Michael Palm Basic Science, Vaccines & Prevention Project Weblog

Peptide-Pulsed Blood as an Immunization Strategy

Don't usually post on weekends but I think this team of Australian researchers deserves recognition for particularly inspired use of the NIH AIDS Reagent Program. While the wider vaccine field wrestles with the challenges of inducing broad and high magnitude T cell responses, this team from Stephen Kent's lab has developed a strategy where multiple peptides obtained from the reagent program are simply mixed with unfractionated peripheral blood mononuclear cels (PBMC) and reinfused:

"All isolated PBMC (on average 24 million cells) were suspended in 0.5 ml of normal saline to which either a pool of 125 SIVmac239 Gag peptides or 823 peptides spanning all SIVmac239 proteins (Gag, Pol, Env, Nef, Vif, Tat, Rev, Vpr, Vpx) were added at 10 µg/ml of each peptide within the pool. Peptides were 15mers overlapping by 11 amino acids at >80% purity kindly provided by the NIH AIDS reagent repository program (catalog numbers 6204, 6443, 6883, 6448-50, 6407, 8762, 6205)."

Using this approach as an immunization strategy in a relatively large study of SIV-infected pigtailed macaques, they induced high-magnitude SIV-specific CD4 and CD8 T cell responses which were associated with durable viral load reductions of close to 1 log compared to controls after cessation of antiretroviral therapy. These are among the best results reported from therapeutic immunization in the SIV model. The viral load data was independently verified in a blinded analysis by Jeff Lison and Michael Piatak from the National Cancer Institute. Kent has now formed a company to develop the approach for human use under the acronym OPAL. According to the discussion section of the paper, the approach may even be viable using whole blood instead of PBMC. Although it would not be ideal in terms of practicality, it is tempting to wonder if this type of approach could be evaluated as a preventive vaccine strategy, particularly given recent data suggesting the importance of inducing a broad response to multiple epitopes in Gag.

The study also offers an opportunity to salute the NIH AIDS Reagent Program itself; when talking to HIV researchers, the program is consistently complimented for the services it provides. At the moment the program website is promoting the biannual feedback campaign, any researchers interested in providing input can fill out a brief online survey (the closing date is tomorrow, May 4).

PLoS Pathog 4(5): e1000055. doi:10.1371/journal.ppat.1000055

Control of Viremia and Prevention of AIDS following Immunotherapy of SIV-Infected Macaques with Peptide-Pulsed Blood

Robert De Rose1, Caroline S. Fernandez1, Miranda Z. Smith1, C. Jane Batten1, Sheilajen Alcântara1, Vivienne Peut1, Erik Rollman1, Liyen Loh1, Rosemarie D. Mason1, Kim Wilson2, Matthew G. Law3, Amanda J. Handley1,4¤, Stephen J. Kent1

1 Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia2 National Serology Reference Laboratory, Fitzroy, Victoria, Australia3 National Centre for HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, New South Wales, Australia4 Opal Therapeutics Pty Ltd, Melbourne, Victoria, Australia

Abstract

Effective immunotherapies for HIV are needed. Drug therapies are life-long with significant toxicities. Dendritic-cell based immunotherapy approaches are promising but impractical for widespread use. A simple immunotherapy, reinfusing fresh autologous blood cells exposed to overlapping SIV peptides for 1 hour ex vivo, was assessed for the control of SIVmac251 replication in 36 pigtail macaques. An initial set of four immunizations was administered under antiretroviral cover and a booster set of three immunizations administered 6 months later. Vaccinated animals were randomized to receive Gag peptides alone or peptides spanning all nine SIV proteins. High-level, SIV-specific CD4 and CD8 T-cell immunity was induced following immunization, both during antiretroviral cover and without. Virus levels were durably ~10-fold lower for 1 year in immunized animals compared to controls, and a significant delay in AIDS-related mortality resulted. Broader immunity resulted following immunizations with peptides spanning all nine SIV proteins, but the responses to Gag were weaker in comparison to animals only immunized with Gag. No difference in viral outcome occurred in animals immunized with all SIV proteins compared to animals immunized against Gag alone. Peptide-pulsed blood cells are an immunogenic and effective immunotherapy in SIV-infected macaques. Our results suggest Gag alone is an effective antigen for T-cell immunotherapy. Fresh blood cells pulsed with overlapping Gag peptides is proceeding into trials in HIV-infected humans.

Homing in on the Cause of Disrupted Hematopoiesis in HIV Infection

A new study in the free access Journal of Clinical Investigation makes a compelling case that the HIV protein Nef is responsible for the hematopoietic abnormalities (anemia, granulocytopenia, and thrombocytopenia) that have been reported in people with HIV infection. Researchers led by Stéphane Prost report that Nef interacts with a nuclear receptor called PPARγ, causing suppression of two signaling proteins, STAT5A and STAT5B, known to be important for the development of hematopoietic stem cells (HSCs). The study used multiple different approaches to verify that a specific region of the nef gene which is conserved among different HIV-1 and SIV strains triggered HSC dysfunction. The authors conclude by suggesting that the study “provides new clues for the development of novel drugs targeting PPARγ activity to cure hematopoietic disorders, including those affecting seropositive/AIDS patients.”

In an accompanying commentary, Frank Kirchhoff and Guido Silvestri praise the work but also caution that “much work will be required to define the mechanism by which extracellular Nef induces PPARγ and to determine whether the levels of Nef in blood and bone marrow are sufficient to impair HSC function during HIV infection.” They also note that “as STAT5 signaling plays a critical role in T cell development and function, an additional question is whether Nef also affects T cell homeostasis by suppressing STAT5A/B function in other, more mature T cell subsets.”

J. Clin. Invest. - (2008). doi:10.1172/JCI35487.

Commentary

Is Nef the elusive cause of HIV-associated hematopoietic dysfunction?

