Michael Palm Basic Science, Vaccines & Prevention Project Weblog

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.

Understanding Control of HIV Replication in the Absence of Therapy

Individuals who maintain control of HIV replication in the absence of any therapy may offer important clues to aid the development of both vaccines and novel immune-based therapies. Many research groups are studying such individuals, with the largest and most ambitious research project being the elite controller study helmed by Bruce Walker from Partners AIDS Research Center in Boston. In a recent issue of the Journal of Infectious Diseases, lead investigator Florence Pereyra and colleagues report preliminary data from 126 individuals who have enrolled into the study. Participants are divided into two categories: elite controllers, defined as those with viral load consistently below 50 copies/mL (the limit of detection for the viral load test) and viremic controllers, whose viral loads are consistently below 2000 copies/mL. The JID paper contains data on 66 individuals in the former category and 60 in the latter; 30 individuals with chronic, progressive infection recruited over the same time period are also included for comparative purposes.

The major finding is that controllers (both elite and viremic) are a heterogeneous group with regard to many of the factors that previous studies have associated with viral load control. In terms of host genetics, HLA B*57 alleles were significantly overrepresented among controllers, but the researchers emphasize that the frequency of HLA B*57 was considerably lower than reported previously in smaller cohorts. When the analysis was expanded to any HLA allele that has been associated with a favorable prognosis in HIV infection, 68% of elite controllers and 60% of viremic controllers were found to carry at least one such allele, compared with 37% of the chronic progressors, “leaving almost one-third of HIV controllers without any known relatively protective HLA alleles.” CCR5 and CCR2 polymorphisms that have been associated with slowed disease progression were not overrepresented among controllers. Absolute CD4 counts were different between elite and viremic controllers (884 vs. 602 cells), in line with a recent paper showing that CD4 declines can still occur – albeit very slowly - despite low or even undetectable viral loads.

Randomly selected subsets from each group of study participants were further evaluated for HIV-specific immune responses. In terms of CD8 T cell responses (as measured by interferon gamma ELISpot), elite controllers displayed the lowest breadth and magnitude of HIV-specific CD8 T cells. Median breadth was 15, 19 and 27 epitopes among elite controllers, viremic controllers and chronic progressors respectively while average magnitude was 5428, 6253 and 8300 spot-forming cells (SFC) per million PBMC (peripheral blood mononuclear cells). The simplistic interpretation of these data is that the CD8 T cell response cannot be important, but the broader, larger responses associated with higher viral loads are to be expected due to the persistent recruitment from the naïve CD8 T cell pool that can occur in the setting of chronic infection, and because interferon gamma production is the last function to be lost by exhausted cells (so a large, broad response does not necessarily equate to an effective response). When the researchers looked at IL-2 and interferon gamma production together, responses were significantly higher in elite controllers than viremic controllers or progressors (both for HIV-specific CD4 T cells and CD8 T cells), but there was still heterogeneity in that these responses were not detectable in some individuals. Interestingly, elite controllers had the highest ratio of IL-2+interferon-gamma-producing HIV-specific CD4 T cells to HIV-specific CD8 T cells while chronic progressors had the lowest, suggesting – as have many prior studies - that CD4 T cell help plays an important, perhaps undervalued, role in the immune response to HIV. Adding to the heterogeneity, elite controllers also displayed the widest range of CD8 T cell interferon gamma ELISpot responses in terms of both breadth and magnitude, with responses ranging from 2-101 epitopes in breadth and from less than 500 SFC to over 25,000 SFC in magnitude. Despite the range of responses, the vast majority of both elite and viremic controllers displayed responses to many more CD8 T cell epitopes than have been reported to date in recipients of T cell-based HIV vaccine candidates (the average response in the recent Merck trial was to three epitopes).

Consistent with previously published studies, the HIV Gag protein was preferentially targeted among controllers. In progressors, Gag, Pol and Nef were targeted equally and more targeting of Env was observed than in controllers. The researchers also looked at neutralizing antibody responses. Plasma samples from both viremic controllers and progressors showed neutralizing activity against viruses sampled from the same groups as well as against laboratory HIV strains. There were no differences in neutralizing antibody titers between these two groups. Elite controllers had significantly lower neutralizing antibody responses overall but, again, heterogeneity was seen: some individuals had broad neutralizing antibody responses whereas others had very low or undetectable responses.

