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[The AIDS Reader 10(9):526-537, 2000. © 2000 Cliggott Publishing Co., Division of SCP/Cliggott Communications, Inc.]
Several approaches to HIV prevention are being pursued, including identification of drug regimens that are practical, inexpensive, and effective in preventing maternal- infant transmission; education and behavioral modification; drug abuse treatment; provision of condoms; use of topical microbicides; treatment of other sexually transmitted diseases; and immunization. History suggests that effective immunization will prove to be the most effective, affordable, long-term approach to stopping the spread of HIV. In addition, the potential for vaccines to have a therapeutic benefit in HIV-infected persons continues to be explored, even though no vaccine has demonstrated significant impact on established infection.
A number of approaches to HIV vaccine design are being pursued. These include recombinant HIV proteins, synthetic peptides, recombinant viral vectors, recombinant bacterial vectors, DNA vaccines, synthetic HIV-like particles, and whole-killed and live-attenuated HIV. The latter 2, however, have not progressed into clinical trials because of unfavorable benefit-risk considerations. Recombinant vectors are harmless viruses or bacteria that are genetically engineered to contain and express selected HIV genes. In no case has any candidate HIV vaccine tested in humans caused HIV infection.
The significance of this conundrum is that AIDS vaccine designers do not know what specific immune responses or what levels of these responses will be necessary to provide protection. Data from the early events in acute HIV infection suggest that cytotoxic T lymphocytes (CTLs) are primarily responsible for controlling the initial peak in viremia. For example, when researchers experimentally depleted the CD8+ T cells from monkeys who were infected with simian immunodeficiency virus (SIV), viremia rose significantly and disease progression was accelerated.[1,2] Thus, several investigators have attempted to create HIV vaccine immunogens that induce high levels of CTLs.
The role of antibody in the control of HIV infection remains somewhat controversial. Control of viremia in natural HIV infection and antibody levels do not correlate as they do for CTLs. Moreover, antibodies produced in an infected person at any given time are specific for viral isolates derived from the person some months earlier. However, infusion of potent neutraliz- ing monoclonal antibodies protected monkeys from SIV infection, demonstrating that such antibodies can indeed have a protective effect.[3-5]
Other types of cellular immunity have also been invoked as potential means by which the immune system can control HIV. Some HIV-infected persons whose disease has failed to progress to AIDS have potent antigen-specific CD4+ T-cell proliferation, or helper T-cell responses.[6] In certain animal model studies, protection correlated best with secretion of b-chemokines by CD8+ T cells.[7] At this time, most AIDS vaccine development efforts aim to induce 1 or more of these 4 responses, with an emphasis on CTL and/or neutralizing antibody responses. In addition, of consummate importance in any vaccine-induced immunity is the induction of long-lasting and quick-acting memory responses.
At least 13 different envelope candidates have been evaluated for safety and immunogenicity. Importantly, these candidate vaccines have been safe, with side effects typical of most immunizations, such as sore arm and redness at the site of injection, which have resolved within a day or two without intervention. These candidates were immunogenic in diverse populations and induced neutralizing antibody in nearly 100% of recipients. Mammalian-derived envelope candidates induced higher titers of neutralizing antibody than candidates produced in yeast, insect cells, or bacteria.
However, the antibodies induced by these early envelope preparations were largely specific for clade B isolates, the subtype of HIV that predominates in the United States. In addition, antibodies induced by these early envelope preparations rarely neutralized primary isolates of HIV derived from patient blood with minimal manipulations. In addition, recombinant proteins alone rarely induced CD8+ CTLs, which recognize and kill cells that have been infected with HIV.[8,9]
Based on these results and the evaluation of HIV infections that occurred in high-risk immunized volunteers in a phase II trial, bivalent preparations of glycoprotein (gp)120, AIDSVAX, based on the envelope of 1 laboratory (B) and of 1 primary isolate (B or E) of HIV, were developed by VaxGen, which is now sponsoring phase III efficacy trials of these candidates in the United States and Thailand. Rabbit sera against these bivalent preparations and at least some vaccinee sera have been reported to neutralize at least some primary HIV isolates. Results from the US trial will be available late in the year 2001. Results from the trial in Thailand likely will not be available before 2002.
