Single-dose vaccination of HD-Ad-FS protected hamsters against SARS-CoV-2 in the upper airway and lungs. (A) Schematic timeline of single-dose hamster experiment. Hamsters were immunized with a single-dose of HD-Ad-FS or HD-Ad-control at 5x109 vp via intranasal route. Three weeks after immunization, hamsters were intranasally challenged with SARS-CoV-2 at 1x105 TCID50. Animal samples were harvested at 4 and 14 dpi. (B, C) Body weight was monitored at the indicated days after challenging with SARS-CoV-2. For (C), the change of weight in male and female hamsters were analyzed separately. (D, E) RNA levels of SARS-CoV-2 from oropharyngeal swabs were determined with RT-qPCR. Swabs were collected at the indicated time points. For (E), the levels of viral RNA in male and female hamsters were analyzed separately. (F) RNA levels of SARS-CoV-2 in lungs were determined with RT-qPCR at 4 dpi. (G) The titers of infectious SARS-CoV-2 in lungs were determined with TCID50 assays at 4 dpi. (H) RNA levels of SARS-CoV-2 in spleens (left) and hearts (right) were measured with RT-qPCR at 4 dpi. (I) The titers of FS-specific IgG in sera were measured with ELISA at 4 dpi. The starting dilution factor was 1:1000. (J) Serum NAb activities against SARS-CoV-2 and variants were determined with neutralization assays at 14 dpi. Dots represent individual hamsters (4 dpi, n=6; 14 dpi, n=9 or 10). For (D-J), the horizontal dotted lines represent the LOD of the assays. For (B, C), statistical analyses were performed by two-way ANOVA; error bars represent mean ± s.e.m. For (D, E), statistical analyses were performed by two-way ANOVA; error bars represent geometric mean with geometric SD. For (F-J), statistical analyses were performed by Mann-Whitney test, two-tailed. Error bars represent geometric mean with geometric SD. For (C), the asterisk signs represent statistical analyses in single-FS hamsters, and the pound signs represent statistical analyses in single-control hamsters. */# p<0.05, **/## p<0.01, ***<0.001, ****/####p<0.0001, and ns, not significant. Data represent one independent animal experiment with indicated biological replicates.