Heterologous saRNA Prime, DNA Dual-Antigen Boost SARS-CoV-2 Vaccination Elicits Robust Cellular Immunogenicity and Cross-Variant Neutralizing Antibodies.

We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (AAHI-SC2) delivered by a nanostructured lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression and an N antigen modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment and increase MHC class I and II stimulation potential. The S sequence in the AAHI-SC2 vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to confer protease resistance. CD-1 mice received vaccination by homologous and heterologous prime > boost combinations. Humoral responses to S were the highest with any regimen that included the AAHI-SC2 vaccine, and IgG bound to wild type and Delta (B.1.617.2) variant S1 at similar levels. An AAHI-SC2 prime followed by an AdS+N boost particularly enhanced CD4+ and CD8+ T-cell responses to both wild type and Delta S peptides relative to all other vaccine regimens. Sera from mice receiving AAHI-SC2 homologous or heterologous vaccination were found to be highly neutralizing for all pseudovirus strains tested: Wuhan, Beta, Delta, and Omicron strains. The findings here, taken in consideration with the availability of both vaccines in thermostable formulations, support the testing of heterologous vaccination by an AAHI-SC2 > AdS+N regimen in animal models of SARS-CoV-2 infection to assess its potential to provide increased protection against emerging SARS-CoV-2 variants particularly in regions of the world where the need for cold-chain storage has limited the distribution of other vaccines.

Intranasal plus subcutaneous prime vaccination with a dual antigen COVID-19 vaccine elicits T-cell and antibody responses in mice.

We have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity-including mucosal immunity-against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.