In an effort to induce both CTL and antibody responses and thereby have immune responses active against both infected cells and free viral particles, attention has turned to evaluating a combination approach, "prime-boost," where a few doses of a recombinant viral vector (the "prime") are followed by or combined with several doses of a recombinant protein (the "boost"). Several recombinant attenuated vaccinia vectors and recombinant canarypox vectors have been evaluated in phase I trials alone and in combination with a recombinant protein envelope boost (Table 1).
In general, vaccinia-immune individuals (ie, those who received the smallpox vaccine) have not responded as well to vaccinia vectors as vaccinia-naive individuals have, although there has been no difference in the response of these groups to recombinant canarypox vectors.[10,11] For this reason, as well as the fact that canarypox does not replicate completely in human cells and is therefore considered safe, canarypox vectors have been the focus of recent clinical studies. Interestingly, at least some CTLs induced by recombinant canarypox vectors based on clade B HIV and directed against the Gag protein were able to kill cells infected with HIV from other clades, because of the more conserved nature of Gag and other internal proteins.[12]
All recombinant viral vectors have been safe and immunogenic to date and have been shown to prime the immune response to an envelope boost, thereby necessitating fewer doses of recombinant protein to reach maximum antibodies titers. However, the antibodies elicited in prime-boost protocols so far have a limited breadth of reactivity.[13]
A phase II trial of a recombinant canarypox vector, vCP205, and an envelope vaccine, gp120 (SF2), was concluded in 1999 and demonstrated the safety and immunogenicity of that vaccine combination in persons at higher risk for HIV infection. This trial also demonstrated that risk-taking behavior did not increase overall among trial volunteers, all of whom received repeated counseling on how to minimize their risk of HIV infection.
A phase II trial of a canarypox vector, vCP1452, and AIDSVAX, the bivalent gp120, will soon be under way in the United States to expand safety information on that combination and to address schedule questions. Canarypox vectors may also enter phase I/II trials in Haiti, Trinidad, and Brazil later this year. An efficacy trial of the best available clade B-based canarypox vector and envelope boost is under development and could begin in late 2001.
Because canarypox is the first candidate HIV vaccine that has induced functional CTL-possessing activity against diverse HIV subtypes, the first phase I trial of a candidate vaccine in Africa was launched early in 1999. This trial will determine the safety and immunogenicity of vCP205 in Ugandan volunteers and the extent to which immunized Ugandan volunteers have CTLs that are active against the subtypes A and D of HIV, which are prevalent in Uganda. For comparison, a similar product containing a clade A envelope (vCP1452-A) could be available for phase I trials later this year.
Other strategies that have progressed to phase I trials in uninfected persons include HIV peptides, HIV lipopeptides, DNA expressing 1 or more HIV proteins, an attenuated Salmonella vector expressing envelope, p24, and others (Table 2). To date, none has proved as effective in eliciting human CTLs and/or antibody as the recombinant canarypox-gp120 combination. Recently, Merck advanced a candidate DNA vaccine containing a codon-optimized gag gene to phase I trials. Other approaches to increasing the immunogenicity of DNA vaccines are being pursued and may enter phase I trials in the next few years.
In summary, clinical trials of candidate HIV vaccines have been informative. All candidate vaccines have been safe and immunogenic in diverse populations. Unfortunately, the specific types and levels of immune responses needed to provide protection are not known. So, larger trials of the most promising candidates will be needed to determine whether any of these candidate vaccines protect individuals from HIV infections.
Many of the early candidate envelope vaccines were evaluated in HIV-infected persons, and in most cases new immune responses were induced. However, therapeutic benefit as measured by delay in disease or a significant drop in viral load was not detected. As a result, interest in therapeutic HIV vaccines waned for several years.
With the advent of HAART, which drops viral loads to undetectable levels, there is now renewed interest in determining whether immunization with candidate vaccines provides additional advantages, particularly in persons who are given HAART soon after infection and before the virus has an opportunity to severely impair the immune system. Studies are now under way to determine whether candidate vaccines, administered to persons after they have been receiving HAART for months or years, induce immune responses capable of preventing or diminishing HIV rebound if/when HAART is halted.