Frank Kirchhoff1 and Guido Silvestri2

1Institute of Virology, University of Ulm, Ulm, Germany.
2Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

HIV-associated hematological abnormalities involve all lineages of blood cells, thus implying that the virus impairs the function of early HSCs. However, the underlying mechanisms of this defect are unknown, particularly since HSCs are largely resistant to HIV-1 infection. In this issue of the JCI, Prost and colleagues show that the viral accessory protein Negative factor (Nef) plays a potentially critical role in the pathogenesis of HIV/SIV-associated hematopoietic dysfunction by affecting the clonogenic potential of HSCs. Soluble Nef induces PPARγ in uninfected HSCs, thereby suppressing the expression of STAT5A and STAT5B, two factors necessary for proper HSC function. The identification of this novel activity of extracellular Nef defines a new mechanism of HIV/SIV pathogenesis and suggests that approaches aimed at increasing STAT5A and STAT5B expression may be considered in HIV-infected individuals with prominent hematological abnormalities. The results also raise the question of whether dysregulation of hematopoiesis by extracellular Nef plays a role in the development of T cell immunodeficiency and the high levels of chronic immune activation associated with AIDS.

J. Clin. Invest. doi:10.1172/JCI33037.

Research Article

Human and simian immunodeficiency viruses deregulate early hematopoiesis through a Nef/PPARγ/STAT5 signaling pathway in macaques

Stéphane Prost1,2,3, Mikael Le Dantec1, Sylvie Augé4,5, Roger Le Grand1, Sonia Derdouch1, Gwenaelle Auregan1,6, Nicole Déglon6, Francis Relouzat2,3, Anne-Marie Aubertin7, Bernard Maillere8, Isabelle Dusanter-Fourt4,5 and Marek Kirszenbaum1

1Immunovirology Division and
2Innovative Therapy Division, Institute of Emerging Diseases and Innovative Therapies, CEA, Fontenay-aux-Roses, France.
3INSERM-CEA-Paris XI, UMR U733, CEA, Fontenay-aux-Roses, France.
4Institut Cochin, Université Paris Descartes, CNRS (UMR8104), Paris, France.
5INSERM U567, Paris, France.
6CEA, I2BM et programme MIRCen, Orsay, France.
7Laboratoire de Virologie, Université Louis Pasteur, Strasbourg, France.
8CEA, IBITEC-S, Service d’Ingénierie Moleculaire des Protéines, Saclay, France.

Infection of primates by HIV-1 and SIV induces multiple hematological abnormalities of central hematopoietic origin. Although these defects greatly contribute to the pathophysiology of HIV-1 infection, the molecular basis for altered BM function remains unknown. Here we show that when cynomolgus macaques were infected with SIV, the multipotent potential of their hematopoietic progenitor cells was lost, and this correlated with downregulation of STAT5A and STAT5B expression. However, forced expression of STAT5B entirely rescued the multipotent potential of the hematopoietic progenitor cells. In addition, an accessory viral protein required for efficient SIV and HIV replication and pathogenicity, “Negative factor” (Nef), was essential for SIV-mediated impairment of the multipotent potential of hematopoietic progenitors ex vivo and in vivo. This newly uncovered property of Nef was both conserved between HIV-1 and SIV strains and entirely dependent upon the presence of PPARγ in targeted cells. Further, PPARγ agonists mimicked Nef activity by inhibiting STAT5A and STAT5B expression and hampering the functionality of hematopoietic progenitors both ex vivo and in vivo. These findings have extended the role of Nef in the pathogenicity of HIV-1 and SIV and reveal a pivotal role for the PPARγ/STAT5 pathway in the regulation of early hematopoiesis. This study may provide a basis for investigating the potential therapeutic benefits of PPARγ antagonists in both patients with AIDS and individuals with hematopoietic disorders.

After the Fact: Merck’s Safety & Immunogenicity Data Published in CID

Data from phase I safety & immunogenicity studies of Merck’s adenovirus serotype 5 (Ad5) HIV vaccine has just been published online in Clinical Infectious Diseases. The data has been presented at various conferences but this is the first relatively comprehensive report to appear in the literature. The paper reinforces several of the observations that were made during the development of the vaccine: first and foremost, it represented a great leap forward in terms of the proportion of recipients that actually developed a detectable CD8 T cell response to vaccine antigens. Previous bests were around 20-30% of recipients (with an ALVAC canarypox vector). What seems to have been entirely lost in recent coverage of the Merck vaccine failure is that, before this Ad5 construct came along, no one had the luxury of worrying about the impact of issues like the magnitude, breadth, function & specificity of the CD8 T cell response because, for the most part, there was no CD8 T cell response there to analyze!

Secondly, although the issue is only mentioned in passing in the paper and would benefit from a more detailed presentation of the data, it is clear that the T cell responses induced by the vaccine were heavily biased toward CD8 T cell responses and the importance of a balanced CD4/CD8 T cell response is now better recognized. Merck’s decision to drop the DNA priming immunization – criticized at the time by Jerry Sadoff and Bette Korber during an NIAID AIDS Vaccine Research Working Group meeting – may have exacerbated this problem, as DNA vaccines typically bias toward CD4 T cell responses.

Thirdly, the tendency of for Ad5 vectors to persist and thus maintain an activated effector memory CD8 T cell population – which has been reported in small animal models over the past few years – may have been mirrored in humans: the figures in the paper showing the magnitude of HIV-specific CD8 T cell responses over time do not provide much evidence of a contraction phase (click on the image to see the figure), normally a prerequisite for the development of a “central memory” CD8 T cell population endowed with robust proliferative capacity.

Finally, the paper unfortunately offers little data on the breadth of the vaccine-induced responses and yet the authors suggest that it was “broad” – this seems inconsistent with the data from the STEP trial showing that recipients only responded to an average of one epitope from each vaccine-encoded protein.

