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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.)

The practical use of cell-based seasonal influenza vaccines is currently being considered in Japan. From the perspective of adventitious virus contamination, we assessed the suitability of NIID-MDCK cells (NIID-MDCK-Cs) as a safe substrate for the isolation of influenza viruses from clinical specimens. We first established a sensitive multiplex real-time PCR system to screen for 27 respiratory viruses and used it on 34 virus samples that were isolated by passaging influenza-positive clinical specimens in NIID-MDCK-Cs. Incidentally, the limit of detection (LOD) of the system was 100 or fewer genome copies per reaction. In addition to influenza viruses, human enterovirus 68 (HEV-D68) genomes were detected in two samples after two or three passages in NIID-MDCK-Cs. To further investigate the susceptibility of NIID-MDCK-Cs to adventitious viruses, eight common respiratory viruses were subjected to passages in NIID-MDCK-Cs. The genome copy numbers of seven viruses other than parainfluenza 3 decreased below the LOD by passage 4. By passaging in NIID-MDCK-Cs, the genome numbers of the input HEV-D68, 1 × 10 8 copies, declined to 10 2 at passage 3 and to under the LOD at passage 4, whereas those of the other six viruses were under the LOD by passage 3. These results implied that during the process of isolating influenza viruses with NIID-MDCK-Cs, contaminating viruses other than parainfluenza 3 can be efficiently removed by passages in NIID-MDCK-Cs. NIID-MDCK-Cs could be a safe substrate for isolating influenza viruses that can be used to develop cell-based influenza vaccine candidate viruses., (© 2022 The Societies and John Wiley & Sons Australia, Ltd.)

In Japan, the practical application of completely cell-based seasonal influenza vaccines is under consideration. Considering the good correlation between the immunogenicity of egg-based influenza vaccines and the hemagglutinin (HA) content determined by the single radial immunodiffusion (SRD) assay, we determined the potency of the first cell-based quadrivalent vaccine experimentally generated in Japan using the SRD assay in this study. A primary liquid standard (PLS) and reference antigen were generated from the purified vaccine virus, and a sheep antiserum was produced against the HA of the vaccine virus. Since the purity of the PLS affects the reliability of vaccine potency testing, the purification steps are significant. We successfully prepared a purified PLS nearly free of cell debris. The HA content in the PLS was first estimated from the total amount of viral protein and the percentage of HA content determined by SDS-PAGE analysis. The HA content in the reference antigen was calibrated to that in the PLS via the SRD assay. The vaccine potency, that is, the HA content in each vaccine, was finally measured using the corresponding reference antigen. Ultimately, the measured vaccine potency of the monovalent vaccine was similar to that of the quadrivalent vaccine., (Copyright © 2020. Published by Elsevier Ltd.)

Antibodies against influenza virus neuraminidase (NA) protein prevent releasing of the virus from host cells and spreading of infection foci and are considered the 'second line of defence' against influenza. Haemagglutinin inhibition antibody-low responders (HI-LRs) are present among influenza split vaccine recipients. The NA inhibition (NAI) antibody response in vaccinees is worth exploring, especially those in the HI-LRs population. We collected pre- and post-vaccination sera from 61 recipients of an inactivated, monovalent, split vaccine against A/H1N1pdm09 and acute and convalescent sera from 49 unvaccinated patients naturally infected with the A/H1N1pdm09 virus during the 2009 influenza pandemic. All samples were subjected to haemagglutinin inhibition (HI), NAI and neutralisation assays. Most paired sera from naturally infected patients exhibited marked elevation in the NAI activity, and seroconversion rates (SCR) among HI-LRs and HI-responders (HI-Rs) were 60% and 87%, respectively; however, those from vaccinees displayed low increase in the NAI activity, and the SCR among HI-LRs and HI-Rs were 0% and 12%, respectively. In both HI-LRs and HI-Rs, vaccination with the inactivated, monovalent, split vaccine failed to elicit the NAI activity efficiently in the sera of the naive population, compared with the natural infection. Hence, the improvement of influenza vaccines is warranted to elicit not only HI but also NAI antibodies., Competing Interests: The authors have declared that no competing interests exist.