Based on clinical results of candidate vaccines to date, a key focus of preclinical studies is identification of candidate vaccines that induce broadly neutralizing antibodies. To this end, over the past few years researchers have determined the 3-dimensional structure of the HIV envelope, which interacts with host surface receptors and coreceptors (CD4 and chemokine receptors), elucidated how the structure might impact viral tropism, and predicted how the envelope assumes a trimeric configuration. These basic research findings have informed vaccine designers, and new design concepts are now entering animal studies. The instability and flexibility of the envelope protein and its tendency to disassemble into monomers are thought to contribute to the poor immunogenicity of certain conformationally determined epitopes of the envelope, thus forcing the immune response to be directed against variable sequential determinants. Promising approaches include genetically engineered, stabilized, trimeric forms of the protein to better mimic what the immune system may "see" on encounter with HIV virions. For example, investigators have introduced stabilizing disulfide bonds into the envelope structure in hopes of "cementing" the relatively unstable trimer into a less dissociable complex.[15,16] Other groups have genetically removed portions of the envelope structure in an attempt to expose critical regions that could induce more broadly reactive immune responses.[17]
Recent breakthroughs in determining the 3-dimensional structure of the envelope revealed specific structural characteristics that enabled investigators to discern the process employed by gp41/gp120 during fusion of the virus and cell membranes and subsequent entry of virus into the cell. Based on these structural determinations, synthetic peptides were designed that prevented HIV from fusing with CD4+ cells.[18] Efforts are under way to determine whether these peptides might be useful as therapeutic interventions or prophylactic vaccines.
In other efforts to interfere with the fusion process, investigators at the University of Montana designed a "fusion-competent" immunogen by mixing cells expressing envelope with cells expressing both CD4 and CCR5 receptors.[19] After permitting fusion to take place for about 5 hours, the cells were fixed with formaldehyde and then injected into mice transgenic for human CD4 and CCR5. The murine sera neutralized primary HIV isolates from different clades, suggesting that intermediate structure(s) that might be common to the fusion of all HIV envelopes was present in the immunogen. The investigators are now trying to reproduce this finding in nonhuman primates and are exploring means to prepare fusion-competent immunogens in practical ways more amenable to vaccine production. Still other investigators are attempting to form and immunize with complexes of envelope, CCR5, and CD4 that may mimic the molecular complexes formed during the fusion process.
One challenge associated with envelope-based vaccine approaches is the tremendous heterogeneity of the envelope sequences from viruses among clades and the viral evolution during the course of HIV infection within an individual. HIV has been characterized into at least 10 clades, or subtypes, that differ from each other by 30% to 35% in the env gene and to a somewhat lesser extent even in the more conserved internal proteins. In addition, there is no direct correlation between clade classification and neutralization or serogroup. Immunizing with any one envelope may not reproduce enough of the common static or fusion-induced structures common among all envelope proteins.
While approaches like the fusion-competent concept mentioned above could identify such common structures, researchers at St Jude's Children's Research Hospital have proposed an alternative approach.[20] Their immunogen, polyEnv1, is based on the concept of simultaneous delivery of multiple envelopes (in this case, via vaccinia virus vectors). They choose envelope proteins based on clades, binding patterns to a panel of antibodies, and sequential viral samples from infected persons in an effort to present to the immune system as many as possible of the common structures of the envelope that an individual might encounter during initial infection and through evolution of virus during chronic infection. Initial studies in mice revealed that simultaneous immunization with several envelopes elicited antibodies capable of neutralizing virus not present in the vaccine. PolyEnv1 has entered clinical studies, and further evaluation in nonhuman primates is being performed as well.
While soluble forms of the envelope protein are the primary way by which vaccine designers aim to induce anti-HIV antibody responses, intracellular delivery of proteins is generally necessary to induce cellular responses. Currently, DNA vaccines and live-virus vectors are the major vehicles for programming host cells to manufacture and process vaccine immunogens for induction of CTL responses.
Some years ago, vaccine researchers studying influenza virus demonstrated that plasmid DNA vaccine encoding a flu antigen was immunogenic in mice, suggesting that the DNA vaccine entered cells, was transcribed and translated, and the resulting protein was appropriately presented to the immune system. Unfortunately, while DNA vaccines induced potent cellular immunity in mice, the responses observed to date are less robust in nonhuman primates and very poor in humans.