Clinical Infectious Diseases 2008;46:000–000
DOI: 10.1086/587993

MAJOR ARTICLE

Safety and Immunogenicity of a Replication-Incompetent Adenovirus Type 5 HIV-1 Clade B gag/pol/nef Vaccine in Healthy Adults

Frances H. Priddy,1 Deborah Brown,2 James Kublin,3 Kathleen Monahan,2 David P. Wright,4 Jacob Lalezari,5 Steven Santiago,7 Michael Marmor,8 Michelle Lally,9 Richard M. Novak,10 Stephen J. Brown,6 Priya Kulkarni,2 Sheri A. Dubey,2 Lisa S. Kierstead,2 Danilo R. Casimiro,2 Robin Mogg,2 Mark J. DiNubile,2 John W. Shiver,2 Randi Y. Leavitt,2 Michael N. Robertson,2 Devan V. Mehrotra,2 and Erin Quirk,2 for the Merck V520-016 Study Groupa

1Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; 2Merck Research Laboratories, West Point, Pennsylvania; 3Fred Hutchinson Cancer Research Center, Seattle, Washington; 4Central Texas Clinical Research Center, Austin; 5Quest Clinical Research Laboratories, San Francisco, and 6AIDS Research Alliance, West Hollywood, California; 7Care Resource, Miami, Florida; 8New York University School of Medicine, New York; 9Warren Alpert Medical School of Brown University, Providence, Rhode Island; and 10University of Illinois at Chicago, Chicago

Background. The safety and immunogenicity of the MRK adenovirus type 5 human immunodeficiency virus type 1 clade B gag/pol/nef vaccine, a replication-incompetent adenovirus type 5–vectored vaccine designed to elicit cell-mediated immunity against conserved human immunodeficiency virus proteins, was assessed in a phase 1 trial.

Methods. Healthy adults not infected with human immunodeficiency virus were enrolled in a multicenter, dose-escalating, blind, placebo-controlled study to evaluate a 3-dose homologous prime-boost regimen of the trivalent MRK adenovirus type 5 human immunodeficiency virus type 1 vaccine containing from to viral particles per 1-mL dose administered on day 1, during week 4 and during week 26. Adverse events were recorded for 29 days after each intradeltoid injection. The primary immunogenicity end point was the proportion of study participants with a positive unfractionated Gag-, Pol-, or Nef-specific interferon-γ enzyme-linked immunosorbent spot response measured 4 weeks after administration of the last dose.

Results. Of 259 randomized individuals, 257 (99%) received 1 dose of vaccine or placebo and were included in the safety analyses. Enzyme-linked immunosorbent spot results were available for 217 study participants (84%) at week 30. No serious vaccine-related adverse events occurred. No study participant discontinued participation because of vaccine-related adverse events. The frequency of injection-site reactions was dose dependent. Vaccine doses of viral particles elicited positive enzyme-linked immunosorbent spot responses to 1 vaccine component in >60% of recipients. High baseline antibody titers against adenovirus type 5 diminished enzyme-linked immunosorbent spot responses at all doses except the viral particle dose.

Conclusions. The vaccine was generally well tolerated and induced cell-mediated immune responses against human immunodeficiency virus type 1 peptides in most healthy adults. Despite these findings, vaccination in a proof-of-concept trial with use of this vaccine was discontinued because of lack of efficacy.

Mannose Y Mannose – Two Attempts to Mimic the Anti-HIV Activity of the 2G12 Antibody

Two new papers in the online first section of J. Virology describe attempts to generate anti-HIV antibodies that can mimic the broad neutralizing activity of the monoclonal antibody 2G12. This antibody is unusual in that it targets the clusters of high-mannose glycans that typically help shield HIV’s envelope from antibody-mediated attack (a glycan is a large carbohydrate molecule composed of many smaller sugar molecules linked together).

Rena Astronomo and colleagues utilized bovine serum albumin (BSA) as the building block for an immunogen designed to induce anti-mannose antibodies that mimic 2G12. Unfortunately the approach proved unsuccessful: immunization of rabbits induced antibodies that failed to even bind HIV’s gp120 protein. The researchers suspect that the molecules of the immunogen were spaced too widely apart and failed to mimic the dense clustering of glycans on gp120; strategies that may overcome the problem – including the use of non-infectious, virus-like particles – are now being explored.

In the second paper, a team headed by Robert Luallen took a different tack. They developed a genetically engineered yeast (Saccharomyces cerevisiae) that encodes proteins containing high density glycan clusters and used heat-killed preparations of the yeast to immunize rabbits. The approach successfully induced antibodies capable of binding gp120 from a clade B and clade C primary isolate as well as SIV 239 and 1A11 and recognition increased after several immunizations. However, when the concentrations of antibodies in the ELISA assay were taken into account, the binding activity was far lower (50-100 fold) than that seen for 2G12 against clade B g120s. The researchers suggest that the lower activity relates to the fact that their approach induced a polyclonal antibody response in which the antibodies mimicking 2G12 are a minority population. Consistent with this possibility, the authors note that preliminary results from neutralization assays have mostly been negative.

Despite the limitations, these data may still represent an important first step toward the design of more effective antibody-based immunogens, as the elicitation of 2G12-like antibodies capable of recognizing HIV and SIV Envs has (to the best of my knowledge) not previously been reported. In discussing their findings, Luallen and colleagues outline a number of strategies they are pursuing in order to induce the closest 2G12 mimics possible, and suggest that the yeast platform is particularly well suited to the task.

