Optimization of A(H1N1)pdm09 vaccine seed viruses: The source of PB1 and HA vRNA as a major determinant for antigen yield.

Vaccination prevents and reduces the severity of influenza virus infections. Continuous evolution of influenza hemagglutinin (HA) and neuraminidase (NA) supports the virus to evade pre-existing immunity, which demands vaccines to be reformulated every year. Incorporation of polymerase basic protein 1 (PB1) viral RNA (vRNA) of the same origin of HA and NA vRNA has been observed in previous pandemic viruses and occasionally reported for influenza A vaccine prototype strains of prior seasons. At this point, it remains to be explored whether this phenomenon translates into an improved growth phenotype. In this work, we showed that the HA vRNA of A(H1N1)pdm09 is generally incorporated with the PB1 vRNA of the same origin, establishing the beneficial effect of the presence of PB1 and the pattern of the PB1-HA co-incorporation in the A(H1N1)pdm09 model. We further investigated the putative interplay between PB1 and antigenic proteins regarding the vRNA composition of the progeny and observed that vRNA segregation does not appear to be mainly determined by protein-protein interactions; while vRNA-vRNA interactions can be suggested as the main driving force. Our data also indicate an increase in the hemagglutination capacity and neuraminidase activity due to incorporation of PB1, HA and NA from A(H1N1)pdm09, in comparison with the recombinant virus incorporating only HA and NA from A(H1N1)pdm09 - which have the potential to improve current limitations regarding antigenicity and immunogenicity of influenza vaccines. Further knowledge of the complex vRNA-vRNA interaction network between PB1 and HA will additionally contribute to improve current vaccine formulation, and to gradually optimize the production of A(H1N1)pdm09 reverse genetics vaccine seed virus towards a higher cost-effectiveness.