However, immunization of monkeys with a DNA vaccine followed by inoculation with a live-virus vector encoding the same antigen led to a better cellular response than that obtained with the live vector or DNA alone. Interestingly, this effect was not seen if the immunizations were given in opposite order, suggesting that the DNA was somehow priming the immune response in a special way beyond simply providing a source of antigen. Moreover, while humoral responses generated by nucleic acid vaccination have been modest, booster immunizations with purified subunit protein have led to higher antibody responses than the subunit alone.
As described above, the most studied live-virus vectors in human vaccine trials belong to the poxvirus family, which includes vaccinia and canarypox. Efforts to improve these as well as other virus vectors are aimed at increasing the level of expression of the encoded antigen(s) and increasing the length of time that antigen is produced.
Significant resources are being invested in the generation of other potentially improved live-vector systems, including herpesvirus, adenovirus, the alphaviruses, modified vaccinia Ankara (MVA), adeno-associated virus (AAV), measles virus, yellow fever virus, varicella-zoster virus, and poliovirus. Each presents specific advantages and disadvantages. Of late, the alphaviruses, and specifically Venezuelan equine encephalomyelitis (VEE) virus, have received considerable attention. VEE appears to possess characteristics that make it a promising vector; VEE directs very high levels of protein production and targets lymphoid tissue, specifically dendritic cells, which are the professional antigen- presenting cells of the immune system. Initial studies in animals demonstrated that VEE recombinant vaccines generated cellular and antibody responses and lowered viral load in monkeys challenged with a pathogenic immunodeficiency virus.[21] MVA, a further attenuated derivative of a vaccinia vaccine strain, has induced potent immune responses and protection from challenge with pathogenic SIV.[22-24]
Canarypox, VEE, and MVA vectors are not persistent vectors and may require multiple immunizations to achieve high and durable levels of immunity. In contrast, AAV and herpesvirus, among others, can establish a persistent infection in the host. For example, herpesvirus remains dormant and reactivates periodically, during which time heterologous genes encoded by the recombinant virus will also be expressed and will likely boost the immune response. Preliminary data in nonhuman primates showed that single immunizations with AAV led to detectable immune responses for more than 1 year after administration. On the other hand, administration of a persistent virus vector in a healthy person raises safety concerns, particularly if the vector integrates into the host genome; these concerns will need to be carefully addressed before human trials are initiated.
Other "live" vectors under preclinical study include recombinant bacteria, such as Salmonella, Listeria, and Shigella. Attenuated Salmonella typhi offers the benefit of oral delivery, mucosal targeting, ease of manufacture, and intracellular deposition of antigen. Salmonella can also be engineered to deliver DNA encoding HIV proteins. The use of bacteria as a highly specific targeted delivery vehicle for DNA delivery is an attractive option for elaborately processed proteins, such as the HIV envelope, which has a complex pattern of glycosylation and a complicated 3-dimensional structure. Manufacture of the envelope in vivo carries the potential advantage of achieving proper folding and post-translational processing. Implicit in the use of these bacterial vectors is the concern for safety, since all are derived from human pathogens. Indications from initial phase I human trials have demonstrated that Salmonella in its attenuated form was well tolerated by vaccinees. However, concern remains that bacterial vectors may not be safe in immunocompromised persons and that issues of plasmid stability and maintenance of nonbacterial sequences in the vaccine strains will be difficult challenges to meet.
In addition to different methods of delivering antigen intracellularly to induce cellular immune responses, researchers are also varying the specific immunogen being delivered. Because the HIV gag gene can self-assemble, vectors that express this protein release viruslike particles. For example, a Gag-based "p55" particle is currently under development and will likely enter phase I trials later in 2000.
