Objective:To construct a VP8-mRNA vaccine using human rotavirus spike protein VP8 domain as the immunogen and analyze its immunogenicity in mice.Methods:The VP8-mRNA sequence was designed, optimized, and synthesized. The VP8 gene of rotavirus G1P[8] type was used to construct the plasmid pUC57-VP8-Kan-SapⅠ, which was then sequenced. The plasmid confirmed by sequencing was subjected to large-scale amplification and extraction, followed by linearization, in vitro transcription, and capping. The purified capped products were encapsulated with lipid nanoparticles using a microfluidic control apparatus. The encapsulated VP8-mRNA vaccine was administered intramuscularly to mice at 10, 15, and 20 μg. Serum samples were collected for antibody detection by ELISA. Cellular immune responses were detected by flow cytometry and ELISPOT. Statistical analysis was performed using one-way or two-way analysis of variance and Tukey-Kramer test. Results:The encapsulated VP8-mRNA vaccine was rounded and spherical, with a particle size of about 100 nm, a polymer dispersion index of 0.088, and an encapsulation rate of 92.3%. Two doses of VP8-mRNA vaccine immunization could induce a good immune response in mice. The level of IgG antibody induced after immunization in the 15 μg group was comparable to that of the 20 μg group, and there was no statistical difference ( P>0.05), but the antibody levels in the two groups were significantly higher than that in the 10 μg group ( P<0.000 1). VP8-mRNA vaccine could induce neutralizing antibodies against rotavirus G1 and G9 types. The highest level of neutralizing antibodies against rotavirus type G1 was observed in the 15 μg group, which was significantly higher than that in the 10 μg group ( P<0.05). All immunization groups exhibited good neutralizing ability against rotavirus G9 type. The results of ELISPOT showed that lymphocytes from mice in each vaccine group were able to secrete IFN-γ when stimulated with VP8 peptide. Flow cytometry showed that the proportions of CD8 + T cell subsets in the vaccine groups were higher than that in the control group. Conclusion:The VP8-mRNA vaccine has good immunogenicity in mice and can induce good humoral and T-cell immune responses.

Objective:To construct a VP8-mRNA vaccine using human rotavirus spike protein VP8 domain as the immunogen and analyze its immunogenicity in mice.Methods:The VP8-mRNA sequence was designed, optimized, and synthesized. The VP8 gene of rotavirus G1P[8] type was used to construct the plasmid pUC57-VP8-Kan-SapⅠ, which was then sequenced. The plasmid confirmed by sequencing was subjected to large-scale amplification and extraction, followed by linearization, in vitro transcription, and capping. The purified capped products were encapsulated with lipid nanoparticles using a microfluidic control apparatus. The encapsulated VP8-mRNA vaccine was administered intramuscularly to mice at 10, 15, and 20 μg. Serum samples were collected for antibody detection by ELISA. Cellular immune responses were detected by flow cytometry and ELISPOT. Statistical analysis was performed using one-way or two-way analysis of variance and Tukey-Kramer test. Results:The encapsulated VP8-mRNA vaccine was rounded and spherical, with a particle size of about 100 nm, a polymer dispersion index of 0.088, and an encapsulation rate of 92.3%. Two doses of VP8-mRNA vaccine immunization could induce a good immune response in mice. The level of IgG antibody induced after immunization in the 15 μg group was comparable to that of the 20 μg group, and there was no statistical difference ( P>0.05), but the antibody levels in the two groups were significantly higher than that in the 10 μg group ( P<0.000 1). VP8-mRNA vaccine could induce neutralizing antibodies against rotavirus G1 and G9 types. The highest level of neutralizing antibodies against rotavirus type G1 was observed in the 15 μg group, which was significantly higher than that in the 10 μg group ( P<0.05). All immunization groups exhibited good neutralizing ability against rotavirus G9 type. The results of ELISPOT showed that lymphocytes from mice in each vaccine group were able to secrete IFN-γ when stimulated with VP8 peptide. Flow cytometry showed that the proportions of CD8 + T cell subsets in the vaccine groups were higher than that in the control group. Conclusion:The VP8-mRNA vaccine has good immunogenicity in mice and can induce good humoral and T-cell immune responses.