Fig. 5: Infection, immunity, and protection of LNP-vRNA vaccine.

A Composition of R3-UpA vRNA-LNP vaccine. B Timeline of vaccination and challenge studies, and course of viremia in vaccinated rats. C Vaccinated rats were transiently depleted for CD8α + (n = 3) or CD4 (n = 3) T cells by antibody (Ab) or isotype control (n = 5) before challenge with RHV-rn1 to assess the role of T cell subsets in vaccine-induced immunity. D Serial analysis of serum ALT values in 12 vaccinated rats before and after RHV-rn1 challenge. Rats that developed chronic infection are shown in red-filled shapes. Two-tailed unpaired t-test and p-value for Pre-Challenge vs Post D14 (**,0.0047), Pre-Challenge vs Post-D21 (****, <0.0001), Post-D14 vs Post-D21, (*,0.0242) and Post-D21 vs Post-D28 (****, < 0.0001). Data points are presented as individual values and error bars at mean ± SEM. E Serial analysis of virus-specific T cells in PBMC of rats using IFN-γ ELISPOT assay. The frequencies of IFN-γ secreting T cells, shown as SFU, in ELISPOT assay in LILs stimulated with a pool of peptides representing the RHV T cell epitopes. The mean value of SFU counts in triplicate wells is shown. The number of animals whose PBMC samples were available for analysis is shown in parentheses; UpA 1st day28 (n = 21), UpA 2nd day7 (n = 6), UpA 2nd day35 (n = 21), Rn-1 day 7 (n = 20), and Rn-1 day21 (n = 20). Two-tailed unpaired t-test and p-value between UpA 2nd day 7 vs UpA 2nd day 35 (*, 0.0284); UpA-2nd day 35 vs Rn-1 day 7 (***, 0.0006); Rn-1 day 7 vs Rn-1 day 21 (*, 0.0327). F, G Serial analysis of the frequencies of NS5B₂₅₁₁- and E1₁₉₁-specific CD8 T cells in the PBMC of unvaccinated (grey) and vaccinated rats that cleared (green) or developed chronic the RHV-rn1 challenge infection (red). FC=folds change. Two-tailed unpaired t-test and p-values between pre-challenge and day 10 post for cleared (**, 0.0081); and between unvaccinated and chronic (**, 0.0046). In Fig. 5F, p-values for Pre-challenge, Day 10 post, and Day 35 post (cleared vs chronic) were ns (0.5562, 0.8372, and 0.1492, respectively). In Fig. 5G p-values for Pre-challenge, Day 10 post, and Day 35 post (cleared vs chronic) were ns (0.8372, 0.1740, and 0.1581, respectively). H Frequencies of cells producing IFN-γ in intracellular cytokine production assay (ICS) of liver infiltrating leukocytes isolated from vaccinated rats with cleared (n = 4, green) and chronic (n = 3, red) RHV-rn1 infection. For ICS, cells were stimulated for 5-h with pools of peptides representing RHV T cell epitopes at 10 µg/mL concentration. Data points are presented as individual values and error bars at mean ± SEM. P-values for % IFN-γ of CD8 and CD4 between cleared and chronic were ns (0.0665,0.8703, respectively). I Viremia titers on day 7 after the first (n = 14) and second (n = 12) RHV-rn-1 challenge infection. Two-tailed unpaired t-test was performed, and a significant p-value was shown as *(0.0371). Data points are presented as individual values and error bars at mean ± SD. J Protective immunity against reinfection in rats that cleared the primary RHV-rn1 challenge infection. Viremia after RHV-rn1 reinfection in IgG-isotype control (green, n = 7), anti-CD8α (blue, n = 4), and anti-CD4 (brown, n = 4) cell-depletion antibody-treated rats. Source data are provided as a Source Data file.