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The HIV vaccine pipeline, from preclinical to phase III

2001; Lippincott Williams & Wilkins; Volume: 15; Linguagem: Inglês

10.1097/00002030-200100005-00018

1473-5571

Alan M. Schultz, James A. Bradac,

Hepatitis B Virus Studies

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 particles. Vaccinia The smallpox vaccine was the first vector developed to express HIV genes [39]; it has a large capacity for expressing multiple genes in the same construct [40]. Recombinant vaccinia grows to high titers and can be produced in large amounts, and immunizing with recombinant vector followed by a protein boost intensifies the antibody response. Inclusion of gag and pol genes improves efficacy of the vaccine against disease progression after pathogenic SIV challenge [41]. There are two ongoing phase I trials of gag-pol and env vaccinia, both alone and boosted with gp120 protein. One tests the Therion Biologics Corporation (Cambridge, MA, USA) product in combination with VaxGen MN gp120 and the other trial tests a multivalent envelope [42]. Both trials were held up until very recently by a shortage of anti-vaccinia globulin, which is needed as a safeguard in case the vaccinia vector replicates to high titer in a volunteer. There have been safety concerns surrounding the use of replication competent vaccinia [43] but, since this virus class exhibits several advantages for use as vectors, attention has shifted to poxviruses that replicate poorly in mammalian cells: either deletion variants of vaccinia that replicate extremely poorly in mammalian hosts but well in avian cells (in which they can be grown in large quantities); or to the avian poxviruses themselves, which are replication incompetent in mammalian cells. Attenuated poxviruses Despite their inability to produce progeny in mammalian cells, these viruses still attach to cells and initiate infection, expressing their early genes. HIV genes placed under control of early promoters are surprisingly immunogenic. However, producing these recombinants in large amounts at high titers has been problematic. Modified vaccinia virus Ankara This spontaneous variant of vaccinia was developed late in the smallpox eradication campaign [44], and has proven popular with academic researchers (Table 1) since it is freely available and has shown encouraging results in primates. Although macaques immunized with modified vaccinia virus Ankara (MVA)-SIV env were not protected from challenge with highly pathogenic SIV, they had lower virus loads and prolonged survival relative to controls following infection [45]. Likewise, immunization with recombinant MVA expressing gag-pol failed to protect from infection but did result in a significant reduction in plasma viremia versus controls following challenge [46,47]. Most significantly, the reduction in virus load directly correlated with an increase in median and cumulative survival of the immunized animals.Table 1: Poxvirus vectored candidate vaccines.NYVAC This proprietary product developed by Aventis Pasteur is probably equivalent to MVA, but was created by specifically deleting 16 open reading frames from vaccinia [48] to achieve extreme attenuation in mammalian cells. It has been tested in several primate studies [49,50] and will be compared with MVA in European trials planned by the Eurovac consortium (Table 1). Fowlpox Avian retrovirus candidates are being developed by Therion and independently by the University of New South Wales, leading a consortium of Australian universities and organizations. The University of New South Wales Consortium proposes to use the fowlpox immunogens as boosts following priming with DNA immunogens. Canarypox (ALVAC) This product developed by Aventis Pasteur is a successful licensed vaccine for immunizing canaries and has been developed as a vaccine vector through several iterations. The possibility for phase III trials of these products (Table 1) has already been mentioned. The full variety of vaccine candidates under development in this category is listed in Table 1. 'Replicon' technology Safety concerns dominate planning for domestic and international trials of HIV vaccines, and much effort has gone into designing vaccine vectors that cannot replicate at all. Packaging systems for several viruses have been devised that provide structural and/or regulatory proteins in trans, so that vector virion 'replicons', completely defective for replication, are produced that package the vaccine gene inside the vector virion coat. These replicons retain any cellular targeting derived from the properties of the parent virus because the physical form of the virion remains. Deleting most of the viral genes from the packaged DNA also increases the coding capacity of the replicon for vaccine antigens. Similar to the defective poxviruses, genes delivered by these systems are still remarkably immunogenic despite the lack of a replication cycle. These replicon systems do have drawbacks. They require continuous, transformed cell lines for the packaging systems, and this can be an impediment to regulatory approval. Only now is regulatory guidance being worked out for characterizing and approving products made in such cell lines for human use. Simply harvesting medium containing the replicons from such cells is unlikely to be acceptable, and extensive purification of the replicons must be worked out to achieve the status of viral vectors. Highly sensitive assays must also be developed to prove that no replicating progeny has escaped the design and manufacturing processes. Finally, yield of replicons can be a manufacturing problem. Alphaviruses The alphavirus family is attractive because of the enormous amplification of the