In concluding, the authors state that: “although elite and viremic controllers share some immunologic features and are distinct from persons with chronic progressive infection, the elite controllers are a distinct subgroup. However, even within these groups there is substantial heterogeneity in all of the parameters studied, which suggests that there are as-yet-undefined viral or host factors or combinations of factors that contribute to this remarkable phenotype.” They also cite the elite controller study linked to above and their hope that this large collaborative effort will better define the pathways that lead to durable control of HIV replication.

Two other recent studies offer additional perspectives on the same topic. In the first, Shiv Ghandi and colleagues show that the antiretroviral activity of APOBEC proteins does not appear to explain elite control of HIV replication. In the second, recently presented at CROI, researchers from Barbara Shacklett’s lab at UC Davis demonstrate that controllers have significantly more polyfunctional HIV-specific CD4 and CD8 T cells in their rectal mucosa, a novel finding indicating that analyses of additional compartments beyond the blood may add to the understanding of the elite controller phenomenon.

The Journal of Infectious Diseases 2008;197:563–571
DOI: 10.1086/526786
MAJOR ARTICLE

Genetic and Immunologic Heterogeneity among Persons Who Control HIV Infection in the Absence of Therapy

Florencia Pereyra,1,2 Marylyn M. Addo,1 Daniel E. Kaufmann,1 Yang Liu,5 Toshiyuki Miura,1 Almas Rathod,1 Brett Baker,1 Alicja Trocha,1,4 Rachel Rosenberg,1 Elizabeth Mackey,1 Peggy Ueda,1 Zhigang Lu,1 Daniel Cohen,3 Terri Wrin,5 Christos J. Petropoulos,5 Eric S. Rosenberg,1 and Bruce D. Walker1,4

1Partners AIDS Research Center, Massachusetts General Hospital and Division of AIDS, Harvard Medical School, 2Brigham and Women’s Hospital, Division of Infectious Diseases, and 3Fenway Community Health Care Center, Boston, Massachusetts; 4Howard Hughes Medical Institute, Chevy Chase, Maryland; and 5Monogram Biosciences, South San Francisco, California

Background: Spontaneous control of human immunodeficiency virus (HIV) infection has been documented in a minority of HIV-infected individuals. The mechanisms behind this outcome remain largely unknown, and a better understanding of them will likely influence future vaccine strategies. Methods: HIV-specific T cell and antibody responses as well as host genetics were examined in untreated HIV-infected patients who maintain comparatively low plasma HIV RNA levels (hereafter, controllers), including those with levels of <50 RNA copies/mL (elite controllers, n=64), those with levels of 50–2000 copies/mL (viremic controllers, n=60); we also examined HIV-specific T cell and antibody responses as well as host genetics for patients with levels of >10,000 copies/mL (chronic progressors, n=30). Results: CD8+ T cells from both controller groups preferentially target Gag over other proteins in the context of diverse HLA class I alleles, whereas responses are more broadly distributed in persons with progressive infection. Elite controllers represent a distinct group of individuals who have significantly more CD4 and CD8 T cells that secrete interferon-γ and interleukin-2 and lower levels of HIV-neutralizing antibodies. Individual responses were quite heterogeneous, and none of the parameters evaluated was uniquely associated with the ability to control viremia. Conclusions: Elite controllers are a distinct group, even when compared to persons with low level viremia, but they exhibit marked genetic and immunologic heterogeneity. Even low-level viremia among HIV controllers was associated with measurable T cell dysfunction, which has implications for current prophylactic vaccine strategies.

Journal of Virology, March 2008, p. 3125-3130, Vol. 82, No. 6
doi:10.1128/JVI.01533-07

Role of APOBEC3G/F-Mediated Hypermutation in the Control of Human Immunodeficiency Virus Type 1 in Elite Suppressors

Shiv K. Gandhi,1 Janet D. Siliciano,1 Justin R. Bailey,1 Robert F. Siliciano,1,2 and Joel N. Blankson1

Department of Medicine, Johns Hopkins University School of Medicine,1 Howard Hughes Medical Institute, Baltimore, Maryland 212052

While many studies show that the APOBEC3 family of cytidine deaminases can inhibit human immunodeficiency virus type 1 (HIV-1) replication, the clinical significance of this host defense mechanism is unclear. Elite suppressors are HIV-1-infected individuals who maintain viral loads below 50 copies/ml without antiretroviral therapy. To determine the role of APOBEC3G/F proteins in the control of viremia in these patients, we used a novel assay to measure the frequency of hypermutated proviral genomes. In most elite suppressors, the frequency was not significantly different than that observed in patients on highly active antiretroviral therapy. Thus, enhanced APOBEC3 activity alone cannot explain the ability of elite suppressors to control viremia.

