Fig. 2: Characteristics, immunogenicity, and efficacy of F protein variants with multiple proline substitutions in a mouse model.

a Locations of identified beneficial proline substitutions from proline scanning of the L0–8 loops. The RSV prefusion F structure was generated based on PDB ID: 4JHW. b PreF (left) and postF (right) expression levels of variants with combined proline substitutions. At 72 h post-transfection, preF concentration in the cell culture supernatant was assayed via sandwich ELISA using prefusion-specific AM14 as coating mAbs and DS-cav2, to plot a standard curve. The binding activity of the cell culture supernatant to postfusion-specific 4D7 was determined using ELISA. Data are presented as mean values +/- SEM from three independent experiments. c Representative of gel-filtration chromatograms of F1-2P-based variants with additional proline substitutions. d Representative of gel-filtration chromatograms of F1-2P-based variants with additional proline substitutions. For c and d Gel-filtration profile for each protein, representative of at least two experiments, was assessed on a Superose 6 10/300 GL SEC column. e–h Groups of BALB/c mice (n = 9) were immunized twice with placebo or F variants adjuvanted with Alum plus CpG1826 at a three-week interval. Serum samples collected at five weeks post initial vaccination were assessed for neutralizing antibody titers against RSV Long strain (e) and RSV BA9 strain (f) by live virus microneutralization assays. At five weeks post initial vaccination, the mice were challenged with 1 × 10⁵ PFU of RSV A (long strain) administered via the i.n. route. Lung samples obtained at 5 dpi were subjected to virus titration by PFU assays (g) and RT-PCR (h). i New groups of BALB/c mice (n = 8) were immunized with placebo, preF7P, or preF6P that was adjuvanted with Alum plus CpG1826, at weeks 0 and 3. At week 5, the mice were challenged with 1 × 10⁵ PFU of the RSV BA9 strain. Lung samples obtained at 5 dpi were subjected to virus titration by RT-PCR. j, k BALB/c mice (n = 10 per group) received two immunizations of placebo, DS-cav1, preF7P, or preF6P at either 2 μg or 12 μg doses, adjuvanted with Alum plus CpG1826 at a three-week interval. Serum neutralizing antibody titers against RSV Long strain (j) and RSV BA9 strain (k) were measured at five weeks post-initial vaccination by live virus microneutralization assays. For e–k, data are presented as mean values +/- SEM. P-values were analyzed with one-way ANOVA and Tukey’s multiple comparison test (n.s. P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). The dashed line indicates the limit of detection. Source data are provided as a Source Data file.