Long-term Immunity of a Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Irradiated by Gamma Rays in Mice.

COVID-19 vaccines effectively prevent symptomatic infection and severe disease, including hospitalization and death. However, unequal vaccine distribution during the pandemic, especially in low- and middle-income countries, has led to the emergence of vaccine-resistant strains. This underscores the need for alternative, safe, and thermostable vaccine platforms, such as dissolved microneedle array patches (MAP) delivering a subunit vaccine, which eliminate the need for cold chain and trained healthcare personnel. This study demonstrates that the SARS-CoV-2 S1 monomer with RS09, a TLR-4 agonist peptide, serves as an optimal protein subunit immunogen. This combination stimulates a stronger S1-specific immune response, resulting in binding to the membrane-bound spike on the cell surface and ACE2-binding inhibition, compared to the monomer S1 alone or trimer S1, regardless of RS09. MAP delivery of the rS1RS09 subunit vaccine elicited higher and longer-lasting immunity compared to conventional intramuscular injection. S1-specific IgG levels remained significantly elevated for up to 70 weeks post-administration. Additionally, different doses of 5, 15, and 45 g of MAP vaccines induced robust and sustained Th2-prevalent immune responses, suggesting a dose-sparing effect and inducing significantly high neutralizing antibodies against the Wuhan, Delta, and Omicron variants at 15 and 45 g dose. Moreover, gamma irradiation as a terminal sterilization method did not significantly affect immunogenicity, with irradiated vaccines maintaining comparable efficacy to non-irradiated ones. The stability of MAP vaccines was evaluated after long-term storage at room temperature and refrigeration for 19 months, showing minimal protein degradation. Further, after an additional one-month of storage at elevated temperature (42°C), rS1RS09 in both non-irradiated and irradiated MAP degraded less than 3%, while the liquid subunit vaccine degraded over 23%. Overall, these results indicate that gamma irradiation sterilized MAP-rS1RS09 vaccines maintain stability during extended storage without refrigeration, supporting their potential for mass production and widespread use in global vaccination efforts.