HIV/AIDS: Vaccines and Alternate Strategies for Treatment and Prevention

Web Sites

Center for HIV/AIDS Vaccine Immunology
The Center for HIV/AIDS Vaccine Immunology (CHAVI) is a consortium of universities and academic medical centers established by the National Institute of Allergy and Infectious Diseases (NIAID) under Barton Haynes, MD, of Duke University. The Center's goal is to solve major problems in HIV vaccine development and design.

The Collaboration for AIDS Vaccine Discovery (CAVD)
The Collaboration for AIDS Vaccine Discovery (CAVD) is an international network of scientists and experts dedicated to designing a variety of novel HIV vaccine candidates and advancing the most promising candidates to clinical trials.

GeoVax Labs Inc.
GeoVax Labs Inc., is a company dedicated to the development of an HIV vaccine.

Global Advocacy for HIV Prevention (formerly AIDS Vaccine Advocacy Coalition)
Founded in 1995, AVAC is an international non-profit organization that uses education, policy analysis, advocacy, and community mobilization to accelerate the ethical development and eventual global delivery of AIDS vaccines and other new HIV prevention options as part of a comprehensive response to the pandemic.

Global HIV Vaccine Enterprise
The Global HIV Vaccine Enterprise is a unique global alliance of independent organizations working together to accelerate the development of safe and effective HIV vaccines.

HIV Vaccine Trials Network (HVTN)
The HIV Vaccine Trials Network (HVTN) is an international collaboration of scientists and educators searching for an effective and safe HIV vaccine. The HVTN's mission is to facilitate the process of testing preventive vaccines against HIV/AIDS. Our organization conducts all phases of clinical trials, from evaluating experimental vaccines for safety and the ability to stimulate immune responses, to testing vaccine efficacy.

IAVI: International AIDS Vaccine Initiative
IAVI's mission is to ensure the development of safe, effective, accessible, preventive HIV vaccines for use throughout the world.

South African AIDS Vaccine Initiative (SAAVI)
The South African AIDS Vaccine Initiative (SAAVI) was formed in 1999 as a lead programme of the Medical Research Council (MRC) of South Africa. SAAVI was established to co-ordinate the research, development and testing of AIDS vaccines in South Africa.

The Step Study
Home site for the Step HIV vaccine study.

Theraclone Sciences
Theraclone Sciences is a company involved in antibody screening efforts.

U.S. Military HIV Research Program
In 1985, the U.S. military recognized the emerging HIV-1 epidemic as a new threat to U.S. and allied forces worldwide. A military directive emerged to develop effective preventive measures to include prevention education, vaccine development and implementation of novel anti-viral therapies and clinical management tools for the Department of Defense (DoD). The United States Congress mandated the formation of a U.S. Army-led HIV/AIDS research unit in 1986.

Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases
The mission of the Vaccine Research Center (VRC) is to conduct research that facilitates the development of effective vaccines for human disease. The primary focus of research is the development of vaccines for AIDS.

Monogram Biosciences
The mission of Monogram Biosciences is to improve healthcare by advancing individualized medicine through the discovery and commercialization of innovative products to guide and improve the treatment of HIV, cancer, and other serious diseases.

Journal Articles

Dan H. Barouch

Barouch DH. 2008. Challenges in the development of an HIV-1 vaccine. Nature 455: 613-619. Full Text

Barouch DH, O'Brien KL, Simmons NL, et al. 2010. Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys. Nat. Med. 16:319-323.

Fischer W, Perkins S, Theiler J, et al. 2007. Polyvalent vaccines for optimal coverage of potential T-cell epitopes in global HIV-1 variants. Nat. Med. 13: 100-106.

Liu J, O'Brien KL, Lynch DM, et al. 2009. Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature 457: 87-91. Full Text

O'Brien KL, Liu J, King SL, et al. 2009. Adenovirus-specific immunity after immunization with an Ad5 HIV-1 vaccine candidate in humans. Nat. Med. 15: 873-875. Full Text

Santra S, Liao HX, Zhang R, et al. 2010. Mosaic vaccines elicit CD8+ T lymphocyte responses that confer enhanced immune coverage of diverse HIV strains in monkeys. Nat. Med. 16: 324-328.

