Heterologous prime–boost reshapes antibody responses enhancing levels of site II–specific antibodies.

(A) Heterologous prime–boost study groups. Three mouse cohorts were immunized with either 1x RSVF (“prime-only”), 3x RSVF (“homologous boost”), or 1x RSVF followed by two boosts with NRM (“heterologous boost”). (B) Antibody titers directed against prefusion RSVF. Mice receiving homologous boosting immunizations show slightly higher RSVF-specific serum titers compared to the prime-only cohort, whereas heterologous boosting yielded statistically comparable titers to the prime-only group. The difference between the homologous and heterologous boost cohorts was statistically significant. (C) Site II–specific titers measured by ELISA showed that the heterologous boost significantly increases site II–specific titers compared to both prime and homologous boost groups. Albeit not statistically significant (p = 0.06), mice receiving a homologous boost had lower levels of site II–specific antibodies compared to prime-only group. (D) SPR competition assay with motavizumab on a prefusion RSVF-coated sensor chip. Sera from indicated groups were diluted 1:100, and RSVF binding responses were quantified. Site II was then blocked with motavizumab, and the remaining serum response was quantified. The heterologous (“heterol.”) boost induced a significantly higher fraction of site II–directed antibodies that competed with motavizumab for RSVF binding, as compared to both prime-only and homologous (“homol.”) boost groups. (E) Quantification of site II–specific responses in a competition ELISA. Binding was measured against prefusion RSVF, and the AUC was calculated in the presence of NRM competitor, normalized to the AUC in the presence of RSVN as a control competitor. Compared to the prime-only group, the homologous boost resulted in significantly lower site II–specific serum titers, confirming the trend observed in (C). The heterologous boost increased the fraction of site II–targeting antibodies within the pool of prefusion RSVF-specific antibodies compared to both control groups. Data presented are from at least two independent experiments, with each sample assayed in duplicates. Statistical comparisons were calculated using two-tailed Mann-Whitney U tests. *p p p p S1 Data. AUC, area under the curve; RSVF, RSV fusion protein; RSVN, RSV nucleoprotein; SPR, surface plasmon resonance.