The HIV vaccine pipeline, from preclinical to phase III

Introduction The AIDS epidemic continues to move inexorably around the globe, making its most recent inroads in China and Southeast Asia, and re-asserting itself in young gay men in places like New York City, after a period of successful behavior modification that had slowed the rate of new infections for a time. The United Nations acknowledges that HIV/AIDS could be a threat to world peace [1]. Worldwide, nearly 12 million men, women and children have become infected in just the past 2 years, and population life expectancy is actually going down in parts of sub-Saharan Africa. The developing world harbors 95% of these new infections, where there is little access to treatments that have prolonged life in industrialized countries. An effective and safe preventive vaccine remains the best hope for ending this pandemic. Vaccines have been cost-effective public health weapons against infectious disease, but some new road maps may be needed to achieve success against HIV. The favored tools of the past, live-attenuated strains and killed vaccines, have not provided a direct pathway to an AIDS vaccine. A truly attenuated and safe HIV that is still immunogenic has proven frustratingly elusive [2], and the difficulty in retaining the envelope glycoprotein on purified HIV particles has interfered with the manufacture of an adequate product to test the utility of the whole-killed approach. In addition, the challenge of producing millions of doses without allowing a single case of HIV from an improperly inactivated production lot, as would be required in the present climate, is daunting to say the least. Safely mimicking HIV infection as the modus operandi for the vaccine, using either the killed or the attenuated approach, has been frustrated so far by the biology and physical chemistry of HIV. Therefore, HIV vaccine design tilts heavily toward the 'modern' school, using the power of genetic engineering to take parts of HIV and create vaccines that could never cause AIDS. But will these 'modern' vaccines be immunogenic enough? While it is still not clear what facets of the immune response are most important for protection from infection or disease, it is now widely believed that an efficacious vaccine will need to induce both cellular and humoral immunity. This review will attempt to show the strengths and weaknesses of various approaches towards inducing and maintaining such immunity. Although each vaccine platform will have its special feature, let us not forget that all the candidates necessarily share one inescapable commonality - they must include the viral genes that contain the protective epitopes. Learning what antigens are protective, or what immune responses (or at which level of intensity or duration) are useful, can be obtained in one system, and that knowledge may be immediately transferable to the other approaches. Phase III timetable While increasingly valuable information is emerging from experiments in primates, an efficacy trial in humans under conditions of natural exposure to HIV is needed to truly prove the worth of a vaccine. In 1998, a large trial of recombinant gp120 (in a bivalent formulation combining a laboratory strain glycoprotein with a primary virus glycoprotein) began in the United States (also with sites in Canada and the Netherlands), mostly among gay men [3]. Another trial in Thailand, among intravenous drug users, completed enrollment in autumn 2000. Using gp120 to generate protective neutralizing antibody was among the earliest 'modern' vaccine concepts to emerge in the drive for an HIV vaccine [4,5], and it is now finally being put to the test. A first look at the protection data from the US trial will occur halfway through the trial in November 2001, according to the manufacturer (VaxGen, Brisbane, CA, USA). If the trial has a statistically valid lower bound of 30% efficacy at that time, the hypothesis that monomeric gp120 can prevent infection by a significant proportion of prevalent HIVs will have been proven. If the primary endpoint is not achieved in November 2001, the trial will proceed to completion. In that case, the utility of gp120 as a vaccine will have to wait until early 2003 for an answer. A second HIV vaccine is now in position for phase III clinical testing. In the years since work on gp120 began, a 'second generation' of HIV vaccines has developed, emphasizing cellular immune responses in addition to antibody and also investigating the role that proteins other than gp120 can play as protective antigens. Canarypox vaccines (ALVAC) of Aventis Pasteur (Lyon, France) are now in final phase II human trials after years of development work, devoted to learning the best way to express multiple antigens in a single recombinant vector and the most appropriate way to assess cellular immune responses. The National Institutes of Health HIV Vaccine Trials Network (HVTN) is testing a subtype B ALVAC that expresses the gag and pol genes as well as the env gene, plus epitopes from nef and int. The Walter Reed Army Institute of Research (WRAIR)/Henry M. Jackson