The Matrix Gene Segment Destabilizes the Acid and Thermal Stability of the Hemagglutinin of Pandemic Live Attenuated Influenza Virus

The threat of future influenza pandemics and their potential for rapid spread, morbidity, andmortality has led to the develop-ment of pandemic vaccines. We generated seven reassortant pandemic live attenuated influenza vaccines (pLAIVs) with the hem-agglutinin (HA) and neuraminidase (NA) genes derived from animal influenza viruses on the backbone of the six internal pro-tein gene segments of the temperature sensitive, cold-adapted (ca) A/Ann Arbor/60 (H2N2) virus (AA/60 ca) of the licensed seasonal LAIV. The pLAIV viruses were moderately to highly restricted in replication in seronegative adults; we sought to deter-mine the biological basis for this restriction. Avian influenza viruses generally replicate at higher temperatures than human in-fluenza viruses and, although they shared the same backbone, the pLAIV viruses had a lower shutoff temperature than seasonal LAIV viruses, suggesting that the HA and NA influence the degree of temperature sensitivity. The pH of HA activation of highly pathogenic avian influenza viruses was greater than human and low-pathogenicity avian influenza viruses, as reported by others. However, pLAIV viruses had a consistently higher pH of HA activation and reduced HA thermostability compared to the corre-sponding wild-type parental viruses. From studies with single-gene reassortant viruses bearing one gene segment from the AA/60 ca virus in recombinant H5N1 or pH1N1 viruses, we found that the lower HA thermal stability and increased pH of HA activation were associated with the AA/60M gene. Together, the impaired HA acid and thermal stability and temperature sensi-tivity likely contributed to the restricted replication of the pLAIV viruses we observed in seronegative adults