Influenza vaccines of H7N9 subtype are consistently less immunogenic in humans than vaccines developed for other subtypes. Although prior immunoinformatic analysis identified T-cell epitopes in H7 hemagglutinin (HA) which potentially enhance regulatory T cell response due to conservation with the human genome, the links between the T-cell epitopes and low immunogenicity of H7 HA remains unknown due to the lack of animal models reproducing the response observed in humans. Here, we utilized a humanized mouse model to recapitulate the low immunogenicity of H7 HA. Our analysis demonstrated that modification of a single H7 epitope by changing 3 amino acids so that it is homologous with a known H3 immunogenic epitope sequence significantly improved the immunogenicity of the H7 HA in the humanized mouse model, leading to a greater than 4-fold increase in HA-binding IgG responses. Thus, we provide experimental evidence for the important contribution of this H7-specific T cell epitope in determining the immunogenicity of an influenza vaccine. Furthermore, this study delineates strategies that can be used for screening and selecting vaccine strains using immunoinformatics tools and a humanized mouse model.

To improve the hemagglutinin (HA) antigen yield of influenza A(H1N1)pdm09 candidate vaccine viruses, we generated 7:1, 6:2, and 5:3 genetic reassortant viruses between wild-type (H1N1)pdm09 (A/California/7/2009) (Cal7) and a high-yielding master virus, A/Puerto Rico/8/34 (PR8). These viruses contained the HA; HA and neuraminidase (NA); and HA, NA, and M genes, respectively, derived from Cal7, on a PR8 backbone. The influence of the amino acid residue at position 223 in Cal7 HA on virus growth and HA antigen yield differed between these reassortant viruses. NIIDRG-7, a 7:1 virus possessing arginine at position 223, exhibited a 10-fold higher 50% egg infectious dose (EID(50)) (10.0 log(10)EID(50)/ml) than the 5:3 and 6:2 viruses. It also had 1.5- to 3-fold higher protein (13.8 μg/ml of allantoic fluids) and HA antigen (4.1 μg/ml of allantoic fluids) yields than the 5:3 and 6:2 viruses, which possessed identical Cal7 HA proteins. However, the HA antigen yield of the other 7:1 virus, which possessed glutamine at position 223 was 60% of that of NIIDRG-7. In addition, a novel 6:2 virus possessing Cal7 HA and the NA of A/Wisconsin/10/98 (a triple reassortant swine-like H1N1 virus), produced 107% of the HA yield of NIIDRG-7. In this study, we showed that the balance between HA and NA in the influenza A(H1N1)pdm09 virus affects its protein and antigen yield.

How the antibodies of individual convalescent human sera bind to each amino acid residue at the antigenic sites of hemagglutinin (HA) of influenza viruses, and how the antigenic drift strains of influenza viruses are selected by human sera, is not well understood. In our previous study, it was found by a binding assay with a chimeric HA between A/Kamata/14/91 (Ka/91) and A/Aichi/2/68 that convalescent human sera, following Ka/91 like (H3N2) virus infection, bind to antigenic site A of Ka/91 HA. Here using chimeric HAs possessing single amino acid substitutions at site A, it was determined how those human sera recognize each amino acid residue at antigenic site A. It was found that the capacity of human sera to recognize amino acid substitutions at site A differs from one person to another and that some amino acid substitutions result in all convalescent human sera losing their binding capacity. Among these amino acid substitutions, certain ones might be selected by chance, thus creating successive antigenic drift. Phylogenetic analysis of the drift strains of Ka/91 showed amino acid substitutions at positions 133, 135 and 145 were on the main stream of the phylogenetic tree. Indeed, all of the investigated convalescent sera failed to recognize one of them., (© 2012 The Societies and Blackwell Publishing Asia Pty Ltd.)

