Live-attenuated pandemic H1N1 influenza vaccines expressing computationally optimized broadly reactive antigens (COBRAs) are immunogenic and protective in mice and ferrets.

Computationally optimized broadly reactive antigens (COBRAs) induce broad and protective immune responses across multiple viral vaccine platforms. However, their suitability for incorporation into live attenuated influenza vaccines (LAIVs) remains uncertain, as antigen modifications could potentially impact LAIV generation, replication, stability, or immunogenicity. In this study, COBRA hemagglutinin (HA) and neuraminidase (NA) antigens designated Y2 and N1I, respectively, of the influenza H1N1 subtype were reverse-engineered into A/Puerto Rico/8/1934 (PR8)-based LAIVs. The impact of HA stability and NA composition on LAIV properties was evaluated in cell culture, mice, and ferrets. COBRA LAIV yields were higher in MDCK cells compared to Vero cells, and a higher HA activation pH was associated with increased LAIV growth in cell culture. The COBRA LAIVs elicited broad antibody responses against pandemic H1N1 viruses and provided robust protection in both mice and ferrets. The standard COBRA LAIV, containing unmodified HA Y2 and NA N1I, had virus inactivation pH and HA activation pH values of 5.4 and 5.6, respectively. In contrast, a modified COBRA LAIV, containing an HA2-K153E mutation and NA from the vaccine strain A/Hawaii/70/2019 (HI19), had a virus inactivation pH of 5.3 and an elevated HA activation pH of 6.0. This modified LAIV had improved growth in cell culture and greater protection from challenge virus lung titers in elderly ferrets. These studies demonstrate the successful integration of COBRA antigen engineering into a LAIV platform. Furthermore, fine-tuning HA stability and NA composition appears to be a promising strategy to enhance LAIVs containing modifications to computationally optimized antigens.