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. 2003 Jan;77(1):642-58.
doi: 10.1128/jvi.77.1.642-658.2003.

Fine mapping of the interaction of neutralizing and nonneutralizing monoclonal antibodies with the CD4 binding site of human immunodeficiency virus type 1 gp120

Ralph Pantophlet  1 , Erica Ollmann Saphire, Pascal Poignard, Paul W H I Parren, Ian A Wilson, Dennis R Burton
Affiliations

Fine mapping of the interaction of neutralizing and nonneutralizing monoclonal antibodies with the CD4 binding site of human immunodeficiency virus type 1 gp120

Ralph Pantophlet et al. J Virol. 2003 Jan.

Abstract

Alanine scanning mutagenesis was performed on monomeric gp120 of human immunodeficiency virus type 1 to systematically identify residues important for gp120 recognition by neutralizing and nonneutralizing monoclonal antibodies (MAbs) to the CD4 binding site (CD4bs). Substitutions that affected the binding of broadly neutralizing antibody b12 were compared to substitutions that affected the binding of CD4 and of two nonneutralizing anti-CD4bs antibodies (b3 and b6) with affinities for monomeric gp120 comparable to that of b12. Not surprisingly, the sensitivities to a number of amino acid changes were similar for the MAbs and for CD4. However, in contrast to what was seen for the MAbs, no enhancing mutations were observed for CD4, suggesting that the virus has evolved toward an optimal gp120-CD4 interaction. Although the epitope maps of the MAbs overlapped, a number of key differences between b12 and the other two antibodies were observed. These differences may explain why b12, in contrast to nonneutralizing antibodies, is able to interact not only with monomeric gp120 but also with functional oligomeric gp120 at the virion surface. Neutralization assays performed with pseudovirions bearing envelopes from a selection of alanine mutants mostly showed a reasonable correlation between the effects of the mutations on b12 binding to monomeric gp120 and neutralization efficacy. However, some mutations produced an effect on b12 neutralization counter to that predicted from gp120 binding data. It appears that these mutations have different effects on the b12 epitope on monomeric gp120 and functional oligomeric gp120. To determine whether monomeric gp120 can be engineered to preferentially bind MAb b12, recombinant gp120s were generated containing combinations of alanine substitutions shown to uniquely enhance b12 binding. Whereas b12 binding was maintained or increased, binding by five nonneutralizing anti-CD4bs MAbs (b3, b6, F105, 15e, and F91) was reduced or completely abolished. These reengineered gp120s are prospective immunogens that may prove capable of eliciting broadly neutralizing antibodies.

