Figure 3. ID immunization with nucleoside-modified trivalent mRNA-LNP protects against genital HSV-2 in guinea pigs.

(a-f) Guinea pigs were immunized three times monthly IM with 10μg PolyC RNA-LNP; ID with 20μg each trivalent nucleoside-modified mRNA-LNP; IM with 10μg each trivalent subunit protein-CpG/alum; or left unimmunized (n=10/group). One month after the final immunization, all immunized animals were infected intravaginally with 5×10⁵ HSV-2 (50 LD₅₀), while unimmunized animals remained uninfected. (a) Survival curves: P<0.0001 comparing PolyC RNA with each other group. P values calculated by the log-rank test. (b) The mean weight loss each day post-infection of animals in the trivalent mRNA, trivalent subunit protein, or uninfected group was compared with PolyC RNA controls; P<0.0001. (c) Cumulative mean genital disease score per guinea pig is shown for each day post-infection. No animal developed genital lesions in the trivalent mRNA, trivalent subunit protein or uninfected group. Comparing trivalent mRNA, trivalent subunit protein or uninfected with PolyC RNA, P<0.0001. The P value in (b) and (c) was calculated by two-way ANOVA followed by Tukey for significance. (d-e) P values shown on the figures are for the primary endpoint comparison between trivalent mRNA and trivalent protein. Other P values are noted below. (d) Day 2 and day 4 vaginal titers: Day 2, P=0.0013 and day 4 P=0.0010 comparing PolyC RNA with trivalent mRNA. Dotted line indicates the limit of assay detection at 3.3 PFU/ml. (e) Day 2 and day 4 vaginal DNA copy number by qPCR. P value in (d) and (e) was calculated by the two-tailed Mann-Whitney, except the day 4 comparison between trivalent mRNA and trivalent subunit proteins was performed by the two-tailed Fisher’s exact test. (f) Vaginal shedding of HSV-2 DNA over 21 days from day 28–50 post-infection. One animal in the trivalent subunit protein group had replication competent virus recovered on a day HSV-2 DNA shedding was detected (marked in green). Comparing days of shedding in the trivalent mRNA and trivalent subunit protein group, P=0.0052, calculated by the two-tailed Fisher’s exact test. (g) HSV-2 DNA copy number/10⁶ copies of GAPDH in spinal cord and DRG at the end of the experiment; n=2 in PolyC RNA and n=10 in the 3 other groups. (h) Table summarizing key results of guinea pig studies.