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. 2011 Aug;85(16):8217-26.
doi: 10.1128/JVI.00756-11. Epub 2011 Jun 8.

Direct antibody access to the HIV-1 membrane-proximal external region positively correlates with neutralization sensitivity

B K Chakrabarti  1 , L M Walker, J F Guenaga, A Ghobbeh, P Poignard, D R Burton, R T Wyatt
Affiliations

Direct antibody access to the HIV-1 membrane-proximal external region positively correlates with neutralization sensitivity

B K Chakrabarti et al. J Virol. 2011 Aug.

Abstract

On the prereceptor-engaged HIV-1 envelope glycoprotein (Env) spike, epitope access by the membrane-proximal external region (MPER)-directed broadly neutralizing antibodies 2F5 and 4E10 remains unresolved. Data on binding to cell surface Env and entry data using primary isolates suggest inaccessibility of the 2F5 and 4E10 epitopes on the viral spike prior to receptor engagement, but trimer gel shift analysis and slow kinetics of shedding induced by 2F5 and 4E10 indicate otherwise. Therefore, it remains unclear if the epitopes themselves are formed in their antibody-bound state (or at least sampled) prior to receptor/coreceptor engagement or if receptor interactions both expose and form the MPER epitopes, presumably in the putative prefusion transitional intermediate. Here, we performed antibody-virus "washout experiments" using both lab-adapted and a panel of clade B primary isolates to analyze MPER accessibility. The neutralization activity of 2F5 and 4E10 against lab-adapted viruses and sensitive and moderately resistant viruses was largely unaffected by relatively rapid antibody-virus washing, suggesting direct interaction with the "static" spike. However, for more neutralization-resistant viruses, the 2F5 and 4E10 antibodies could neutralize only under the "no antibody-virus wash" conditions, implying that the MPER epitopes were not accessible prior to receptor engagement. Accessibility in the washout conditions could be precisely predicted by the relative resistance to neutralization in a standard neutralization format. These data are consistent with a model in which the local MPER antibody epitope conformations may be sampled on the native spike but are occluded to antibody by local steric or distal quaternary constraints adopted by highly resistant HIV-1 isolates.

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Figures

Fig. 1.
Fig. 1.
Antibody binding to cell surface JR-FL Env monitored by FACS. Mean fluorescence intensity (MFI) values of both neutralizing (b12, 2G12, 2F5, and 4E10) and nonneutralizing (F105, 7B2, 22B) antibodies are shown for cleavage-competent JR-FL Env expressed on 293T cells. The data were derived from representative experiments performed in duplicate. The standard errors between duplicates were minimal (less than 0.01) and are not shown since the error bars are obscured by the symbols.
Fig. 2.
Fig. 2.
Virus entry into TZM-bl cells with and without inhibitory antibodies and with and without ligand-virus washing. (A) Schematics of “no antibody-virus” wash and “antibody-virus wash” of the antibody-virus mixture in the assay used in this study. (B) Positive- and negative-controls for the JR-FL virus, with and without washing, are shown as bar graphs. Relative luciferase units (RLU) generated from target cell lysates are shown on the vertical axis. Left to right: virus with no T-20 added to target cells; virus incubated with T-20 at 100 μg/ml prior to target cell interaction; virus incubated with T-20 followed by washing prior to target cell interaction; no virus added to target cell (negative control).
Fig. 3.
Fig. 3.
Neutralization of JR-FL and HXBc2 virus by the antibodies b12, 2F5, and 4E10 with and without washing. Left panels (b12, 2F5, and 4E10), preincubation with JR-FL virus with or without washing prior to incubation with target cells. Right panels (b12, 2F5, and 4E10), preincubation with HXBc2 virus with or without washing prior to incubation with target cells. The red curves and the blue curves indicate “no antibody-virus” wash and “antibody-virus” wash, respectively.
Fig. 4.
Fig. 4.
Resistance to washing of antibodies 2F5 and 4E10 from the viruses, MN, ADA, MW965.26, and HIV2-C3 after “antibody-virus” complex formation. Antibodies 2F5 and 4E10 preincubated with MN with or without washing prior to incubation with target cells are shown in the top row. The same antibody-virus wash experimental data are shown in the middle panels for the primary isolate ADA. The bottom panels show HIV2-C3 virus with 2F5 and MW965.26 with 4E10 only.
Fig. 5.
Fig. 5.
Accessibility of WT JRCSF, V3, and V1/V2 mutant JRCSF for 2F5, 4E10, and T-20 under “no antibody-virus” and “antibody-virus” wash conditions. (A) Results for the antibodies 2F5, 4E10, and b12 preincubated with either JRCSF or mutant JRCSF virus with or without washing prior to incubation with the target cells are shown. (A) Left, data for WT JRCSF; middle, V3 mutant JRCSF virus or JRCSF-I309A data; right, V1/V2 mutant JRCSF virus or JRCSF-Y177A data. The red curves depict “no antibody-virus wash” data sets, and the blue curves depict “antibody-virus wash” data sets. (B) Percent inhibition mediated by T-20 peptide preincubated with JRCSF-I309A virus with or without washing prior to incubation with target cells (TZM-bl). The red curves and the blue curves represent “no antibody-virus” and “antibody-virus” wash, respectively. (C) Percent inhibition of the entry of WT and mutant virus to the target cells in the presence of differing concentrations of the anti-CD4 antibody.
Fig. 6.
Fig. 6.
Model depicting MPER neutralizing antibody access to primary isolate and lab-adapted virus functional spikes before and after CD4 engagement. Upper panels: left, the more tightly packed primary isolate spike does not permit access of neutralizing ligands before CD4 engagement (top view); right, the more open architecture of the lab-adapted virus allows direct binding to the functional spike by neutralizing antibodies, implying that the epitopes are formed prior to CD4 engagement (top view). Lower panels: left, the more tightly packed primary isolate spike does not allow MPER-directed antibodies to bind before receptor engagement (side view); right, the more open architecture of the primary isolates after receptor engagement and repositioning of primary isolate Env elements (perhaps variable regions) allow accessibility of neutralizing ligands to already preformed MPER determinants (side view).
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