Dual-Route H5N1 Vaccination Induces Systemic and Mucosal Immunity in Murine and Bovine Models

Since its discovery in U.S. dairy cattle in early 2024, the highly pathogenic H5N1 avian influenza (clade 2.3.4.4b) has spread widely among herds, causing major economic losses. This zoonotic event emphasizes the urgent need for H5 vaccines that elicit strong, durable, cross-reactive immune responses in cattle, especially young calves. To address this, we immunized mice and cattle with a centralized consensus H5 vaccine, designed to localize to the central node of the human H5 phylogenetic tree. The vaccine was delivered using serotype-switched adenoviral vectors in a prime-boost regimen, combined with intramuscular and intranasal coadministration to target systemic and mucosal immunity and elicit strong humoral and cellular immune responses. This approach strategically integrates multiple innovative features: centralized consensus immunogens, mucosal targeting, and vector serotype switching that are aimed at maximizing immune protection against H5N1 viruses. Our results show that vaccination elicited strong humoral and cellular immunity in both mice and calves. In challenge experiments, vaccinated mice were fully protected against lethal infection with multiple divergent H5N1 strains, including the 2024 bovine isolate (A/bovine/Ohio/B24OSU-439/2024). Given that vaccine induced immunity was consistent across species, these results support the translatability of the mouse model findings to cattle. Overall, our findings represent a promising approach for immunizing cattle and other key livestock against HPAI H5N1, mitigating agricultural losses, and reducing the risk of zoonotic transmission. Significance StatementH5N1 influenza A virus is a serious pathogen recently detected in many mammals, including cattle. It transmits sporadically to humans and causes major economic losses in dairy and poultry industries, raising global concern. No H5N1 vaccines are currently licensed for cattle. To address this gap, we tested a centralized consensus H5 (H5CC) vaccine, previously effective in mice and swine. Using multiple Adenovirus vectors, we delivered H5CC intramuscularly and intranasally to one-week-old calves. The vaccines induced strong humoral and cellular immune responses, essential for preventing infection and limiting transmission. These findings highlight the vaccines potential to reduce agricultural losses and remove cattle as a newly established reservoir for zoonotic spread, providing a promising strategy for mitigating pandemic risk.