Influenza vaccine viruses and the development of seasonal vaccines: A Japanese perspective.

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.

Identification of the properties of H5 influenza vaccine viruses with high hemagglutinin yields.

Manufactured influenza vaccines have to contain a defined amount of hemagglutinin (HA) antigen. Therefore, vaccine viruses with a high HA antigen yield (HAY) are preferable for manufacturing vaccines, particularly vaccines in response to a pandemic, when vaccines need to be rapidly produced. However, the viral properties associated with a high HAY have not yet been fully clarified.

Suitability of NIID-MDCK cells as a substrate for cell-based influenza vaccine development from the perspective of adventitious virus susceptibility.

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.

Determination of the potency of a cell-based seasonal quadrivalent influenza vaccine using a purified primary liquid standard.

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.

Cell-Based Influenza A/H1N1pdm09 Vaccine Viruses Containing Chimeric Hemagglutinin with Improved Membrane Fusion Ability.

The H1N1 influenza pandemic vaccine has been developed from the A/California/07/09 (Cal) virus and the well-known high-yield A/Puerto Rico/8/34 (PR8) virus by classical reassortment and reverse genetics (RG) in eggs. Previous studies have suggested that Cal-derived chimeric hemagglutinin (HA) and neuraminidase (NA) improve virus yields. However, the cell-based vaccine of the H1N1 pandemic virus has been less investigated.

Low response in eliciting neuraminidase inhibition activity of sera among recipients of a split, monovalent pandemic influenza vaccine during the 2009 pandemic.

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.

Publisher Correction: A humanized mouse model identifies key amino acids for low immunogenicity of H7N9 vaccines.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A humanized mouse model identifies key amino acids for low immunogenicity of H7N9 vaccines.

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.

Generation of a Genetically Stable High-Fidelity Influenza Vaccine Strain.

Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus.

Development of an Influenza A Master Virus for Generating High-Growth Reassortants for A/Anhui/1/2013(H7N9) Vaccine Production in Qualified MDCK Cells.

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.

Development of a high-yield reassortant influenza vaccine virus derived from the A/Anhui/1/2013 (H7N9) strain.

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.

Host Adaptation and the Alteration of Viral Properties of the First Influenza A/H1N1pdm09 Virus Isolated in Japan.

A/Narita/1/2009 (A/N) was the first H1N1 virus from the 2009 pandemic (H1pdm) to be isolated in Japan. To better understand and predict the possible development of this virus strain, the effect of passaging A/N was investigated in Madin-Darby canine kidney cells, chicken eggs and mice. A/N that had been continuously passaged in cells, eggs, or mice obtained the ability to grow efficiently in each host.

Epitope mapping of the hemagglutinin molecule of A/(H1N1)pdm09 influenza virus by using monoclonal antibody escape mutants.

Unlabelled: We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb, and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A. J.

The host protease TMPRSS2 plays a major role in in vivo replication of emerging H7N9 and seasonal influenza viruses.

Unlabelled: Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s) in vivo. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and is capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site.

Composition of hemagglutinin and neuraminidase affects the antigen yield of influenza A(H1N1)pdm09 candidate vaccine viruses.

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.

Genetics and infectivity of H5N1 highly pathogenic avian influenza viruses isolated from chickens and wild birds in Japan during 2010-11.

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.

Reactivity of human convalescent sera with influenza virus hemagglutinin protein mutants at antigenic site A.

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.

Prediction of probable mutations in influenza virus hemagglutinin protein based on large-scale ab initio fragment molecular orbital calculations.

Ab initio electronic-state calculations for influenza virus hemagglutinin (HA) trimer complexed with Fab antibody were performed on the basis of the fragment molecular orbital (FMO) method at the second and third-order Møller-Plesset (MP2 and MP3) perturbation levels. For the protein complex containing 2351 residues and 36,160 atoms, the inter-fragment interaction energies (IFIEs) were evaluated to illustrate the effective interactions between all the pairs of amino acid residues. By analyzing the calculated data on the IFIEs, we first discussed the interactions and their fluctuations between multiple domains contained in the trimer complex.

Sialic acid recognition of the pandemic influenza 2009 H1N1 virus: binding mechanism between human receptor and influenza hemagglutinin.

Quantum mechanical fragment molecular orbital calculations have been performed for receptor binding of the hemagglutinin protein of the recently pandemic influenza 2009 H1N1, A/swine/Iowa/1930, and A/Puerto Rico/8/1934 viruses to α2-6 linked sialyloligosaccharides, as analogs of human receptors. The strongest receptor binding affinity was observed for the 2009/H1N1pdm. The inter-fragment interaction energy analysis revealed that the amino acid mutation of 2009/H1N1pdm, Ser145Lys, was a major cause of such strong binding affinity.

Effects of single-point amino acid substitutions on the structure and function neuraminidase proteins in influenza A virus.

In order to clarify the effect of amino acid substitutions on the structure and function of the neuraminidase (NA) protein of influenza A virus, we introduced single-point amino acid substitutions into the NA protein of the A/Tokyo/3/67 (H2N2) strain using PCR-based random mutation. The rate of tolerant random one amino acid substitutions in the NA protein was 47%. Rates of tolerant substitutions for the stalk and for the surface and inner portion of the head region of the NA protein were 79, 54, and 19%, respectively.

Protective effect of nasal immunization of influenza virus hemagglutinin with recombinant cholera toxin B subunit as a mucosal adjuvant in mice.

To develop an efficient nasal influenza vaccine, influenza A and B virus HA with rCTB as a mucosal adjuvant were administered to mice intranasally. Serum anti-HA IgG and IgA antibody responses for both HA vaccines were significantly increased in the presence of rCTB. Higher HI and neutralizing antibody titers and higher mucosal IgA antibody responses in the respiratory tract were detected when rCTB was added than without rCTB.

[Analysis of the amino acid changes of the hemagglutinin of H5 avian influenza virus].

We introduced 38 single-point amino acid changes into the hemagglutinin (HA) protein of the reassortmented A/Duck/Mongolia/54/01 (H5N2) strain by a PCR random mutation method. The percentage of amino acid changes on the HA domain that did not abrogate hemadsorption activity was calculated to be 89%. Changes in the amino acids of the HA2 domain were observed to be about half of those in the HA1 domain of these mutants.

Comparison of epitope structures of H3HAs through protein modeling of influenza A virus hemagglutinin: mechanism for selection of antigenic variants in the presence of a monoclonal antibody.

Starting with nine plaques of influenza A/Kamata/14/91(H3N2) virus, we selected mutants in the presence of monoclonal antibody 203 (mAb203). In total, amino acid substitutions were found at nine positions (77, 80, 131, 135, 141, 142, 143, 144 and 146), which localized in the antigenic site A of the hemagglutinin (HA). The escape mutants differed in the extent to which they had lost binding to mAb203.

[Accumulation of amino acid substitutions promotes irreversible structural changes in the hemagglutinin of human influenza AH3 virus during evolution].

During protein evolution the amino acid substitutions accumulate with time. However, the effect of accumulation of the amino acid substitutions to structural changes has not been estimated well. We will propose that the discordance of amino acid substitution on the HA protein of influenza A virus is useful for the assessment of structural changes during evolution.