
66. Moore JP, Ho DD: HIV-1 neutralization: the consequences of viral adaptation to growth on transformed T cells. AIDS 1995, 9(suppl A):S117 - S136.
67. Moore JP, Cao Y, Qing L, et al.: Primary isolates of human immundodeficiency virus type 1 are relatively resistant to neutralization by monoclonal antibodies to gp120, and their neutralization is not predicted by studies with monomeric gp120. J Virol 1995, 69:101 - 109.
68. Sattentau QJ, Moore JP: Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer. J Exp Med 1995, 182:185 - 196.
69. Sullivan N, Sun Y, Li J, Hofmann W, Sodroski J: Replicative function and neutralization sensitivity of envelope glycoproteins from primary and T-cell line-passaged human immunodeficiency virus type 1 isolates. J Virol 1995, 69:4413 - 4422.
70. Sawyer LSW, Terri Wrin M, Crawford-Miksza L, et al.: Neutralization sensitivity of human immunodeficiency virus type 1 is determined in part by the cell line in which the virus is propagated. J Virol 1994, 68:1342 - 1349.
71. Stott J, Almond N: Assessing animal models of AIDS. Nature Medicine 1995, 1:295 - 297.
72. Stott EJ: Anti-cell antibody in macaques (letter). Nature 192, 353:393.
73. Arthur LO, Bess JW, Urban RG, et al.: Macaques immunized with HLA-DR are protected from challenge with simian immunodeficiency viruses. J Virol 1995, 69:3117 - 3124.
74. Putkonen P, Nilsson C, Hild K, Benthin R, Cranage M, Aubertin AM, Biberfeld G: Whole inactivated SIV vaccines grown on human cells fails to protect against homologous SIV grown on simian cells. J Med Primatol 1993, 22:100 - 103.
75. de Vries P, Heeney JL, Boes J, et al.: Protection of rhesus macaques from SIV infection by immunization with different experimental SIV vaccines. Vaccine 1994, 12:1443 - 1452.
76. Giavedoni LD, Planelles V, Haigwood NL, et al.: Immune responses of rhesus macaques to recombinant simian immunodeficiency virus gp130 does not protect from challenge infection. J Virol 1993, 67:577 - 583.
77. Israel ZR, Edmonson PF, Maul DH, et al.: Incomplete protection, but suppression of virus burden, elicited by subunit simian immunodeficiency virus vaccines. J Virol 1994, 68:1843 - 1853.
78. Siegel F, Kurth R, Norley S: Neither whole inactivated virus immunogen nor passive immunoglobulin transfer protects against SIVAGM infection in the african green monkey natural host. J Acquir Immune Defic Syndr Human Retrovirol 1995, 8:217 - 226.
79. Daniel MD, Mazzara GP, simon MA, et al.: High-titer immune responses elicited by recombinant vaccinia virus priming and particle boosting are ineffective in preventing virulent SIV infection. AIDS Res Hum Retroviruses 1994, 10:839 - 851.
80. Hu S-L, Abrams K, Barber GN, et al.: Protection of macaques against SIV infection by subunit vaccines of SIV envelope glycoprotein gp160. Science 1992, 255:456 - 459.
81. Hu S-L, Polacino P, Stallard V, et al.: Transmembrane protein and core antigens in protecting against SIV infection. In Vaccines 95, Cold Spring Harbor Laboratory Press. 1995:167 - 173.
82. Hu S-l, Polacino P, Stallard V, et al.: Is subunit envelope antigen our best bet for an AIDS vaccine? In Retroviruses of Human AIDS and Related Animal Diseases, 9e Colloque des Cent Gardes. Edited by Girard M, Valette L. Marnes-La-Coquette, Paris: Fondation Marcel Meux; 1994:275 - 281.
83. Desrosiers RC: HIV with multiple gene deletions as a live attenuated vaccine for AIDS. AIDS Res Hum Retroviruses 1992, 8:411 - 421.
