Fig. 1: Norovirus VP1 mRNA-LNP vaccines elicit potent and sustained humoral immune responses.

Expi293F cells were transfected with mRNA-encoding (A) norovirus GI.1 or (B) GII.4 VP1 and analyzed for protein expression by western blot as described in Methods. The supernatant was collected, clarified, ultrafiltered, and visualized via NSEM. Representative micrographs are shown from cells transfected with (C) GI.1 or (D) GII.4, with n = 5 micrograph images per group. E Experimental schematic of the study. Balb/c mice (n = 10 per group) were immunized on days 0 and 28 with 10 μg of mRNA-LNP vaccine encoding Norwalk1968/GI.1 or CapeTown2012/GII.4 VP1, or empty LNP as control at an equivalent dose. Sera were collected at various time points (4, 8, 14, 22, 26, and 34 weeks post-prime) and screened against (F) Norwalk1968/GI.1 and (G) CapeTown2012/GII.4 VLPs to assess their ability to block VLP binding to its carbohydrate ligand in a surrogate neutralization assay. The data was analyzed using GraphPad Prism, and the dilution at which 50% of VLP-ligand binding was blocked (ID50) was calculated. Sera that did not block at least 50% of VLP-ligand binding were assigned a titer of 0.5X the lower limit detection (ID50 = 25) and marked below this limit (dashed line). The nAb titer is presented as 50% inhibitory dilution (ID50) values (Mean ± SD), n = 10 per group (5 male and 5 female). The nAb titers were compared with the values from Day 56 for each strain, ∗p < 0.05 F:(D56 vs D28 p = 0.0135) G:(D56 vs D28 p = <0.0001, D56 vs D150 p = <0.0001, D56 vs D180 p = <0.0001, d56 vs d240 p = <0.0001), one-way ANOVA, Tukey’s post hoc tests. A red square symbol indicates nAb titers in a serum sample collected on Day 14 from a patient infected with the GII.4 norovirus strain. nAb titer values for empty LNP are not shown because they were below the level of detection. Schematic for Fig. 1E created with Biorender.