Jerome Kim

Nitayaphan S, Pitisuttithum P, Karnasuta C, et al. 2004. Safety and immunogenicity of an HIV subtype B and E prime-boost vaccine combination in HIV-negative Thai adults. J. Infect. Dis. 190: 702-706. (PDF, 92 KB) Full Text

Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, et al. 2009. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N. Engl. J. Med. 361: 2209-2220. Full Text

Thongcharoen P, Suriyanon V, Paris RM, et al. 2007. A phase I/II comparative vaccine trial of the safety and immunogenicity of CRF01 AE (subtype E) candidate vaccines: ALVAC-HIV (vCP1521) prime with oligomeric gp160 (92TH023/LAI-DID) or bivalent gp120 (CM235/SF2) boost. J. Acquir. Immune Defic. Syndr. 46: 48-55.

Julie McElrath

Buchbinder SP, Mehrotra DV, Duerr A, et al. (Step Study Protocol Team) 2008. Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial. Lancet 372: 1881-1893. Full Text

Fellay J, Ge D, Shianna KV, et al. (NIAID Center for HIV/AIDS Vaccine Immunology (CHAVI)) 2009. Common genetic variation and the control of HIV-1 in humans. PLoS Genet. 5: e1000791. Full Text

Flynn NM, lynn NM, Forthal DN, et al. (rgp120 HIV Vaccine Study Group) 2005. Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection. J. Infect. Dis. 191: 654-665. (PDF, 229 KB) Full Text

Gilbert PB, Peterson ML, Follmann D, et al. 2005. Correlation between immunologic responses to a recombinant glycoprotein 120 vaccine and incidence of HIV-1 infection in a phase 3 HIV-1 preventive vaccine trial. J. Infect. Dis. 191: 666-677. (PDF, 479 KB) Full Text

McElrath MJ, De Rosa SC, Moodie Z, et al. (Step Study Protocol Team) 2008. HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case-cohort analysis. Lancet 372: 1894-1905. Full Text

Pitisuttithum P, Gilbert P, Gurwith M, et al. (Bangkok Vaccine Evaluation Group) 2006. Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand. J. Infect. Dis. 194: 1661-1671. (PDF, 1.41 MB) Full Text

Russell ND, Graham BS, Keefer MC, et al. (National Institute of Allergy and Infectious Diseases HIV Vaccine Trials Network) 2007. Phase 2 study of an HIV-1 canarypox vaccine (vCP1452) alone and in combination with rgp120: negative results fail to trigger a phase 3 correlates trial. J. Acquir. Immune Defic. Syndr. 44: 203-212. Full Text

Chris Miller

Genescà M, Skinner PJ, Bost KM, et al. 2008. Protective attenuated lentivirus immunization induces SIV-specific T cells in the genital tract of rhesus monkeys. Mucosal Immunol. 1: 219-228.

Genescà M, Skinner PJ, Hong JJ, et al. 2008. With minimal systemic T-cell expansion, CD8+ T Cells mediate protection of rhesus macaques immunized with attenuated simian-human immunodeficiency virus SHIV89.6 from vaginal challenge with simian immunodeficiency virus. J. Virol. 82: 11181-11196. Full Text

Genescà M, Miller CJ. 2010. Use of nonhuman primate models to develop mucosal AIDS vaccines. Curr. HIV/AIDS Rep. 7: 19-27. Full Text

Stone M, Ma ZM, Genescà M, et al. 2009. Limited dissemination of pathogenic SIV after vaginal challenge of rhesus monkeys immunized with a live, attenuated lentivirus. Virology 392: 260-270.

Sanjay Phogat

Corti D, Langedijk JP, Hinz A, et al. 2010. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. PLoS One 5: e8805. Full Text

Li Y, Migueles SA, Welcher B, et al. 2007. Broad HIV-1 neutralization mediated by CD4-binding site antibodies. Nat. Med. 13: 1032-1034.

Scheid JF, Mouquet H, Feldhahn N, et al. 2009. Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals. Nature 458: 636-640.

