Jen Gommerman @jengommerman@qoto.org

While mRNA vaccines have been highly effective over the past 2 years in combating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), waning of vaccine-induced antibody responses and lack of induction of respiratory tract immunity contribute to ongoing infection and transmission. However, intranasally (i.n.) administered vaccines may induce mucosal immunity at the site of respiratory virus infection and may thus boost protection. In this work, we present an i.n. administered SARS-CoV-2 self-amplifying RNA (saRNA) vaccine, delivered by a nanostructured lipid carrier (NLC), which induces both potent respiratory mucosal and systemic immune responses. Following prime-boost immunization in C57BL/6 mice, i.n. vaccination induces serum neutralizing antibody titers, bone marrow resident IgG-secreting cells, and robust systemic polyfunctional T cells, similar to intramuscular (i.m.) vaccination. The intranasal vaccine additionally induces key SARS-CoV-2-reactive lung-resident polyfunctional memory and lung-homing T cell populations. As a booster following i.m. administration, the i.n. vaccine also elicits robust mucosal and systemic immunity, exceeding an i.m. booster, durable for at least 4 months. The potent mucosal and systemic immunogenicity of this i.n. saRNA vaccine may be key for combating SARS-CoV-2 and other respiratory pathogens. ### Competing Interest Statement A.G. and E.A.V. declare no Competing Non-Financial Interests but the following Competing Financial Interests. A.G. and E.A.V. are co-inventors on PCT patent application PCT/US21/40388, Co-lyophilized RNA and Nanostructured Lipid Carrier, and related national filings, as well as U.S provisional patent application 63/345,345, Intranasal Administration of Thermostable RNA Vaccines, and 63/144,169, A thermostable, flexible RNA vaccine delivery platform for pandemic response. All other authors declare they have no competing interests.