A Strategy for Eliciting Antibodies against Cryptic, Conserved, Conformationally Dependent Epitopes of HIV Envelope Glycoprotein

Novel strategies are needed for the elicitation of broadly neutralizing antibodies to the HIV envelope glycoprotein, gp120. Experimental evidence suggests that combinations of antibodies that are broadly neutralizing in vitro may protect against challenge with HIV in nonhuman primates, and a small number of these antibodies have been selected by repertoire sampling of B cells and by the fractionation of antiserum from some patients with prolonged disease. Yet no additional strategies for identifying conserved epitopes, eliciting antibodies to these epitopes, and determining whether these epitopes are accessible to antibodies have been successful to date. The defining of additional conserved, accessible epitopes against which one can elicit antibodies will increase the probability that some may be the targets of broadly neutralizing antibodies.We postulate that additional cryptic epitopes of gp120 are present, against which neutralizing antibodies might be elicited even though these antibodies are not elicited by gp120, and that many of these epitopes may be accessible to antibodies should they be formed. We demonstrate a strategy for eliciting antibodies in mice against selected cryptic, conformationally dependent conserved epitopes of gp120 by immunizing with multiple identical copies of covalently linked peptides (MCPs). This has been achieved with MCPs representing 3 different domains of gp120. We show that some cryptic epitopes on gp120 are accessible to the elicited antibodies, and some epitopes in the CD4 binding region are not accessible. The antibodies bind to gp120 with relatively high affinity, and bind to oligomeric gp120 on the surface of infected cells.Immunization with MCPs comprised of selected peptides of HIV gp120 is able to elicit antibodies against conserved, conformationally dependent epitopes of gp120 that are not immunogenic when presented as gp120. Some of these cryptic epitopes are accessible to the elicited antibodies

Modulation of Antibody Responses to the V1V2 and V3 Regions of HIV-1 Envelope by Immune Complex Vaccines

Prophylactic HIV vaccines must elicit antibodies (Abs) against the virus envelope glycoproteins (Env) to effectively prevent HIV infection. We investigated a vaccine platform that utilizes immune complexes made of Env proteins gp120 and monoclonal Abs (mAbs) against different gp120 epitopes. We previously observed alterations in V3 antigenicity upon formation of certain gp120/mAb complexes and demonstrated the ability of these complexes to modulate the elicitation of V3 Ab responses. However, the effects on the V1V2 domain, an important target for Abs that correlate with vaccine-induced protection against HIV, have not been studied, nor have immune complex vaccines made with non-B subtype Env. This study compared subtypes B (JRFL) and CRF_01.AE (A244) Env gp120 proteins in complex with selected gp120-specific mAbs. Allosteric and antigenic changes were detected on these immune complexes, indicating that gp120/mAb interaction induces alterations on the Env surface that may modify the Env immunogenic properties. To evaluate this idea, mice were immunized with gp120/mAb complexes or their uncomplexed gp120 counterparts. The overall serum IgG titers elicited against gp120 were comparable, but a marked skewing toward V1V2 or V3 was evident and dependent on the gp120 strain and the specificity of the mAb used to form the complexes. Compared with uncomplexed gp120JRFL, gp120JRFL complexed with CD4bs or V1V2 mAbs, but not with C2 or V3 mAbs, elicited V3 Abs of greater titers and breadth, and Abs more capable of neutralizing tier 1 virus. Epitope mapping revealed a shift to a more conserved site in the V3 crown. However, the complexes did not enhance V1V2 Ab response, and the elicited V1V2 Abs were not cross-reactive. This profile contrasts with Ab responses to gp120A244/mAb complexes. Notably, gp120A244/mAb complexes induced higher levels of V1V2 Abs with some cross-reactivity, while also stimulating weak or strain-specific V3 Abs. Sera from gp120A244/mAb complex-immunized animals displayed no measurable virus neutralization but did mediate Ab-dependent cellular phagocytosis, albeit at levels similar to that induced by gp120A244 alone. These data indicate the potential utility of immune complexes as vaccines to shape Ab responses toward or away from Env sites of interest

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

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

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

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 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