mRNA-based VP8* nanoparticle vaccines against rotavirus are highly immunogenic in rodents

Fig. 1. mRNA encoding P2-VP8* P[8] induced robust levels of both humoral and cellular immune responses in mice. Female BALB/c mice (n = 6/group) were vaccinated IM with 6 µg of alum-adjuvanted P2-VP8* P[8] protein or 4 µg of P2-VP8* P[8] mRNA vaccine on day 0 and day 21. Buffer (0.9% NaCl) vaccinated animals served as negative controls. Sera were collected on day 21, day 42 and day 56 and splenocytes were isolated on day 56. a P2-VP8* P[8]-specific binding antibodies are displayed as endpoint titers for IgG1 and IgG2a in serum upon one (day 21) or two vaccinations (day 42 and day 56). b P[8] genotype (Wa strain)-specific rotavirus-neutralizing antibody titers in serum upon two vaccinations (day 42 and day 56). c Multifunctional IFN-γ/TNF-positive CD4+ and CD8+ T cells were analyzed in splenocytes stimulated with VP8*-specific peptides followed by intracellular cytokine staining and detection by flow cytometry. Each diamond symbol represents an individual animal and bars depict geometric means with geometric standard deviation (SD) in (a, b) and means with SD in panel c. Dotted lines indicate the lower limit of quantification (LLOQ). Values below the LLOQ (<50 for (a) and <10 for (b))…

Fig. 2. Proteins translated from an mRNA encoding LS-P2-VP8* P[8] were secreted and self-assembled into 60-mer nanoparticles. a Schematic drawings of mRNA encoded P2-VP8* and LS-P2-VP8* proteins. b HEK 293 T cells were transfected with two independent mRNA batches each of P2-VP8* P[8] or LS-P2-VP8* P[8]. VP8* expression in cell lysates and supernatants were analyzed via western blotting 48 h post transfection. Tubulin was employed as loading control. c Cell supernatants of HeLa cells transfected with LS-P2-VP8* P[8] or LS only mRNA were subjected to density gradient ultracentrifugation using an isopycnic iodixanol density gradient 48 h post transfection. After ultracentrifugation, equivalent volumes of five density gradient fractions with increasing iodixanol concentrations (F1 to F5) or unpurified supernatants (SN) were analyzed for VP8* and/or LS expression via western blotting. A supernatant from HeLa cells transfected with water served as negative control (Neg) and 100 ng of P2-VP8* P[8] protein loaded to the gel was used as positive control (Protein). d Negative stain EM image of LS-P2-VP8* P[8] nanoparticles derived from HeLa cell culture supernatants. White is high density.…

Fig. 3. mRNA encoding LS-P2-VP8* P[8] induced high and long-lasting levels of humoral immunity in guinea pigs. Female Dunkin Hartley guinea pigs (n = 8/group) were vaccinated IM with 20 µg of alum-adjuvanted P2-VP8* P[8] protein or 25 µg each of P2-VP8* P[8] or LS-P2-VP8* P[8] mRNA vaccines on day 0 and day 21. Buffer (0.9% NaCl) vaccinated animals served as negative controls. Sera were collected on different days as indicated. a P2-VP8* P[8]-specific binding antibodies are displayed as endpoint titers for total IgG in serum at the indicated time points. Each dot represents the geometric mean with geometric standard deviation (SD) of a group at a given time. Syringe symbols indicate the vaccination time points. b P[8] genotype (Wa strain), P[4] genotype (DS-1 strain) or P[6] genotype (1076 strain)-specific rotavirus-neutralizing antibody titers in serum upon two vaccinations (day 56 and day 196). Each diamond symbol represents an individual animal and bars depict geometric means with geometric SD. Dotted lines indicate the lower limit of quantification (LLOQ). Values below the LLOQ (<50 for (a) and <10 for (b)) were set to half of the respective LLOQ. Individual readouts and time points…

Fig. 4. Trivalent LS-P2-VP8* mRNA vaccine encoding genotypes P[8], P[6] and P[4] of VP8* elicited high titers of binding antibodies against the P2-VP8* P[8] protein in guinea pigs. Female Dunkin Hartley guinea pigs (n = 7/group) were vaccinated IM either three times on day 0, day 21, and day 42 or twice on day 21 and day 42 with different doses of monovalent and trivalent alum-adjuvanted P2-VP8* protein vaccines or monovalent and trivalent P2-VP8* or LS-P2-VP8* mRNA vaccines as indicated. The genotype(s) of the encoded VP8* employed for each group, the doses used as well as the number of immunizations administered (indicated by the number of syringe symbols) are displayed beneath each group. Trivalent vaccines are labeled P[8,6,4]. Buffer (0.9% NaCl) vaccinated animals served as negative controls. P2-VP8* P[8]-specific binding antibodies are displayed as endpoint titers for total IgG in serum collected on day 21 (a), day 42 (b), or day 70 (c). Each diamond symbol represents an individual animal and bars depict geometric means with geometric SD. Dotted lines indicate the lower limit of quantification (LLOQ). Individual time points were statistically analyzed using an ordinary one-way…

Fig. 5. Trivalent LS-P2-VP8* mRNA vaccine encoding genotypes P[8], P[6] and P[4] of VP8* elicited high titers of functional antibodies against P[8], P[6] and P[4] rotavirus genotypes in guinea pigs. Female Dunkin Hartley guinea pigs (n = 7/group) were vaccinated IM either three times on day 0, day 21, and day 42 or twice on day 21 and day 42 with different doses of monovalent and trivalent alum-adjuvanted P2-VP8* protein vaccines or monovalent and trivalent P2-VP8* or LS-P2-VP8* mRNA vaccines as indicated. The genotype(s) of the encoded VP8* employed for each group, the doses used as well as the number of immunizations administered (indicated by the number of syringe symbols) are displayed beneath each group. Trivalent vaccines are labeled P[8,6,4]. Buffer (0.9% NaCl) vaccinated animals served as negative controls. Sera were collected on day 70. a P[8] genotype (Wa strain), (b) P[6] genotype (1076 strain) or c P[4] genotype (DS-1 strain)-specific rotavirus-neutralizing antibody titers in serum. Each diamond symbol represents an individual animal and bars depict geometric means with geometric SD. Dotted lines indicate the lower limit of quantification (LLOQ). Values below the LLOQ (<10)…