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      Thermostability of Well-Ordered HIV Spikes Correlates with the Elicitation of Autologous Tier 2 Neutralizing Antibodies

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          Abstract

          In the context of HIV vaccine design and development, HIV-1 spike mimetics displaying a range of stabilities were evaluated to determine whether more stable, well-ordered trimers would more efficiently elicit neutralizing antibodies. To begin, in vitro analysis of trimers derived from the cysteine-stabilized SOSIP platform or the uncleaved, covalently linked NFL platform were evaluated. These native-like trimers, derived from HIV subtypes A, B, and C, displayed a range of thermostabilities, and were “stress-tested” at varying temperatures as a prelude to in vivo immunogenicity. Analysis was performed both in the absence and in the presence of two different adjuvants. Since partial trimer degradation was detected at 37°C before or after formulation with adjuvant, we sought to remedy such an undesirable outcome. Cross-linking (fixing) of the well-ordered trimers with glutaraldehyde increased overall thermostability, maintenance of well-ordered trimer integrity without or with adjuvant, and increased resistance to solid phase-associated trimer unfolding. Immunization of unfixed and fixed well-ordered trimers into animals revealed that the elicited tier 2 autologous neutralizing activity correlated with overall trimer thermostability, or melting temperature (T m). Glutaraldehyde fixation also led to higher tier 2 autologous neutralization titers. These results link retention of trimer quaternary packing with elicitation of tier 2 autologous neutralizing activity, providing important insights for HIV-1 vaccine design.

          Author Summary

          As the sole determinant exposed on the viral surface to the host B cells, development of native-like HIV-1 envelope glycoprotein (Env) functional spikes has been a major initial objective in HIV-1 vaccine design. As immunogens, these trimer mimetics should remain stable in a native-like conformation to preferentially present conserved neutralizing epitopes, as opposed to non-neutralizing epitopes, to better elicit neutralizing B cell responses and antibodies in vivo during the immune response. We assessed SOSIP or NFL trimers displaying a range of stabilities, including chemical fixation. We demonstrate that increased resistance to high temperature-induced unfolding correlated with enhanced elicitation of tier 2 autologous neutralizing antibodies that are capable of penetrating this well-shielded viral pathogen, an important consideration for HIV vaccine development.

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          Most cited references28

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          Molecular architecture of native HIV-1 gp120 trimers.

          The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection. Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with three-dimensional image classification and averaging, we report the three-dimensional structures of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells.
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            The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens.

            The human immunodeficiency virus-type 1 (HIV-1) envelope glycoproteins interact with receptors on the target cell and mediate virus entry by fusing the viral and cell membranes. The structure of the envelope glycoproteins has evolved to fulfill these functions while evading the neutralizing antibody response. An understanding of the viral strategies for immune evasion should guide attempts to improve the immunogenicity of the HIV-1 envelope glycoproteins and, ultimately, aid in HIV-1 vaccine development.
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              HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites.

              The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120-receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor-antibody thermodynamic cycles suggested a receptor-binding-site 'conformational masking' mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus-receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                3 August 2016
                August 2016
                : 12
                : 8
                : e1005767
                Affiliations
                [1 ]IAVI Neutralizing Center at TSRI, Department of Research and Development, International AIDS Vaccine Initiative, La Jolla, California, United States of America
                [2 ]Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America
                [3 ]The Scripps CHAVI-ID, The Scripps Research Institute, La Jolla, California, United States of America
                [4 ]Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, United States of America
                [5 ]IAVI Design and Development Lab, Department of Research and Development, International AIDS Vaccine Initiative, Brooklyn, New York, United States of America
                University of Zurich, SWITZERLAND
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: YF KT RTW. Performed the experiments: YF KT NdV SB RW JG. Analyzed the data: YF KT NdV SB JG. Contributed reagents/materials/analysis tools: SK HA JD ABW. Wrote the paper: YF KT RTW.

                Author information
                http://orcid.org/0000-0003-1564-4098
                Article
                PPATHOGENS-D-16-00497
                10.1371/journal.ppat.1005767
                4972253
                27487086
                b876f8ee-2d7e-4aa8-be79-8bff7364423f
                © 2016 Feng et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 8 March 2016
                : 24 June 2016
                Page count
                Figures: 8, Tables: 2, Pages: 26
                Funding
                Funded by: HIVRAD
                Award ID: AI104722
                Award Recipient :
                Funded by: CHAVI-ID
                Award ID: AI100663
                Award Recipient :
                Funded by: CHAVI-ID
                Award ID: AI100663
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000866, International AIDS Vaccine Initiative;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000866, International AIDS Vaccine Initiative;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000866, International AIDS Vaccine Initiative;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000866, International AIDS Vaccine Initiative;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000866, International AIDS Vaccine Initiative;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000866, International AIDS Vaccine Initiative;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000866, International AIDS Vaccine Initiative;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000866, International AIDS Vaccine Initiative;
                Award Recipient :
                This work was funded by the HIVRAD grant number P01 AI104722 (RTW), CHAVI-ID grant number AI100663 (ABW and RTW) and by the International AIDS Vaccine Initiative (IAVI) and its generous donors (YF, KT, JG, SK, HA, JD, ABW and RTW). IAVI's work is made possible by generous support from many donors including: the Bill & Melinda Gates Foundation; the Ministry of Foreign Affairs of Denmark; Irish Aid; the Ministry of Finance of Japan; the Ministry of Foreign Affairs of the Netherlands; the Norwegian Agency for Development Cooperation (NORAD); the United Kingdom Department for International Development (DFID), and the United States Agency for International Development (USAID). The full list of IAVI donors is available at www.iavi.org. This study is made possible by the generous support of the Bill & Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery and the American people through USAID. The contents are the responsibility of the International AIDS Vaccine Initiative and do not necessarily reflect the views of USAID or the United States Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files except for the coordinates of the EM models which are deposited in the Electron Microscopy Data Bank, http://www.emdatabank.org/. EM maps have been deposited in the EMDB as follows: unliganded BG505 NFL Wt (EMD-8270) or X-link (EMD-8271) and VRC01-bound BG505 NFL Wt (EMD-8269) or X-link (EMD-8268).

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