ABSTRACT
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. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417–427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177, and 180 in the Sa site, residues 170, 173, 202, 206, 210, 211, and 212 in the Sb site, residues 151, 154, 156, 157, 158, 159, 200, and 238 in the Ca2 site, and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb), and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain.
IMPORTANCE The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating with swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope.
INTRODUCTION
The first influenza pandemic of the 21st century occurred in 2009. The pandemic strain, a novel swine-derived, triple reassortant A(H1N1)pdm09 (pdm09) virus, contained hemagglutinin (HA) that genetically originated with the 1918 Spanish influenza virus (1). Although the pdm09 virus was predominant in the world in the 2009/2010 and 2010/2011 influenza seasons, the A(H3N2) virus became predominant during the 2011/2012 and 2012/2013 seasons (2, 3) (see also the “Influenza virus activity in the world” website [http://www.who.int/influenza/gisrs_laboratory/updates/summaryreport_20120706/en/] and the “FluNet Summary” website [http://www.who.int/influenza/gisrs_laboratory/updates/summaryreport/en/]). The H1N1 virus was the second virus to originate with the 1918 virus, following the Russian influenza virus in 1977 (4). In the case of the Russian influenza in early 1978, most of the isolates in South America exhibited antigenic drift away from the prototype virus, A/USSR/90/77 (5). However, from 2009 to 2013, no antigenic drift was observed for the pdm09 virus, although isolates with amino acid substitutions in their antigenic sites were detected (6, 7). In 2010, viruses with double mutations in HA (N142D/E391K) were found with increased frequency in the Southern Hemisphere (7), and it was suggested that the double mutations N142D/E391K and N142D/N173K might be associated with a reduction in the ability of vaccine sera to recognize the pdm09 virus (7, 8). Furthermore, the N173K mutation has been shown to emerge under vaccine-induced immune pressure in a ferret model of contact transmission (9). However, such viruses had not been dominant until 2013.
Antigenic mapping of H1 subtype HA was performed on A/PR/8/34 HA (PR8 HA) using variants selected by monoclonal antibodies (MAbs), revealing the existence of four major antigenic sites, Sa, Sb, Ca, and Cb, in HA1 (10, 11). HAs of the pdm09 and A/PR/8/34 viruses originate with the Spanish influenza virus. However, pdm09 HA is directly derived from an American “triple reassortant” possessing the HA of classical swine influenza viruses (12); therefore, the antigenic regions of pdm09 HA and PR8 HA are not necessarily identical. To take precautions against antigenic drift of the pdm09 virus in the near future, it is important to determine the precise antigenic structure of pdm09 HA. In response to the 2009 pandemic, several groups elucidated the antigenic region of pdm09 HA using HA MAbs; however, a systemic analysis of the epitopes has not previously been performed (13–16).
In this study, to precisely identify antigenic regions, we have selected escape mutants of A/Narita/1/2009, the first isolate of the pdm09 virus in Japan, using 16 anti-HA MAbs. For systemic analysis of the epitopes of each MAb, we generated several parent viruses, as we did in our previous study, by considering the mutation rate during the growth of a plaque (17, 18). Thus, by using one MAb, we obtained a maximum of nine escape mutants possessing a single mutation at different positions. Finally, we isolated 599 escape mutants and identified the components of the epitopes of the 16 MAbs at four antigenic sites by cross-reactions of the escape mutants with anti-A/Narita/1/2009 HA MAbs.
MATERIALS AND METHODS
Viruses and cells.
A/Narita/1/2009, a pdm09 virus, was isolated from MDCK cells and embryonated chicken eggs, which were infected with a clinical sample. The amino acid sequences of HA genes of the MDCK isolate (accession no. ACR09395) and egg isolate (accession no. ACR09396) viruses are identical. In this study, the MDCK isolate virus was propagated in MDCK cells in Dulbecco's modified Eagle medium supplemented with 0.3% bovine serum albumin and 2 μg/ml acetyl-trypsin (Sigma). Four different clones, P1, P2, P3, and P4, were obtained from well-isolated plaques of MDCK cells infected with A/Narita/1/2009 and used as parent viruses. The amino acid sequence of each parent HA was identical to that in the database (accession number ACR09395). Subsequently, seven pdm09 viruses isolated from MDCK cells from clinical samples were used for antigenic analysis: A/Yamagata/232/2009 (accession no. AB601602), A/Yamagata/752/2009 (AB601604), A/Yamagata/143/2010 (AB601605), A/Yamagata/203/2011 (AB898075), A/Yamagata/206/2011 (AB898076), A/Yamagata/264/2012 (AB898077), and A/Yamagata/87/2013 (AB898078). To examine the cross-reactivity of MAbs with seasonal H1N1 virus HA, purified HA proteins of A/Solomon Islands/3/2006 and A/Brisbane/59/2007 (Denka Seiken Co., Ltd., Tokyo, Japan) were used.
