Figure 4. DS-Cav1 immunization activates pneumovirus cross-reactive B cells.

HMPV pre-F probe-binding IgG⁺ B cells were assessed in 50 of 95 participants. Memory B cells were gated (mononuclear cells/singlets/live/CD19⁺/CD20⁺CD38⁻/IgD⁻/IgG⁺/CD27⁺) and frequencies of probe-binding populations were determined. (A) Representative plots from a single participant show probe-binding frequencies of HMPV and RSV cross-reactive (yellow) and HMPV-specific (green) B cells at weeks 0, 1, 2, and 44. (B) Longitudinal probe-binding frequencies of cross-reactive and HMPV-only B cells are shown. Data are presented as mean ± SEM. (C) Probe-binding frequencies in cross-reactive and HMPV-specific B cells were measured at week 0 and week 2. Significance was calculated using a Wilcoxon paired t-test (pairs with zero probe-binding events at W0 were excluded). ****p<0.0001; ns, not significant. (D) The correlation of RSV pre-F-binding with HMPV F-cross-reactive probe-binding frequencies at week 2 is shown. (E) Representative flow plots from one participant show cross-reactive (yellow) and HMPV-specific (green) B cells overlaid on total memory B cells (IgG⁺CD27⁺). Phenotypes were defined based on expression of CD21 and CD71. The relative frequencies of IgG⁺CD27⁺ (F) cross-reactive and (G) HMPV-specific B cells are shown for individuals who received one (n=31) or two (n=19) immunizations. Probe-binding frequencies were normalized to 100.