JVI Accepts, published online ahead of print on 23 April 20
J. Virol. doi:10.1128/JVI.00412-08

An engineered Saccharomyces cerevisiae strain binds the broadly neutralizing HIV-1 antibody 2G12 and elicits mannose-specific, gp120 binding antibodies

Robert J. Luallen, Jianqiao Lin, Hu Fu, Karen K. Cai, Caroline Agrawal, Innocent Mboudjeka, Fang-Hua Lee, David Montefiori, David F. Smith, Robert W. Doms, and Yu Geng

ProSci Incorporated, 12170 Flint Place, Poway, CA 92064; Department of Microbiology, University of Pennsylvania, 225 Johnson Pavilion, Philadelphia, PA 19104; Department of Surgery, Duke University Medical Center, Box 2926, DUMC, Durham, NC 27710; Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd NE, Room 4035, Atlanta, GA 30322

Abstract

The glycan shield of the HIV-1 envelope (Env) protein serves as a barrier to antibody-mediated neutralization and plays a critical role in transmission and infection. One of the few broadly neutralizing HIV-1 antibodies, 2G12, binds to a carbohydrate epitope consisting of an array of high mannose glycans exposed on the surface of the gp120 subunit of the Env protein. To produce proteins with exclusively high mannose carbohydrates, we generated a mutant strain of Saccharomyces cerevisiae by deleting three genes in the N-glycosylation pathway, Och1, Mnn1, and Mnn4. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man8GlcNAc2, and four endogenous glycoproteins were identified that were efficiently recognized by 2G12. These yeast proteins, like HIV-1 gp120, contain a large number and high density of N-linked glycans, with glycosidase digestion abrogating 2G12 cross-reactivity. Immunization of rabbits with whole och1mnn1mnn4 yeast produced sera that recognized a broad range of HIV-1 and SIV Env glycoproteins, despite no HIV/SIV-related proteins being used in the immunization procedure. Analyses of one of these sera on a glycan array showed strong binding to glycans with terminal Man1,2Man residues, and binding to gp120 was abrogated by glycosidase removal of high mannose glycans and terminal Man1,2Man residues, similar to 2G12. The fact that yeast are genetically pliable, and can be grown easily and inexpensively, will make it possible to produce new immunogens that recapitulate the 2G12 epitope, and may make the glycan shield of HIV Env a practical target for vaccine development.

JVI Accepts, published online ahead of print on 23 April 2008
J. Virol. doi:10.1128/JVI.00293-08

A glycoconjugate antigen, based on the recognition motif of a broadly-neutralising HIV antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the ‘self’ glycans of gp120

Rena D. Astronomo, Hing-Ken Lee, Christopher N. Scanlan, Ralph Pantophlet, Cheng-Yuan Huang, Ian A. Wilson, Ola Blixt, Raymond A. Dwek, Chi-Huey Wong, and Dennis R. Burton

Departments of Immunology and Microbial Science, Molecular Biology, The Skaggs Institute for Chemical Biology, Chemistry, and the Glycan Array Synthesis Core D, Consortium for Functional Glycomics, The Scripps Research Institute, La Jolla, California 92037; The Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU

Abstract

The glycan shield of HIV-1 gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly-neutralizing antibody, 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing antibodies (Abs) and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man4) that corresponds to the D1 arm of Man9GlcNAc2 inhibits 2G12 binding to gp120 as efficiently as Man9GlcNAc2 itself, indicating the potential use of Man4 as a building block for creating immunogens. Here, we describe the development of neoglycoconjugates displaying variable copy numbers of Man4 on bovine serum albumin (BSA) molecules by conjugation to Lys residues. The increased valency enhances the apparent affinity of 2G12 for Man4 up to a limit which is achieved at 10 copies per BSA, beyond which no further enhancement is observed. Immunization of rabbits with BSA-(Man4)14 elicits significant serum Ab titres to Man4. However, these Abs are unable to bind gp120. Further analysis reveals that the elicited Abs bind a variety of unbranched and, to a lesser extent, branched Man9 derivatives, but not natural N-linked oligomannose containing the chitobiose core. These results suggest that Abs can be readily elicited against the D1 arm; however, potential differences in the presentation of Man4 on neoglycoconjugates, as compared to glycoproteins, poses challenges for eliciting anti-mannose Abs capable of cross-reacting with gp120 and HIV-1.

Transmission of CD8 T Cell Escape Mutants is Associated with Lower Viral Loads in Newly Infected Individuals

A new study by Paul Goepfert and colleagues offers compelling evidence that CD8 T cell responses can pressure HIV into mutating in ways that compromise viral fitness. CD8 T cells target tiny slices of viral proteins called epitopes, which are displayed by infected cells as a sort of alarm signal; CD8 T cells that recognize a particular epitope (via a docking bay-type structure called a T cell receptor or TCR) can mediate destruction of an infected cell by releasing cell-destroying substances such as perforin and granzyme B. HIV mutations that impact the structure of an epitope can abrogate CD8 T cell recognition and this phenomenon is called immune escape (it is loosely analogous to the way mutations can allow the virus to resist the effects of antiretroviral drugs). If a particular epitope-specific CD8 T cell response is effective, viruses with escape mutations in that epitope are at a selective advantage because they can persist despite the presence of the immune response. However, certain parts of HIV can tolerate mutations more easily than others, and studies have shown that the Gag protein is so vital to replication that mutations affecting epitopes in Gag can impair the ability of the virus to replicate in a lab dish (in vitro).

To assess whether these observations are relevant in people, Goepfert et al analyzed data from 114 epidemiologically linked transmission pairs in Zambia (the individuals had been participants in a larger cohort of “discordant” couples in which one partner was HIV-infected; despite counseling and increased condom use in the cohort, transmission still occurred at a rate of approximately 8% per year). The researchers looked for evidence of CD8 T cell escape mutations in Gag and Nef and then analyzed whether the transmission of HIV containing mutations impacted viral load in the newly infected individual. The timepoint for the analysis was 6 months after infection, because 579 of the 610 mutations documented in the transmitting partners were still present in recipients at this time point. The results showed that higher numbers of CD8 T cell escape mutations in the Gag protein of transmitted viruses were significantly associated with lower viral loads in the newly infected individuals (no such effect was seen for Nef). Further analyses revealed that the effect was most consistent for Gag epitopes targeted by HLA-B-restricted CD8 T cells (HLA genes manufacture the CD8 T cell TCR and thus govern the epitope structures that a CD8 T cell can recognize). The researchers hypothesized that the impact on HIV replication of mutations in epitopes targeted by HLA-B-restricted CD8 T cells would be most prominent in individuals lacking the same HLA-B genes themselves, and indeed this turned out to be the case: when the analysis was restricted to only these study participants the associations between more mutations in Gag and lower viral load became much stronger (p=0.0003).

