Distinct mechanisms regulate exposure of neutralizing epitopes in the V2 and V3 loops of HIV-1 envelope

Abstract

Broadly neutralizing antibodies targeting the HIV-1 envelope (Env) are key components for protection against HIV-1. However, many cross-reactive epitopes are often occluded. This study investigates the mechanisms contributing to the masking of V2i (variable loop V2 integrin) epitopes compared to the accessibility of V3 epitopes. V2i are conformation-dependent epitopes encompassing the integrin α4β7-binding motif on the V1V2 loop of HIV-1 Env gp120. The V2i monoclonal antibodies (MAbs) display extensive cross-reactivity with gp120 monomers from many subtypes but neutralize only few viruses, indicating V2i's cryptic nature. First, we asked whether CD4-induced Env conformational changes affect V2i epitopes similarly to V3. CD4 treatment of BaL and JRFL pseudoviruses increased their neutralization sensitivity to V3 MAbs but not to the V2i MAbs. Second, the contribution of N-glycans in masking V2i versus V3 epitopes was evaluated by testing the neutralization of pseudoviruses produced in the presence of a glycosidase inhibitor, kifunensine. Viruses grown in kifunensine were more sensitive to neutralization by V3 but not V2i MAbs. Finally, we evaluated the time-dependent dynamics of the V2i and V3 epitopes. Extending the time of virus-MAb interaction to 18 h before adding target cells increased virus neutralization by some V2i MAbs and all V3 MAbs tested. Consistent with this, V2i MAb binding to Env on the surface of transfected cells also increased in a time-dependent manner. Hence, V2i and V3 epitopes are highly dynamic, but distinct factors modulate the antibody accessibility of these epitopes. The study reveals the importance of the structural dynamics of V2i and V3 epitopes in determining HIV-1 neutralization by antibodies targeting these sites.

Importance: Conserved neutralizing epitopes are present in the V1V2 and V3 regions of HIV-1 Env, but these epitopes are often occluded from Abs. This study reveals that distinct mechanisms contribute to the masking of V3 epitopes and V2i epitopes in the V1V2 domain. Importantly, V3 MAbs and some V2i MAbs display greater neutralization against relatively resistant HIV-1 isolates when the MAbs interact with the virus for a prolonged period of time. Given their highly immunogenic nature, V3 and V2i epitopes are valuable targets that would augment the efficacy of HIV vaccines.

FIG 1

Influence of CD4 engagement on virus neutralization by V2i and V3 MAbs. (A) Neutralization of JRFL pseudovirus by V2i, V3, and control MAbs was tested following treatment with or without sCD4. Virus was pretreated with sCD4 at one designated concentration for 30 min at 37°C or left untreated and then incubated with serially diluted MAb for 1 h at 37°C. The mixture was added to TZM.bl target cells, and after 48 h, virus infection was measured based on β-galactosidase activity. (B, C) Comparison of neutralization by V2i, V3, and control MAbs in the presence or absence of sCD4 against JRFL and BaL.ec1. “sCD4 alone” denotes virus neutralization by sCD4 alone (no MAbs). Means and standard errors from two independent experiments (each in duplicate) are shown. Statistical analyses were performed on neutralization curves reaching ≥50%. *, P < 0.001 for JRFL (B) and P < 0.05 for BaL (C), based on the two-way analysis of variance (ANOVA) test showing significant synergistic difference above the calculated sum of percent neutralization by titrated MAb plus sCD4.

FIG 2

Changes in N-glycan sugar composition affect virus neutralization by V3 MAbs but not V2i MAbs. (A) V2i, V3, and control MAbs were titrated and tested for neutralizing activity against JRFL produced in the presence or absence of kifunensine. Virus and MAb were incubated for 1 h and then added to TZM.bl target cells. Virus infectivity was measured 48 h later based on β-galactosidase activity. (B, C) Comparison of neutralization by V2i, V3, and control MAbs against JRFL or BaL.01 produced with or without kifunensine. Means and standard errors calculated from three different experiments (each in duplicate) are shown. *, P < 0.0001 for JRFL and BaL.01, based on the two-way ANOVA test comparing the neutralization curves of each MAb tested against virus produced with or without kifunensine.