Simek MD, Rida W, Priddy FH, et al. 2009. Human immunodeficiency virus type 1 elite neutralizers: individuals with broad and potent neutralizing activity identified by using a high-throughput neutralization assay together with an analytical selection algorithm. J. Virol. 83: 7337-7348. Full Text

Walker LM, Phogat SK, Chan-Hui PY, et al. 2009. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326: 285-289.

Louis Picker

Hansen SG, Powers CJ, Richards R, et al. 2010. Evasion of CD8+ T cells is critical for superinfection by cytomegalovirus. Science 328: 102-106.

Hansen SG, Vieville C, Whizin N, et al. 2009. Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge. Nat. Med. 15: 293-299. Full Text

Harriet L. Robinson

Amara RR, Villinger F, Altman JD, et al. 2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292: 69-74.

Amara RR, Smith JM, Staprans SI, et al. 2002. Critical role for Env as well as Gag-Pol in control of a simian-human immunodeficiency virus 89.6P challenge by a DNA prime/recombinant modified vaccinia virus Ankara vaccine. J. Virol. 76: 6138-6146. Full Text

Amara RR, Villinger F, Staprans SI, et al. 2002. Different patterns of immune responses but similar control of a simian-human immunodeficiency virus 89.6P mucosal challenge by modified vaccinia virus Ankara (MVA) and DNA/MVA vaccines. J. Virol. 76: 7625-7631. Full Text

Buge SL, Ma HL, Amara RR, et al. 2003. Gp120-alum boosting of a Gag-Pol-Env DNA/MVA AIDS vaccine: poorer control of a pathogenic viral challenge. AIDS Res. Hum. Retroviruses 19: 891-900.

Ellenberger D, Otten RA, Li B, et al. 2006. HIV-1 DNA/MVA vaccination reduces the per exposure probability of infection during repeated mucosal SHIV challenges. Virology 352: 216-225.

Smith JM, Amara RR, Campbell D, et al. 2004. DNA/MVA vaccine for HIV type 1: effects of codon-optimization and the expression of aggregates or virus-like particles on the immunogenicity of the DNA prime. AIDS Res. Hum. Retroviruses 20: 1335-1347.

Wyatt LS, Earl PL, Vogt J, et al. 2008. Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines. Vaccine 26: 486-493. Full Text

Michael Worobey

Wertheim JO, Worobey M. 2009. Dating the age of the SIV lineages that gave rise to HIV-1 and HIV-2. PLoS Comput. Biol. 5: e1000377. Full Text

Worobey M, Gemmel M, Teuwen DE, et al. 2008. Direct evidence of extensive diversity of HIV-1 in Kinshasa by 1960. Nature 455: 661-664.

Gilbert MT, Rambaut A, Wlasiuk G, et al. 2007. The emergence of HIV/AIDS in the Americas and beyond. Proc. Natl. Acad. Sci. 104: 18566-18570. Full Text

Susan Zolla-Pazner

Almond D, Kimura T, Kong X, et al. 2010. Structural conservation predominates over sequence variability in the crown of HIV type 1's V3 loop. AIDS Res. Hum. Retroviruses 26: 717-723.

Gorny MK, Stamatatos L, Volsky B, et al. 2005. Identification of a new quaternary neutralizing epitope on human immunodeficiency virus type 1 virus particles. J. Virol. 79: 5232-5237. Full Text

Hioe CE, Wrin T, Seaman MS, et al. 2010. Anti-V3 monoclonal antibodies display broad neutralizing activities against multiple HIV-1 subtypes. PLoS One 5: e10254. Full Text

Jiang X, Burke V, Totrov M, et al. 2010. Conserved structural elements in the V3 crown of HIV-1 gp120. Nat. Struct. Mol. Bio. [Epub ahead of print]

Robinson JE, Franco K, Elliott DH, et al. 2010. Quaternary epitope specificities of anti-HIV-1 neutralizing antibodies generated in rhesus macaques infected by the simian/human immunodeficiency virus SHIVSF162P4. J. Virol. 84: 3443-3453.

Zolla-Pazner S, Cardozo T. 2010. Structure-function relationships of HIV-1 envelope sequence-variable regions refocus vaccine design. Nat. Rev. Immunol. 10: 527-535.