84. World Health Organization Working Group: Feasibility of developing live attenuated HIV vaccines: conclusions and recommendations. AIDS 1994, 10:221 - 222.
85. Clements JE, Montelaro RC, Zink MC, et al.: Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus. J Virol 1995, 69:2737 - 2744.
86. Almond N, Kent K, Cranage M, Rud E, Clarke B, Stott EJ: Protection by attenuated simian immunodeficiency virus in macaques against challenge with virus-infected cells. Lancet 1995, 345:1342 - 1344.
87. Ruprecht RM, Baba TW, Greene MF; Almond N, Cranage M, Stott J: Attenuated vaccines for AIDS? Lancet 1995, 346:177 - 179.
88. Baba TW, Jeong YS, Penninck D, Bronson R, Greene MF, Ruprecht RM: Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science 1995, 267:1820 - 1825.
89. Beale J: An attenuated vaccine for AIDS? Lancet 1995, 345:1318 - 1319.
90. Marthas ML, Van Rompay KKA, Otsyula M, et al.: Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques. J Virol 1995, 69:4198 - 4205.
91. Putkonen P, Bjng E, erblom L, et al.: Long-standing protection of macaques against cell-free HIV-2 with a HIV-2 Iscom vaccine. J Acquir Immune Defic Syndr 1994, 7:551 - 559.
92. Franchini G, Robert-Guroff M, Tartaglia J, et al.: Highly attenuated HIV 2 recombinant poxviruses, but not HIV-2 recombinant Salmonella vaccines, induce long-lasting protection in rhesus macaques. AIDS Res Hum Retroviruses 1995, 11:909 - 920.
93. Abimiku AG, Franchini G, Tartaglia J, et al.: HIV-1 recombinant poxvirus vaccine induces cross-protection against HIV-2 challenge in rhesus macaques. Nature Medicine 1995, 1:321 - 329.
94. Travers K, Mboup S, Marlink R, et al.: Natural protection against HIV-1 infection provided by HIV-2. Science 1995, 268:1612 - 1615.
95. Berman PW, Murthy KK, Wrin T, et al.: Protection of MN-rgp 120 immunized chimpanzees from heterologous infection with a primary isolate of HIV-1. J Infect Dis 1996, 173:52 - 59.
96. Girard M, Meignier B, Barrinoussi, et al.: Vaccine-induced protection of chimpanzees against infection by a heterologous human immunodeficiency virus type 1. J Virol 1995, 69:6239 - 6248.
97. Bruck C, Thiriart C, Fabry L, et al.: HIV-1 envelope-elicited neutralizing antibody titres correlate with protection and virus load in chimpanzees. Vaccine 1994, 12:1141 - 1148.
98. Fultz PN, Wei Q, Yue L, Barrinoussi F, Girard M: HIV-1 dynamics after superinfection of chimpanzees with strains from two different clades. Annual Meeting of the Laboratory of Tumor Cell Biology. Bethesda, MD, August 1995, Abstract # 283. AIDS Res Hum Retroviruses 1995, 11 (suppl 1):S135.
99. Esparza J, Osmanov S: The development and evaluation of HIV vaccines. Curr Opin Infect Dis 1993, 6:218 - 229.
100. Walker MC, Fast PE: Clinical trials of experimental AIDS vaccines. AIDS 1994, 8(suppl 1):S213 - S236.
101. Fast P: Phase I and II trials of candidate HIV-1 vaccines: current status and future directions. In Retroviruses of Human AIDS and Related Animal Diseases, 9e Colloque des Cent Gardes. Edited by Girard M, Valette L. Marnes-La-Coquette, Paris: Fondation Marcel Meux; 1994:293 - 299.
102. Belshe RB, Bolognesi D, Clements ML, et al.: Studies on HIV-1 vaccines by the AIDS Vaccine Evaluation Group (AVEG). Annual Meeting of the Laboratory of Tumor Cell Biology. Bethesda, MD, August 1995, Abstract # 296. AIDS Res Hum Retroviruses 1995, 11 (suppl 1):S135.
