MAb 11A8 detects HIV on the surface of cells infected by HIV isolates from clades A, B, D, and E.

*<p>Murine IgG₁<i>κ</i> was used as an isotypic control for MAb 11A8.</p><p>CEM-4 cells were infected with HIV isolates from different clades. Cells were incubated with the antibodies at saturating concentrations and the antibody binding was determined by flow cytometry using the appropriate secondary antibodies. Results are expressed as % of cells fluorescing.</p

MCPs elicit antibodies to cryptic epitopes of gp120.

<p>(A) Binding of MAb 11A8 to gp120 is not inhibited by antisera to gp120. Competition for gp120_MN binding of biotinylated MAb 11A8 and murine sera was evaluated in ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008555#s4" target="_blank">Materials and Methods</a>. All competing sera were obtained from C57Bl/10 mice prior to immunization (preimmune), from mice immunized with 426–441 MCP (130, 000) or with rgp120MN (550, 000). Titers of sera antibodies recognizing gp120 are listed in parenthesis. Results are expressed as % inhibition of biotinylated 11A8 binding to gp120 by competing antibodies. (B) HIVIG does not inhibit the binding to gp120 of murine antibodies elicited by MCPs 419–439, 363–384 or 105–117. Competition between HIVIG and anti-MCP sera for binding to gp120 was assayed by ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008555#s4" target="_blank">Materials and Methods</a>. Gp120 was incubated with HIVIG at the indicated concentrations and the binding of anti-MCP sera (colored lines) from C3H/HeJ mice (anti-MCPs 105–117 and 363–384), C57Bl/10 mice (419–439 MCP) or of HIVIG (shown as black line) was determined. Results are expressed as absorbance at 405 nm.</p

MAb 11A8 neutralizes SF162 pseudovirus.

<p>(A) Concentration curve of 11A8 and the murine isotypic control anti-TNP for SF162 pseudovirus neutralization. (B) Neutralization of SF162 by 11A8 is abolished by preincubation with the homologous MCP. MAb 11A8 was incubated before neutralization assay for 1 h at 37° with 426–441 MCP or 105–117 MCP or with medium (control). The final concentration of 11A8 in neutralization assay was 0.1 µM and of the MCPs was 1 µM.</p

Antibodies elicited by MCPs 419 and 426 partially compete for CD4 binding to gp120.

<p>(A) CD4 inhibition of antibodies binding to gp120. (B) MAb 11A8 weakly inhibits CD4 binding to gp120. Reciprocal competition of CD4 and antibodies for binding to gp120 was evaluated by ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008555#s4" target="_blank">Materials and Methods</a> with gp120 captured on the antibody to the C-terminal sequence of gp120.</p

Antibodies elicited by MCPs fail to recognize denatured gp120.

<p>Recombinant gp120_MN or RCM gp120_MN were either maintained at 4° (control) or were heated for 5 minutes at 95° with 1 mM DTT, 0.1% SDS (denatured). Gp120 was then immobilized in ELISA wells using Ab D7324 and binding of antibodies was determined. Antigens captured on the plate were as follows: (A) gp120 (control); (B) gp120 (denatured). MAbs: 11A8 and 2D3, antisera to MCP 419 and to MCP 426 were all derived from C57Bl/10 immunized mice and antisera to gp120 was derived from C3H/HeJ mice. All sera and MAb preparations were diluted as indicated in the Figure. Protein concentration of the stock of undiluted MAb preparations used was, respectively: 1.96 mg/ml (MAb 11A8), and 0.5 mg/ml (MAb 2D3).</p

MAb 11A8 does not recognize gp120 passively bound to uninfected CEM-4 cells.

<p>CEM-4 cells were incubated for 2 h at 4°C with gp120_MN (0.24 µg/ml) or with no gp120 (control), washed, then incubated with the primary antibodies (10 µg/ml) listed in the Table. Antibody binding to cells was determined by flow cytometry. Results are expressed as % of cells fluorescing. Secondary antibodies:</p>*<p>anti-mouse.</p>**<p>anti-human.</p

11A8 binding to HIV on infected cells is blocked by gp120 and by homologous MCP.

<p>11A8 was incubated for 30 min at 37° with 426–441 MCP, 363–384 MCP or with gp120 in 2% FCS RPMI and subsequently the binding of 11A8 to HIV-1 clade B infected CEM-4 cells was assayed. Final concentration of 11A8 in the binding assay was 0.5 µM, and concentrations of competing antigens were as indicated. MAb 11A8 binding to cells was determined by flow cytometry and % inhibition of MAb binding by antigens was calculated.</p

MAbs 11A8 and 2D3 compete for binding to gp120.

<p>Binding to gp120 of biotinylated 2D3 in the presence of unconjugated MAbs 2D3 or 11A8 was evaluated in ELISA. Concentration of biotinylated 2D3 in the assay was 1.2 nM, concentrations of competing, unconjugated MAbs 2D3 and 11A8 were as indicated.</p

Antibodies to MCPs are not elicited by immunization with gp120 or by HIV infection.

<p>ELISA wells were coated with gp120_MN or with MCPs listed in the Table, and antibody titers were determined.</p>*<p>Sera were obtained from C3H/HeJ mice immunized with gp120_MN.</p>**<p>HIV Immunoglobulin, purified from pooled HIV+ human plasma.</p

Recognition of native gp120_MN and of RCM^** gp120_MN by antisera elicited by MCPs.

*<p>gp120 was denatured as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008555#pone-0008555-g002" target="_blank">Figure 2</a>.</p>**<p>RCM, reduced, carboxymethylated gp120_MN.</p><p>Sera to 363–384 MCP were harvested after the third boost, and sera to 419–439 MCP or 426–441 MCP after the second boost. Antisera to MCPs 363 and 419 were obtained from C3H/HeJ mice and to 426 MCP from C57Bl/10 mice. ELISA wells were coated directly with gp120_MN native, gp120 denatured (as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008555#pone-0008555-g003" target="_blank">Figure 3</a>) or with RCM gp120_MN and antibody titers were determined.</p