Zolla-Pazner S, Cohen S, Pinter A, et al. 2009. Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope. Virology 392: 82-93. Full Text

Sponsors

Academy Friends

• Mabtech
• NeED Pharmaceuticals

Grant Support

This program is supported by an educational grant from Merck & Co., Inc.

Speakers:
Jerome Kim, Walter Reed Army Institute of Research
Julie McElrath, Fred Hutchinson Cancer Research Center
Alan Bernstein, Global HIV Vaccine Enterprise

Highlights

• A successful vaccine may need to elicit both a cellular response by T-cells and a humoral, antibody response.
• There have been few large-scale clinical efficacy trials of potential HIV vaccines and only one statistically significant positive result, but the modest protection from acquisition it showed has energized the field.
• Analysis of vaccine-induced immune responses can augment the scientific value of trials.

Introduction

On May 19, 2010, the Vaccine Science Discussion Group joined with the Global HIV Vaccine Enterprise to host a day-long symposium called "HIV/AIDS: Vaccines and Alternate Strategies for Treatment and Prevention." Although behavioral changes and strategies such as condom use, circumcision, microbicides, and prophylactic antiviral therapy have key roles to play in prevention, most of the discussion centered on vaccines, in part because the meeting came just a day after HIV Vaccine Awareness Day and in part because of the known effectiveness of vaccines in stopping many other infectious pathogens.

In spite of extensive scientific understanding of how the HIV-1 virus infects people, the prospects for an effective preventive vaccine remain unclear, because the immune correlates of protection are not known. Researchers have long known of candidate targets for an immune response, such as the viral glycoprotein called Env, which forms spikes on the virus that bind with the CD4 receptor to gain entry into helper T-cells. Other viral genes, including gag, pol, tat, and nef, encode other internal proteins that may also make good targets. Nonetheless, early efforts using gp120 protein were disappointing, stimulating B-cells to produce antibodies to the virus but not blocking infection. A major challenge is the extreme variability of many HIV-1 virus components, notably Env. Many researchers expect that a successful vaccine will need to elicit both a cellular response by T-cells and a humoral, antibody response.

Rather than using proteins or peptide fragments directly to elicit a response, recent efforts aim to produce them locally from exogenous DNA in the cells. The vaccine can supply this DNA in the form of a plasmid that cells take up, or as part of a safe, genetically engineered virus. This strategy works particularly well when an initial "prime" inoculation is followed some time later by a "boost." Much current HIV vaccine research centers on "heterologous" prime-boost regimens, in which subsequent inoculations use different vectors.

The RV144 "Thai trial"

To date, three different HIV vaccine concepts have been carried to large-scale trials of their effectiveness. Only one showed a modest positive result in preventing acquisition. This trial, the RV144 "Thai trial," used a heterologous prime/boost regimen, which combined a canarypox-vector prime with a gp120 subunit boost. This regimen appeared to reduce the risk of infection by about one third (31.2%) in placebo-controlled study involving 16,000 Thai volunteers. While only providing modest protection, the results have provided some scientific clues for vaccine scientists and fuelled considerable enthusiasm that a more effective vaccine is possible.

The "Thai Trial," RV144, showed that a heterologous prime/boost regimen reduced HIV acquisition by about one third. This is the first positive human HIV/AIDS vaccine result. While the primary statistical analysis was statistically significant, two others were not due to loss of endpoints. However, they support the trend towards protection as shown above.

"We're very excited by the result," said Jerome Kim of the U.S. Military HIV Research Program (MHRP), centered at the Walter Reed Army Institute of Research, "and by the follow-on to the result." Indeed, the study of factors that co-exist with protection, the "immune correlates," may ultimately be more important than the modest protection the vaccine provides.

Correlates research is underway at MHRP and with nearly 20 other collaborating organizations from around the world, and samples from the study participants have been systematically distributed. Due to budgetary limitations, collection of PBMC was limited, so MHRP and NIAID developed a review process to ensure the most efficient and effective use of the samples. Kim said, "While we hope to identify the correlates, it should be noted that immune correlates of protection do not yet exist for some vaccines that have been licensed for years.”