In discussing their results, the study authors note that there was a ~10-fold difference in viral loads when individuals infected with viruses containing less than two escape mutations in Gag were compared to those with more than six such mutations, suggesting that infection with these multiple escape mutants may slow disease progression (although longer term follow up of a subset of participants indicates that viral load is, as is typical, increasing over time). They also state that: “these data imply that for CTL-based HIV vaccines to effectively control viral load, they must simultaneously target multiple Gag epitopes, thereby ensuring that fitness constraints prevent the virus from easily mutating.” In an accompanying news brief, JEM editor Hema Bashyam speculates that the study “might explain why a T cell vaccine that induces immune responses against two Gag epitopes failed in a recent trial.” In fact, recipients of the Merck HIV vaccine developed responses to an average of just one Gag epitope.

The Journal of Experimental Medicine
Published online 21 April 2008
doi:10.1084/jem.20072457

BRIEF DEFINITIVE REPORT

Transmission of HIV-1 Gag immune escape mutations is associated with reduced viral load in linked recipients

Paul A. Goepfert1,2, Wendy Lumm4, Paul Farmer4, Philippa Matthews5, Andrew Prendergast5, Jonathan M. Carlson6,7, Cynthia A. Derdeyn4,8, Jianming Tang1,2, Richard A. Kaslow3, Anju Bansal1, Karina Yusim10, David Heckerman6, Joseph Mulenga11, Susan Allen9, Philip J.R. Goulder5,12,13, and Eric Hunter4,8

1 Department of Medicine, 2 Department of Microbiology, and 3 Department of Epidemiology, University of Alabama at Birmingham, Birmingham, AL 35294
4 Emory Vaccine Center at Yerkes National Primate Research Center, Atlanta, GA 30322
5 Department of Pediatrics, The Peter Medawar Building for Pathogen Research, University of Oxford, Oxford OX1 3SY, England, UK
6 Microsoft Research, Redmond, WA 98052
7 Department of Computer Science and Engineering, University of Washington, Seattle, WA 98195
8 Department of Pathology and Laboratory Medicine and 9 Department of Global Health, Emory University, Atlanta, GA 30322
10 Los Alamos National Laboratory, Los Alamos, NM 87545
11 Zambia-Emory HIV Research Group, Lusaka, Zambia
12 HIV Pathogenesis Program, Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban 4013, South Africa
13 Partners AIDS Research Center, Massachusetts General Hospital, Charlestown, MA 02129

CORRESPONDENCE Paul A. Goepfert: paulg@uab.edu

In a study of 114 epidemiologically linked Zambian transmission pairs, we evaluated the impact of human leukocyte antigen class I (HLA-I)–associated amino acid polymorphisms, presumed to reflect cytotoxic T lymphocyte (CTL) escape in Gag and Nef of the virus transmitted from the chronically infected donor, on the plasma viral load (VL) in matched recipients 6 mo after infection. CTL escape mutations in Gag and Nef were seen in the donors, which were subsequently transmitted to recipients, largely unchanged soon after infection. We observed a significant correlation between the number of Gag escape mutations targeted by specific HLA-B allele–restricted CTLs and reduced VLs in the recipients. This negative correlation was most evident in newly infected individuals, whose HLA alleles were unable to effectively target Gag and select for CTL escape mutations in this gene. Nef mutations in the donor had no impact on VL in the recipient. Thus, broad Gag-specific CTL responses capable of driving virus escape in the donor may be of clinical benefit to both the donor and recipient. In addition to their direct implications for HIV-1 vaccine design, these data suggest that CTL-induced viral polymorphisms and their associated in vivo viral fitness costs could have a significant impact on HIV-1 pathogenesis.

HLA Class II Associations with Resistance & Susceptibility to HIV Infection

Following a similar theme to a recently posted study indicating that certain class II HLA genes – which impact antigen presentation to CD4 T cells – are associated with elite control of HIV replication in infected individuals, a new paper just published in the journal AIDS adds to the literature showing significant associations between class II HLA genes and resistance/susceptibility to HIV infection. The data comes from a large cohort study of sex workers in the Pumwani district of Nairobi, approximately 10% of whom persistently resist HIV infection despite an estimated ~60 unprotected exposures per year.

AIDS. 22(7):807-816, April 23, 2008.

BASIC SCIENCE

Human leukocyte antigen-DQ alleles and haplotypes and their associations with resistance and susceptibility to HIV-1 infection.

Hardie, Rae-Anne; Luo, Ma; Bruneau, Brigitte; Knight, Erin; Nagelkerke, Nico JD; Kimani, Joshua; Wachihi, Charles; Ngugi, Elizabeth N; Plummer, Francis A

Abstract:

Objectives: To determine the association of DQ antigens with resistance and susceptibility to HIV-1.

Design: Despite repeated exposure to HIV-1, a subset of women in the Pumwani Sex Worker cohort established in Nairobi, Kenya in 1985 have remained HIV-1 negative for at least 3 years and are classified as resistant. Differential susceptibility to HIV-1 infection is associated with HIV-1 specific CD4+ and CD8+ T cell responses. As human leukocyte antigen-DQ antigens present viral peptides to CD4+ cells, we genotyped human leukocyte antigen -DQ alleles for 978 women enrolled in the cohort and performed cross-sectional and longitudinal analyses to identify associations of human leukocyte antigen -DQ with resistance/susceptibility to HIV-1.

