Inhibition of MN-rgp120 binding to α4β7 by monoclonal antibodies to the V2 domain.

<p>Biotinylated MN-rgp120 was incubated with monoclonal antibodies to the V2 domain of gp120 for 1 hr at room temperature. Antibodies representative of the five V2 competition groups were tested: Group A, 1019 and 1027; Group B, 1022; Group C, 1088; Group D, 1025; Group E, 6E10. The mixture was then added to cells and incubated for 1 hr at 4°C. After washing, the cells were incubated with APC-conjugated streptavidin to detect gp120 binding. The amount of gp120 binding to cells was measured by flow cytometry and statistical analysis was carried out by one way ANOVA including a Bonferroni correction. The binding of MN-rgp120 to α4β7 without added MAbs (control) is indicated by the black bar. Statistically significant inhibition of MN-rgp120 binding (p<0.05) is indicated by gray bars; inhibition that does not reach statistical significance is indicated by white bars. Inhibition of MN-rgp120 binding by the FIB504.64 MAb to α4β7 was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#s4" target="_blank">Materials and Methods</a>.</p

Relative binding of V2 MAbs to MN-rgp120 V2 mutants.

<p>Data represent ratios of ELISA results where MAb binding to envelope proteins with the mutations indicated was compared to binding of the wildtype MN-rgp120. Significant differences in MAb binding are represented by underlined numbers. Percentage inhibition is calculated according to the following equation: (1 – binding to mutant/binding to MN-rgp120)×100.</p

Properties of V2 region monoclonal antibodies.

<p>RCM-rgp120, indicates the ability of MAbs to bind to denatured (reduced and carboxymethylated) gp120. HIV rgp120 binding, indicates the ability of MAbs to bind to recombinant gp120 from the MN, IIIB and JRCSF strains of HIV in an ELISA format. CD4 blocking represents a summary of the data provided in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045.s001" target="_blank">Figure S1</a>. Neutralization data represents the MAb concentration (µg/mL) required for 50% inhibition (IC50) of infectivity by the MN-3 strain of HIV-1 in the TZM-bl neutralization assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#s4" target="_blank">Materials and Methods</a>) with the exception of the 6E10 MAb (*) where neutralization titers were measured using the IIIB strain of gp120 as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Nakamura1" target="_blank">[27]</a>. Controls for the assays (italic) included MAbs 1026, b12 and PG9. ND, indicates not done. Epitope localization, summarizes the minimal gp120 structure required for antibody binding based on MAb reactivity with the gp120 fragments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone-0039045-g001" target="_blank">Figure 1</a>.</p

Summary of V2 epitopes recognized by Murine MAbs to MN-rgp120.

<p>Summary of V2 epitopes recognized by Murine MAbs to MN-rgp120.</p

Fragments of gp120 used for epitope mapping studies.

<p>Fragments of the gp120 gene were selected based on the two-dimensional structure of Leonard et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Leonard1" target="_blank">[54]</a> and fused to the signal sequence and 27 N terminal amino acids of glycoprotein D from type 1 herpes simplex virus. The fragments were expressed in 293 cells, and cell culture supernatants containing the secreted fragments were screened by ELISA for MAb binding. MAb binding to fragments A–E localized epitopes outside of the V1 and V2 domains; MAb binding to fragments F–I localized epitopes that depended on the V1 and V2 domain.</p

Frequency of sequence polymorphisms at positions in the V2 domain recognized by monoclonal antibodies to MN-rgp120.

<p>Letters represent standard single letter amino acid codes. GAP indicates the frequency of a deletion at a given position. Subscripts represent the percentage of sequence polymorphism at a single position in each dataset. Positions are numbered according to the HXB2 reference sequence. The VAX004 dataset includes 1047 clade B sequences from 349 individuals. The Keele data set represents 2744 clade B sequences from 102 individuals. Residues circled in blue indicate amino acids present in the MN_GNE strain of HIV-1. VAX004 data obtained from Pérez-Losada et al., 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-PerezLosada1" target="_blank">[60]</a>, using data listed in the GSID HIV Data Browser <a href="http://www.gsid.org/gsid_hiv_data_browser.html" target="_blank">http://www.gsid.org/gsid_hiv_data_browser.html</a>. Sequences of clade B envelope proteins from new infections were obtained from Keele et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Keele1" target="_blank">[29]</a> using data listed in the Los Alamos HIV Sequence Database <a href="http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html" target="_blank">http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html</a>.</p

Listing of V2 region mutations incorporated into MN-rgp120.

<p>Listing of V2 region mutations incorporated into MN-rgp120.</p

Competitive binding of monoclonal antibodies.

<p>Each monoclonal antibody was labeled with horseradish peroxidase (HRPO). The ability of a large excess of each unlabeled antibody to inhibit the binding of HRPO MAbs was measured. Inhibition of greater than 50% was considered significant and was defined as directly competitive. Isotype matched antibodies to the V3 domain (1034) and the C4 domain (1024) served as negative controls. Data represent percentage of inhibition: red, 90–100%; orange, 70–89%; yellow, 50–69%; green, <50%.</p

Location of amino acids critical for the binding of monoclonal antibodies to the V2 domain of gp120.

<p>A diagram of the gp120 V2 domain, incorporating amino acids from the MN_GNE strain of HIV-1, was created based on the disulfide structure of Leonard et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Leonard1" target="_blank">[54]</a>. The locations of the amino acids required for the binding of the MAbs to the V2 domain described in this paper, as well as 697-D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Gorny2" target="_blank">[43]</a>, 2909 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Honnen1" target="_blank">[47]</a> and the potent PG9 and 16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Walker2" target="_blank">[22]</a> neutralizing antibodies are indicated by colored dots. Group, indicates competition group and epitopes from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone-0039045-t004" target="_blank">Table 4</a>. Arrows, indicate the location of three radical amino acid polymorphisms found in MN-rgp120 compared to consensus clade B clinical isolates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-PerezLosada1" target="_blank">[60]</a> and Keele <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Keele1" target="_blank">[29]</a>. Data for this figure was derived from previously published results <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Honnen1" target="_blank">[47]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Walker2" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-ZollaPazner1" target="_blank">[33]</a>.</p