Cotton rats (N = 6) were immunized twice with 25 μg mRNA/LNP vaccine or DS-Cav1 formulated with Adju-Phos®. a Sera collected 2 weeks following the second immunization were assayed for binding to the prefusion DS-Cav1 protein (white circle) or postfusion F protein (white triangle) by ELISA. Endpoint titers are shown for each animal and the center bar indicates the geometric mean and error bars show the 95% confidence interval for each group. The dotted line indicates the limit of detection for the assay. Binding titers to prefusion versus postfusion F protein were compared for each group by two-sided unpaired t-test using GraphPad Prism software. Cotton rats immunized with mDS-Cav1, sDS-Cav1, or DS-Cav1 protein had significantly higher binding to the DS-Cav1 prefusion F protein (p = 0.01, 0.04, <0.0001, respectively). b Sera from each animal was assayed for the ability to neutralize RSV Long infection in vitro. The 50% neutralizing titer (NT50) was defined as the reciprocal of the serum dilution required to neutralize 50% of input virus as determined by four-parameter sigmoidal curve fit. Measurements for each animal are indicated (black circle). The center bar indicates the geometric mean titers and error bars show the 95% confidence interval. The limit of detection titer of 4 is indicated by a dashed line. Neutralizing titers for each immunized group were compared to the control group given RSV A2 intranasally by two-sided unpaired t-test using GraphPad Prism software and significant differences are shown on the graph. Immunization with mRNA vaccines or DS-Cav1 protein led to significantly higher neutralizing antibody titers than intranasal RSV A2 (sDS-Cav1, p = 0.04; mDS-Cav1 p = 0.0093; sF, p = 0.04; mF, p = 0.02; DS-Cav1 protein, p = 0.04). No other significant differences between vaccinated groups were identified. c Animals were challenged with RSV A2 4 weeks after the second immunization. Nose and lung tissue were collected 4 days following challenge and assayed for the presence of RSV by plaque assay. Lung titers (∆) and nose titers (•) are shown for each animal. Geometric mean titer with 95% CI is shown for each group. The assay limit of detection (LOD) is 100 for lung titers and 40 for nose titers; each is indicated on the graph with a dotted line. Animals with no RSV infection measured were plotted at one-half the LOD. Because the viral titers in the lungs of the unvaccinated, RSV-challenged animals were lower than expected, qPCR to measure RSV RNA levels was conducted on the animals in this study. The qPCR data confirmed that the animals received an effective RSV challenge and the mRNA vaccines reduced RSV titer in the lung and nose, comparable to the RSV A2 group (Supplementary Fig. 4).