viral message that occurs after infection. Antigen loads can be very high using these vectors, and three different replicon packaging systems have been developed. The Venezuelan equine encephalitis virus (VEE) replicon has induced some disease amelioration against pathogenic SIV in macaque studies [51]. Wild-type VEE and some vector variants have the potential to target dendritic cells for infection. This vector is being developed by AlphaVax (Durham, NC, USA). Phase I trial is planned in 2002 for a prototype vaccine containing a clade C (South Africa) gag gene. Additional VEE vectors containing HIV-1 clade C env and pol components are in preclinical development. Chiron Corporation has developed a Sindbis virus vector packaging system and likewise has plans to produce a South African clade C candidate; mutations placed into the Sindbis glycoprotein can also target dendritic cells [52]. A third alphavirus, Semliki forest virus (SFV), has been developed in loose association with Aventis Pasteur [53] and is earlier in development. These alphaviruses are all relatively small, even with wild-type genes removed. Because of this, a vaccine product expressing gag, env and pol will require combining three separate replicons. Adenovirus This is a large virus whose replication is dependent on the E1 gene. Deletion of E1, and supplying it in trans, provides a packaging system that yields a non-infectious vector. Merck (West Point, PA, USA) has recently announced testing this vector encoding gag in humans. Adeno-associated virus This virus, despite the name, is quite different from adenovirus. Its major advantage as an HIV vaccine vector is the amplification and persistence of adenoassociated virus (AAV) episomes in the cell for extremely long times, and it is being used in humans for experimental gene therapies. It attaches to and penetrates a broad range of cells, and establishes itself in non-dividing cells. Wild-type AAV, which is widely found in humans and is totally harmless, integrates into a specific site on chromosome 19 in addition to the episomal forms. The AAV vectors developed for human use are defective for chromosome 19 integration. If persistence of vaccine antigen is a key to a successful HIV vaccine, this vector system provides a unique platform. Its coding capacity is similar to alphaviruses. A packaging system has been developed [54] and a prototype vaccine is under development, with IND submission planned for 2003. Herpes simplex An amplicon system has also been devised for herpes simplex virus (HSV) [55,56]. This system is in early preclinical development for both gene therapy and HIV vaccine uses. Attenuated viral vectors Ingenious research on the potential of several families of virus has been the basis for a variety of proposed recombinant live vectors for HIV vaccines. Since developing packaging systems for replicon creation is so time consuming, many groups have begun with wild-type vectors in animal tests for proof-of-concept experiments, or on attenuated strains. There is a sense that attenuated vectors that nonetheless replicate will be better, more immunogenic vectors than non-replicating replicons. However, 'attenuation' is often difficult to assess in advance of human testing, and this strategy will face substantially higher regulatory/safety hurdles than the replicon approaches. Retroviruses Just as cowpox gave Edward Jenner the cross-reacting key to a smallpox vaccine for humans, the attenuated vpu-minus hybrid SHIV (SIV/HIV hybrid virus) [57] has been proposed as such a cross-reactive vaccine for humans. A similar proposal was made for caprine arthritis-encephalitis virus, based on some apparent cross-reactivity with HIV [58]. These are at the research stage. Rhabdoviruses Recombinant vesicular stomatitis viruses (VSV) have been developed and shown to be high-level expression vectors and effective vaccine vectors in a small primate experiment. VSV vectors expressing HIV gag and env genes were shown to produce HIV virus-like particles containing HIV envelope protein [59]. VSV vectors expressing HIV env have been shown to induce neutralizing antibodies in small animals [60]. Interesting immunogenicity studies in small animals have been presented using rabies virus vectors expressing HIV env[61]. Picornaviruses The Sabin vaccine strain of poliovirus has been proposed as an HIV vaccine vector acceptable to the regulatory authorities. In a recently published study, two poliovirus serotypes were engineered to express SIV gene fragments encompassing a large portion of the genome. Immunization of cynomologous macaques by the nasal route resulted in full or partial protection of a majority of the animals from vaginal challenge with a pathogenic SIV [62]. The coding capacity of this replicating virus is limited, and the vaccine used in the primate study was actually a mixture of 20 different constructs of each serotype, expressing a total of 24 different genetic fragments of the virus. The complexity of such a vaccine could be an extreme problem for manufacturing and formulation. Flaviviruses Early work has begun on the yellow fever virus attenuated vaccine strain as an HIV vaccine vector candidate [63]. This vector has drawn interest because it has proven to be very safe (over 200 million people have been immunized with the attenuated yellow fever vaccine), it is inexpensive to produce, and it is highly effective at eliciting protective immunity with long duration. Orthomyxoviruses Two groups have worked with influenza as a vector for HIV [64-66]. An oral or nasal administration could be convenient for final distribution, but the potential for air-borne dissemination from volunteers during the research phase would certainly raise regulatory concerns. H