Methods: DQA1 and DQB1 were genotyped using taxonomy-based sequence analysis. SPSS 13.0 was used to determine associations of DQ alleles/haplotypes with HIV-1 resistance, susceptibility, and seroconversion rates.

Results: Several DQB1 alleles and DQ haplotypes were associated with resistance to HIV-1 infection. These included DQB1*050301 (P = 0.055, Odds Ratio = 12.77, 95% Confidence Interval = 1.44-112), DQB1*0603 and DQB1*0609 (P = 0.037, Odds Ratio = 3.25, 95% Confidence Interval = 1.12-9.47), and DQA1*010201-DQB1*0603 (P = 0.044, Odds Ratio = 17.33, 95% Confidence Interval = 1.79-168). Conversely, DQB1*0602 (P = 0.048, Odds Ratio = 0.68, 95% Confidence Interval = 0.44-1.05) and DQA1*010201-DQB1*0602 (P = 0.039, Odds Ratio = 0.64, 95% Confidence Interval = 0.41-1.03) were overrepresented in the HIV-1 infected population. DQA1*0504-DQB1*0201, DQA1*010201-DQB1*0201, DQA1*0402-DQB1*0402 and DQA1*0402-DQB1*030101 genotypes were only found in HIV-1 positive subjects (Odds Ratio = 0.30-0.31, 95% Confidence Interval = 0.03-3.70), and these women seroconverted rapidly. The associations of these DQ alleles and haplotypes with resistance and susceptibility to HIV-1 were independent of the previously reported human leukocyte antigen-DRB*01, human leukocyte antigen A2/6802, and human leukocyte antigen-A*2301.

Conclusion: The associations of DQ alleles and haplotypes with resistance and susceptibility to HIV-1 emphasize the importance of human leukocyte antigen-DQ and CD4 in anti-HIV-1 immunity.

CTLA-4 Blockade Can Increase SIV Replication

The expression of the molecule CTLA-4 by T cells is typically associated with suppression of T cell activation. Prior studies have shown that CTLA-4 is elevated in individuals with progressing HIV infection compared to healthy controls. Furthermore, levels of CTLA-4 on HIV-specific CD4 T cells are significantly higher in individuals with progressive disease compared to "elite controllers," suggesting that expression of the molecule may contribute to the failure of the immune response to control HIV replication. These data have led to the suggestion that blockade of CTLA-4 may deserve evaluation as a therapeutic approach to treating HIV. One prior study in macaques with longstanding (>96 weeks) SIV infection reported that two doses of an anti-CTLA-4 antibody (MDX-010, manufactured by a company called Medarex) slightly reduced SIV RNA in lymphoid tissue compared to antiretroviral therapy (ART) alone.

However, a new paper by the same research group now reports that MDX-010 increases immune activation and consequently increase SIV replication in gut-associated lymphoid tissue of macaques (see abstract below). In this study, MDX-010 was administered immediately prior to SIV infection and, in a second group of animals, during early SIV infection (four doses given at weeks 22, 27, 32 & 37 after infection). The authors do not offer much in the way of explanation for the differing study outcomes, beyond noting that mucosal sites were not evaluated in the prior work. Notably, there was no evidence of enhancement of SIV-specific T cell responses in either this or the previous study. Because regulatory T cells (Tregs) preferentially express CTLA-4, the authors argue that their findings suggest "a limited contribution of Treg to suppression of (SIV-specific) immune responses in vivo." However, the relative specificity of MDX-010 for Tregs versus other T cells that may transiently express CTLA-4 (such as effector T cells) remains controversial and uncertain. The findings imply that MDX-010 is unlikely to be a useful HIV treatment; one human phase I safety study has been conducted by Medarex but the only reference to the results to be found online is in an IAVI Report article on Tregs, in which the clinician responsible for the trial states that the treatment was "safe and well tolerated." The company does not appear to be conducting further trials in HIV infection.

The Journal of Immunology, 2008, 180: 5439-5447.

Immune Activation Driven by CTLA-4 Blockade Augments Viral Replication at Mucosal Sites in Simian Immunodeficiency Virus Infection

Valentina Cecchinato2,*, Elzbieta Tryniszewska2,*,, Zhong Min Ma¶, Monica Vaccari*, Adriano Boasso, Wen-Po Tsai*, Constantinos Petrovas||, Dietmar Fuchs#, Jean-Michel Heraud3,*, David Venzon, Gene M. Shearer, Richard A. Koup||, Israel Lowy**, Christopher J. Miller¶ and Genoveffa Franchini4,*

* Animal Models and Retroviral Vaccines Section, Experimental Immunology Branch, and Biostatistics and Data Management Section, National Cancer Institute, Bethesda, MD 20892; Department of Microbiology Diagnostics, Medical University of Bialystok, Bialystok, Poland; ¶ California National Primate Research Center, University of California, Davis, CA 95616; || Immunology Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892; # Division of Biological Chemistry Biocentre, Innsbruck Medical University, Innsbruck, Austria; and ** Medarex, Bloomsbury, NJ 08804

The importance of chronic immune activation in progression to AIDS has been inferred by correlative studies in HIV-infected individuals and in nonhuman primate models of SIV infection. Using the SIVmac251 macaque model, we directly address the impact of immune activation by inhibiting CTLA-4, an immunoregulatory molecule expressed on activated T cells and a subset of regulatory T cells. We found that CTLA-4 blockade significantly increased T cell activation and viral replication in primary SIVmac251 infection, particularly at mucosal sites, and increased IDO expression and activity. Accordingly, protracted treatment with anti-CTLA-4 Ab of macaques chronically infected with SIVmac251 decreased responsiveness to antiretroviral therapy and abrogated the ability of therapeutic T cell vaccines to decrease viral set point. These data provide the first direct evidence that immune activation drives viral replication, and suggest caution in the use of therapeutic approaches for HIV infection in vivo that increase CD4+ T cell proliferation.