15th Conference on Retroviruses & Opportunistic Infections
Abstract #355

Polyfunctional HIV-specific T Cells in Rectal Mucosa of HIV Controllers

April Ferre*1, P Hunt2, D Young1, J Garcia1, H Yee2, R Pollard1, S Deeks2, and B Shacklett1

1Univ of California, Davis, US and 2Univ of California, San Francisco, US

Background: Among the HIV-infected population exists a unique group of individuals who achieve control over HIV replication in the absence of ART. The study of such individuals is crucial to understanding how the immune system may effectively control viral replication and limit progression to AIDS. Methods: In this study we examined cell-mediated immune responses in 26 HIV controllers (viral load <2000 copies/mL), 14 non-controllers (viral load >10,000 copies/mL), and 10 HAART-suppressed individuals (viral load <50 copies/mL) in peripheral blood mononuclear cells (PBMC) and rectal mucosa. Of the controllers, 14 were classified as elite controllers, individuals who maintain plasma viral load <75 copies/mL in the absence of ART. This group is believed to encompass <1% of all HIV-infected individuals. In intracellular cytokine assays, we measured the production of 3 cytokines (interferon-γ, interleukin-2, tumor necrosis factor-α), 1 chemokine (MIP-1β), and the cytolytic granule marker CD107 in response to stimulation by HIV-1 Gag peptides. We hypothesized that “polyfunctional” T cells, capable of producing multiple antiviral factors, are critical in limiting viral replication and disease progression. Results: Mucosal CD8 T cell responses in controllers were significantly stronger and more complex than those in HAART-suppressed individuals (p = 0.0006). Differences between controllers and non-controllers were more subtle, but included a higher frequency of 4-function HIV-specific CD8 T cells in rectal mucosa of controllers than in non-controllers (p = 0.002). CD4 T cell responses were less complex and of lower magnitude than CD8 responses, but several controllers had unusually strong, polyfunctional mucosal CD4 responses. Conclusions: These findings demonstrate that many controllers mount strong and complex HIV-specific T cell responses in mucosal tissues. These polyfunctional cells may play an important role in immune surveillance of gut mucosa, as suggested by their relative enrichment among individuals who appear to be controlling HIV replication in absence of therapy.

Nabbing Broadly Neutralizing Antibodies from People with HIV Infection

In the advance online section of the Nature Medicine website, a group of researchers from the Vaccine Research Center at the National Institutes of Health report new findings suggesting that the induction of broadly neutralizing antibodies against HIV may be possible. The researchers evaluated the ability of serum sampled from a cohort of 32 HIV-infected individuals to neutralize a variety of HIV isolates from clades A, B & C. Sera from two individuals displayed broad neutralizing activity against most viruses tested. Further analyses revealed that these antibodies were directed against a conserved part of HIV's gp120 envelope protein that binds to the human CD4 molecule. Although additional work will be needed to try and home in on the specific epitope(s) that these antibodies are targeting, the researchers note that, encouragingly, "these data show that it is possible for B cells in the normal human repertoire to effectively target epitopes located in the gp120 CD4-binding site."

Nat Med. 2007 Aug 26; [Epub ahead of print]

Broad HIV-1 neutralization mediated by CD4-binding site antibodies.

Li Y, Migueles SA, Welcher B, Svehla K, Phogat A, Louder MK, Wu X, Shaw GM, Connors M, Wyatt RT, Mascola JR.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

We have identified several patient sera showing potent and broad HIV-1 neutralization. Using antibody adsorption and elution from selected gp120 variants, the neutralizing specificities of the two most broadly reactive sera were mapped to the primary receptor CD4-binding region of HIV-1 gp120. Novel antibodies to the CD4-binding site are elicited in some HIV-1-infected individuals, and new approaches to present this conserved region of gp120 to the immune system may result in improved vaccine immunogens.