103. Belshe RB, Graham BS, Keefer MC, et al.: Neutralizing antibodies to HIV-1 in seronegative volunteers immunized with recombinant gp120 from the MN strain of HIV-1. JAMA 1994, 272:475 - 480.
104. Kahn JO, Sinangil F, Baenziger J, et al.: Clinical and immunological responses to human immunodeficiency virus (HIV) type 1SF2 gp120 subunit vaccine combined with MF59 adjuvant with or without muramyl tripeptide dipalmitoyl phosphatidylethanolamine in non-HIV-infected human volunteers. J Infect Dis 1994, 170:1288 - 1291.
105. Belshe RB, Clements ML, Dolin R, et al.: Safety and immunogenicity of a fully glycosylated recombinant gp160 human immunodeficiency virus type 1 vaccine in subjects at low risk of infection. J Infect Dis 1993, 168:1387 - 1395.
106. Gorse GJ, Rogers JH, Perry JE, et al.: HIV-1 recombinant gp160 vaccine induced antibodies in serum and saliva. Vaccine 1995, 13:209 - 214.
107. Gorse GJ, Schwartz DH, Graham BS, et al.: HIV-1 recombinant gp160 vaccine given in accelerated dose schedules. Clin Exp Immunol 1994, 98:178 - 184.
108. Gorse GJ. Phase I/II trials of preventive HIV vaccine candidates. Dose and schedule: summary. AIDS Res Hum Retroviruses1994, 10(suppl 2):S141 - S143.
109. Gorse GJ, Frey SE, Patel G, Newman FK, Belshe RB, NIAID AIDS Vaccine Clinical Trials Network: Vaccine-induced antibodies to native and recombinant human immunodeficiency virus type 1 envelope glycoproteins. Vaccine 1994, 12: 912 - 918.
110. Graham BS, Gorse GJ, Schwartz DH, et al.: Determinants of antibody response after recombinant gp160 boosting in vaccinia-naive volunteers primed with gp160-recombinant vaccinia virus. J Infect Dis 1994, 170:782 - 786.
111. McElrath MJ, Corey L, Berger D, et al.: Immune responses elicited by recombinant vaccinia-human immunodeficiency virus (HIV) envelope and HIV envelope protein: analysis of the durability of responses and effect of repeated boosting. J Infect Dis 1994, 169:41 - 47.
112. Plotkin SA, Cadoz M, Meignier B, et al.: The safety and use of canarypox vectored vaccines. Dev Biol Stand 1995, 84:165 - 170.
113. Egan MA, Pavlat WA, Tartaglia J, et al.: Induction of human immunodeficiency virus type 1 (HIV-1)-specific cytolytic T lymphocyte responses in seronegative adults by a nonreplicating, host-range-restricted canarypox vector(ALVAC) carrying the HIV-1MN env gene. J Infect Dis 1995; 171:1623 - 1627.
114. Pialoux G, Excler J-L, Rivi Y, et al.: A prime-boost approach to HIV preventive vaccine using a recombinant canarypox virus expressing glycoprotein 160 (MN) followed by a recombinant glycoprotein 160 (MN/LAI). AIDS Res Hum Retroviruses 1995, 11:373 - 381.
115. Excler J-L, Salmon D, Sicard D, et al.: Safety and immunogenicity of a live recombinant canarypox virus vaccine expressing gp120/gag/protease boosted by a p24-V3 peptide. Annual Meeting of the Laboratory of Tumor Cell Biology. Bethesda, MD, August 1995, Abstract # 295. AIDS Res Hum Retroviruses 1995, 11 (supl 1):S138.
116. OHagan DT, McGee JP, Wang CY, Potts B, Koff WC: The UBI multicomponent HIV® vaccine rgp120SF2 subunit vaccines: the advantages of controlled release microparticles. In Retroviruses of Human AIDS and Related Animal Diseases, 8e Colloque des Cent Gardes. Edited by Girard M, Valette L. Marnes-La-Coquette, Paris: Fondation Marcel Meux; 1993:309 - 313.