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Figures

FIG. 1.
FIG. 1.
Locations of amino acid substitutions on gp120, spatial relationships of epitopes on gp120, and variability of gp120 residues among primate immunodeficiency viruses and HIV-1. (A) Ribbon amino acid sequence diagram of gp120JR-CSF indicating the locations of the alanine substitutions in this study. Arrows indicate amino acids that were mutated to alanine. Variable-loop deletions are indicated by white letters encircled by blue spheres. Light blue dots indicate the positions of amino acid mutations in b12 neutralization escape mutants (38, 54). Red and yellow dots indicate primary and secondary b12 contact residues, respectively, based on a computational docking model of b12 and the gp120HxB2 core (44). Primary b12 contacts are gp120 residues that, based on our docking model, contact MAb b12; secondary contacts are gp120 residues within 3 to 5 Å of MAb b12. (B) Locations of mutations (shown in blue) mapped onto the structure of the gp120 core of HIV-1HxB2. The view is from the perspective of CD4. (C) Approximate locations of the faces of the gp120 core, defined by the interactions of gp120 and antibodies (30, 76, 78). The region in the CD4bs that is accessible to neutralizing ligands on primary HIV-1 isolates (i.e., MAbs CD4-IgG2 and b12), termed the neutralizing face, is shown in yellow. The region that is believed to be poorly accessible on oligomeric gp120 and that elicits nonneutralizing antibodies is shown in cyan. The location of the immunologically silent face, which encompasses the epitope recognized by broadly neutralizing MAb 2G12 (59, 62, 73), is shown in magenta. The coreceptor binding site is shown in light grey. Modeled carbohydrate chains are shown in dark grey and black. The approximate areas that are believed to be covered by the V2 and V3 loops (primarily the coreceptor binding site) are indicated. The locations of the Phe43 cavity (30), involved in CD4 binding, and the stem of the V1/V2 loop are also indicated. (D) Molecular surface of gp120 depicting the sequence variability of the amino acid residues among primate immunodeficiency viruses and HIV-1: green, residues that are conserved among all primate immunodeficiency viruses; yellow, residues that are conserved among all HIV-1 isolates but not among all primate immunodeficiency viruses; grey, residues that are variable among HIV-1 isolates. Amino acid conservation is defined as in reference .
FIG. 2.
FIG. 2.
Apparent affinity of MAbs for alanine mutants of gp120JR-CSF relative to the wild type. Numbering is based on the sequence of HIV-1HxB2 (26). (A) b3 binding (green bars). (B) b6 binding (grey bars). (C) b12 binding (blue bars). On the x axis, only every second amino acid residue listed in Table 1 is numbered. Orange bars represent CD4 binding. A schematic of the conserved and variable regions of HIV-1 gp120 is also shown. Numbers indicate amino acid residues (HxB2 numbering). C, conserved domain. V, variable region.
FIG. 3.
FIG. 3.
Effects of alanine substitutions on antibody binding mapped onto the structure of the gp120 core of HIV-1HxB2. The view is from the perspective of CD4. Only substitutions that affected antibody binding are colored and labeled. Alanine substitutions of residues colored yellow significantly enhanced MAb binding (>200% affinity relative to the wild type), whereas those colored blue significantly reduced MAb or CD4 binding (<50% affinity relative to the wild type). Amino acid substitutions in the V2 loop that affected antibody or CD4 binding are indicated by colored circles to the left of each structure.
FIG. 4.
FIG. 4.
Epitope maps of the unique effects of alanine substitutions on MAb binding affinity. Color scheme and labeling are as described in the legend to Fig. 3, except that amino acid mutations that did not significantly affect antibody binding are also indicated (colored black). (Top panels) Differential maps of alanine point mutations for which the effect on MAb binding was unique compared to the effects on the binding of the other two MAbs. (Bottom panels) Differential maps of the unique effects of alanine substitutions on binding by MAbs b3 and b6 in comparison to MAb b12.
FIG. 5.
FIG. 5.
Alanine mutants that were selected for neutralization assays. Labeled amino acids colored grey indicate mutant pseudovirions for which there was a reasonable correlation between b12 binding to monomeric gp120 and neutralization efficiency. Residues labeled and colored yellow indicate pseudovirions which were neutralized equally as well as or better than wild-type virus despite a decrease in the b12 affinity for monomeric gp120 of the respective mutant. Residues labeled and colored red indicate pseudovirions which were not neutralized as well as the wild-type virus despite an increase in the b12 affinity for monomeric gp120 of the respective mutant.
FIG. 6.
FIG. 6.
Antibody binding in the context of the functional envelope trimer. (A) Trimeric gp120 model depicted as proposed by Kwong et al. (31). Here, gp120 is depicted as viewed from the virus. (B) Model of docking of MAb b12 (yellow) to gp120. (C and D) Models of how two hypothetical nonneutralizing anti-CD4bs MAbs (pink and green) may interact with gp120. Note that b12 and the nonneutralizing anti-CD4bs MAbs are able to interact with monomeric gp120 but that only b12 binds at an orientation that also allows an interaction with gp120 in the context of the functional envelope trimer. For clarity, only antibody Fab fragments are shown.
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