AEX-1: A Vero cell variant with enhanced permissiveness for influenza virus replication

Background Continuous cell substrates with improved permissiveness for influenza virus replication are of considerable interest for vaccine development and manufacturing. Vero cells have an excellent regulatory track record; however, their relatively low influenza virus productivity limits their broader application. Methods We developed the AEX-1 cell line by co-cultivating Vero cells with chicken embryo amniotic membrane fragments. Cellular morphology, intracellular acidification, influenza virus replication, transcriptomic profile, genetic stability, and tumorigenicity were evaluated. Results AEX-1 cells displayed distinct morphology and enhanced endosomal/lysosomal acidification compared with parental Vero cells. At 72 h post-infection, a commonly used harvest time for influenza vaccine virus production, AEX-1 cells produced higher infectious titres of WHO-recommended egg-derived A/Sydney/5/2021 (H1N1)pdm09-like and B/Austria/1359417/2021 vaccine viruses, with titres approaching those obtained in embryonated chicken eggs and exceeding Vero cell yields by 0.9–1.7 log 10 EID 50 /ml. No replication advantage was observed for the egg-derived A/Croatia/10136RV/2023 (H3N2) vaccine virus, carrying the egg-adaptive HA G53D substitution, or for the egg-derived A/duck/Vietnam/NCVD-1584/2012 (H5N1) vaccine strain. However, the A/Darwin/9/2021-like Vero-derived primary isolate A/Austria/1516645/2022 (H3N2), lacking G53D, reached infectious titres up to 3.0 log 10 TCID 50 /ml higher than in Vero and 2.3 log 10 TCID 50 /ml higher than in MDCK cells. H1N1 UniFluVec-based influenza virus vectors also replicated efficiently in AEX-1 cells. Transcriptomic analysis revealed changes in genes involved in proton transport, vesicular acidification, and intracellular pH regulation. AEX-1 remained genetically stable after prolonged passaging and showed no evidence of increased tumorigenicity in vitro or in vivo. Conclusions Enhanced intracellular acidification may contribute to improved influenza virus replication in AEX-1 cells, suggesting that modulation of endosomal acidification may provide a rational strategy for developing improved cell substrates for influenza vaccine production.