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

<div><p>The swine-origin pandemic A(H1N1)2009 virus, A(H1N1)pdm09, is still circulating in parts of the human population. To monitor variants that may escape from vaccination specificity, antigenic characterization of circulating viruses is important. In this study, a hybridoma clone producing human monoclonal antibody against A(H1N1)pdm09, designated 5E4, was prepared using peripheral lymphocytes from a vaccinated volunteer. The 5E4 showed viral neutralization activity and inhibited hemagglutination. 5E4 escape mutants harbored amino acid substitutions (A189T and D190E) in the hemagglutinin (HA) protein, suggesting that 5E4 recognized the antigenic site Sb in the HA protein. To study the diversity of Sb in A(H1N1)pdm09, 58 viral isolates were obtained during the 2009/10 and 2010/11 winter seasons in Osaka, Japan. Hemagglutination-inhibition titers were significantly reduced against 5E4 in the 2010/11 compared with the 2009/10 samples. Viral neutralizing titers were also significantly decreased in the 2010/11 samples. By contrast, isolated samples reacted well to ferret anti-A(H1N1)pdm09 serum from both seasons. Nonsynonymous substitution rates revealed that the variant Sb and Ca2 sequences were being positively selected between 2009/10 and 2010/11. In 7,415 HA protein sequences derived from GenBank, variants in the antigenic sites Sa and Sb increased significantly worldwide from 2009 to 2013. These results indicate that the antigenic variants in Sb are likely to be in global circulation currently.</p> </div

Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus

<div><p>Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus.</p> </div

Characterization of HuMAb 5E4.

<p>(A) Nucleotide and amino acid sequences of the V_H and V_L of HuMAb 5E4. The closest germline sequences to the V_H and V_L of HuMAb 5E4 in the NCBI database, found by IgBLAST software, were aligned. Complementarity-determining regions (CDRs) are indicated in red, blue and pink (CDRs 1, 2 and 3, respectively). V-D and D-J junctions in VH and V-J junctions in VL are highlighted in white, gray and yellow, respectively. (B) Epitope region of HuMAb 5E4 in structural model of HA in A(H1N1)pdm09. A trimer complex is shown in surface representation with the antigenic sites highlighted: Sa in yellow, Sb in blue, Ca1 in cyan, Ca2 in orange and Cb in green. The epitope regions, A189 and D190, are shown in pink. The receptor binding pocket is circled. (C) AC-ELISA using wild-type viruses (open bars) and escape mutants (solid bars) of A/Suita/1/2009 (left panel) and A/Osaka/63/2009 (right panel). MuMAb C179 and HuMAb 5E4 were used as the coating and detecting antibodies, respectively. All data are represented as the means ± s.d. of three independent experiments. Asterisks denote <i>P</i><0.01.</p

Genetic analysis of HA sequences of 7,415 A(H1N1)pdm09 isolates collected throughout the world, obtained from GenBank between April 2009 and January 2013.

<p>(A) The distribution of epidemics of A(H1N1)pdm09 globally. A total of 7,415 HA sequences of A(H1N1)pdm09 were aligned individually according to their date of isolation (between April 2009 and January 2013). They are divided into six periods: Periods 1 to 6. (B) The ratios of amino acid mutants (bar charts) and the Shannon indices (line charts) at each of the antigenic sites (Sa, Sb, Ca1, Ca2, and Cb) in the HA protein during Periods 1 to 6. The amino acid sequences in these antigenic sites were aligned and their mutation rates estimated (with respect to the dominant sequence in Period 1). *<i>P</i><0.05 and **<i>P</i><0.01.</p

The rates of nonsynonymous and synonymous substitutions of the coding region of HA1 derived from viral isolates during 2009 to 2011 in Japan.

<p>(A) The HA1 gene in the 58 viral strains for which data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077892#pone-0077892-g002" target="_blank">Figure 2A</a> was direct-sequenced and aligned with HA1 in A/California/7/2009 as a control. The rates of nonsynonymous and synonymous substitutions of the coding region of the HA1 gene were calculated for each 51 bp window (sliding in 3 bp increments) and are indicated by red and black colors, respectively. Colored bars mark the antigenic sites, as follows: Sa (Sa-1 and -2) in yellow, Sb in blue, Ca1 (Ca1-1, -2 and -3) in cyan, Ca2 (Ca2-1 and -2) in orange and Cb in green. (B) The nonsynonymous (red) and synonymous (black) substitution rates within nine regions of five antigenic sites were also calculated.</p

The reactivity of viral isolates in Japan to HuMAb 5E4 and anti-A(H1N1)pdm09 ferret serum.

<p>(A) Thirty and twenty-eight viral isolates obtained in the 2009/10 and 2010/11 winter seasons, respectively, in Osaka, Japan were titrated for HI and VN₅₀ activities using 5E4 and ferret serum. Colored dots correspond to the individual viral isolates for which kinetic data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077892#pone-0077892-g002" target="_blank">Figure 2B</a>. The y-axis shows the reciprocal antibody dilutions. Asterisks denote <i>P</i><0.01. (B) Viral replication kinetics were examined in six randomly selected viral isolates. MDCK cells were infected with the isolates at a multiplicity of infection (MOI) of 1 (left panel) or 0.01 (right panel). At 5, 10, 24 and 48 hours post-infection, the supernatant was titrated by focus-forming assay.</p