Abstract
In 2013, the first case of human infection with an avian influenza A virus (H7N9) was reported in China, and the human infection with this virus has continued as of 2016. At the request of the WHO, we have successfully developed candidate reassortant vaccine virus using A/Anhui/1/2013 and the high egg-growth master virus A/PR/8/1934. Recent plans regarding influenza vaccine production include using cell-cultured systems in Japan and several other countries. However, egg-based vaccine viruses are not always suitable for cell-cultured vaccine production due to potential issues with growth, protein yield and antigenic stability. Therefore, in this study, we have developed a high-growth master virus (hg-PR8) adapted to qualified NIID-MDCK cells that are competent for vaccine production. The virus hg-PR8 was obtained after 20 serial passages of A/Puerto Rico/8/1934 (PR8) in NIID-MDCK cells. The viral titer of hg-PR8 was 108.6 plaque-forming units per milliliter (PFU/mL). Seven amino acid substitutions were identified in the amino acid sequences of PB2, PB1, PA, NA, M and NS of hg-PR8 compared to the sequence of the original PR8 (org-PR8) strain. The growth capacities of the reassortant viruses, which possess heterologous internal genes from hg-PR8 or org-PR8, indicated that the amino acid changes in PB2 and NS2 similarly affected growth capacity in NIID-MDCK cells. To assess the suitability of hg-PR8 as a master virus, we generated 6:2 reassortant viruses possessing the HA and NA segments from A/Anhui/1/2013 (H7N9) and the remaining segments from hg-PR8. The virus titers of the reassortant strains were 107−108 PFU/mL. The antigenicity of the viruses was stable during ten passages of the viruses in NIID-MDCK cells. In comparison with the egg-based reassortant vaccine viruses with identical HA and NA segments, the hg-PR8-based viruses showed 1.5- to 2-fold higher protein yields in NIID-MDCK cells.
Highlights
Influenza vaccination is the primary strategy for influenza prevention and control, and it has been used for more than 60 years
Because of the pandemic threat posed by this virus, we have developed egg-grown candidate 6:2 reassortant vaccine viruses using reverse genetics (RG) techniques at the request of the World Health Organization (WHO) [18]
The org-Puerto Rico/8/1934 (PR8) virus with a virus titer of 107.1 PFU/mL was subjected to multiple passages in NIID-Madin Darby canine kidney (MDCK) cells as described in the Materials and Methods
Summary
Influenza vaccination is the primary strategy for influenza prevention and control, and it has been used for more than 60 years. Frequent amino acid substitutions in hemagglutinin (HA) have been reported during the propagation of the viruses in eggs [4,5,6,7,8,9] These egg-adapted mutations often influence the receptor specificity and the antigenicity of the viruses. Such mutations in vaccine viruses may reduce the efficacy of the influenza vaccines [10] To avoid these antigenic changes in vaccine viruses, propagation of these viruses in mammalian cell lines instead of in eggs has been suggested. The antigenic stability and antigen yields of the vaccine viruses should be considered In this context, Donis et al investigated the characteristics of the cell lines qualified for influenza virus isolation and propagation in terms of their cell-cultured influenza vaccine production [11]. Several groups have taken alternative approach of attempting to improve the virus growth and protein yields of the cell-cultured vaccine viruses, i.e., development of a high-growth master virus for creating reassortant viruses and genetic modification of the viral genome to improve the virus growth and/or yields [12,13,14,15,16]
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