FIG 3

Time-dependent increase of virus neutralization by V2i and V3 MAbs. (A, B) Virus neutralization was measured after JRFL or BaL.ec1 was incubated with serially diluted MAbs for the designated period of time at 37°C prior to the addition of TZM-bl target cells. The CD4bs MAb NIH45-46 and the irrelevant control MAb 1418 were tested in parallel for comparison. (C, D) Comparison of neutralization by V2i, V3, CD4bs, and control MAbs against JRFL and BaL.ec1 after various times of virus-MAb preincubation. Means and standard errors from two to three experiments are shown. *, P < 0.05 for V2i and P < 0.001 for V3 MAbs, from the two-way ANOVA test comparing neutralization curves observed with virus-MAb preincubation of 1 h versus 18 h and 1 h versus 24 h. (E) JRFL neutralization achieved after a 24-h preincubation with V2i MAbs correlated with gp120 JRFL binding as measured by ELISA. Correlation was also observed with an 18-h incubation (P = 0.033; r = −0.75) but not with a 1-h incubation (not shown). (F) JRFL neutralization after a 24-h incubation with V2i MAb displayed virus dose dependency, while no neutralization was achieved with a 1-h incubation regardless of the amount of input virus. Virus was titrated, treated with one fixed concentration of V2 MAb 2158 or control MAb 1418 (100 μg/ml) for 1 h or 24 h, and then added to the TZM.bl target cells. Virus infection was assessed 48 h later based on β-galactosidase activity. At high virus dilutions yielding exceedingly low infectivity, some neutralization values reached below 0% and are denoted in the graph as 0%.

FIG 4

Time-dependent increase of Env spike binding by V2i and V3 MAbs. Relative levels of MAb binding to BaL (A) or JRFL (B) Env spikes on transfected 293T cells were measured by flow cytometry. Cells were transfected with gp160Δc^BaL-IRES-GFP for 48 h and treated with V2i, V3, or control MAbs at 4°C for various times from 30 min to 24 h. MAb binding was detected with APC-conjugated anti-human IgG. Fold increase of MAb binding was calculated relative to mean fluorescence intensity measured at 30 min (normalized to 1). Means and standard errors from two to four independent experiments are shown. *, P < 0.05 based on the one-tail t test comparing fold increase at 24 h versus 30 min; #, P = 0.059; ND, 2 h and 8 h time points not tested for JRFL.

FIG 5

Effect of pH and temperature on virus neutralization by V2i and V3 MAbs. (A) To measure the effect of pH on virus infectivity, BaL.ec1 pseudovirus was incubated for 1 h in medium adjusted to pH 4.2, 5.6, 6.6, or 7.4 prior to the addition of TZM.bl target cells. Virus infectivity was measured 48 h later based on β-galactosidase activity. (B) To compare BaL.ec1 neutralization by V2i MAb 2158 and control MAb 1418 at pH 6.6 versus pH 7.4, virus-MAb mixtures were incubated at the designated pH for 1 h or 5 h before adding the target cells. Means and standard errors from representative experiments (each in duplicate) are shown. (C) To evaluate the effect of higher temperature on virus neutralization, 5-fold serial dilutions of V2i and V3 MAbs were incubated with JRFL and BaL.ec1 at 37°C versus 38.5°C for 1 h before the addition of TZM-bl target cells. The irrelevant control MAb 1418 was used as a negative control. The cells were then cultured at 37°C for 48 h and infection measured based on β-galactosidase activity. Means and standard errors of two experiments are shown.

FIG 6

Time-dependent increase of neutralization against different viruses by V2i and V3 MAbs. Virus neutralization was measured after REJO (A), YU2 (B), SHIV C.1157ipEL-p (C), HIV-1 92TH023 (D), and CM244.OR (E) viruses were incubated with serially diluted MAbs for 1 h versus 24 h at 37°C prior to the addition of TZM-bl target cells. Means and standard errors from two to three experiments are shown. Statistical analyses were done on neutralization data reaching ≥50%. *, P < 0.001 for REJO, YU2, and SHIV and P < 0.05 for CM244.OR and HIV-1 92TH023.

FIG 7

Virus neutralization by V2p and V2q MAbs after prolonged virus-MAb incubation. Virus neutralization by V2p (CH58 and CH59) and V2q (PG9) MAbs was measured after 1 h versus 24 h of virus-MAb preincubation. REJO (A), YU2 (B), SHIV C.1157ipEL-p (C), 92TH023 (D), and CM244.OR (E) were incubated with MAbs for the designated period of time at 37°C prior to the addition of TZM-bl target cells. Means and standard errors from two to three experiments are shown. Statistical analyses were done as described in the legend to Fig. 6. *, P < 0.001 for REJO, YU2, SHIV (except P < 0.05 for PG9), HIV-1 92TH023, and CM244.OR.