Antibodies.
BALB/c mice were subcutaneously primed with 20 μg of inactivated A/Narita/1/2009 virus twice within a 3-week interval, and splenocytes were fused with Sp2/O myeloma cells at day 3 after the boosting. After limiting serial dilutions, hybridoma cells binding to A/Narita/1/2009 HA but not to A/Brisbane/59/2007 HA (H1N1) were selected by enzyme-linked immunosorbent assay (ELISA). This study was approved by the Institutional Animal Care and Use Committee of the National Institute of Infectious Diseases, Japan, and all mice were used in accordance with their guidelines.
In this study, we characterized the epitopes of the following 16 monoclonal antibodies (MAbs): NSP18 (n2), NSP30 (n3), NSP21 (n4), NSP2 (n5), NSP19 (n6), NSP22 (n7), NSP24 (n8), NSP20 (n9), NSP6 (n10), NSP26 (n11), NSP11 (n12), NSP8 (n13), NSP29 (n15), NSP27 (n16), NSP17 (n17), and NSP10 (n18). Postinfection ferret antiserum against A/California/7/2009 was used for serological assays.
Neutralization test.
The virus neutralization test was performed using 6-well microplates. Mixtures of 2-fold serial dilutions of each MAb and 100 PFU of A/Narita/1/2009 were incubated for 30 min at 37°C and used to inoculate MDCK cells. After 1 h, an agar overlay medium was added, and the cells were incubated at 37°C for 3 days. Neutralization titers are presented as reciprocals of the highest antibody dilution causing a >50% reduction of plaque number in 100 PFU of A/Narita/1/2009 virus.
Selection of escape mutants.
The escape mutants were selected by incubating each parent virus with MAbs against A/Narita/1/2009 HA, essentially following the procedure of a previous study (17, 19). Briefly, a 10-fold serial dilution of each parental virus (P1, P2, P3, and P4) was mixed with an equal volume of a 1:10 dilution of ascites fluid containing MAbs. After incubation for 30 min at room temperature, the virus-antibody mixture was inoculated onto the MDCK cells and an agar overlay medium was added. Ten nonneutralized plaque viruses per experiment were isolated and amplified in MDCK cells. The nucleotide sequence of each mutant HA gene was determined, and the deduced amino acid sequence was compared with that of A/Narita/1/2009 HA. The antigenic character of each isolate was examined using a hemagglutination inhibition (HI) test.
Nucleotide sequencing.
The nucleotide sequences of the HA genes were directly determined from RT-PCR products using the ABI Prism 3130 sequencer (Applied Biosystems). The sequences of HA primers will be provided upon request.
Radioisotope labeling and immunoprecipitation.
Monolayers of MDCK cells infected with stock virus were incubated with (+TM) or without (−TM) tunicamycin (2 μg/ml) at 37°C. At 7 h postinfection, the cells were pulsed for 30 min (−TM) or 60 min (+TM) with 4 MBq/ml [35S]methionine (ARC) and then disrupted as described previously (19). Immunoprecipitates of MAb n17 were analyzed by SDS-PAGE on 15% gels containing 4 M urea and processed for analysis by fluorography.
RESULTS
Selection of escape mutants of A/Narita/1/2009.
We have generated 16 MAbs with neutralizing activity against A/Narita/1/2009 HA. The HI titers and neutralization titers of the MAbs are shown in Table 1. To localize the antigenic sites on pdm09 HA, we selected escape mutants of the A/Narita/1/2009 strain using these MAbs. Four parent viruses (P1, P2, P3, and P4) were neutralized with each of the 16 MAbs, and nonneutralized plaque viruses were then analyzed as described in Materials and Methods. Escape mutants resistant to each MAb were isolated with a frequency ranging from 10−2.97 to 10−4.84 (Table 1). Consequently, we isolated 599 escape mutants with a single amino acid substitution in the HA1 domain; 157, 147, 148, and 147 mutants were derived from P1, P2, P3, and P4, respectively. These mutants were classified into 43 groups, which had different amino acid substitutions at 24 positions. By neutralizing the parent viruses derived from more than one plaque, we were able to obtain various mutants carrying different mutations (Table 2).
