Emerging Antigenic Variants at the Antigenic Site Sb in Pandemic A(H1N1)2009 Influenza Virus in Japan Detected by a Human Monoclonal Antibody

Yasugi, Mayo; Kubota‐Koketsu, Ritsuko; Yamashita, Akifumi; Kawashita, Norihito; Du, Anariwa; Misaki, Ryo; Kuhara, Motoki; Boonsathorn, Naphatsawan; Fujiyama, Kazuhito; Okuno, Yoshinobu; Nakaya, Takaaki; Ikuta, Kazuyoshi

doi:10.1371/journal.pone.0077892

Cited by 20 publications

(25 citation statements)

References 46 publications

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“…These findings concur with other studies where 129D and 186D were found in H1N1pdm09 viruses already egg adapted at 222/223 [1,11,12] or 191 [12, 13], and we conclude that (like reside 119) residues 129 and 186 are not independent egg adaptation loci for A/Christchurch/16/2010.…”

Section: Discussionsupporting

confidence: 94%

The Ability of a Non-Egg Adapted (Cell-Like) A(H1N1)pdm09 Virus to Egg-Adapt at HA Loci Other than 222 and 223 and Its Effect on the Yield of Viral Protein

et al. 2016

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Previous studies on influenza A(H1N1)pdm09 candidate vaccine viruses (CVVs) that had adapted to growth in embryonated chicken eggs by the acquisition of amino acid substitutions at HA positions 222 or 223 showed that improved protein yield could be conferred by additional amino acid substitutions in the haemagglutinin (HA) that arose naturally during passaging of the virus in eggs. In this study we investigated, by means of reverse genetics, the ability of a non-egg adapted (cell-like) A(H1N1)pdm09 virus to egg-adapt at HA loci other than 222/223, introducing amino acid substitutions previously identified as egg adaptations in pre-H1N1pdm09 H1N1 viruses and assessing their effect on protein yield and antigenicity. We also investigated the effect on the protein yield of these substitutions in viruses that had A(H1N1)pdm09 internal genes rather than the traditional PR8 internal genes of a CVV. The data show that a cell-like A/Christchurch/16/2010 can be egg-adapted via amino acid substitutions in at least three alternative HA loci (187, 190 and 216), in viruses with either PR8 or A/California/7/2009 internal genes, but that the effects on protein yield vary depending on the amino acid substitution and the internal genes of the virus. Since CVVs need to produce high protein yields to be suitable for vaccine manufacture, the findings of this study will assist in the future characterisation of both wild type viruses and lab-derived CVVs for vaccine use.

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Section: Discussionsupporting

confidence: 94%

The Ability of a Non-Egg Adapted (Cell-Like) A(H1N1)pdm09 Virus to Egg-Adapt at HA Loci Other than 222 and 223 and Its Effect on the Yield of Viral Protein

et al. 2016

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“…Notably, these 5 novel positions are comparable to those in antigenic site B in H3 virus (24)(25)(26). This site, equivalent to antigenic site Sa and partial site Sb (27,28), is close to the HA receptor binding site and is probably under greater immune pressure than other antigenic sites (29)(30)(31). We examined 1,036 full-length H9 sequences available in GenBank (as of 19 July 2013) by using multiple sequence comparison by log expectation (MUSCLE) (32) and found variations at each of the 9 amino acid positions ( Table 4).…”

mentioning

confidence: 77%

Antigenic Mapping of the Hemagglutinin of an H9N2 Avian Influenza Virus Reveals Novel Critical Amino Acid Positions in Antigenic Sites