Foundation has an ALVAC with gag and pol genes containing subtype E env in a phase II trial in Thailand. Each ALVAC vaccine probably would be tested in combination with a VaxGen gp120 boost, although none of the phase III trial protocols in development is final. If either the National Institutes of Health or WRAIR proceed with an efficacy test of the ALVAC/gp120, such a trial could begin in 2002 and not yield results until at least 2005. It is sobering to reflect on the time it takes to lead up to and then plan, mount, and enroll such trials, which then typically will run for 3 years after the last volunteer is enrolled, to assess the duration of any protective effect. This chapter will attempt to characterize what may come next for phase III clinical testing, but nothing that will be covered next is likely to be ready for phase III until 2004 or later. It will be a long wait. Envelope protein antigens (the neutralization conundrum) Monomeric gp120, the only candidate AIDS vaccine to advance to large-scale efficacy trials so far, was initially developed from laboratory strains of HIV and shown early on to be the most effective vaccine at inducing neutralizing antibodies against the laboratory strains available at the time. It was subsequently learned that HIV grown long-term in culture (i.e. a 'laboratory strain') was much more sensitive to neutralization than HIV freshly isolated from patients and only briefly adapted to culture (so-called 'primary strains') [6]. The antibodies induced in human volunteers by the original gp120 vaccines were incapable of neutralizing 'primary' HIV [7,8]. The consequences of this limited breadth of neutralization can be shown experimentally in a primate challenge study, where vaccination with a laboratory strain gp120 prevented infection if animals were challenged with a virus homologous to the vaccine but proved inadequate in protection against challenge with a heterologous virus based on a primary strain [9]. The current phase III trials of this vaccine approach will soon settle whether this research analysis is predictive of its protective potential in the real world. While it is beyond the scope of this review to address the details of HIV envelope structure and why getting vaccines to perform better in these laboratory analyses has been so hard [10], we digress to provide a short summary of attempts to improve envelope immunogens. One avenue for improvement is to design and produce immunogens that mimic 'native' HIV as closely as possible, on the assumption that the trimeric glycoprotein spike structure on the surface of primary HIV contains neutralization epitopes that cannot be duplicated on monomeric, recombinant gp120. Alternatively, on the assumption that primary HIV is intrinsically neutralization resistant because it has evolved mechanisms to hide neutralizing epitopes, another approach is to modify the structure of gp120 to expose those hidden and putatively effective neutralization epitopes. 'More native' envelope immunogens An early hypothesis to improve monomeric gp120 was to base the vaccine not on a laboratory strain, but on a primary strain. In fact, the gp120 vaccines currently in phase III trial have adapted this approach by adding a primary isolate gp120 to the original HIVMN gp120 in bivalent formulations, one with an added subtype E for Thailand and another with a primary subtype B for North America/Europe. Although the hypothesis that such a gp120 will necessarily induce antibodies capable of neutralizing primary isolates did not pan out in one case [11], other workers have discovered that some primary HIV envelopes can induce antibodies capable of neutralizing primary isolates, albeit only the homologous strain [12]. It has been suggested that envelope immunogens made from certain virus strains (those derived from infected individuals who developed broadly reactive neutralizing antibodies) may be significantly better than others at inducing breadth of neutralization, although this has yet to be shown experimentally [13]. The concept of mixing more than one variant of a diverse pathogen to create a polyvalent vaccine is well established. A preliminary attempt to broaden anti-HIV neutralization by mixing six different gp120s in a primate challenge experiment failed to significantly broaden the neutralization response [14]. A human phase I trial is underway testing a mixture of 23 different recombinant vaccinia expressing HIV-1 envelope genes [15], with plans to test in combination with DNA and/or envelope protein. In addition, Advanced BioSciences Laboratories Inc. is developing a polyvalent vaccine to contain nine env-expressing DNA and three env proteins. For a polyvalent approach to be successful, identification of gp120s with much broader than 'average' neutralization-inducing capability will probably be required. The native envelope complex on the HIV virion surface contains gp120 molecules bundled together in groups of three, held in this conformation by non-covalent association with a bundle of three transmembrane gp41 molecules. These bundles, whose exact structure is not known, are