Intriguingly, although the study was not designed to explore this issue, "this vaccine appeared to work best in people at the lowest risk of HIV infection" as judged by their reported behavior, Kim noted. The data also hint that the protective effect was highest at 6-12 months and was not durable, decreasing to 31.2% after 3.5 years. MHRP is planning two follow-on small clinical studies with an additional boost to see if they can extend the immune responses. These studies allow them to better characterize the immune response profile of ALVAC/AIDSVAX using state-of-the-art immune assessment platforms.

Although the vaccine appeared to prevent infection in some recipients, in those who nonetheless acquired the virus it did not reduce viral replication or prevent the decline of CD4+ T cells.

The Step trial

Such post-hoc analysis can be very informative, said Julie McElrath of the Fred Hutchinson Cancer Research Center. She reviewed results from the earlier Step trial, which in 2007 found no significant vaccine-induced protection.

The initial strategy was to elicit the CD8+ T-cell immune response, McElrath said, and early studies showed that "viral vectors that encoded HIV genes were the real winners." The researchers abandoned a canarypox vector like that used in the Thai trial after it showed a rather small effect. "Fortunately, others didn't," McElrath noted.

Instead, the Step trial used the more promising-looking adenovirus vector, Ad5, expressing the HIV-1 genes gag, pol, and nef. "The study did not lower HIV infection rates or post-infection plasma viremia," McElrath concluded, although 90% of vaccinees had detectable HIV-specific responses dominated by CD8+ T cells.

One problem is that many people have already been exposed to Ad5 and developed neutralizing antibodies before vaccination. "Those individuals who had a baseline Ad5 seropositivity in general had a blunted response to the HIV vaccine inserts," McElrath noted.

"We need to stop thinking about trials as built solely on acquisition or protection."

The CD8+ T cell response had antiviral activity, McElrath said, but the response may not have been adequate to suppress replication. More importantly, "it is unlikely that we were able to achieve sufficient breadth to be able to recognize incoming viral strains," she said. The vaccine did appear to impede viruses whose proteins had the amino-acid sequences in the vaccine, because "viruses infecting vaccinees were more likely to have epitope sequences that differed from those in the vaccine," McElrath said. Unfortunately, many subjects were infected by viruses with these differing sequences.

The experience with these trials shows that scientific goals should be emphasized earlier in study design, said Alan Bernstein, of the Global HIV Vaccine Enterprise. "We need to stop thinking about trials as solely built on acquisition or protection, and think of trials also as a way of perturbing the human immune system and therefore both an opportunity to try to understand what's going on and the best route to product licensure."

The integration of scientific inquiry with product development, as well as faster turnaround, is one priority of the enterprise's new strategic plan. The second priority, Bernstein said, is to "harness the full potential of preclinical models and the ongoing revolution in biomedical science." He stressed that the plan provides guidance on achieving synergies, but not specific tasks, for researchers and organizations. The full plan will be made public in coming months.

Speakers:
Michael Worobey, University of Arizona
Sanjay Phogat, International AIDS Vaccine Initiative
Susan Zolla-Pazner, New York University School of Medicine
Dan Barouch, Beth Israel Deaconess Medical Center

Highlights

• Biogeographical analysis suggests that SIV is at least 100,000 years old.
• The extraordinary natural variability of HIV proteins makes targeted immunity difficult, but some patients have antibodies that neutralize a very broad range of viruses.

Looking into the past

Michael Worobey of the University of Arizona provided a historical perspective on the diversity of HIV. He and his collaborators have been collecting "samples that take us back in time." From this perspective, the pandemic strain is intermingled in the evolutionary tree with various related strains found in non-human primates called simian immunodeficiency viruses (SIV). "HIV-1/M looks like a chimpanzee virus," he said. Some analyses had suggested it arose only within the past 200 years or so.

But the SIV found in mandrill-like drills on Bioko island in the Republic of Equatorial Guinea shares features with mainland SIV variants, even though the island was isolated by sea-level rise 12,000 years ago. "This biogeographical calibration I think is much more solid," Worobey said. Based on this reanalysis, "it's clear that SIV is old, very conservatively 100,000 years old.

The virus has probably been making forays into humans for a long time, Worobey suggested, so its recent success probably reflects higher population density in Africa. But samples from central Africa in 1959 and 1960 show that "you had a huge diversity and a suggestion of many decades of evolutionary change leading up to even that early time point." He traces the origin of the pandemic HIV-1/M to around the beginning of the twentieth century.