Wan

et al. 2014

J Virol

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H9N2 influenza virus is undergoing extensive genetic and antigenic evolution, warranting detailed antigenic mapping of its hemagglutinin (HA). Through examining antibody escape mutants of an Asian avian H9N2 virus, we identified 9 critical amino acid positions in H9 antigenic sites. Five of these positions, 164, 167, 168, 196, and 207, have not been reported previously and, thus, represent novel molecular markers for monitoring the antigenic change of H9N2 virus. H9N2 influenza virus is circulating in poultry worldwide. H9N2 virus infection is usually mild in nature but may lead to higher mortality if it is associated with secondary infection (1, 2). In Asia, since its introduction into land-based poultry in the late 1980s, H9N2 virus has been spreading to various avian and mammalian species, including pigs (3, 4). Due to the Q226L mutation (change of Gln to Leu at position 226) in the hemagglutinin (HA) (5-7), a significant proportion of H9N2 isolates have acquired human virus-like receptor specificity (5,8). Consistent with this receptor specificity change, multiple human cases of H9N2 virus infection have been reported (9-12). Moreover, H9N2 virus has provided internal genes for the highly pathogenic H5N1 (9, 13, 14) and novel H7N9 (15) viruses. These have put H9N2 virus high on the list of influenza viruses with pandemic potential.Although the crystal structure of H9 has been solved (16), no details for H9 antigenic epitopes have been elucidated. Previous investigations by other groups have identified multiple amino acids in H9 antigenic sites (17, 18). These are nevertheless far from being sufficient for understanding the H9 antigenic structure. To identify more amino acids constituting H9 antigenic sites, we performed an antigenic mapping of the HA of an avian H9N2 virus A/Chicken/Jiangsu/X1/2004 (hereinafter called X1) (GenBank nucleotide sequence accession number KF688983) with monoclonal antibodies (MAbs).H9-specific MAbs were generated through the fusion of myeloma Sp2/0 cells with splenocytes from a BALB/c mouse immunized with X1 virus (19). The immunization included 3 intraperitoneal inoculations at 2-week intervals and a final boost with live X1 virus (on day 3 before the fusion). Hybridomas were screened by indirect immunofluorescence assay using chicken embryo fibroblast cells infected with X1 virus as the antigen, followed by screening with a hemagglutination inhibition (HI) assay using 4 hemagglutination units of X1 virus (20). Ascitic fluid of each selected hybridoma was generated in mice and used directly (e.g., without further purification or treatment with receptor-destroying enzyme) in the characterization of each MAb. All animal experiments were done in accordance with the institutional animal care guidelines, and the protocol (number 06R015) was approved by the Animal Care Committee at Yangzhou University. A microneutralization (MN) assay was performed in Madin-Darby canine kidney (MDCK) cells, following a previous protocol (21), except that 100 median tissue infectious doses (TCID 50 ) o...

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“…Most heterosubtypic crossneutralizing HuMAb have been reported to recognize epitopes located mainly in the most conserved HA stem region [7,10,14,23]. Also, a few HuMAbs that target relatively conserved epitopes adjacent to or overlapping the RBS in the head region were shown to be subtype-specific [17,25,26]. To our knowledge, 5D7 is the first heterosubtypic neutralizing HuMAb that targets a conserved epitope distinct from the RBS in the HA globular head region.…”

Section: Discussionmentioning

confidence: 94%

“…Escape mutants were isolated by culturing A/Suita/1/2009 in the presence of HuMAb, as described previously [17], with minor modifications. Briefly, the virus was incubated with 10-fold diluted HuMAb (to give final concentrations of 5, 0.5 and 0.05 lg/ml) at 37°C for 1 h. MDCK cells in 24-well plates were then inoculated with the mixtures and cultured with DMEM/F12+GlutaMAX-1 (Invitrogen) supplemented with 0.4% BSA, 2 lg/ml acetylated trypsin and antibiotics.…”

Section: Selection Of Escape Mutantsmentioning

confidence: 99%

“…In contrast, we have reported a cell-to-cell fusion method using a new fusion partner cell line, SPYMEG, and donor-derived peripheral blood mononuclear cells (PBMCs) [9,[15][16][17][18]. In this study, we report the characterization of an anti-influenza HuMAb generated from the PBMCs of a volunteer vaccinated with seasonal influenza vaccine.…”

Section: Introductionmentioning

confidence: 99%

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A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

Boonsathorn

Panthong

Koksunan

et al. 2014

Biochemical and Biophysical Research Communications

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Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.

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Emerging Antigenic Variants at the Antigenic Site Sb in Pandemic A(H1N1)2009 Influenza Virus in Japan Detected by a Human Monoclonal Antibody

Cited by 20 publications

References 46 publications

The Ability of a Non-Egg Adapted (Cell-Like) A(H1N1)pdm09 Virus to Egg-Adapt at HA Loci Other than 222 and 223 and Its Effect on the Yield of Viral Protein

The Ability of a Non-Egg Adapted (Cell-Like) A(H1N1)pdm09 Virus to Egg-Adapt at HA Loci Other than 222 and 223 and Its Effect on the Yield of Viral Protein

Antigenic Mapping of the Hemagglutinin of an H9N2 Avian Influenza Virus Reveals Novel Critical Amino Acid Positions in Antigenic Sites

A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

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