TABLE 1.
Characterization of MAbs and selection frequencies of escape mutants
| MAba | HI titerb | NT50c | Selection frequency (−log10)d |
|||
|---|---|---|---|---|---|---|
| P1 | P2 | P3 | P4 | |||
| n2 | 1,280 | 8,000 | 4.41 | 4.54 | 4.29 | 4.06 |
| n3 | 25,600 | 80,000 | 4.15 | 4.64 | 4.57 | 4.11 |
| n4 | 25,600 | 80,000 | 3.14 | 4.30 | 4.30 | 3.83 |
| n5 | 12,800 | 40,000 | 3.11 | 4.27 | 4.23 | 4.22 |
| n6 | 3,200 | 20,000 | 3.13 | 4.40 | 3.99 | 4.21 |
| n7 | 640 | 2,000 | 3.05 | 4.40 | 4.02 | 3.57 |
| n8 | 160 | 400 | 2.97 | 4.22 | 4.09 | 3.75 |
| n9 | 12,800 | 40,000 | 3.28 | 4.13 | 3.79 | 3.70 |
| n10 | 6,400 | 20,000 | 3.41 | 3.85 | 3.89 | 3.62 |
| n11 | 1,600 | 10,000 | 3.07 | 3.86 | 3.76 | 3.60 |
| n12 | 25,600 | 100,000 | 4.08 | 3.96 | 4.32 | 4.13 |
| n13 | 640 | 4,000 | 3.30 | 3.77 | 3.44 | 3.58 |
| n15 | 25,600 | 1,600 | 4.02 | 4.40 | 3.03 | 3.38 |
| n16 | 25,600 | 16,000 | 4.19 | 4.69 | 3.74 | 3.27 |
| n17 | 25,600 | 100,000 | 4.00 | 4.84 | 4.37 | 4.02 |
| n18 | 12,800 | 80,000 | 4.00 | 4.31 | 4.06 | 4.76 |
Nomenclatures for each MAbs are described in Text.
An HI test was performed on A/Narita/1/2009 with 0.5% turkey erythrocytes. The HI titer was expressed as the reciprocal of the highest antibody dilution which completely inhibited hemagglutination.
Fifty percent neutralization titers (NT50s) are presented as reciprocals of the highest antibody dilution causing a >50% plaque reduction, as described in the text.
Tenfold serial dilutions of each parent virus (P1 to P4) were mixed with 10-fold dilutions of each MAb. Escape mutants were isolated at the indicated frequency.
TABLE 2.
Amino acid mutations in HAs of escape mutants with their isolation efficiencies and their effects on reactivity with each MAb
| Amino acid change of escape mutants | No. of escape mutants derived from parent virus |
MAb(s) used for selection of escape mutants | Hemagglutination inhibition titer of MAba or of ferret antiserum against pdm09 virus |
||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MAb at site: |
Ferret antiserum against A/California/7/2009 | ||||||||||||||||||||||
| Sa |
Sb |
Ca2 |
Pa; n2 | ||||||||||||||||||||
| P1 | P2 | P3 | P4 | Total | n3 | n4 | n5 | n6 | n7 | n8 | n9 | n10 | n11 | n12 | n13 | n15 | n16 | n17 | n18 | ||||
| None (parent virus [P3]) | 12,800 | 12,800 | 12,800 | 3,200 | 640 | 160 | 12,800 | 6,400 | 2,560 | 25,600 | 640 | 25,600 | 25,600 | 25,600 | 25,600 | 640 | 1,280 | ||||||
| P141T | 3 | 2 | 5 | n9, n10, n11, n13 | — | —/0 | — | — | — | — | 0 | 0 | 0 | 0 | 0 | — | — | — | — | — | NTb | ||
| P141L | 3 | 3 | 6 | n3, n8, n12 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | — | — | NT | ||
| N142D | 10 | 3 | 10 | 2 | 25 | n3, n4, n6, n7, n9, n11, n12 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | — | — | 1,280 |
| H143Y | 2 | 2 | n7 | — | 0 | 0 | 0 | — | — | — | — | — | — | — | — | — | — | — | — | NT | |||
| K171E | 1 | 3 | 11 | 15 | n4–n9, n11, n12 | —/0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | — | — | NT | |
| K171N | 8 | 8 | 25 | 41 | n3–n13 | —/0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | — | — | 1,280 | |
| K171T | 2 | 1 | 6 | 9 | n3, n4, n9, n10, n12, n13 | —/0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | — | — | NT | |
| K171Q | 5 | 5 | n12, n13 | — | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | — | — | NT | |||
| G172E | 40 | 71 | 45 | 52 | 208 | n2, n3–n13 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | — | —/0 | 1,280 |
| G172V | 3 | 4 | 7 | n12 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | — | NT | ||
| S174L | 51 | 1 | 1 | 53 | n4–n11 | — | 0 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | — | — | — | — | — | NT | |