117. Rubinstein A, Goldstein H, Pettoello-Mantovani M, et al.: Safety and immunogenicity of a V3 loop synthetic peptide conjugated to purified protein derivative in HIV-seronegative volunteers. AIDS 1995, 9:243 - 251.
118. Cohen J: Thailand weighs AIDS vaccine tests. Science 1995, 270:904 - 907.
119. Belshe RB, Bolognesi DP, Clements ML, et al.: HIV infection in vaccinated volunteers (letter). JAMA 1994, 272:431.
120. Kahn JO, Steimer KS, Baenziger J, et al.: Clinical, immunologic, and virologic observations related to human immunodeficiency virus (HIV) type 1 infection in a volunteer in an HIV-1 vaccine clinical trial. J Infect Dis 1995, 171:1343 - 1347.
121. Esparza J: Development of WHO-sponsored sites for HIV vaccine evaluation. AIDS Res Hum Retroviruses 1993, 9(suppl 1):S133 - S134.
122. Esparza J, Osmanov S, Kallings LO, Wigzell H: Planning for HIV vaccine trials: the World Health Organization perspective. AIDS 1991, 5(suppl 2):S159 - S163.
123. Esparza J, Osmanov S, Clements ML, Heymann D: Preparation for efficacy trials of preventive HIV candidate vaccines: the role of the World Health Organization. In Retroviruses of Human AIDS and Related Animal Diseases, 7e Colloque des Cent Gardes. Edited by Girard M, Valette L. Marnes-La-Coquette, Paris: Fondation Marcel Meux; 1992:217 - 222.
124. Hoff R: Preparation for HIV vaccine trials: moving from baseline studies to efficacy trials. AIDS Res Hum Retroviruses 1994, 10(suppl 2):S191 - S193.
125. Heyward WL, Osmanov S, Saba J, et al.: Preparation for Phase III HIV vaccine efficacy trials: methods for the determination of HIV incidence. AIDS 1994, 8:1285 - 1291.
126. Chesney MA, Lurie P, Coates TJ: Strategies for addressing the social and behavioural challenges of prophylactic HIV vaccine trials. J Acquir Immune Defic Syndr Human Retrovirol 1995, 9:30 - 35.
127. Jenkins RA, Temoshok LR, Virochsiri K: Incentives and disincentives in prophylactic HIV vaccine research. J Acquir Immune Defic Syndr Human Retrovirol 1995, 9:36 - 42.
128. Temoshok LR: Behavioural research contributions to planning and conducting HIV vaccine efficacy trials. AIDS Res Hum Retroviruses 1994, 10(suppl 2):S277 - S280.
129. Wasi C, Herring B, Raktham S, et al.: Determination of HIV-1 subtypes in injecting drug users in Bangkok, Thailand, using peptide-binding enzyme immunoassay and heteroduplex mobility assay: evidence of increasing infection with HIV-1 subtype E. AIDS 1995, 9:843 - 849.
130. Kalish ML, Baldwin A, Raktham S, et al.: The evolving molecular epidemiology of HIV-1 envelope subtypes in injecting drug users in Bangkok, Thailand: implications for HIV vaccine trials. AIDS 1995, 9:851 - 857.
131. Esparza J, Osmanov S, Heyward WL, Piot P: Ethical aspects of trials of candidate vaccines against the human immunodeficiency virus (HIV). In Proceedings of the 4th International Seminar on Immunizations in Africa. Yamoussoukro, CdIvoire, March 1994: 330 - 331.
132. Burke DS: Human trials of experimental HIV vaccines. AIDS 1995, 9(suppl A):S171 - S180.
133. Johnston MI, Noe JS, Killen JY: Recent advances in AIDS vaccine research and development. AIDS Res Hum Retroviruses 1994, 10(suppl 2):S317 - S323.
134. Haynes BF: Scientific and social issues of Human Immunonodeficiency Virus vaccine development. Science 1993, 260:1279 - 1286.