Targeting DNA Vaccines to Dendritic Cells

A new study from Ralph Steinmans's laboratory indicates that targeting DNA vaccines to dendritic cells can substantially boost their immunogenicity. The approach involves fusing the antigen of interest (in this study, HIV-1 Gag) with a single chain antibody targeting the dendritic cell receptor DEC205 (also called CD205). Combined with delivery via electroporation, the researchers report that the approach boosted vaccine potency 100-fold in a mouse challenge system. Steinman recently won a Lasker award for his work on dendritic cells; writing about his work in Nature Medicine, he noted sadly that most of the research he conducted (and on which the Lasker award was based) would probably not have been funded if the NIH grant payline had been at the low level it is today:

"The vision of Mary Lasker was to urge the government to sponsor basic research to understand and treat disease. Her vision remains critical to meet ongoing challenges to the scientific enterprise. One challenge is our declining ability to support independent scientists. I probably could not have carried out my research under the current funding environment for basic research, because many of my grant applications only ranked in the top quartile, whereas funding by the US National Institutes of Health is currently only sufficient for less than 10% of basic proposals."

In another recent paper, Jinyan Liu and colleagues show that electroporation alone results in a 77-fold enhancement in the recruitment of dendritic cells to the site of DNA vaccine inoculation, likely accounting for the significant enhancements in immunogenicity that have been reported using this delivery method. The question mark hanging over viral vectors due to the Merck HIV vaccine results is likely to refocus attention on these and other strategies for improving DNA vaccine immunogenicity.

J Clin Invest. 2008 Apr 1;118(4):1427-1436. (free full text access)

The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells.

Nchinda G, Kuroiwa J, Oks M, Trumpfheller C, Park CG, Huang Y, Hannaman D, Schlesinger SJ, Mizenina O, Nussenzweig MC, Uberla K, Steinman RM.

Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York, USA. Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany. The Aaron Diamond AIDS Research Center, New York, New York, USA. Ichor Medical Systems, San Diego, California, USA. Laboratory of Molecular Immunology, Chris Browne Center for Immunology, The Rockefeller University, New York, New York, USA.

DNA vaccines promote an immune response by providing antigen-encoding DNA to the recipient, but the efficacy of such vaccines needs improving. Many approaches have considerable potential but currently induce relatively weak immune responses despite multiple high doses of DNA vaccine. Here, we asked whether targeting vaccine antigens to DCs would increase the immunity and protection that result from DNA vaccines. To determine this, we generated a DNA vaccine encoding a fusion protein comprised of the vaccine antigen and a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Following vaccination of mice, the vaccine antigen was expressed selectively by DCs, which were required for the increased efficacy of MHC class I and MHC class II antigen presentation relative to a control scFv DNA vaccine. In addition, a DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein induced 10-fold higher antibody levels and increased numbers of IFN-gamma-producing CD4(+) and CD8(+) T cells. After a single i.m. injection of the DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein, mice were protected from an airway challenge with a recombinant vaccinia virus expressing the HIV gag p41, even with 1% of the dose of nontargeted DNA vaccine. The efficacy of DNA vaccines therefore may be enhanced by inclusion of sequences such as single-chain antibodies to target the antigen to DCs.

J Virol. 2008 Mar 19 [Epub ahead of print]

Recruitment of Antigen-Presenting Cells to the Site of Inoculation and Augmentation of HIV-1 DNA Vaccine Immunogenicity by In Vivo Electroporation.

Liu J, Kjeken R, Mathiesen I, Barouch DH.

Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; Inovio Biomedical, San Diego, CA 92121.

In vivo electroporation (EP) has been shown to augment the immunogenicity of plasmid DNA vaccines, but its mechanism of action has not been fully characterized. In this study, we show that in vivo EP augmented cellular and humoral immune responses to an HIV-1 Env DNA vaccine in mice and allowed a 10-fold reduction in vaccine dose. This enhancement was durable for over 6 months, and re-exposure to antigen resulted in anamnestic effector and central memory CD8+ T lymphocyte responses. Interestingly, in vivo EP also recruited large mixed cellular inflammatory infiltrates to the site of inoculation. These infiltrates contained 45-fold increased numbers of macrophages and 77-fold increased numbers of dendritic cells as well as 2- to 6-fold increased numbers of B and T lymphocytes as compared with infiltrates following DNA vaccination alone. These data suggest that recruiting inflammatory cells including APCs to the site of antigen production substantially improves the immunogenicity of DNA vaccines. Combining in vivo EP with plasmid chemokine adjuvants that similarly recruited antigen-presenting cells (APCs) to the injection site, however, did not result in synergy.

NIAID Issues "Requests for Information" on Vaccine Research

The National Institute of Allergy and Infectious Diseases (NIAID) has released two requests for information, one to solicit comments to inform the "Development of a Funding Opportunity Announcement on Highly Innovative Tactics to Interrupt Transmission of HIV" and the other to "Solicit Input and Ideas on Priorities in Basic Vaccine Discovery Research."

Hints of Correlations Between Immune Responses and Viral Load Emerge from the Merck HIV Vaccine Trial

The Merck HIV vaccine efficacy trial was conducted in collaboration with the National Institutes of Health-sponsored HIV Vaccine Trials Network (HVTN). At Keystone, the head of the HVTN, Larry Corey, provided an update on the ongoing analyses of data from the study and offered his thoughts on the implications for future research. Most intriguingly, Corey reported that some hints of correlations between vaccine-induced CD8 T cells and viral load setpoints have emerged, suggesting that the magnitude of the response may be important. Specifically, among the participants with anti-Ad5 antibody titers of less than 1:18 units, the magnitude of CD8 T cell responses to Gag (as measured by interferon gamma ELISpot four weeks after the second immunization) showed an inverse correlation with viral load setpoint (p=0.01). Responses to Pol displayed a similar trend (p=0.07) while those targeted against Nef did not (p=0.22). It must be emphasized that Corey cautioned that the numbers are small and the significance is fragile (additional data points may alter the findings), but the data is consistent with prior SIV challenge studies demonstrating inverse correlations between the magnitude of pre-challenge SIV-specific T cell responses and post-challenge viral load levels. However, no such correlation was seen among individuals with anti-Ad5 titers over 1:18 and exactly how pre-existing immunity would impact the relationship between CD8 T cell responses and viral load is unclear. Additional analyses of relationships between pre-infection HIV-specific CD4 T cell responses and post-infection viral load setpoints might shed light on this question, as one speculative possibility is that individuals with pre-existing immunity developed weaker HIV-specific CD4 T cell responses due to interference from vector-specific memory CD4 T cells.

