Present Status of Human HIV Vaccine Development

Sandra A. Calarota; David B. Weiner

Disclosures

AIDS. 2003;17(18s) 

In This Article

Progress in Non-human Primate Models

Since the excitement generated in rhesus macaques a few years ago, when IL-2/Ig adjuvanted DNA vaccines (see below) or DNA prime followed by MVA boosts elicited control over viral replication and protection from CD4 T-cell loss, macaque models have been viewed with renewed interest in pre-clinical vaccine development. The macaque model serves several important purposes in current HIV vaccine research. It allows analysis of vaccine safety and proof of immunogenicity in a species more closely related to humans that is more demanding than rodent or rabbit model systems. Furthermore there are a few interesting options for testing the mettle of a vaccine in a mucosal or intravenously pathogenic challenge system such as the SIV or SHIV challenges that as discussed above may be flawed. Recently, another caveat has come to focus regarding the influence of the Mamu-A*01 MHC allele in vaccine challenge outcomes. Originally, Pal et al.[22] suggested that animals with a Mamu-A*01 haplotype appeared to be naturally resistant to disease progression compared to non Mamu-A*01 animals. Among the most impressive protection studies,[23,24,25] the groups that received active vaccine frequently were biased towards a higher percentage of Mamu-A*01 animals in order to allow for a more thorough evaluation of vaccine-induced cellular responses by focusing on analysis of Mamu-A*01 epitope based T-cell responses.[26,27,28]

However, a recent study by Shiver et al. from Merck,[29] testing their same Ad5 gag vaccine in non Mamu-A*01 animals, elicited substantially less viral control than the observed in their original study performed in Mamu-A*01 animals. Not only was the viral control poorer but the magnitude of the anti-gag response was also lower illustrating that Mamu-A*01 animals have a dramatic dominate response that may not be representative of human responses to candidate HIV cellular vaccines. It will be important to keep these caveats in mind going forward in the primate model as direct comparisons of results from Mamu-A*01 animals cannot be compared with results in non-Mamu-A*01 animals. The protection observed in these animals should be viewed cautiously as this story unfolds. However, other findings in the macaque model have continued to generate important pre-clinical progress. In particular these advances have come in the area of prime-boost studies as well as the further cytokine adjuvantation of DNA vaccines.

The concept of prime-boost in HIV vaccines is not new although it has been rejuvenated in the form of DNA or recombinant vector prime-boost combinations. In the context of an expanding health problem, malaria infection, McConkey et al.[30] reported the first demonstration in humans that a heterologous prime-boost regimen of DNA followed by rMVA induced T-cell responses that produce partial protection after sporozite challenge. Such heterologous prime-boost immunization approaches may provide the basis for both therapeutic and prophylactic HIV vaccination in humans. Such studies conducted by Merck have focused on a prime-boost approach of plasmid DNA followed by Ad5 vector (both expressing SIV-gag). This regimen elicited strong immunogenicity in rhesus macaques; after challenge with SHIV-89.6P, these animals exhibited pronounced attenuation of the virus infection.[25] The same group compared immunogenicity of plasmid DNA, rMVA, and Ad5 vectors expressing HIV-1 gag gene in rhesus macaques. Of the regimens tested, DNA formulated with the chemical adjuvant CRL1005 primed T-cell responses more effectively and provided the strong immune responses after boosting with Ad5-gag.[31] Based on these studies, clinical trials were initiated to determine the safety and immunogenicity of DNA and Ad5 vectors expressing HIV-1 gag in humans (see below). More recent macaque studies in a collaboration between Merck and Aventis Pasteur tested the utility of Ad5 priming followed by a canary pox vector boost (ALVAC vCP205). ALVAC vectors have been studied for years as potential stand-alone vaccines or as a prime for a subunit boost. In general ALVAC vectors induce modest CD8 T-cell immunity in non-human primates or humans on their own. Interestingly, ALVAC as a boosting agent induced high T-cell boosting, in fact ELISPOT results were as high and perhaps higher than DNA prime/Ad5 boosting, considered to be the former gold standard for induction of cellular immunity in macaques. Studies from the laboratory of Letvin in collaboration with investigators from the VRC also reported that several different poxviral vectors could all function quite well as boosting agents in such prime-boost studies.[32] Another important new recombinant vector that is under investigation by John Rose and colleagues is vesicular stomatitis virus (VSV). Recently, this group reported that VSV delivered as a mucosal vaccine vector can prime for strong CD8+ T-cell responses and impact viral load in the macaque model system.[33] However, recent studies from this group have demonstrated that rVSV vectors containing SIV antigens can be effectively primed by recombinant poxviral vectors.[34] Collectively, it is clear that poxviral vectors including ALVAC, which may not have been the most potent priming vehicles, appear to be exciting choices as recombinant boosting agents. Importantly, many of these including the combination of Ad5/ALVAC are now being moved to clinical evaluation. The Merck-Aventis collaboration is an important milestone that builds on the prior large corporate collaborations aimed at testing DNA vaccine concepts that include Aventis-Chiron collaborative studies of poxviral prime-subunit boosts and the Aventis-Wyeth DNA prime-poxviral boost studies. This is an important trend that represents the collaborative effort at all levels seeking to find an effective and safe vaccine for HIV.

