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

Abstract

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 gp120_JRFL, gp120_JRFL 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 gp120_A244/mAb complexes. Notably, gp120_A244/mAb complexes induced higher levels of V1V2 Abs with some cross-reactivity, while also stimulating weak or strain-specific V3 Abs. Sera from gp120_A244/mAb complex-immunized animals displayed no measurable virus neutralization but did mediate Ab-dependent cellular phagocytosis, albeit at levels similar to that induced by gp120_A244 alone. These data indicate the potential utility of immune complexes as vaccines to shape Ab responses toward or away from Env sites of interest.

Keywords: HIV; antibody; envelope; immune complex; vaccine.

Figure 1

Changes in antigenicity of gp120_JRFL upon immune complex formation. (A) Immune complexes were prepared by incubating gp120_JRFL with different gp120-specific mAbs (molar ratio of 1:2). Serially diluted complexes were then coated onto ELISA plates and probed with biotinylated mAbs. Representative data are shown depicting the binding of biotinylated anti-V3 mAb 694/98D to gp120_JRFL in complex with CD4bs mAb 654 or V2 mAb 2158 as compared with gp120_JRFL treated with an irrelevant parvovirus-specific mAb 1418 or no mAb. (B) The immune complexes were also tested by BLI using Fortebio Octet for their relative reactivity with mAb 694/98D that was immobilized on the biosensor tip. (C) This panel summarizes ELISA data showing fold changes in mAb reactivity to different gp120_JRFL/mAb complexes vs. uncomplexed gp120_JRFL (gp120_JRFL plus control mAb 1418). AUC: area under titration curve.

Figure 2

Changes in antigenicity of gp120_A244 upon immune complex formation. Immune complexes made with gp120_A244 and gp120-specific mAbs (molar ratio of 1:2) were tested in ELISA for reactivity with biotinylated mAb probes as described in Figure 1. (A–B) Representative data depicting altered reactivity of biotinylated anti-V2q mAbs PG9 (A) and CH01 (B) to gp120_A244 complexed with V2i mAb 2158, V2i mAb 697, or V2q mAb PG9 relative to reactivity with uncomplexed gp120_JRFL mixed with control mAb 1418 are shown. (C) This panel summarizes fold changes in mAb reactivity to different gp120_A244/mAb complexes vs. uncomplexed gp120_A244 (gp120_A244 + control mAb 1418). RLU: relative luminescence unit from ELISA with PhosphaGLO AP substrate. AUC: area under titration curve.

Figure 3

Serum Ab responses induced by vaccination with gp120_JRFL/mAb complexes. BALB/c mice were immunized with gp120 B.JRFL complexed with human IgG1 mAbs of defined specificity—C2 (1006-30D), V2i (2158), CD4bs (654), V3 (1006-15D)—or with no mAb. Immune complexes were administered 4 times subcutaneously in the presence of adjuvant MPL/DDA. Mice immunized with PBS and adjuvant (no gp120) served as negative controls. (A–C) Pooled sera collected 2 weeks after the last immunization were tested in ELISA for IgG reactivity against gp120 (A), V1V2 (B), or V3 (C). (D) Sera from individual mice immunized with gp120_JRFL vs. gp120_JRFL/CD4bs mAb 654 were also tested for ELISA reactivity against V3. AUC: area under the titration curve of each serum sample; OD₄₀₅: optical density at 405 nm obtained from designated serum dilution in ELISA with p-nitrophenyl phosphate substrate. ^*p < 0.05.

