Immunogenicity and quantification of site II–specific antibody responses.

(A) Immunization scheme. Balb/c mice were immunized three times on days 0, 21, and 42, and blood was drawn 14 days after each vaccination. (B) Serum antibody titers elicited by FFLM and NRM at different time points measured by ELISA against the respective immunogen. NRM shows significantly increased immunogenicity at days 14, 35, and 56 relative to FFLM. (C) SPR competition assay with motavizumab. Day 56 sera of mice immunized with RSVF, FFLM, or NRM were diluted 1:100, and SPR RU were measured on sensor chip surfaces containing the respective immunogen. Motavizumab binding sites were then blocked by saturating amounts of motavizumab, and the residual serum response was measured to calculate the serum fraction competed by motavizumab binding. Mice immunized with FFLM or NRM show significantly higher levels of serum antibodies that are competed by motavizumab binding. (D) Site II–specific serum titers at day 56 from mice immunized with RSVF, FFLM, and NRM, measured by ELISA against site II peptide. Three immunizations with prefusion RSVF elicited low levels of site II–specific antibodies, whereas FFLM and NRM vaccinations yielded significantly higher peptide-specific serum titers. Data shown are derived from at least two independent experiments, with each sample assayed in duplicate. Statistical comparisons were calculated using two-tailed Mann-Whitney U tests. **p < 0.01, ***p < 0.0001, ****p < 0.0001. Data are available in S1 Data. RSVF, respiratory syncytial virus fusion protein; RU, response units; SPR, surface plasmon resonance.