As mentioned above recombinant MVA constructs are being explored as candidate AIDS vaccine. Studies performed by Amara et al.[24,35] have demonstrated that DNA priming followed by rMVA boost (expressing SIV gag-pol and HIV env) elicited reasonable frequencies of CD8 T-cell responses in monkeys and the subsequent control of the SHIV-89.6P challenge; again two or more animals per group were Mamu-A*01. A considerable question is the role of the HIV envelope in this model. Robinson observed that inclusion of Env as a gene vaccine and in the boost had an effect on the challenge outcome. Animals vaccinated without Env exhibited less control of viral replication and greater CD4+ T-cell loss of a SHIV-89.6P challenge than did gag-pol-env immunizations.[35] These results suggest that the inclusion of several genes in a vaccine will elicit broad immune responses and possibly better control of viral infection. These studies also establish a role for Env and possibly antibodies that bind to this protein may provide a better protection in this animal model. On the other hand, Horton et al.[36] used a DNA prime/MVA boost regimen to immunize macaques against nearly all SIV proteins. The animals were challenged with SIVmac239. Despite the induction of virus-specific CD8+ and CD4+ T-cell responses and reduced peak viral loads in vaccinated animals, the regimen did not prevent CD4 depletion or disease progression. However, other studies appear to support the observation of an Env effect. Specifically Letvin reported an Env effect in a prime-boost study.[32] In addition, a SIV challenge study also appears in agreement with this concept. Specifically, Muthumani et al.[37] studied the effect of a multi-component DNA vaccine in the control of pathogenesis and viral replication in a mucosal SIVmac251 model system. Following challenge, rhesus macaques immunized with plasmids encoding Gag/Pol plus Env/Rev exhibited increased neutralizing antibody titers and significant improvement in control of viral challenge as well as protection from CD4 T-cell decline compared to control animals. However, this study was limited in that animals were only followed for 6 months before the study was terminated so conclusions on the long-term effects in this study are not available. However, these collective results suggest that vaccines that have more antigens are probably important for improving control and preventing CD4 T-cell loss at least in macaque model systems. Additional strategies aimed at including multi-epitope antigens include the development of a multi-antigen subunit protein[38] and a mucosal priming approach with Salmonella Type III secretion system followed by boost with rMVA.[39] A summary of these approaches and others are listed in Table 2 .

