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We established a qualified Madin-Darby canine kidney cell line (qMDCK-Cs) and investigated its suitability for source virus isolation to develop cell-based seasonal influenza vaccine viruses using vaccine manufacturer cells (Manuf-Cs). When inoculated with 81 influenza-positive clinical specimens, the initial virus isolation efficiency of qMDCK-Cs was exceeded 70%. Among the qMDCK-C isolates, 100% of the A/H1N1pdm09, B/Victoria and B/Yamagata strains and >70% of the A/H3N2 strains showed antigenicity equivalent to that of the contemporary vaccine or relevant viruses in haemagglutination inhibition (HI) or virus neutralization (VN) tests using ferret antisera. These qMDCK-C isolates were propagated in Manuf-Cs (MDCK and Vero cells) (Manuf-C viruses) to develop vaccine viruses. In reciprocal antigenicity tests, ferret antisera raised against corresponding reference viruses and Manuf-C viruses recognized 29 of 31 Manuf-C viruses and corresponding reference viruses, respectively at HI or VN titres more than half of the homologous virus titres, which is the antigenicity criterion for cell culture seasonal influenza vaccine viruses specified by the World Health Organization. Furthermore, ferret antisera against these Manuf-C viruses recognized ≥95% of the viruses circulating during the relevant influenza season with HI or VN titres greater than one-quarter of the homologous virus titres. No cell line-specific amino acid substitutions were observed in the resulting viruses. However, polymorphisms at positions 158/160 of H3HA, 148/151 of N2NA and 197/199 of B/Victoria HA were occasionally detected in the qMDCK-C and Manuf-C viruses but barely affected the viral antigenicity. These results indicated that qMDCK-Cs are suitable for isolating influenza viruses that can serve as a source of antigenically appropriate vaccine viruses. The use of the qMDCK-C isolates will eliminates the need for clinical sample collection, virus isolation, and antigenicity analysis every season, and is expected to contribute to the promotion of vaccine virus development using manufacturer cells., Competing Interests: Declaration of competing interest Takao Fujimoto, Fumiaki Horikoshi, and Shuhei Chida were employees of BIKEN Co., Ltd., Kenji Tanaka is an employee of Daiichi Sankyo Biotech Co., Ltd., Kenji Minari, and Yoshimi Tanimoto are employees of Takeda Pharmaceutical Company Limited., The other coauthors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

