INTRODUCTION. The low physicochemical stability of mRNA and the dependence of current mRNA vaccines on the successful encapsulation into lipid nanoparticles and adherence to mandatory cold chain requirements limit their storage and transportation. A promising approach is the lyophilization of nonencapsulated mRNA with its subsequent delivery by jet injection. However, the preservation of its structure and functional activity during long-term storage has been insufficiently studied. AIM. To evaluate the structural and functional stability of lyophilized nonencapsulated mRNA during long-term storage and its immunogenicity as a component of a trivalent mRNA vaccine against seasonal influenza administered to mice by jet injection. MATERIALS AND METHODS. mRNA encoding green fluorescent protein (GFP) was lyophilized with various cryoprotectants and stored at −20, 4, and 20 °C. Its functional activity was determined by GFP expression after transfection of HEK293 cells, and its integrity was analyzed by microchip electrophoresis. The immunogenicity of the lyophilized and nonlyophilized mRNA-Vector-Flu vaccine was evaluated in female inbred BALB/c mice after two jet immunizations by enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HAI) assay, virus neutralization assay, and interferon gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay. RESULTS. Based on the results of a preliminary screening, a 10% trehalose formulation was selected for further studies because it preserved the functional activity of GFP mRNA at the level closest to that of the control preparation. The lyophilized GFP mRNA retained its functional activity after 12 months of storage at all tested temperatures; no statistically significant differences were found compared with the mRNA synthesized immediately before use ( p >0.05). The major mRNA fraction was preserved, although minor fractions of partially degraded mRNA were detected after 6–12 months, particularly at 20 °C. After 3 months of storage of the vaccine at 4 °C, the geometric mean antibody titers against hemagglutinin subtype 1 (H1), hemagglutinin subtype 3 (H3), and influenza B hemagglutinin (HB) were 1:382,000, 1:628,000, and 1:273,000, respectively, compared with 1:710,000, 1:628,000, and 1:328,000 in the nonlyophilized vaccine group. HAI titers were 1:37–1:80, virus-neutralizing antibody titers were 1:50–1:90; and the numbers of IFN-γ-secreting cells were 440 and 500 spot-forming cells (SFC) per 1×10 ⁶ splenocytes. No significant between-group differences were identified. CONCLUSIONS. Lyophilization with 10% trehalose preserved the functional activity of nonencapsulated mRNA for 12 months despite signs of partial degradation at 20 °C. After 3 months of storage at 4 °C, the trivalent vaccine retained its ability to induce humoral and T-cell immune responses in mice. These findings support the potential of combining lyophilization and jet injection for the development of stable mRNA vaccines.

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