The escape of the virus from Africa is more recent, probably within a few years of 1969, Worobey said. "The common ancestor of subtype B [or clade B] as a whole looks like it existed in Haiti, where the virus diversified for several years before it stepped elsewhere into the U.S., Canada, and other countries."

"Across the scales that we look at we keep seeing evidence of extreme genetic bottlenecks," Worobey said. The observed separation of subtypes probably reflects the founder effects, he said. "You see the same pattern when the virus transmits from one host to the next: a real collapse in the genetic diversity."

The good news is that these bottlenecks show that the virus only rarely moves between groups or between people. "HIV-1 lives close to the edge of extinction," Worobey said. Reducing transmission, whether by vaccine or other means, could have a major impact on the epidemic.

The search for broad neutralizing antibodies

To accomplish this with vaccines, said Sanjay Phogat of the International AIDS Vaccine Initiative (IAVI), "we need neutralizing antibodies that can really cover the entire variability." In the past few years researchers have made significant progress in screening infected people for "broad neutralizing antibodies." Identifying the conserved molecular regions, or epitopes, that these antibodies target, may let researchers design antigens that elicit these antibodies in unexposed people.

"We need neutralizing antibodies that can cover the entire variability of the HIV virus."

The key to the recent progress is screening for antibodies that neutralize viral activity, rather than simply binding to viruses, Phogat said. Recent advanced techniques, requiring just a few antibody-producing B cells from a patient's serum, enable screening of many viral variants simultaneously for neutralizing ability.

Phogat reviewed the improved antibody screening approaches used by several labs. The NIH Vaccine Research Center (VRC) looks for binding to a target that was designed to expose specific regions of the envelope glycoproteins. "They got a very broad neutralizing antibody, VRC-01, that is dedicated to the CD4 binding site on the virus," Phogat noted. In parallel, the Collaboration for AIDS Vaccine Discovery (CAVD) used binding followed by a neutralization screen to find broad neutralizing antibodies, notably one designated HJ16.

Phogat and his colleagues at IAVI and The Scripps Research Institute took pains to collect, and to characterize well, samples from around the world. One patient from their "Protocol G" yielded two excellent antibodies, denoted PG9 and PG16. "These are very potent and broad neutralizing antibodies." Phogat said.

To move from these broad antibodies to vaccines that elicit them, researchers need to identify and mimic the epitopes the antibodies bind. Many of the broad neutralizing antibodies bind to previously known sites, such as the CD4 binding site that facilitates viral entry into the cell, or to one of the "variable loops" in the envelope glycoprotein.

The PG9 and PG16 antibodies, however, bind to epitopes in two different variable loops. As discussed by Susan Zolla-Pazner of the New York University School of Medicine, these antibodies primarily recognize "quaternary neutralizing epitopes," or QNEs, which form only when multiple subunits of the envelope protein arrange into the quaternary structure that makes them biologically active. For PG9, PG16, and 13 other QNE-specific human and macaque antibodies, this structure is a trimer of gp120 glycoproteins that forms the surface spikes of the virus.

Newly identified antibodies neutralize a wide range of variants of the glycoproteins gp120 and gp41. These glycoproteins, coded by the env gene, trimerize to form the spikes that interact with CD4 receptor and CCR5 coreceptors on T cells to enter the cells.

The broad neutralizing capability, Zolla-Pazner emphasized, "means that there must be conserved structures in the second and third 'variable' regions," known as V2 and V3. She and her colleagues studied the amino-acid sequences of V2 and V3 from diverse variants, and found little variation at many positions in each of these regions. This conservation appears to be related to the biological functions of these portions of the envelope, such as infectivity and glycosylation.

Zolla-Pazner also showed crystallographic structures for many V3 antibodies in complexes with V3 peptides, and found that the structure was conserved and sequence variability was confined to particular portions of the V3 loop. "This begins to tell you how to design an immunogen that will induce broadly neutralizing antibodies, what areas you want to target and what areas you want to avoid." She and her colleagues have spliced the critical region into cholera toxin B, and shown that in rabbits it induces neutralizing antibodies to diverse variants of the envelope glycoprotein.

