The C3/465 glycan hole cluster in BG505 HIV-1 envelope is the major neutralizing target involved in preventing mucosal SHIV infection

Abstract

Stabilized HIV-1 envelope (Env) trimers elicit tier 2 autologous neutralizing antibody (nAb) responses in immunized animals. We previously demonstrated that BG505 SOSIP.664.T332N gp140 (BG505 SOSIP) immunization of rhesus macaques (RM) provided robust protection against autologous intra-vaginal simian-human immunodeficiency virus (SHIV) challenge that was predicted by high serum nAb titers. Here, we show that nAb in these protected RM targeted a glycan hole proximal to residue 465 in gp120 in all cases. nAb also targeted another glycan hole at residues 241/289 and an epitope in V1 at varying frequencies. Non-neutralizing antibodies directed at N611-shielded epitopes in gp41 were also present but were more prevalent in RM with low nAb titers. Longitudinal analysis demonstrated that nAb broadened in some RM during sequential immunization but remained focused in others, the latter being associated with increases in nAb titer. Thirty-eight monoclonal antibodies (mAbs) isolated from a protected RM with an exceptionally high serum neutralization titer bound to the trimer in ELISA, and four of the mAbs potently neutralized the BG505 Env pseudovirus (PV) and SHIV. The four neutralizing mAbs were clonally related and targeted the 465 glycan hole to varying degrees, mimicking the serum. The data demonstrate that the C3/465 glycan hole cluster was the dominant neutralization target in high titer protected RM, despite other co-circulating neutralizing and non-neutralizing specificities. The isolation of a neutralizing mAb family argues that clonotype expansion occurred during BG505 SOSIP immunization, leading to high titer, protective nAb and setting a desirable benchmark for HIV vaccines.

Fig 1. Mapping of serum neutralizing activity in high titer, protected RM using BG505 Env PV mutants.

Serum neutralizing activity on the day of challenge (week 84) was evaluated using the TZM-bl assay (A—E) and ID₅₀ titers were calculated using GraphPad prism. Neutralization activity for protected vs. infected RM against (A) BG505 Env PV (p = 0.0057), and BG505.SHIV produced in (B) 293T cells (p = 0.0120) and (C) rhesus CD4⁺ T cells (p = 0.0187) is shown. In each case, nAb ID₅₀ titer was higher in protected RM using the Mann-Whitney test (*p<0.05, **p<0.01). For (A) through (C), the horizontal bar represents the median nAb ID₅₀ titer for each group. Day of challenge serum neutralizing activity was also mapped using mutant BG505 Env PVs. Infectivity curves are depicted in (D) with the key indicating the color for each BG505 mutant PV as follows. The parental BG505 Env PV with T332N is shown with black closed symbols, while the BG505 Env PV with T332 is shown with black open symbols. Mutant PVs that had a reduction or increase in ID₅₀ titer of 0.5log10 or greater compared to the parental BG505 are represented in color and symbol as listed in the key. The gray infectivity curves represent mutant PVs that did not reach the pre-defined threshold of a 0.5log10 fold increase or decrease in ID₅₀ compared to the parental BG505 Env. The reciprocal of the serum dilution is plotted on the x axis on a log10 scale and the percent of viral infectivity is plotted on the y axis relative to the virus only control at 100%. Each serum-PV combination was run in duplicate, and each assay was repeated independently at least twice. The error bars represent the SEM for each data point. (E) the relative change in ID₅₀ titer for each BG505 mutant PV compared to the parental BG505 PV is shown. The second row represents whether a log fold decrease or increase in ID₅₀ titer is expected. The third row identifies each mutant. The ID₅₀ titer against the parental BG505 containing T332N is shown in the third column and is the comparator for all mutants. The number of RM that targeted a particular region/mutant is shown in the last row. Mutants with a substantial decrease (red) or increase (green) in ID₅₀ are highlighted, and correspond to the neutralization curves shown in (D).

Fig 2. Visualization of antibody binding to BG505 SOSIP.

nsEMPEM of BG505 SOSIP in complex with Fabs derived from serum IgG at week 82 is shown. Eight RM were selected for analysis based on their vaccine group, neutralization ID₅₀ titer, and challenge outcome, as indicated in (A—C). The code for each RM and the neutralization ID₅₀ titer against BG505 Env PV is shown. Previously defined epitopes on BG505 SOSIP are coded by color in the key. Dashed ovals indicate antibody specificities detected by serum nAb mapping but not nsEMPEM; white text indicates antibody specificities that were detected by serum nAb mapping and nsEMPEM.

