Egg‐ or cell culture‐derived hemagglutinin mutations impair virus stability and antigen content of inactivated influenza vaccines

Abstract

Egg-derived viruses are the only available seed material for influenza vaccine production. Vaccine manufacturing is done in embryonated chicken eggs, MDCK or Vero cells. In order to contribute to efficient production of influenza vaccines, we investigate whether the quality of inactivated vaccines is influenced by the propagation substrate. We demonstrate that H3N2 egg-derived seed viruses (A/Brisbane/10/07, IVR147, and A/Uruguay/716/07) triggered the hemagglutinin (HA) conformational change under less acidic conditions (0.2-0.6 pH units) than antigenically similar primary isolates. This phenotype was associated with HA1 (A138S, L194P) and HA2 (D160N) substitutions, and strongly related to decreased virus stability towards acidic pH and elevated temperature. The subsequent propagation of H3N2 and H1N1 egg-derived seed viruses in MDCK and Vero cells induced HA2 N50K (H1N1) and D160E (H3N2) mutations, improving virus growth in cell culture but further impairing virus stability. The prevention of the loss or recovery of stability was possible by cultivation at acidified conditions. Viruses carrying less stable HAs are more sensitive for HA conformational change during concentration, purification and storage. This results in decreased detectable HA antigen content - the main potency marker for inactivated influenza vaccines. Thus, virus stability can be a useful marker for predicting the manufacturing scope of seed viruses.

Influenza Virus M2 Protein Ion Channel Activity Helps To Maintain Pandemic 2009 H1N1 Virus Hemagglutinin Fusion Competence during Transport to the Cell Surface

“…The reduced stability of (H1N1)pdm09 HA likely makes it more susceptible to pH or other environmental stresses. Several groups have shown that the stability of HA varies greatly among different virus strains that have adapted to different environmental conditions and hosts (21,(58)(59)(60)(61)(62)(63)(64)(65)(66) (58). Pathogenic H7 HA proteins can vary up to 0.6 pH unit in their pH of fusion activation (9).…”

“…H3, H5, and H7 HA proteins with a lysine-to-isoleucine substitution at position 58 (K58I; H3 numbering) in HA2 have been shown to decrease the pH for membrane fusion by 0.6 to 0.7 pH unit (12,(64)(65)(66). In addition, HA mutants with less stability in the presence of acid were rapidly selected when viruses passaged in mammalian cells were subsequently passaged in embryonated chicken eggs (62,63). An H5 HA mutation with increased acid stability rendered an H5N1 virus more efficient in airborne transmission in ferrets (68,69).…”

Влияние Конформационной Стабильности Гемагглютинина Вируса Гриппа На Качество Инактивированных Вакцин H5N1

“…Ранее нами показано, что мутации в HA сезонных штаммов вируса гриппа человека, возникающие при адаптации вирусов, приводят к повышению значения порога рН-активации HA, а это снижает стабильность вируса и тем самым обусловливает низкое содержа-ние HA-антигена в инактивированных вирусных пре-паратах [4]. На основании этих данных мы предполо-жили, что высокий порог рН-активации HA (5.6-6.0), характерный для высокопатогенных вирусов гриппа птиц H5N1 [5,6], и есть причина низкой стабильности нативного НА и качества получаемых вакцин.…”

Influenza vaccines manufacturing in continuous cell lines: problems and solutions

“…The next important goal is to prevent the emergence of HA mutations associated with increased pH of HA fusion during the vaccine production. We have demonstrated that the propagation of viruses at acidified conditions in Vero cell line helps to preserve the original HA primary structure and the virus stability [33]. Another possibility to prevent the emergence of adaptation mutations that increase the pH of HA fusion is to cultivate the virus in the presence of Amphotericin B promoting fusion of viral and cellular membranes [68].…”

“…In order to assess the consequences of virus adaptation to various substrates, we performed 5 consecutive passages of H1N1, H3N2, and influenza B viruses in eggs as well as in the MDCK and Vero cell lines and compared the properties of obtained viruses [33]. The egg-derived seed viruses recommended for the vaccine production were selected as the starting material.…”