Murine Antibody Responses to Cleaved Soluble HIV-1 Envelope Trimers Are Highly Restricted in Specificity

Abstract

Generating neutralizing antibodies (nAbs) is a major goal of many current HIV-1 vaccine efforts. To be of practical value, these nAbs must be both potent and cross-reactive in order to be capable of preventing the transmission of the highly diverse and generally neutralization resistant (Tier-2) HIV-1 strains that are in circulation. The HIV-1 envelope glycoprotein (Env) spike is the only target for nAbs. To explore whether Tier-2 nAbs can be induced by Env proteins, we immunized conventional mice with soluble BG505 SOSIP.664 trimers that mimic the native Env spike. Here, we report that it is extremely difficult for murine B cells to recognize the Env epitopes necessary for inducing Tier-2 nAbs. Thus, while trimer-immunized mice raised Env-binding IgG Abs and had high-quality T follicular helper (Tfh) cell and germinal center (GC) responses, they did not make BG505.T332N nAbs. Epitope mapping studies showed that Ab responses in mice were specific to areas near the base of the soluble trimer. These areas are not well shielded by glycans and likely are occluded on virions, which is consistent with the lack of BG505.T332N nAbs. These data inform immunogen design and suggest that it is useful to obscure nonneutralizing epitopes presented on the base of soluble Env trimers and that the glycan shield of well-formed HIV Env trimers is virtually impenetrable for murine B cell receptors (BCRs).

Importance: Human HIV vaccine efficacy trials have not generated meaningful neutralizing antibodies to circulating HIV strains. One possible hindrance has been the lack of immunogens that properly mimic the native conformation of the HIV envelope trimer protein. Here, we tested the first generation of soluble, native-like envelope trimer immunogens in a conventional mouse model. We attempted to generate neutralizing antibodies to neutralization-resistant circulating HIV strains. Various vaccine strategies failed to induce neutralizing antibodies to a neutralization-resistant HIV strain. Further analysis revealed that mouse antibodies targeted areas near the bottom of the soluble envelope trimers. These areas are not easily accessible on the HIV virion due to occlusion by the viral membrane and may have resulted from an absence of glycan shielding. Our results suggest that obscuring the bottom of soluble envelope trimers is a useful strategy to reduce antibody responses to epitopes that are not useful for virus neutralization.

FIG 1

BG505 SOSIP.664 trimers are immunogenic and elicit Tier-1 neutralizing antibodies in mice. (A) BALB/c mice were immunized at weeks 0, 4, and 16 with 10, 10, and 20 μg of BG505 SOSIP trimers per mouse, respectively, in 0.2 μg of AbISCO-100 per mouse via footpads. Mouse serum was collected before and after immunizations as indicated. (B) GC B cell frequencies within the CD19⁺ CD4⁻ CD8⁻ CD11c⁻ F4/80⁻ Gr-1⁻ B cell population in draining popliteal lymph nodes (PLNs) at week 18 (2 weeks after 3rd immunization) and week 0. (C) Fluorescence-activated cell sorter (FACS) plots of GC B cell frequencies gated on B cells at week 0 and week 18. Shown are frequencies of BG505 probe-specific GC B cells gated on Fas⁺ GL7⁺ CD19⁺ CD4⁻ CD8⁻ CD11c⁻ F4/80⁻ Gr-1⁻ B cells. (D) GC Tfh cell frequencies within the CD4⁺ CD44⁺ CD62L⁻ CD19⁻ CD4 T cell population in draining PLNs at week 18 (2 weeks after 3rd immunization) and week 0. (E) FACS plots of GC Tfh cell frequencies gated on activated CD4 T cells at week 0 and week 18. (F) Kinetic of BG505 gp140-specific IgG in mouse serum. (G) Tier-1 HIV-1, Tier-2 HIV-1, and control VSV pseudovirus neutralization titers were measured at week 18 using mouse serum IgG from BG505-immunized mice compared to control mice receiving adjuvant alone or immunization with an irrelevant antigen, KLH. Human CD4 IgG2 was used as a positive control for binding the Tier-1 and Tier-2 HIV-1. (H) Data from neutralization assays showing percent inhibition of pseudovirus infection of TZM-bl cells over a series of dilution factors. Dilution factors of purified mouse IgG were calculated to represent dilution factors of original serum IgG. (I) Tier-1 and Tier-2 neutralization titers were assayed using purified mouse IgG from various immunization strategies. Strategies shown include the use of different mouse strains, antigen dosing, and adjuvants and the use of drug treatments. Data shown are from immunization strategies marked with an asterisk in Tables S1 to S6 in the supplemental material. (J) Validation of V3 Fc competition ELISA. V3 Fc incubation with V3-specific MAb 14e reduces BG505 SOSIP.664 trimer binding titers by ∼5,000-fold. V3 Fc incubation with CD4 binding site-specific bnAb VRC01 shows negligible reduction in BG505 SOSIP.664 binding titers. (K) V3 Fc competition ELISA shows a 10-fold drop in BG505 SOSIP.664 trimer-specific IgG titers in mouse serum after incubation with V3 Fc.