Fig 3. Longitudinal serum nAb mapping.

Neutralization activity against the BG505 parental and mutant PVs was assessed using serum collected from five RM at two to four weeks after the second, third, and fourth SOSIP immunizations (weeks 26, 42/44, or 84) using the TZM-bl assay. Neutralization in serum from two RM (RAb16 and RPc16) was mapped using a panel of seven mutants at all timepoints because the nAb ID₅₀ titers against the parental BG505 Env PV exceeded 1:100. The remaining RM had titers against the parental BG505 PV below 1:100 at week 26 and were only mapped against N611A at this timepoint. The RM is indicated in the first row, and the SOSIP immunizations, 2, 3, and 4, are indicated in the second row. The nAb titer for the parent BG505 PV is shown in the third row, with the log fold reduction or increase for each mutant PV in the rows below. Each mutant is indicated in the third column, and whether there is an expected decrease or increase in ID₅₀ in the first column. Red shading indicates a log fold reduction of 0.5log10 of greater; green shading indicates a log fold increase of 05.log10 or greater. N/A indicates not analyzed due to low titer.

Fig 4. Isolation and characterization of mAbs derived from RUp16.

Cryopreserved PBMC from RUp16 collected at week wk44 (four weeks after the third SOSIP immunization) were single-cell sorted as follows: size, live/dead, CD14^-, CD3^-, CD20⁺, IgG⁺, BG505 gp120⁺ or SOSIP⁺. Representative flow cytometry plots showing gates for (A) BG505 SOSIP-specific B cells and (B) BG505 gp120-specific B cells along with naïve RM controls, are shown. Variable regions were PCR amplified, sequenced, and germlines were assigned by analyzing the nucleotide sequences using IgBlast (https://www.ncbi.nlm.nih.gov/igblast/). The proportion of VH and VL kappa or lambda families are shown for (C) SOSIP-sorted and (D) gp120-sorted B cells with the gene families indicated by colors shown in the adjacent keys. CIRCOS diagrams were generated to illustrate VH and VK/L germline gene usage and pairing for (E), 18 SOSIP-sorted and 20 gp120-sorted mAbs from RUp16 that bound to BG505 SOSIP in ELISA. The VH and VL kappa or lambda germlines are shown on the left and right halves of the circle, respectively. The ribbons extending from VH to VL are color-coded based on VH family with kappa or lambda shown as shades of gray, as indicated in the key. The width of the band is proportional to the number of mAbs that were derived from the same VH and VL combination. (F) ELISA positive SOSIP-sorted (left) or gp120-sorted (right) mAbs were tested for neutralization of the BG505 Env PV in the TZM-bl assay. Infectivity is plotted against the concentration of mAb in μg/ml. The four neutralizing mAbs, as well as negative control anti-influenza HA mAb EM4C04 and positive control HIV bnAb VRC01, are shown in color as indicated in the key. Non-neutralizing mAbs are shown in shades of dark (SOSIP-sorted) or light (gp120-sorted) gray. The four neutralizing mAbs were also tested for neutralization of BG505.SHIV produced in (G) 293T cells and (H) RM CD4⁺ T cells in the TZM-bl assay. Each assay was run with duplicate wells and repeated independently at least twice. The error for each data point is indicated by the SEM. (I) The IC₅₀ titers for each mAb against the BG505 Env PV and the BG505.SHIVs are shown in μg/ml. 25 μg/ml was the highest concentration tested.

Fig 5. Mapping of mAb neutralizing activity.

Neutralizing activity against the parental BG505 and mutant PVs was evaluated using the TZM-bl assay in (A—F). The mAb is indicated above each graph, which displays percent infectivity plotted against mAb concentration in μg/ml. mAbs VRC01 (E) and EM4C04 (F) were used as positive and negative controls, respectively. (G) The log10 fold increase or decrease in IC₅₀ titer of each mutant PV relative to the parental BG505 PV is shown. An increase in IC₅₀ of greater than 0.5log10 is highlighted red.