Mosaic antigens

Dan Barouch of the Beth Israel Deaconess Medical Center described a different approach to inducing broad immunologic coverage. With Bette Korber of Los Alamos National Labs, he explored "mosaic antigens," which are "polyvalent in-silico recombined antigen sequences that aim to optimize coverage of global virus diversity," Barouch said.

The researchers decided that including just two of the computationally generated mosaic antigens provided a good balance between increasing coverage and practical constraints. Testing this bivalent vaccine in monkeys confirmed much of the theoretical promise. In terms of the breadth of the cellular immune response against a panel of HIV peptides from various clades, the mosaic vaccine was superior to both naturally occurring and consensus sequences. In addition, the mosaic vaccine elicited a greater depth of response, meaning that there was increased more viral variants.

The increased breadth "may improve coverage of global virus diversity, leading to the possibility of a globally relevant T-cell based vaccine," Barouch said, while the greater depth may make a vaccine more effective. "We feel that the 2-valent mosaic antigens are attractive for clinical evaluation."

Speakers:
Dan H. Barouch, Beth Israel Deaconess Medical Center
Harriet L. Robinson, GeoVax Inc.
Chris Miller, University of California, Davis
Louis J. Picker, Oregon Health & Science University

Highlights

• Modified vaccinia Ankara virus shows promising results in monkey and early human trials.
• Computationally generated "mosaic antigens" can elicit very broad response to natural virus populations.
• Other virus vectors may have other advantages, notably the cytomegalovirus that creates long-term "effector memory" that could rapidly respond to new infections.

Adenovirus and vaccinia vectors

Dan Barouch described early studies of alternative adenovirus vectors, such as Ad26 and Ad35, to which individuals should have less pre-existing immunity than the Merck Ad5 serotype used in the Step trial. In addition to low seropositivity, these viruses are "biologically very different than Ad5," Barouch noted.

In a phase 1 dose-escalation clinical trial, the Ad26 vector proved safe and well tolerated. Surprisingly, even at the lowest dose used in the study, 10⁹ viral particles, subjects developed a fairly consistent immune responses to EnvA antigen that was expressed by the vaccine.

These immune responses included both antibody responses and Env-specific T-cell responses. Interim analysis of this ongoing study "suggests that the Ad26 vaccine was safe and immunogenic at all three doses tested," Barouch said. Moreover, "there is a fairly large dose range" between the dose required for immunogenicity and the dose where there is modest reactogenicity, he added. Ad26 and Ad35 vectors are planned for further clinical evaluation this year.

Harriet Robinson of Geovax and her colleagues at the NIAID have been exploring a different viral vector, the modified vaccinia Ankara virus (MVA). Cells infected with this vaccine express non-infectious HIV-like particles. The products of env are displayed on the surface of the virus-like particles in a transmembrane, trimeric form, just as they are in the HIV-1 virus.

This modified MVA, expressing gag, pol, and env was used in a phase 1 human trial, in repeated doses on its own, and after one or more priming inoculations with a gag/pol/env DNA vaccine. The DNA prime improved the magnitude and breadth of the T-cell response. But in terms of antibody responses, "we found that our MVA-only regimen raised the highest antibody," Robinson noted.

In addition, the homologous MVA regimens had lower levels of CD4+ T-cell responses, relative to the CD8+ T-cell response. "That's important because you do not want your vaccine raising targets for infection," she stressed, and the antiviral CD4+ T cells are preferentially infected by HIV-1.

The researchers also studied the efficacy of these vaccine regimens in macaques against repeated rectal challenges with SIV. Both the heterologous DNA/MVA and homologous MVA/MVA vaccinated animals "are significantly protected relative to the controls," Robinson said, but there was little effect on viral replication once infection occurred. "Overall, it's very much like the Thai trial: we're preventing infection, we're not preventing post-infection viremia."

The researchers are currently pursuing phase 2 trials, to which they are adding a homologous MVA arm. Because the monkey experiments involved very high levels of challenge virus, 40 to 400 times higher than typical human exposure, Robinson expressed hope that the human protection will be better than the significant but incomplete protection afforded to the macaques.

