Cotton rats (N = 10) were immunized twice with 25 μg mF mRNA/LNP, mDS-Cav1 mRNA/LNP, Luciferase mRNA/LNP, LNP only (no RNA), FI-RSV (lot 100), FI-RSV generated using similar methods to lot 100 (FI-RSV (new)) or were left unvaccinated. With the exception of one unvaccinated group, all groups were challenged 4 weeks following the second immunization with RSV A2. Five days following the challenge, the animals were sacrificed. Lungs from each animal were trisected and processed for virus quantification (Supplementary Fig. 5b, c), pathology (a), and cytokine mRNA analysis (b). a Lung pathology following RSV challenge. Lung tissue from each animal was embedded in paraffin, sectioned, stained with hematoxylin and eosin, and given a pathology score from 0 (no pathology) to 4 (severe pathology) by a blinded pathologist. Sections were scored for peribronchiolitis (PB), perivasculitis (PV), interstitial pneumonia (IP), and alveolitis (A). Mean pathology score with standard error is shown. Raw histopathology scores for each animal are shown in Supplementary Table 1. Lung pathology scores were assessed using a two-sided exact permutation test. The mF and mDS-Cav1 mRNA vaccines resulted in significantly lower levels of pathology relative to both FI-RSV controls (for alveolitis, p = 0.0032 for mF versus FI-RSV (new) and 0.0358 for mF versus FI-RSV lot 100; p = 0.0151 for mDS-Cav1 versus FI-RSV (new) and 0.1016 for mDS-Cav1 versus FI-RSV lot 100), and the pathology scores for these two groups are not significantly different from the luciferase mRNA/LNP, LNP alone, unvaccinated and unchallenged negative control groups. b Cytokine gene expression after vaccination and RSV challenge. mRNA was isolated from the lungs of each cotton rat and IL-4, IL-13, IL-5, IFN-γ, and IL-2 mRNA levels were determined by rtPCR. The relative level of each mRNA was determined by normalizing to levels of the housekeeping gene β-actin and plotted for each animal. The geometric mean and 95% confidence interval for the β-actin normalized relative mRNA expression units for each of the five cytokines is shown. Differences between the groups were determined by two-sided t-test using the error term from a one-way ANOVA. Significant differences from unvaccinated, challenged animals are shown on the graph, with p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, and p < 0.0001 = ****. Relative to the animals in the two FI-RSV groups, animals immunized with mF or mDS-Cav1 had lower mRNA expression of IFN-γ (for both mF and mDS-Cav1, p < 0.0001 for both FI-RSV (new) and FI-RSV Lot 100), lower expression of IL-2 (for mF, p < 0.0001 for FI-RSV (new) and p < 0.0001 for FI-RSV (lot100); for mDS-Cav1, p = 0.0006 for FI-RSV (new) and p < 0.0001 for FI-RSV (lot100)); lower expression of IL-13 (for mF, p = 0.0008 versus FI-RSV (new) and 0.0375 for FI-RSV (lot100); for mDS-Cav1 p = 0.0236 versus FI-RSV (new) and p = 0.3625 (no difference) for FI-RSV (lot100)); and lower expression of IL-5 (p < = 0.0001 comparing either mF or mDS-Cav1 with either FI-RSV (new) or FI-RSV (lot100). IL-4 expression was significantly decreased for mF only when compared to FI-RSV (new) (p = 0.0118), while mDS-Cav1 showed a trend toward significance (p = 0.1045). The overall pattern of cytokine gene expression in the lungs of mF and mDS-Cav1 mRNA/LNP immunized cotton rats is most similar to the Group 8 unvaccinated and unchallenged animals, which reflects the complete protection from RSV challenge observed in these animals (Supplementary Fig. 5b, c).