Another particularly exciting approach is the use of "epitope" vaccines designed to mount immune responses against only those small portions of large proteins to which the individual's immune system is genetically programmed to respond -- eg, the epitope. Specifical- ly, when a protein immunogen is processed inside an antigen-presenting cell, the cell clips the protein into fragments of a certain number of amino acids. These fragments are then displayed on the cell surface complexed with a specific class I major histocompatibility complex (MHC) molecule and b2-microglobulin. Other cells of the immune system recognize the presenting cell via this complex. Epitopes can be delivered by DNA, live vector, or synthetic peptide. Investigators are exploring the use of "strings-of-beads" of several epitopes from multiple HIV genes in the same candidate vaccine. One obstacle to vaccines of this type is that epitope responses are specific for certain alleles of the class I MHC; if a given individual does not possess a given allele, then any epitope that specifically associates with that allele will not be recognized. This becomes of great importance when considering an AIDS vaccine for worldwide use, since MHC alleles are racially and geographically diverse. Some groups are addressing this problem by searching for epitopes in HIV gene products that are able to bind to more than 1 class I MHC allele, with the goal of designing a vaccine containing a set of epitopes to which persons of most MHC types will be able to respond -- a truly global AIDS vaccine.
Populations with the highest incidence of HIV infection in the United States and Europe are among the hardest to recruit and retain in vaccine efficacy trials. These include injection drug users and their sexual partners; high-risk men who have sex with men; individuals who frequently acquire other sexually transmitted diseases; and persons in other high-risk populations who are difficult to identify and who are generally distrustful of government and academic researchers. Community education and support have proved essential to the conduct of clinical trials, and the National Institute of Allergy and Infectious Diseases (NIAID)-supported clinical trial sites all have community advisory boards to ensure community input in the design and conduct of such trials.
Developing countries where HIV is spreading offer unique opportunities and a different set of challenges. Few developing countries have all the trained investigators (science, clinical trials, ethics, laboratories, data management, project management) and infrastructure (clinics, laboratories, equipment, supplies) in place to conduct trials of experimental HIV vaccines. Few have the regulatory processes to consider and evaluate proposed experimental vaccine trials. As a consequence of these conditions and the fact that most candidate vaccines are based on clade B HIV, initiating tri-als in developing country settings remains challenging. Finally, few countries are willing to participate in trials without some assurance of access to the final successful vaccine.
These obstacles can be overcome only through years of true collaborative interactions that ensure that the infrastructure is in place and that both the vaccine trial sponsor and the developing country host gain from the conduct of the research. In addition, UNAIDS has promulgated ethical guidelines for the conduct of HIV vaccine trials in developing countries and has international committees on both vaccine science and ethics. These committees give guidance to countries on specific proposed clinical trials.
There are additional challenges to HIV vaccine development that cross into the financial, political, and social realms and that have spawned new organizations, approaches, and legislative proposals to address those challenges. While a reasonable market for an AIDS vaccine may exist in the United States and other industrialized countries, the majority of new infections and the greatest demand for an HIV vaccine will come from developing countries. When the costs and the time frame of vaccine development and the chances for success are weighed against the potential profit, there appears to be little incentive for companies to aggressively pursue HIV vaccine development. To address this, NIAID, Walter Reed Army Institute of Research, and the International AIDS Vaccine Initiative have taken measures to increase support for public-private collaborative efforts. In addition, several legislative proposals to offer additional incentives to the private sector have been promulgated both from within Congress and by activists' organizations. Finally, the World Bank and other philanthropic organizations are working on new ways to help ensure a large, profitable market for the successful HIV vaccine.
Finally, basic research is leading to new clues as to how more broadly neutralizing antibodies and more consistent induction of cellular immune responses and mucosal immune responses can be induced. Animal models continue to be refined and employed to evaluate the potential of new designs to provide protection. Thus, the next 5 years will be an exciting time in AIDS vaccine development, when information on existing products becomes available, and new candidates enter clinical trials. Clinical trials will remain a critical component of the search for safe and effective HIV vaccines, since only through such trials will information about the efficacy of any candidate vaccine be obtained.
Virus Designation Insert Vaccinia HIVAc gp160 TCB-IIIB gp120, Gag, Pol polyEnv1 gp140 (23 clones) Canarypox vCP125 gp160 vCP205 Gag, Pro, gp120/TM vCP300 Gag, Pro, gp120/TM, 5 Pol/Nef epitopes vCP1433 Gag, Pro, gp120/TM, 5 Pol/Nef epitopes vCP1452 Gag, Pro, gp120/TM, 5 Pol/Nef epitopes, vvE3L, vvK3L vCP1521 Gag, Pro, gp120/TM, 5 Pol/Nef epitopes, vvE3L, ccK3L
gp, glycoprotein; TM, transmembrane portion of glycoprotein 41.
