Fig. 7: Preclinical evaluation of RV3 preF (OnlyEcto).

A−D Mice (n = 5 or 8) were immunized with 15 µg adjuvanted preF or postF protein or formulation buffer (Mock) at week 0 and 4. Two weeks later, serum samples were taken and preF (A) and postF (B) binding antibody titers were measured by ELISA or virus neutralization titers (VNT) in preF, postF or Mock pooled serum samples were determined with an VNA (virus neutralization assay) on Vero cells (C) or on differentiated human airway epithelial cell cultures (D) using the RV3 JS strain equipped with a GFP reporter gene. E, F Mice (n = 5 or 8) were immunized with a dose range of 1.5, 5 or 15 µg non-adjuvanted OnlyEcto or formulation buffer (Mock) at week 0 and 4. Two weeks later serum samples were taken and preF binding antibody titers were measured by ELISA (E) or RV3 neutralizing antibody titers by RV3-GFP VNA on Vero cells were determined (F). G, H Mice (n = 3 or 6) were intranasally exposed to RV3 or formulation buffer as control and immunized 19 weeks later with OnlyEcto or formulation buffer. Six weeks later, serum samples were taken and preF binding antibody titers were measured by ELISA (G) or RV3 neutralizing antibody titers by RV3-GFP VNA on Vero cells were determined (H). Analysis of variance (ANOVA; 2-sided t-test) was used for statistical comparisons between groups. Tukey-Kramer (A, B and G, H) or Dunnett adjustments. E, F for multiple comparisons were applied. I−L Cotton rats (n = 5 or n = 6) were immunized with OnlyEcto with or without adjuvant or postF with adjuvant or formulation buffer (Mock) at week 0 and 4. Three weeks after the final immunization, cotton rats were challenged intranasally with RV3. Four days later viral loads in nose and lung tissue were determined (I, J). RV3 neutralizing antibody titers by plaque reduction neutralization test (PRNT) were determined in pre-challenge sera (K) and the correlation between nose viral loads and VNA titers were calculated with a spearman correlation analysis (L). Mean responses per group are indicated with horizontal lines. Statistical comparisons were performed across dose levels using a Tobit model (I−L). P < 0.05 were considered statistically significant.