Fusion peptide priming reduces immune responses to HIV-1 envelope trimer base

Abstract

Soluble "SOSIP"-stabilized envelope (Env) trimers are promising HIV-vaccine immunogens. However, they induce high-titer responses against the glycan-free trimer base, which is occluded on native virions. To delineate the effect on base responses of priming with immunogens targeting the fusion peptide (FP) site of vulnerability, here, we quantify the prevalence of trimer-base antibody responses in 49 non-human primates immunized with various SOSIP-stabilized Env trimers and FP-carrier conjugates. Trimer-base responses account for ∼90% of the overall trimer response in animals immunized with trimer only, ∼70% in animals immunized with a cocktail of SOSIP trimer and FP conjugate, and ∼30% in animals primed with FP conjugates before trimer immunization. Notably, neutralization breadth in FP-conjugate-primed animals correlates inversely with trimer-base responses. Our data provide methods to quantify the prevalence of trimer-base responses and reveal that FP-conjugate priming, either alone or as part of a cocktail, can reduce the trimer-base response and improve the neutralization outcome.

Keywords: HIV vaccine; SOSIP; fusion peptide; immune response; immunization regimen; immunogen cocktail; nanoparticle immunogen; neutralization; prefusion-stabilized trimer; trimer base.

Figure 1.. Immunization regimens of 49 NHPs analyzed for plasma anti-trimer base responses; for all regimens, NHP plasma was analyzed 2 weeks after the second trimer immunization (indicated with a red arrow)

(A) Three groups of NHPs were immunized twice with CH505 DS-SOSIP immunogens as indicated. The CH505 DS-SOSIP immunogens were FP-degly3, with three glycans (N230, N241, and N611) removed around the fusion peptide (FP); FP-degly4, with four glycans (N88, N230, N241, and N611) removed around the FP; and CD4bs-degly4, with four glycans (N197, N276, N462, and N362) removed around the CD4-binding site. (B) One group of five NHPs was immunized twice with BG505 DS-SOSIP. (C) Two groups of eight animals each were primed with FP8-KLH + BG505 DS-SOSIP or FP8-rTTHc nanoparticles + BG505 DS-SOSIP. (D) One group of five animals was immunized with long intervals, primed with FP8-KLH at weeks 0, 4, and 20, followed by an FP7-KLH immunization at week 32, an FP6-KLH immunization at week 44, and two BG505 DS-SOSIP immunizations at weeks 56 and 64. (E) One group of five animals was immunized with short intervals, primed with FP8-KLH at weeks 0, 4, and 8, followed by an FP7-KLH immunization at week 12, an FP6-KLH immunization at week 20, and two BG505 DS-SOSIP immunizations at weeks 24 and 28. See also Figure S1.

Figure 2.. Anti-base Fabs showed base-specific ELISA binding and do not block mAbs targeting the FP, V3 glycan, CD4bs, or V1V2 region

(A) RM19R, RM20A2, and RM19B1 IgG showed reduced binding to BG505 DS-SOSIP base KO, which has glycans added at N502 and N660 of the base region. (B–E) Competition ELISA (OD₄₅₀) for the binding of various epitope-specific mAbs to BG505 DS-SOSIP in the absence (black bars) or presence of base-binding Fabs: RM19R (pink), RM20A2 (blue), RM19B1 (red), or RM20G (green), at concentrations as indicated. (B) FP-specific mAbs DF1W-a.01, DFPH-a.01, 0PV-c.01, vFP16.02, and VRC34.01. (C) CD4-binding-site-specific mAbs VRC01 and N6. (D) V1V2- specific mAb PGT145. (E) V3 glycan-specific mAbs PGT128, 10–1074, and 3074. See also Figure S2.

Figure 3.. Anti-base Fabs block anti-BG505 DS-SOSIP responses in NHP immunized with trimer

(A–D) ELISA for the binding of NHP A13V009 plasma to BG505 DS-SOSIP competing with base-binding Fabs at concentrations as indicated, starting at a dilution of 1:100: (A) RM19R, (B) RM20A2, (C) RM19B1, and (D) RM20G. (E) Summary of the competition ELISA as a percentage of the BG505 DS-SOSIP plasma response blocked by base-binding Fabs. (F) Raw OD values taken at 450 nm for the base-binding Fab RM19R at 2 μg/mL, 1 μg/mL, and 0.4 μg/mL in a competition ELISA with RM19R IgG.