generated after the gp160 polyprotein is cleaved into gp120 and gp41, and they are too unstable to be purified intact. Engineering the envelope protein to prevent cleavage into gp120 and gp41, but eliminating the insoluble transmembrane half of gp41, yields soluble gp140 glycoproteins that assemble into multimeric forms that can be purified [16]. Unfortunately, elicitation of neutralizing antibodies by these oligomeric forms has been disappointing, perhaps because these multimers are not the native trimer [17]. In attempts to make truly trimeric molecules, cross-links to stabilize the trimeric spike structures have been tried. One group has permitted cleavage between gp120 and gp41 but stabilized their association by inserting disulfide bonds into the gp120/gp41 contact region, resulting in a covalently bound gp120-gp41 complex. The antigenic properties of this molecule are quite similar to the native molecule found on the virus [18], but the neutralizing antibodies induced in laboratory animals show little improvement over those induced by gp120 monomer protein. Another group has created a stabilized envelope trimer by preventing gp120/gp41 cleavage and introducing three modifications that stabilize both the gp120/gp41 interaction and the association between gp41 subunits that holds the trimeric bundle together [19]. These latter modifications have been shown to yield a molecule with enhanced immunogenicity properties compared with the analogous gp120 protein, but the improvement is minimal [20]. The ultimate 'native' immunogen is, of course, HIV itself, and the difficulty in getting large quantities of such a product has already been mentioned. Inactivating methods, to make a safe product, are themselves often highly damaging to protein structures. Recently, two rather gentle inactivation methods that do not modify the protein antigens have been developed. One removes essential Zn2+ ions from int and gag gene products [21], and a newly developed, efficient psoralen molecule significantly reduces the amount of damaging ultraviolet radiation needed to specifically inactivate the HIV RNA [22]. It is possible that a gently and safely inactivated HIV that is immunogenic might result from the combination of these two methods. Finally, a genetically inactivated HIV based on a primary isolate was created, produced and purified with great effort by the Canadian subsidiary of Aventis Pasteur. Although this product induces effective neutralizing antibodies against the homologous primary HIV in macaques, broad neutralization of other primary HIV was not found [12]. It is unlikely that this product will be evaluated in humans. Improving on 'native' envelope immunogens HIV envelope proteins are highly glycosylated. Strategically removing glycosyl moieties from the envelope protein not only results in a mutated virus that is more neutralization sensitive, but also yields an infectious immunogen that is antigenically and immunogenically superior to the native envelope [23]. Neutralization epitopes can apparently also be unmasked by removing amino acid stretches (variable loops) from the gp120 molecule [24,25]. Immunization with DNA expressing a deleted form of the envelope (delta V2) followed by boosting with the protein itself has been shown to induce antibodies effective not only at neutralizing homologous neutralization-resistant wild-type virus, but also effective at neutralizing a number of heterologous primary isolates [26]. DNA and protein immunogens from a primary strain of subtype B with deleted V2 loop, being developed by Chiron Corporation (Emeryville, CA, USA), are planned to be ready for human trial within 2 years (Fig. 1).Fig. 1: Timeline for AIDS vaccine clinical trial testing.The HIV envelope structure is fluid and dynamic, to allow interaction with cellular receptors. During the infection process, there are transient conformational changes in the envelope protein that expose additional neutralization epitopes [27]. The probable reason why antibodies to these epitopes are not found in HIV patients is that they are only briefly exposed. Several groups have devised ways to 'freeze' these transient structures. A 'fusion-competent' immunogen was created from cells expressing HIV envelope proteins that were formalin-fixed at the time of envelope-induced cell fusion. It was reported that this whole-cell immunogen induced antibodies in mice capable of neutralizing a wide range of unrelated primary HIV isolates [28]. Similarly, gp120-CD4 receptor protein complexes have been created as a more simple way to expose envelope epitopes that only exist after receptor binding. It has been reported that these complexes elicit broadly reactive neutralizing antibodies [29]. The design has been further refined by producing the complex as a single-chain polypeptide, whereby gp120 is joined to the D1D2 domain of CD4 by an amino acid linker [30]. This fusion protein could actually be produced in reasonable quantities and is proposed for human testing. Finally, viruses can be created that bypass CD4 binding and utilize the CCR5 chemokine receptor directly for infectivity [31,32], behaving as if they were already beyond the CD4-binding stage. These viruses are now neutralization sensitive, and perhaps envelope proteins from these altered viruses will exhibit enhanced envelope immunogenicity [32]. A better understanding of envelope protein structure and function has led to incremental improvements when designing envelope-based immunogens, but has not yet led to a vaccine product with broadly reactive immunogenicity. Combinations of approaches (e.g., V1V2-deleted gp120 in oligomeric form) are also on the drawing boards. Utilizing the technique of 'DNA shuffling' [33] to re-assort domains from multiple gp120s into one molecule that might expose neutralization epitopes or alternatively combine multiple gp120s into one gp120 molecule is another research approach to find a better gp120 immunogen. These experimental immunogens are pure research creations and far from being in a developmental pipeline. The high cost of producing protein immunogens may render this strategy prohibitive but, once a better immunogen design is achieved, expression of these improved molecules by viral vectors or DNA immunization may be a more feasible alternative. Other protein immunogens Immunology has had to deal with a revision in the dictum that protein antigens could not induce cellular responses. Immunization with P55gag protein in the form of virus-like particles was shown to induce long-lived (> 8.5 months) cytotoxic T lymphocytes (CTL) in macaques [34]. An HIV form of Pr55gag, devoid of envelope proteins and with lipids and nucleic acids extracted, is being produced by Protein Sciences Corporation (Meriden, CT, USA) for human trial as a T-cell immunogen. Proteins also can be presented directly into the class II pathway via fusion with inactivated anthrax toxin subunit [35]. This product, expressing HIV gag, is being developed for human trial. Similarly, peptides with fatty acids attached can induce cellular responses, in humans as well as smaller animals. There are plans in the HVTN to build on the small human trial in France [36] and to test these lipopeptides alone and in combination with other modalities. Synthetic peptides were among the first HIV vaccines tried. Wyeth-Lederle, in collaboration with Duke University, is proposing a mixture of multi-epitope peptides containing various targets for cellular immune responses or different HIV-1 envelope variable regions as a booster to their DNA vaccines (Fig. 1). There have been several recent reports of tat protein immunogens providing protection in primate experiments [37,38] and, although these results have proven controversial, there are theoretical reasons why immune responses to the tat protein could be protective. A DNA form of the vaccine has been tested, and a phase I trial has been proposed. Whether the tat protein should be 'native' or altered to remove biological activity before being tried experimentally in humans has not been determined. Viral vectors The use of live viral vectors provides HIV vaccine design with several advantages. To the extent that getting the HIV envelope into a 'native' conformation is key to a successful vaccine, in situ expression of gp120/gp41 in cells of the vaccine recipient, after delivery of the genes encoding them, should provide such expression. Secondly, 'infection' with a vector induces more robust cellular responses than does immunization with proteins or The vaccine was the first vector developed to express HIV genes it has a large for expressing multiple genes in the vaccinia to high and can be produced in large and with recombinant vector followed by a protein the antibody of gag and pol genes efficacy of the vaccine against after challenge There are two phase I trials of and env both alone and with gp120 protein. One the Corporation USA) product in combination with VaxGen gp120 and the other trial a envelope trials were held up until by a of which is needed as a in case the vaccinia vector to high in a There have been the use of vaccinia but, since this virus class several for use as has to that in either of vaccinia that in but well in cells (in which they can be grown in large or to the which are in their to produce in these viruses still to cells and expressing their early HIV genes under of early are producing these in large at high has been vaccinia virus This variant of vaccinia was developed in the and has proven with since it is available and has shown results in Although macaques with vaccinia virus env were not from challenge with highly they had lower virus and prolonged to infection immunization with recombinant expressing failed to from infection but did result in a significant in challenge the in virus directly with an in and of the 1: candidate This product developed by Aventis Pasteur is probably to but was created by specifically from vaccinia to achieve in It has been tested in several primate and will be compared with in trials planned by the 1). candidates are being developed by and by the of New a of and The of New to use the immunogens as with DNA immunogens. 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