In Japan, the Ministry of Health, Labour and Welfare (MHLW) designates one specific virus strain for each component of the quadrivalent seasonal influenza vaccine, and four domestic manufacturers produce egg-based influenza vaccines with the same formulation (inactivated, split-virus) using uniform vaccine strains. Thus, discussions of the development of effective seasonal influenza vaccines so far has focused solely on the antigenic match between the vaccine strains and epidemic viruses. However, in 2017, the Japanese selection system of vaccine viruses demonstrated that even a candidate vaccine virus that is antigenically similar to the predicted circulating viruses is not necessarily suitable for vaccine production, given lower productivity of the vaccine. Taking this experience into account, the MHLW reformed the scheme of vaccine strain selection in 2018, and instructed the Vaccine Epidemiology Research Group created by the MHLW to probe how the virus strains for the seasonal influenza vaccine should be selected in Japan. In this context, a symposium, entitled "Issues of the Present Seasonal Influenza Vaccines and Future Prospects", was held as part of the 22nd Annual Meeting of the Japanese Society for Vaccinology in 2018, and subjects related to the influenza vaccine viruses were discussed among relevant administrators, manufacturers, and researchers. This report summarizes the presentations given at that symposium in order to convey the present scheme of vaccine virus selection, the evaluation of the resulting vaccines, and the efforts at new vaccine formulation in Japan. Notably, from March 2022, the MHLW has launched a discussion of the merits of the seasonal influenza vaccines produced by foreign manufacturers., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘Takao Hozawa is an employee of Denka Co., Ltd. in Tokyo, Japan. All other authors declare that they are free of competing interests.’, (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In April 2013, the first three fatal cases of human infection with an avian influenza A virus (H7N9) were reported in China. Because of a pandemic threat by this virus, we have commenced to develop candidate vaccine viruses (CVVs). Three 6:2 genetic reassortant viruses with different hemagglutinin (HA) sequences, NIIDRG-10, -10.1 and -10.2, were generated by a reverse genetics technique between the high egg-growth master virus, A/Puerto Rico/8/34 (H1N1) and A/Anhui/1/2013 (H7N9), kindly provided by the Chinese Center for Disease Control and Prevention. The different HA gene sequences of the three CVVs were derived from the original virus stock. NIIDRG-10 possesses HA, whose sequence is identical to that of the original A/Anhui/1/2013 (H7N9) in the Global Initiative on Sharing Avian Influenza Data (EPI439507), while NIIDRG-10.1 and -10.2 possess amino acid differences, A125T and N123D/N149D, respectively, compared with NIIDRG-10. NIIDRG-10 replicated in embryonated chicken eggs with low hemagglutination titer 128, whereas NIIDRG-10.1 and -10.2 grew well with hemagglutination titer 1024. These viruses reacted well with a ferret antiserum raised against the original A/Anhui/1/2013 virus. Ferret antiserum against NIIDRG-10.1 reacted well with A/Anhui/1/2013 similar to the homologous virus NIIDRG-10.1. These results indicated that NIIDRG-10.1 passed the two-way test of antigenic identity. In contrast, the ferret antiserum against NIIDRG-10.2 reacted with A/Anhui/1/2013 at an 8-fold lower hemagglutination inhibition titer than with the homologous virus NIIDRG-10.2, indicating an antigenic change. The total and HA protein yields of NIIDRG-10.1 were 14.7 and 6.9 μg/ml, respectively, similar to those levels of high-yield seed viruses of seasonal influenza vaccines. NIIDRG-10.1 was approved as one of the CVVs for H7N9 viruses by the WHO in 2013. The candidate vaccine derived from NIIDRG-10.1 is currently being evaluated in a phase II clinical study in Japan., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Outbreaks of H5N1 subtype highly pathogenic avian influenza virus (HPAIV) were recorded in chickens, domesticated birds and wild birds throughout Japan from November 2010 to March 2011. Genetic analysis of the Japanese isolates indicated that all gene segments, except the PA gene, were closely related to Japanese wild bird isolates in 2008 and belonged to clade 2.3.2.1 classified by the WHO/OIE/FAO H5N1 Evolution Working Group. Direct ancestors of the PA gene segment of all Japanese viruses analyzed in this study can be found in wild bird strains of several subtypes other than H5N1 isolated between 2007 and 2009. The PA gene of these wild bird isolates share a common ancestor with H5N1 HPAIVs belonging to clades 2.5, 7 and 9, indicating that wild birds were involved in the emergence of the current reassortant 2.3.2.1 viruses. To determine how viruses were maintained in the wild bird population, two isolates derived from chickens (A/chicken/Shimane/1/2010, Ck10 and A/chicken/Miyazaki/S4/2011, CkS411) and one from a wild bird (A/mandarin duck/Miyazaki/22M-765/2011, MandarinD11) were compared in their ability to infect and be transmitted to chickens. There was a significant difference in the survival of chickens that were infected with 10(6)EID(50) of CkS411 compared to those with MandarinD11 and the transmission efficiency of CkS411 was greater than the other viruses. The increased titer of CkS411 excreted from infected chickens contributed to the improved transmission rates. It was considered that reduced virus excretion and transmission of MandarinD11 could have been due to adaptation of the virus in wild birds., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In an effort to find efficient DNA vaccine candidates, cDNA of influenza A virus hemagglutinin (HA) gene and several derived mutants were injected into mice using a gene gun. Mice immunized with HA1 DNA, with or without a membrane domain, showed a humoral immune response and the survival rate against homologous virus challenge was comparable to that of mice injected with HA DNA. In order to analyze epitopes recognized by antibodies induced by gene gun immunization, we used a binding assay employing the chimeric HA protein method. Serum antibodies of mice immunized with HA DNA recognized the HA1 domain but not the HA2 domain. In addition, antisera obtained from mice immunized with HA1 DNA reacted with each of the known antigenic sites on the HA1 domain, similar to the results obtained with HA DNA immunization.

We previously reported that the neurovirulent influenza A virus strain, AIWSN/33 has strong affinity for the substantia nigra (SN) in mouse brain. In this study, we used a neuron-glia co-culture system in order to observe the cellular responses to viral infection. Co-cultured cells were infected with the A/WSN/33 strain and were examined immunohistochemically. Viral antigens were strictly confined to the neuronal cell bodies and their neurites. More than 90% of tyrosine hydroxylase (TH)-positive neurons in the SN were also positive to anti-WSN antibody. There was no increase in class I major histocompatibility complex (MHC) expression in virus antigen positive cells. Thus, we can conclude that the virus preferentially infects neurons, mainly TH-positive neurons in the SN. An antigen presenting response mediated by class I MHC was not observed in infected neurons.

Antigenic drift of the haemagglutinin (HA) molecule of type A influenza viruses has been thought to occur by the accumulation of a series of point mutations in the antigenically important regions of the molecule. In order to study the antigenic sites on the HA molecule, an attempt was made to use the site-specific mutagenesis method.