Figure 2. IM immunization with nucleoside-modified trivalent mRNA-LNP protects against genital HSV-2 in mice.

BALB/c mice were immunized as in Figure 1 except one group received 2μg trivalent mRNA. Mice were challenged one month after the final immunization intravaginally with 5×10³ HSV-2 (275 LD₅₀). (a) Survival curves: P<0.0001 comparing PolyC RNA with each other group. P values were calculated by the log-rank test. (b) Weight loss of individual animals: P<0.0001 comparing PolyC RNA with the other three groups. (c) Genital disease: P<0.0001 comparing PolyC RNA with the other three groups. P values in (b-c) were calculated by two-way ANOVA followed by Tukey for significance. Sample size: (a-c), n=10 in PolyC RNA group for (a) and (c), n=5 for (b), n=10 in trivalent subunit protein, n=5 in 2μg trivalent mRNA, and n=10 in 10μg trivalent mRNA. Error bars in (c) represent SEM. P values in (d-e) are shown on the figures for the primary endpoint comparison between trivalent mRNA and trivalent protein or the comparison between 2μg and 10μg trivalent mRNA. Other P values are noted below. (d) Day 2 vaginal titers: comparing 10μg trivalent mRNA or trivalent subunit protein with PolyC RNA, P<0.0001; comparing 2μg trivalent mRNA with PolyC RNA, P=0.2500. Day 4 vaginal titers: comparing the number of animals with positive day 4 cultures in the 10μg trivalent mRNA, the 2μg trivalent mRNA or the trivalent subunit protein group with PolyC RNA, P<0.0001. Sample size: n=15 in PolyC RNA group, n=10 in trivalent subunit protein group, n=5 in the 2μg trivalent mRNA group and n=20 in the 10μg trivalent mRNA group. Dotted line indicates the limit of assay detection at 6.7 PFU/ml. (e) DRG HSV-2 DNA copy number at time of euthanasia of PolyC RNA group or on day 28 in the other groups; P<0.0001 comparing the number of animals with positive HSV-2 DNA copy number in the PolyC RNA with the 10μg trivalent mRNA or the trivalent subunit protein group; P=0.0050 comparing the number of animals with positive HSV-2 DNA copy in the PolyC RNA with the 2μg trivalent mRNA group. Other comparisons were not significant. Sample size: n=9 in the PolyC group, n=10 in the trivalent subunit protein group, n=5 in the 2μg trivalent mRNA group and n=10 in the 10μg trivalent mRNA group. P values for (d-e) were calculated by the two-tailed Fisher’s exact test.