DNA vaccines for HIV-1 appeared on the horizon over a decade ago full of luster and promise.[40,41,42,43,44,45,46] Several years later and after various clinical studies[47,48,49,50,51] supporting the safety of these approaches it has become clear that the potency of even the most engineered of these vaccines could use improvement. Some of the luster has faded. In fact, the replacement in the Merck program of the DNA prime-Ad5 boost regime with the Ad5-ALVAC prime-boost protocol appears to underscore this issue. However, very recent improvements in adjuvanting these vaccines have generated renewed interest and excitement in this approach. Cytokine plasmids, which adjuvant DNA vaccines for HIV-1, have been under investigation for several years.[52,53,54] The first primate challenge study of an adjuvanted DNA vaccine was reported by Barouch et al..[23] These studies demonstrated that immune responses elicited in rhesus macaques by DNA vaccines can be augmented about 2× by the administration of IL-2/Ig fusion protein or a plasmid encoding IL-2/Ig. Moreover, cytokine-augmented DNA vaccine immunized macaques managed to control viral replication to a greater extent than DNA only immunized animals. However, one of these macaques had an eventually vaccine failure, a single mutation within an immunodominant Gag CTL epitope resulted in viral escape from CTL, a burst in viral replication, clinical disease progression and death.[55] A further longitudinal analysis of the immunologic control and viral sequence evolution in a set of DNA-vaccinated macaques was performed.[56] Viral escape occurred in most of these animals over a 3-year study period. These events indicate that viral escape from CTL recognition may be a major restriction of the cellular immune responses elicited by HIV vaccines, representing a mechanism of vaccine failure. For that reason, it will be very important to develop vaccine candidates able to elicit the most potent and broad cellular immune responses.

In this vein studies from our laboratory at the University of Pennsylvania in collaboration with Wyeth focused on identifying particularly strong Th1 cytokine adjuvants for plasmid vaccines. One goal was to develop a DNA vaccine formulation that would provide less dependence on CD4 T-cell help, which likely is compromised during the initiating events of HIV infection. There is now strong evidence that IL-15 plays a key role in regulating homeostatic proliferation of CD8+ memory cells[57] and in mouse studies can function to expand CD8 immunity as a plasmid vaccine.[53] Accordingly, they analyzed the immune responses induced in macaques following immunization with SIV gag plasmid vaccine adjuvanted with an engineered high expressing IL-15 vector. IL-15 was a strong inducer of IFN-γ as measured by ELISPOT assay. Notably after two immunizations with a modest 2 mg dose of vaccine, the mean number of IFN-γ was about 500/million cells in animals receiving IL-15 whereas those immunized with gag alone averaged approximately 100 spots/million cells.[58] Supporting the data observed in mouse studies, IL-15 appears to drive this CD8 immune expansion in the absence of strong CD4 T-cell help. These observations indicate that the use of IL-15 may be particularly beneficial in the context of HIV immune therapy, in which the CD4 impairment is the central issue. John Eldridge and his colleagues at Wyeth as part of this collaboration reported on IL-12 as a plasmid vaccine adjuvant in rhesus macaques.[59] Dramatic enhancement of cellular gag-specific immune responses as well as humoral responses was observed in macaques immunized with SIV-gag encoding DNA together with a plasmid expressing IL-12. Moderate IFN-γ production (mean 300 spots/million cells) was detected in macaques immunized with 5 mg of SIV-gag DNA alone compared to high (mean 1400 spots/million cells) in macaques co-immunized with 5 mg of gag + IL-12 three times. Reasonable antibody responses (>1/1000) were detected in eight out of 10 animals receiving IL-12 compared with three out of 10 animals that did not receive IL-12. Furthermore, boosting these immunized animals with rVSV, expressing SIV gag and HIV env, generated ELISPOT results above 3500/million cells and robust antibody titers (1/100 000).[60] Taken together, the co-administration of DNA vaccines simultaneously with strong Th1 cytokine-expressing plasmid provides several important enhancements to the technology that include lower doses, quicker and less variability in responses, the ability to boost multiple times without vector interference which places their immune potency in line with live vector systems. It is hoped that adjuvanted DNA vaccines will translate these patterns in planned clinical studies.

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