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
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
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.
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