Vaccine type Example DNA DNA plasmids Live viruses Adeno-associated virus Herpesvirus Vaccinia Canarypox Modified vaccinia Ankara Yellow fever Replicons Alphaviruses (VEE, Sindbis, Semiliki) Poliovirus Bacteria Salmonella Listeria Shigella BCG Whole-killed HIV inactivated by multiple methods Live-attenuated HIV with gene deletions Recombinant subunit Envelope (eg, oligomeric, polyvalent) Regulatory proteins (eg, Tat) Epitope-based Peptides Polyepitope Pseudovirions/viruslike particles p55 Gag Adjuvant/immunomodulator
Cytokines (protein or DNA)IL-2 IL-12 IL-15 GM-CSF Costimulatory molecules B7 CD40L Delivery vehicles Synthetic microparticles Mucosa-specific (eg, cholera toxin)
VEE, Venezuelan equine encephalitis; BCG, bacille Calmette-Guérin; IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor.
Obstacles Explanation Possible approaches Immune correlates The exact type and level of immune responses necessary for protection are not known Conduct efficacy trials of promising, safe candidates and attempt to decipher immune correlates Viral diversity HIV exists in at least 10 subtypes, or clades, that differ genetically by 30% - 35%; the relevance of these clades to vaccine development remains unknown Design candidate vaccines based on HIV that circulates in the proposed trial population where feasible; conduct trials of both clade-matched and -mismatched candidate vaccines and determine whether either is superior in inducing potentially protective immune responses; design candidates that induce immune responses active against all HIV clades Animal models There is no practical animal model of HIV disease in which to test candidate vaccines, though there are very good models that use SIV or SHIV infection Construct analogues of candidate vaccines, and evaluate them for immunogenicity and efficacy in animal models that most closely parallel human infection with HIV; use SHIV model to address issues related to envelope diversity Mucosal immunity It is not known whether strong immune responses at mucosal surfaces will be necessary to provide protection from sexual transmission Design candidate vaccines and novel delivery systems that are capable of inducing mucosal immune responses; in efficacy trials, determine whether mucosal immune responses correlate with protection from sexual transmission HIV pathogenesis HIV infects and can remain latent in cells of the immune system that are necessary to mount an immune response If candidate vaccines do not completely protect from infection, they should induce CTLs that can control if not eliminate cells infected with HIV Efficacy trials in the United States Even the highest-risk populations in the United States generally have an annual incidence of 1% - 2%, thus necessitating that 5000 - 10,000 volunteers be enrolled and followed for as long as 3 - 4 years Consider novel trial designs that can distinguish candidate vaccines that have a high, moderate, or very low level of activity; consider trials in other settings; minimize social harms to volunteers; work closely with at-risk of vaccine trials communities in the design and implementation Behavioral component Most exposure to HIV results from risky behaviors; most individuals at risk remain at risk for years, if not decades Volunteers in trials should be counseled repeatedly on how to minimize their exposure to HIV; vaccines may need to induce long-lasting immunity; unless a vaccine is 100% effective, it should be provided in the context of an HIV education and prevention counseling setting Clinical trials in developing countries Few developing countries have the infrastructure, training, and processes to conduct vaccine trials Provide sufficient support over a sufficient period to ensure that clinical trials can be conducted ethically and in full partnership with developing country scientists and communities; design candidate vaccines that have the potential to be affordable in poorer countries; ensure access to successful vaccine Market forces Because of technical difficulties, the uncertainty of success, the time it takes to develop a vaccine versus the perceived profit, few companies are willing to invest their resources heavily in HIV vaccine development Public sector support of private sector activities, tax credits for vaccine research and development expenditures, and a sizable guaranteed market for a successful vaccine have all been suggested to fully engage the private sector
SIV, simian immunodeficiency virus; SHIV, simian HIV; CTL, cytotoxic T lymphocytes.
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