Here, we described an approach to enhance the growth of influenza vaccine seed viruses in Vero cells, which are approved for use in human vaccine production. Influenza vaccine seed viruses that provide robust growth in cell culture are needed to ensure an adequate supply of influenza vaccines as either a supplement to or an alternative method for egg-based vaccine production. Our approach involved introducing a single amino acid mutation (N117D) into the HA2 subunit of HA, which was found in a Vero cell-adapted PR8 virus. The seasonal influenza vaccine seed-like viruses (6:2 reassortants with a PR8 backbone) tested in this study grew poorly in Vero cells. However, the introduction of this HA2 single mutation into these viruses produced mutants that grew to 100 to 1,000 times higher titers in Vero cells than wild-type viruses. This strategy for virus growth enhancement based on the HA2 mutation could thus be feasible for the production of growth-enhanced seasonal or pandemic vaccine seed viruses.
The amino acid at position 117 of HA2 is located on the stem region of HA. One concern is the possibility that changing the amino acid residue at this position may affect the antigenicity of inactivated vaccines, although the major antigenic sites of H1 HA (Ca, Cb, Sa, and Sb), against which most neutralization antibodies elicited by inactivated vaccines are raised, are located on the globular head region that surrounds the receptor binding site (3
). Recent studies revealed that antibodies against the HA stem region confer universal protection from influenza virus infection (5
). However, such neutralizing antibodies are rarely induced by conventional vaccinations (28
). These findings suggest that the HA2 N117D mutation would not affect the antigenicity or efficacy of the vaccines.
Here, we suggest that the growth enhancement was most likely due to broadening of the optimal pH range for virus membrane fusion mediated by HA2. The mutation site is located in the HA stalk region, close to the fusion peptide in the HA trimer (B). To accomplish virus membrane fusion, cleaved HA needs to be exposed to low pH (14
). Previous reports demonstrate that the substitution of the neutral amino acid residue(s) in the HA stalk region for charged amino acids affects the optimal pH for virus membrane fusion (24
). Since the N117D substitution introduces a negative charge, it may change the electrostatic balance between the residue at position 117 and the fusion peptide, possibly resulting in fewer proton-dependent conformational changes in the HA molecule.
We revealed that the endosomal pH was higher in Vero cells than in MDCK cells 15 min after dextran intake. Based on this observation, we assume that this difference in endosomal pH affects virus growth in these cell lines. Indeed, cell-type-dependent endosomal pH kinetics are important for influenza virus infection (21
). In MDCK cells, influenza virus reaches the late endosome (pH 5.0) 10 min after endocytosis (21
). On the other hand, in HeLa cells, which is a nonpermissive cell line for influenza virus infection (6
), it takes 40 min for influenza virus to colocalize with a late endosome marker following endocytosis (36
). However, highly pathogenic H5N1 virus, which requires a higher pH for optimal membrane fusion (pH 5.9) (32
), grows in MDCK, Vero, and HeLa cells with comparable titers (44
). Moreover, vesicular stomatitis virus (VSV) infectivity is much lower in MDCK cells than in Vero cells (11
), possibly due to the higher optimal pH (pH 6.0) for membrane fusion of the VSV G protein (34
). These facts imply that Vero cells may have a higher pH in the early endosome than MDCK cells. A precise assessment of endosomal pH changes is needed to better understand the mechanism of enhanced virus growth in Vero cells.
We used PR8 virus as a reassortant backbone virus in this study because it is attenuated in humans and is approved by the WHO for use as a genetic backbone for vaccine seed viruses (42
). However, introducing the HA2 mutation identified here into any wild-type virus, including seasonal influenza viruses, could also enhance virus replication in Vero cells, providing alternative vaccine seed viruses for the production of inactivated influenza vaccines.
In conclusion, we propose a mutant virus possessing aspartic acid at HA2 position 117 as a seed virus for Vero cell-based influenza vaccine production. A virus with this single amino acid mutation can be produced easily by using reverse genetics. A cell culture-based vaccine strategy with this seed virus would allow the production of more doses of inactivated influenza vaccines in a timely, cost-effective manner, not only for seasonal, but also for pandemic vaccines.