Sera from 27 children and eight older persons, which had been collected in 1998 and 1999 and showed haemagglutination-inhibition (HI) activity against influenza A/Sydney/5/97 (H3N2) strain, were characterized with a binding assay using chimeric haemagglutinin (HA) proteins between A/Aichi/2/68 (A/AI/68) and A/Sydney/5/97 (A/SD/97) strains. Sera from the young children had a tendency to recognize only the antigenic site B1 of the HA1 region. On the other hand, sera of the older individuals were fully reactive to all antigenic sites of HA1 except antigenic site D. Recent epidemic strains, A/Panama/2007/99 (A/PM/99)-like viruses have differences in amino acids in antigenic sites A, C, and B2 but not B1. However, human antisera obtained even from young children had HI activity to Panama-like viruses. The limited epidemic of A/PM/99-like viruses may have been due to the existence of antibody against B1, which had been produced in response to infection by the A/SD/97-like viruses.

Eight convalescent human sera obtained from patients aged 3 to 14 years old, who were infected with influenza A(H3N2) virus during the 1990/1991 influenza season, were characterized by a binding assay with chimeric hemagglutinin (HA) proteins between influenza virus A/Aichi/2/68 and A/Kamata/14/91(H3N2) strains. These sera did not recognize the HA protein of the A/Aichi/2/68 strain but recognized that of the A/Kamata/14/91 strain. The binding assay revealed that these sera recognized only the HA1 domain of A/Kamata/14/91 HA protein. A further assay of the binding of these sera to the chimeric proteins of the HA1 domain revealed that three sera (A-1, A-2, and A-3) from very young patients bound only to region 150-170 (site B1) and one serum (Y-1) bound to regions 96-150 (site A) and 96-170 (sites A and B1). These four sera showed reduced hemagglutination inhibition (HI) activity with the 203v2 strain, a monoclonal variant of the A/Kamata/14/91 strain with two amino acid changes in the HA protein at antigenic sites A and B1. The other four sera (Y-2, G-1, G-2, and A-4) bound to regions 1-96 (site C/E), 96-150 (site A), 96-170 (sites A and B1), and 170-200 (site B2), two of which further bound to region 240-306 (site C); these sera were all fully reactive with the 203v2 strain. All eight sera showed reduced HI reactivity to a drift strain A/Aichi/4/93. Amino acid changes of the A/Aichi/4/93 strain from the A/Kamata/14/91 strain were located at antigenic sites A, B1, B2, and C. We propose a possible model for the emergence of a drift strain A/Aichi/4/93 from an A/Kamata/14/91-like strain by sequential changes during reinfections of individuals starting from A-1-like, next to Y-1-like, and then to Y-2-like populations., (Copyright 2000 Academic Press.)

In order to conduct a survey of influenza viruses entering Japan via travellers arriving by airplanes, gargle solutions were collected from passengers who reported to the quarantine station of Nagoya International Airport complaining of respiratory symptoms. From 504 samples collected between August 1996 and March 1999, 30 influenza virus strains were isolated. Twenty-eight of the isolates were influenza A (H3N2) viruses and two were influenza B viruses. No H1N1 virus was isolated. Among 28 isolates of H3N2 virus, 3 strains were obtained outside the influenza season. Nucleotide sequences of the haemagglutinin (HA) genes of these isolates along with those from domestic patients were analysed in order to determine the influence of imported influenza viruses by travellers on epidemics in Japan. From the phylogenetic and chronological aspects, the possibility was suggested in one case in 1997/8 and two in the 1998/9 season that imported virus by travellers may have influenced the domestic influenza epidemics.

Cleavage of the hemagglutinin (HA) molecule by proteases is a prerequisite for the pathogenicity and even for the neurovirulence of influenza A viruses. WSN, a neurovirulent virus, adapted to mouse brain, grew in vitro in several types of cells including neuroblastoma cells in the absence of trypsin. When mice were intracerebrally inoculated with WSN, the viral antigen was found in the substantia nigra zona compacta and hippocampus. The mice inoculated with viruses isolated from children with acute encephalopathy associated with an influenza virus infection, on the other hand, showed no neurological symptoms. Furthermore, these viruses did not grow in the human neuroblastoma and glioblastoma cells. Since 1991, most of the human influenza A viruses have not agglutinated chicken erythrocytes. Whether this altered receptor binding specificity is related to the occurrence of influenza encephalitis and encephalopathy is now under investigation.