Experiments with non-human primates let researchers test the efficacy of vaccines against intentional viral challenges, and also to explore the immune correlates of protection and the spread of infection in specific tissues. Chris Miller of the University of California, Davis, has looked for unique aspects of protection from live, attenuated vaccines. Although the risk of viral mutations that actually cause disease makes them an unlikely candidate for human use, he said, "in the non-human primate world, live-attenuated vaccines have been the only vaccine modality that have provided reliable protection from vaginal SIV challenge."

Miller's team vaccinated monkeys with a chimeric virus that is a combination of the envelope of HIV, with the rest of the genes from SIV. After vaginal viral challenge months later, he said, "about 60% of monkeys can control viral replication and don't have any CD4 loss," he said. "We have some evidence of complete protection" in these monkeys.

"Vaccines may be a two-edged sword."

To explore the correlates of protection, Miller said, "we wanted to look at immune responses in tissues, because our previous studies trying to look at correlates of protection in blood were not very satisfactory." At various stages after viral challenge, the researchers necropsied unvaccinated monkeys, and both protected and unprotected vaccinated monkeys. When the vaccine works, Miller noted, it seems to stop the virus before it spreads beyond the reproductive tract.

To check whether this protection is afforded by CD8+ T cells, the researchers used a monoclonal antibody to deplete these cells, as well as natural killer cells, in five protected animals. As expected, the depletion opened the monkeys to infection.

But in what Miller called a "sobering observation," the animals that were immunized and then depleted of CD8+ T cells had higher viral loads in their blood than did the unimmunized animals." He attributed this increased susceptibility to an increase in the CD4+ T cells that are targets for the virus, once the CD8+ cells are gone. "Vaccines are a two-edged sword in the case of a virus that actually infects CD4+ target cells," Miller said.

The effector memory T-cell response

Most HIV-vaccine research has expanded from focusing on a pure antibody response to include a CD8+ cellular response as well. But Louis Picker of Oregon Health and Science University cautions that the "central memory" T cell population may expand too slowly to stop acquisition of the virus. "The kinetics of the virus is much faster," he said, and peak replication may occur before memory cells can proliferate and migrate to the site of infection.

"We believe that the major obstacle for a T cell vaccine is not actually the sequence variability of the virus," Picker said, "it's this kinetic mismatch." To address this challenge, Picker proposes exploiting the effector memory T-cell response, which relies on resting, differentiated cells that, although limited in number, remain present in the affected tissues. "They are poised for immediate effector function," he said.

The rapid but limited effector-memory T-cell response could play an important role in slowing the intermediate stages of HIV infection and replication as part of a comprehensive vaccine paradigm.

"To generate those responses, we turned to cytomegalovirus (CMV) because it was known from previous work to be the quintessential inducer of high-frequency, effector-memory-dominant T-cell responses." Over the past eight years, Picker and his team have tracked the response of monkeys to a CMV vector that expresses SIV genes gag, rev, nef, tat, env, and pol.

The CMV infection results in a persistent effector-memory T-cell response to as few as 100 viral particles. These responses are "widely distributed in effector sites and extraordinarily durable," Picker notes, lasting seven years so far.

The response of monkeys to this vaccine was bimodal. "Roughly half of the animals appear to be completely protected" from HIV infection. In addition, Picker said, "this virological control must be very stringent," because the researchers saw no antibody response in the protected animals after challenge, even as they suppressed the infection.

The results "extend the T-cell HIV/AIDS vaccine paradigm, and suggest that effector T cells should be integrated into a vaccine approach," Picker said. "If we're going to be successful, we're going to have a vaccine that incorporates all of these elements."

Will live, attenuated vaccines ever be acceptably safe for use in humans?

Will a successful vaccine need to elicit an effector-memory T cell response, in addition to antibody and central-memory T cell responses?

How important is it that the vaccine-induced response minimize the increase of CD4+ T-cells that are targets for the HIV virus?

Can the immune responses to vaccination be adequately understood using blood samples alone?

How can the scientific information from clinical trials be more efficiently planned for and extracted?

What vectors, antigens, and regimen will provide the best protection from HIV?

Can the HIV epidemic be essentially stopped by a modest reduction in transmission?

What are the best ways of identifying the conserved sequence or structural features of a virus?