Assessment of critical bioprocess parameters for broadly cross-reactive chimeric hemagglutinin influenza virus vaccines.

Antigenic drift of seasonal influenza viruses and unpredictable outbreaks with new influenza virus subtypes are a global concern that requires the development of broadly protective vaccines. We previously showed that chimeric hemagglutinin (cHA) virus vaccines are an effective strategy to elicit cross-protective immune responses to the conserved hemagglutinin (HA) stalk domain. However, the critical bioprocess parameters and vaccine quality attributes for cHA inactivated split constructs remain to be defined. Here, we developed an egg-based bioprocess for inactivated virus vaccine production with special focus on group 2 cHA viruses (cH15/3HK14N2HK14). The egg inoculum dose, incubation temperature and time, the high-speed centrifugation step, reversing the order of virus inactivation and splitting, and incorporating a sterile filtration step were thoroughly investigated. We also demonstrated that the selected virus inactivation conditions result in complete virus inactivation and defined an optimal detergent concentration in the final vaccine preparation. Using the optimal conditions, at least a 2-fold increase in HA yield was achieved for the cH15/3HK14N2HK14 virus in comparison with the unoptimized bioprocess. Vaccines produced using regular bioprocess conditions (virus inactivation first) or reversing the inactivation and splitting steps were equally immunogenic in mice, elicited antibody responses against the HA and neuraminidase (NA), and showed to be antigenically stable in different storage conditions. This study will support the development of cHA virus vaccines in a phase I clinical trial.