Figure 4

V3-specific Ab responses induced by gp120_JRFL/mAb complexes vs. uncomplexed gp120_JRFL. (A) Sera from mice immunized with gp120 B.JRFL in complex with V2i mAb 2158, CD4bs mAb 654, or no mAb were tested in ELISA for IgG reactivity against V3 peptides of HIV-1 subtypes A, B, and C. (B) Overlapping V3 peptides P1 to P5 were used for epitope mapping. Sequence logo depicting V3 amino acid variations is included to show the central V3 regions that are targeted by Abs induced by gp120_JRFL (blue-shaded box) vs. gp120_JRFL/mAb complexes (orange-shaded box). (C) Sera from individual animals were tested for reactivity with overlapping V3 peptides P1 to P5 and demonstrated more uniform recognition of P2 and P3 peptides by all animals that received gp120_JRFL/CD4bs mAb 654 vs. uncomplexed gp120_JRFL.. (D) Sera from mice immunized with gp120_JRFL or gp120_JRFL/mAb complexes were also tested for IgG reactivity with different V1V2 antigens. AUC: area under the titration curve. PBS: Sera from control group that received PBS and adjuvant (no gp120).

Figure 5

Serum Ab responses induced by gp120_A244/mAb complex vaccines. Mice were immunized with gp120 AE.A244 in complex with human IgG1 mAbs specific for V2i (2158), V2i (697), CD4bs (1331E), V3 (2219), or with no mAb in the presence of adjuvant MPL/DDA. (A–C) Sera collected 2 weeks after the fourth immunization were tested in ELISA for IgG reactivity to gp120 (A), V3 (B), and V1V2 (C). (D) Sera were also evaluated for cross-reactivity with V3 and V1V2 from viruses of different HIV-1 subtypes. (E) Further mapping of V2 epitope was performed using overlapping V2 peptides P1 to P9. V2 sequence logo is shown to indicate amino-acid variability within the defined epitope region (orange-shaded box). (F) Sera were subjected to competition ELISA using V1V2 C.ZM109-1FD6 to assess the presence of V1V2-specific serum Abs able to compete with conformation-dependent V2i mAb 830A. ^****p < 0.0001.

Figure 6

Inhibitory activity of immune sera from animals that received gp120/mAb complexes vs. uncomplexed gp120. (A) Pooled sera from mice immunized with gp120_JRFL/mAb complexes or uncomplexed gp120_JRFL were evaluated for neutralizing activity against HIV-1 B.SF162 using TZM.bl target cells. (B) Neutralization activity of individual animal serum from groups that received gp120_JRFL vs. gp120_JRFL/CD4bs mAb 654 was also compared. (C) Neutralization mediated by V3-specific Abs was assessed by measuring neutralization activity of sera pretreated with or without V3 peptide (40 μg/ml). Δ% neutralization was calculated by subtracting % neutralization of untreated sera with that of V3 peptide-treated sera. (D) Likewise, sera from animals immunized with gp120_A244/mAb complex or uncomplexed gp120_A244 were tested for neutralizing activity against HIV-1 C.ZM109 using TZM.bl cells. CD4bs-specific mAb VRC01 was included as a positive control. (E) Sera from all groups of immunized mice were also tested for the capacity to block V2 interaction with the integrin α4β7. The α4-specific mAb HP2/1 served as a positive control. PBS: sera from control group that received PBS and adjuvant (no gp120).

Figure 7

ADCP activity of immune sera from animals that received with gp120_A244 or gp120_A244/mAb complexes. (A–C) Pooled sera from groups of mice immunized with gp120 AE.A244 in complex with different mAbs or no mAb were tested for the ability to mediate ADCP using phagocytic THP-1 cells and fluorescent beads coated with gp120 (A), V1V2-1FD6 (B), or cyclic V2 peptide (C). No significant difference was observed among the groups. Sera from animals that received adjuvant MPL/DDA and PBS (no gp120) were included as negative controls. ADCP score was calculated by multiplying the percentage of bead-bearing cells with the geometric mean intensity of the cells and subtracting the background score. Cutoff value (dotted lines) was determined based on ADCP scores of cells and beads without serum.

Figure 8

Differential modulation of Ab responses following immunization with gp120_JRFL/mAb complexes vs. gp120_A244/mAb complexes.