In addition to the issue of magnitude, Corey also pointed out two other potentially significant limitations of the T cell responses induced by the Merck vaccine that could conceivably have contributed to its failure: breadth and functionality. Biometric data indicates that HIV’s Gag protein contains ~150 potential T cell epitopes that should be present in a substantial proportion of circulating clade B HIVs (>15%), but STEP participants only developed a CD8 T cell response to an average of one epitope from the full-length Gag protein encoded by the vaccine (similarly, participants only responded to one epitope in each of the other two vaccine-encoded proteins, Pol & Nef). Corey cited several strategies that are being pursued to increase breadth, including the use of genes from multiple different HIV variants in prime-boost vaccines (heterologous inserts), protein or peptide boosting and prime-boost with differing vaccine vectors (heterologous vectors). In terms of functionality, Corey offered his own “revisionist bit of history,” pointing out that it is now understood that Ad5 vectors given alone bias the CD8 T cell response toward a terminally differentiated phenotype with poor proliferative capacity. In support of this argument, he showed data comparing the phenotype of CD8 T cells induced by priming and boosting with Ad5 alone compared to DNA/Ad5 and also cited unpublished data from Rafi Ahmed’s lab showing that an Ad5 vector offered poor protection against LCMV due to the induction of T cells with limited proliferative capacity. It is currently unclear if this problem is also seen with other adenovirus serotypes (some recent macaque data indicates that there is some serotype-specific variability in the phenotype of vaccine-induced T cell responses).

Corey also described a suite of studies the HVTN is either conducting or planning to conduct in order to better understand the outcomes of both the STEP and Phambili Merck vaccine trials, including looking at the interaction between Ad5 immunization and pre-existing Ad5-specific immune responses at mucosal sites, sequencing the viruses in individuals that became infected, and analyzing the impact of HLA types on both susceptibility to HIV infection and the immune responses to the vaccine.

Looking at the bigger picture, Corey echoed recent statements about the importance of discovery research for developing neutralizing antibody-based or other approaches that may lead to complete protection against infection. But he also stressed that the evidence still strongly supports pursuing the nearer-term goal of trying to increase the chance that an individual who becomes infected will control the virus immunologically, thus reducing the risk of onward transmission and preventing or slowing progression to AIDS.

Keystone Symposia: HIV Vaccines: Progress and Prospects
Banff, Alberta, Canada, March 27-April 1, 2008

Abstract #002

An Overview of the SteP trial

L. Corey
MRK HVTN Study Team, University of Washington, Seattle, WA

Background: A collaborative study between Merck, the HVTN, and NIH was initiated in 2004 to perform a randomized, multi-center, double-blind, placebo-controlled test-of-concept study in 3000 HIV seronegative volunteers at high risk of acquiring HIV. Volunteers were randomized (1:1) to receive 3 injections of either the MRKAd5 HIV-1 gag/pol/nef vaccine (a replication defective Ad5 vector) or placebo. Volunteers were tested ~every 6 months for acquisition of HIV, in volunteers who became HIV infected, viral load and CD4 cell counts were measured at multiple time points post diagnosis. Results: The study was stopped in September 2007 at the first planned interim analysis; 82 endpoints were noted in men and only 1 in women. Among male volunteers with low (<200) baseline Ad5 neutralizing antibody titers (the primary analysis population), there were 24 infections among the 741 vaccinees compared to 21 infections among the 762 placebo recipients (using a modified “intent-to-treat” approach), and among the volunteers who became HIV infected, the geometric mean plasma vRNA level was ~40,000 copies/mL in the vaccine groups compared to ~26,000 copies/mL in the placebo group. Vaccine and placebo recipients were well-matched in demographic and risk characteristics at baseline within Ad5 strata. In ongoing multivariate analyses, the following factors appear to be more strongly associated with HIV-1 acquisition; treatment (vaccine vs. placebo), hazard ratio (HR) 1.9, 95% CI (1.1, 3.2); region (North America vs. other), HR 3.2 (1.5, 6.7); and unprotected receptive anal sex (yes vs. no), HR 4.4 (2.4, 8.7). With interaction terms in the model, the treatment HR (vaccine vs. placebo) was 3.1 (1.5, 6.5) in Ad5 seropositives vs. HR 1.0 (0.5, 2.0) in Ad5 seronegative; the treatment HR was 4.5 (1.8, 11.4) in uncircumcised men vs. 1.0 (0.6, 1.8) in circumcised men. A 25% sample of all recipients and all persons who subsequently acquired HIV was performed at week 8 after 2 doses of vaccine. The frequency of CD8+ T cell responses to HIV genes (76%) was found in persons who subsequently acquired HIV-1; similar to that seen in persons without HIV acquisition. Similarly the GMT of responses was also similar in vaccinees who acquired and did not acquire HIV. Conclusions: There was no evidence thatvaccination prevented infection or lowered viral setpoint; in fact, there were more infections in the vaccine group. In multivariate analysis of baseline risk factors the Merck Ad5 trivalent vaccine appeared to be associated with an increased risk of HIV-1 acquisition in men with pre-existing Ad5 immunity and in uncircumcised men. The mechanism for potential increased acquisition will be discussed.