Figure 4.. Plasma binding to BG505 DS-SOSIP trimer was mostly blocked by RM19R Fab for NHPs primed with a SOSIP trimer but mostly maintained for NHPs primed with FP

(A–D) Geometric mean ELISA responses (OD₄₅₀) with 95% confidence interval for each group are plotted against plasma dilution factors in the presence (purple) or absence (gray) of RM19R Fab. (A) 18 NHPs primed with CH505 DS-SOSIP trimers that had three or four glycans removed around the FP (FP-degly3 and FP-degly4), or four glycans removed around the CD4 binding site (CD4bs-degly4). (B) 5 NHPs primed with BG505 DS-SOSIP trimers. (C) 16 NHPs primed with a FP + BG505 DS-SOSIP trimer cocktail. (D) 10 NHPs primed with FP8-KLH followed by sequential immunization with FP8–7-6-KLH and two BG505 DS-SOSIP trimers. (E) RM19R mAb as a control. See also Figure S3.

Figure 5.. Immunization regimens with FP in the prime elicited substantial responses targeting regions outside the base of the trimer

(A) Comparison of the total ELISA response to BG505 DS-SOSIP between all NHPs with trimer prime, cocktail prime, or FP prime, CH505 DS-SOSIP versus BG505 DS-SOSIP primed groups, FP8-KLH + BG505 DS-SOSIP cocktail versus FP8-rTTHc np + BG505 DS-SOSIP cocktail primed groups, and FP long interval versus FP short interval primed groups revealed no significant difference among various groups. The ELISA OD₄₅₀ values for total BG505 DS-SOSIP binding at a dilution of 1:500 plasma were plotted for individual animals. (B) Comparison of anti-base Fab competition ELISA response between all NHPs with trimer prime, cocktail prime, and FP only prime groups, CH505 DS-SOSIP versus BG505 DS-SOSIP primed groups, FP8-KLH + BG505 DS-SOSIP cocktail versus FP8-rTTHc np + BG505 DS-SOSIP cocktail primed groups, and FP long interval versus FP short interval primed groups for the percentage of plasma response targeting the base region of the BG505 DS SOSIP trimer. The percentage of BG505 DS-SOSIP responses blocked was calculated by taking the difference between OD₄₅₀ values without and with RM19R Fab at a plasma dilution of 1:500 divided by the OD₄₅₀ values at a dilution of 1:500 without RM19R Fab blocking and multiplied by 100. Statistical analysis was performed with a two-tailed Mann-Whitney nonparametric test to assess p values for means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. Correlation analysis was performed using a two-tailed Pearson correlation coefficiency test. See also Figures S3–S7.

Figure 6.. Anti-base responses for the 10 FP-primed NHPs correlate inversely with the neutralization breadth and the long-interval group showed greater autologous neutralization titers than the short-interval group

(A) Correlation between percentages of anti-base responses based on OD₄₅₀ values at a dilution of 1:500 and neutralization breadth defined as the number of wild-type FP-sensitive HIV-1 viruses neutralized out of a 10-virus panel. (B–D) Neutralization breadth (B) and ID₅₀ titers on BG505 (C) and BG505 Δ611 (D) for the long-interval (black circles) and short-interval (hollow circles) FP-primed NHPs. Statistical analysis was carried out with a two-tailed Mann-Whitney nonparametric test to assess p values for means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. Correlation analysis was performed using a two-tailed Pearson correlation coefficiency test. See also Figure S6.

Figure 7.. Schematic showing that priming with FP, a conserved subregion of the SOSIP trimer, provided a means to reduce the induction of trimer-base-specific antibodies in NHPs

Priming with SOSIP trimers with base-region exposed elicited dominant trimer-base responses (90% of total response, antibodies in gray), and cocktail priming with trimer/FP reduced the anti-base responses (70% of total response), and elicited more antibodies targeting non-base region on the trimer (antibodies in green). Priming with FP elicited anti-FP specific antibodies, including those recognizing FP in trimer context (antibodies in red) and those that can only recognize FP in linear conformation (antibodies in black), and trimer boost after FP prime elicited least anti-base antibodies (30% of total responses) and most non-base region antibodies. Anti-FP specific antibodies are included in the anti-trimer non-base region antibodies. Elicitation of FP⁺trimer⁺ B cells in NHPs was characterized previously (Cheng et al., 2020).