A prefusion-stabilized RSV F subunit vaccine elicits B cell responses with greater breadth and potency than a postfusion F vaccine

Abstract

There is currently no licensed vaccine for respiratory syncytial virus (RSV). Here, we assess the effect of RSV fusion protein (F) conformation on B cell responses in a post hoc comparison of samples from the DS-Cav1 [prefusion (pre-F)] and MEDI7510 [postfusion (post-F)] vaccine clinical trials. We compared the magnitude and quality of the serological and B cell responses across time points and vaccines. We measured RSV A and B neutralization, F-binding immunoglobulin G titers, and competition assays at week 0 (before vaccination) and week 4 (after vaccination) to evaluate antibody specificity and potency. To compare B cell specificity and activation, we used pre-F and post-F probes in tandem with a 17-color immunophenotyping flow cytometry panel at week 0 (before vaccination) and week 1 (after vaccination). Our data demonstrate that both DS-Cav1 and MEDI7510 vaccination robustly elicit F-specific antibodies and B cells, but DS-Cav1 elicited antibodies that more potently neutralized both RSV A and B. The superior potency was mediated by antibodies that bind antigenic sites on the apex of pre-F that are not present on post-F. In the memory (CD27⁺) B cell compartment, vaccination with DS-Cav1 or MEDI7510 elicited B cells with different epitope specificities. B cells preferentially binding the pre-F probe were activated in DS-Cav1-vaccinated participants but not in MEDI7510-vaccinated participants. Our findings emphasize the importance of using pre-F as an immunogen in humans because of its deterministic role in eliciting highly potent neutralizing antibodies and memory B cells.

Trial registration: ClinicalTrials.gov NCT02508194 NCT02289820 NCT03049488.

Figure 1.. Pre-F vaccination elicits a greater magnitude of pre-F-exclusive and highly neutralizing antibodies than post-F vaccination.

(A to C) Conformation-specific neutralization capacity of serum obtained at week 0 (W0) and week 4 (W4) after vaccination with DS-Cav1 (pre-F, N = 30) or MEDI7510 (post-F, N = 52) was measured for (A) RSV subtype A2 without post-F competition or (B) with post-F competition, and (C) subtype B. In (B), excess post-F was used to adsorb post-F-exclusive and dual-binding antibodies to assess the neutralization capacity of pre-F exclusive antibodies in serum. (D to F) Fold-change in neutralization capacity at W4 after vaccination with either DS-Cav1 or MEDI7510 was calculated for RSV subtype A2 (D) without or (E) with post-F competition, and (F) subtype B. Bars represent mean ± standard deviation (SD) of log₂ data. Dotted lines indicate the limit of detection (LOD) for each assay. For all graphs, asterisks denote the P-value, with ****P < 0.0001, ***P = 0.0001 to 0.001, **P = 0.001 to 0.01, *P = 0.01 to 0.05, and ns = not significant as determined using Brown-Forsythe and Welch ANOVA tests (A to C), Kruskal-Wallis tests (D and E), or Mann-Whitney tests (F). Red circles denote participants who received DS-Cav1, blue squares denote participants who received MEDI7510. Geometric mean W4/W0 fold-change is shown in green text.

Figure 2.. Pre-F vaccination elicits pre-F-exclusive binding antibody and higher neutralization potency.

(A) W0 and W4 pre-F binding IgG endpoint titers are shown for serum samples from participants after vaccination with DS-Cav1 (pre-F, N = 30) or MEDI7510 (post-F, N = 52). (B) Pre-F binding IgG endpoint titers are shown as in (A), except excess post-F was used to adsorb post-F-exclusive and dual-binding antibodies from serum to leave pre-F-exclusive binding antibodies. W0 and W4 (C) post-F binding IgG endpoint titers are shown. (D) The fold-change ratio of post-F binding to neutralization is shown for each participant. Fold-change ratio = (W4/W0 fold-change, post-F IgG binding)/(W4/W0 fold-change, RSV A neutralization). A ratio below 1 indicates the increase in neutralization capacity exceeded the increase in binding capacity, demonstrating the elicitation of highly potent neutralizing antibodies. The gray area represents the historical range of this ratio observed in previous clinical trials of post-F subunit vaccines (fold-change ratio = 5 to 8). (E to G) Concentrations of (E) pre-F-binding D25-competing antibodies (DCA), (F) post-F-binding palivizumab-competing antibodies (PCA), and (G) post-F-binding 131–2A-competing antibodies (1CA) in serum are shown for samples collected before (W0) or 4 weeks after (W4) vaccination with DS-Cav1 or MEDI7510. Bars represent mean ± SD of log₁₀ data. Dotted lines indicate the lower limit of quantitation (LLOQ) for (E to G), with undetectable values arbitrarily set to ½ LLOQ. Fold-change and statistics were calculated using values above the LLOQ. For all graphs, asterisks denote the P-value, with ****P < 0.0001, ***P = 0.0001 to 0.001, **P = 0.001 to 0.01, *P = 0.01 to 0.05, and ns = not significant as determined using Brown-Forsythe and Welch ANOVA tests (A to C, G), Mann-Whitney tests (D), or Kruskal-Wallis tests (E and F). Red circles denote participants who received DS-Cav1, blue squares denote participants who received MEDI7510. Geometric mean W4/W0 fold-change is shown in green text.

Figure 3.. Pre-F vaccination elicits pre-F-preferring and dual-binding memory B cells.

Representative pre-F and post-F probe-binding profiles from a single participant vaccinated with DS-Cav1 (pre-F, N = 30) or MEDI7510 (post-F, N = 30) at W0 and W1 are shown. Fluorescently labeled pre-F or post-F probes were used to separate class-switched memory B cells into three compartments (pre-F-preferring, dual-binding, or post-F-preferring). IgG⁺ (A) representative binding profile and (B) quantification of probe-binding B cells are shown. IgA⁺ (C) representative binding profile and (D) quantification of probe-binding B cells are shown. For all graphs, asterisks denote the P-value, with ****P < 0.0001, ***P = 0.0001 to 0.001, **P = 0.001 to 0.01, *P = 0.01 to 0.05, and ns = not significant as determined using Brown-Forsythe and Welch ANOVA tests (B) or Kruskal-Wallis tests (D). Red circles denote participants who received DS-Cav1, blue squares denote participants who received MEDI7510.

Figure 4.. Activation phenotypes of memory B cells following pre-F or post-F vaccination parallels F probe-binding profiles.

Representative pre- and post-F probe-binding profiles are shown for a single participant vaccinated with DS-Cav1 (pre-F, N = 30) or MEDI7510 (post-F, N = 30) at W0 and W1. CD27⁺ B cells were delineated based on expression of surface markers CD27 and CD21 as activated (CD21⁺CD71⁺, CD21⁻CD71⁻, CD21⁻CD71⁺) or resting (CD21⁺CD71⁻). F probe-binding B cells were overlaid for each compartment. IgG⁺ (A) representative activation profiles and (B) activation phenotype by F probe-binding specificity are shown. IgA⁺ (C) representative activation profiles and (D) activation phenotype by F probe-binding specificity are shown.