FIG 2

Antigen delivery via osmotic pumps reduces the immunodominant response to the V3 loop. (A) 129/Sv mice were immunized at weeks 0, 8, and 19 with BG505 SOSIP.664 trimers in Iscomatrix adjuvant in osmotic pumps. Group 1 received 7-day slow-release pumps containing 50 μg BG505 SOSIP.664 trimers in 0.5 U of Iscomatrix , group 2 received 7-day slow-release pumps containing 50 μg BG505 SOSIP.664 trimers in 0.5 U of Iscomatrix plus a bolus injection of 20 μg BG505 SOSIP.664 trimers in 0.5 U of Iscomatrix , and group 3 received 14-day slow-release pumps containing 100 μg BG505 SOSIP trimers in 0.5 U of Iscomatrix. Mouse serum was collected before and after immunizations as indicated. (B) GC B cell frequencies within the CD19⁺ CD4⁻ CD8⁻ CD11c⁻ F4/80⁻ Gr-1⁻ B cell population in draining brachial lymph nodes (BLNs) at week 23 (4 weeks after 3rd immunization). (C) FACS plots of GC B cell frequencies gated on B cells at week 23 in BG505 and pump-immunized mice. GC B cell frequencies at week 24 in mice immunized with adjuvant alone are shown. (D) GC Tfh cell frequencies within the CD4⁺ CD44⁺ CD62L⁻ CD19⁻ CD4 T cell population in draining BLNs at week 23 (4 weeks after 3rd immunization). (E) FACS plots of GC Tfh cell frequencies gated on activated CD4 T cells at week 23 in BG505 SOSIP.664 trimer and pump-immunized mice. GC Tfh cell frequencies at week 24 in mice immunized with adjuvant alone. (F) Kinetic of BG505 SOSIP.664 trimer-specific IgG in mouse serum. (G) Kinetics of BG505 SOSIP.664 trimer binding IgG titers from mice that received 7-day pumps plus bolus immunization. Preincubation of serum with V3-Fc showed a 1.34-fold drop in BG505 gp140-specific IgG titers in mouse serum after incubation with V3 Fc. (H) Fold decrease in BG505 SOSIP.664 trimer binding IgG titers after incubation with V3-Fc. (I) Tier-1 HIV-1, Tier-2 HIV-1, and control VSV pseudovirus neutralization titers were measured at week 23 using mouse serum IgG from mice immunized with 7-day slow-release pumps plus bolus injection. Mouse serum IgG from a mouse receiving adjuvant only was used as a negative control. Human CD4 IgG2 was used as a positive control for binding Tier-1 and Tier-2 HIV-1.

FIG 3

Dissociation rate kinetics as measured by the biosensor binding kinetics assay (Octet). (A) Association and dissociation of bnAbs to BG505 SOSIP.664 trimers. (B) Association and dissociation of non-BG505 neutralizing antibodies to BG505 SOSIP.664 trimers. PGT151 is a BG505 neutralizing Ab used as a comparison. (C) Association and dissociation of 3BC315 at different concentrations. (D) Average off-rate constants of 3BC315 at different concentrations. (E) Association and dissociation of 3BC315 and 35O22 compared to those of other bnAbs. (F) Association and dissociation of purified IgG from mice receiving 7-day pumps. (G) Association and dissociation of purified IgG from mice receiving 14-day pumps. (H) Average off-rate constants of purified IgG from mice receiving immunization with pumps compared to average off rates of bnAbs.

FIG 4

Mouse serum IgG competes for binding to BG505 SOSIP.664 trimers with bnAbs specific to the base of Env trimers. (A) Validation of antibody competition assay. bnAbs and epitope specificities are indicated. Association of PGT128 (Association 2) is shown after no competition (red), competition with PGT125 (orange), or competition with PGT126 (green). Alignment of association of PGT128 with or without competition shows the percent drop in binding. (B) Antibody competition assay was performed using serum IgG from pump-immunized mice as competitors. A drop in association of 35O22 and 3BC315 in the presence of competing mouse serum IgG is shown. (C) Percent drop in binding of bnAbs to BG505 SOSIP.664 trimers in the presence of serum IgG from pump-immunized mice.

FIG 5

BnAbs 35O22 and 3BC315 compete with mouse IgG for binding to BG505 SOSIP.664 trimers. (A) Antibody competition assay was performed using 35O22 and 3BC315 as competitors. A drop in association of serum IgG from pump-immunized mice in the presence of 35O22, 3BC315, or 35O22 and 3BC315 is shown. (B) Percent drop in binding of serum IgG from pump-immunized mice to BG505 SOSIP.664 trimers in the presence of 35O22 and 3BC315.

FIG 6

Structural model of soluble BG505 SOSIP.664 trimers. Shown are the side view, top view, and bottom view of a soluble BG505 SOSIP.664 trimer (4TVP.pdb [Pancera14]) with glycans (aqua) surrounding the gp120 (tan) and gp41 (gray) domains of the trimer protein. Glycans were modeled using Glyprot (http://www.glycosciences.de/modeling/glyprot/) according to Table 1 and Table S2 of Guttman et al. (91).