| K177N | 2 | 1 | 1 | 4 | n10, n11 | — | — | — | — | — | — | —/0 | 0 | — | 0 | — | — | — | — | — | — | NT | |
| K177T | 1 | 2 | 3 | n11–n13 | — | — | — | — | — | — | 0 | 0 | —/0 | 0 | — | — | — | — | — | — | 2,560 | ||
| K177E | 1 | 2 | 3 | n10 | — | — | — | — | — | — | 0 | 0 | —/0 | 0 | — | — | — | — | — | — | NT | ||
| K180E | 1 | 1 | 2 | n3, n7 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | — | — | — | — | — | — | — | 1,280 | ||
| K180N | 1 | 1 | n7 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | — | — | — | — | — | — | — | NT | |||
| K170E | 2 | 3 | 10 | 3 | 18 | n2, n6, n8, n15, n16 | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | — | —/0 | NT |
| K170Q | 1 | 1 | n16 | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | — | — | NT | |||
| K170T | 1 | 1 | n15 | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | — | — | NT | |||
| N173I | 2 | 1 | 3 | n9, n15 | — | — | — | — | — | — | 0 | 0 | 0 | 0 | 0 | 0 | 0 | — | — | — | 640 | ||
| N173D | 1 | 6 | 29 | 5 | 41 | n9–n11, n13, n15, n16 | — | — | — | — | — | — | 0 | 0 | —/0 | — | — | 0 | 0 | — | — | — | NT |
| S202N | 2 | 2 | n16 | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | — | — | 1,280 | |||
| Q206E | 11 | 1 | 12 | n15, n16 | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | — | — | NT | ||
| Q210L | 1 | 1 | n15 | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | — | — | NT | |||
| Q210E | 1 | 1 | n11 | — | — | — | — | — | — | — | — | — | — | — | 0 | — | — | — | — | NT | |||
| N211D | 9 | 9 | n10, n11 | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | — | — | NT | |||
| A212E | 17 | 5 | 3 | 18 | 43 | n15, n16 | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | — | — | 2,560 |
| A151V | 1 | 1 | n17 | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | — | — | NT | |||
| A151G | 2 | 3 | 5 | n17, n18 | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | 640 | ||
| P154S | 1 | 2 | 1 | 4 | n18 | — | — | — | — | — | — | — | — | — | — | — | — | — | —/0 | 0 | — | NT | |
| P154T | 2 | 2 | n18 | — | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | — | NT | |||
| A156V | 1 | 1 | n17 | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | — | — | NT | |||
| A156T | 1 | 4 | 5 | n17 | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | — | — | NT | ||
| A156D | 5 | 4 | 4 | 13 | n17, n18 | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | NT | |
| G157E | 15 | 8 | 4 | 3 | 30 | n17, n18 | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | NT |
| G157R | 1 | 1 | n18 | — | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | — | NT | |||
| A158E | 2 | 2 | n17, n18 | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | — | 1,280 | |||
| A158V | 1 | 1 | n18 | — | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | — | NT | |||
| K159N | 2 | 2 | n17 | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | 0 | 0 | 1,280 | |||
| S200P | 1 | 1 | 2 | n17, n18 | — | — | — | — | — | — | — | — | — | — | — | — | — | 0 | —/0 | — | 1,280 | ||
| R238K | 2 | 2 | n18 | — | — | — | — | — | — | — | — | — | — | — | — | — | — | —/0 | — | NT | |||
| K147N | 2 | 1 | 3 | n2 | — | — | — | — | — | — | — | — | — | — | — | — | — | — | —/0 | 0 | NT | ||
| K147Q | 4 | 4 | n2 | — | — | — | — | — | — | — | — | — | — | — | — | — | — | —/0 | 0 | 2,560 | |||
| Total no. of mutants | 157 | 147 | 148 | 147 | 599 | ||||||||||||||||||
An HI test was performed with 0.5% turkey erythrocytes. The HI titer was expressed as the reciprocal of the highest antibody dilution which completely inhibited hemagglutination. —, 2- to 4-fold-lower or -higher HI titer than that with the parent virus. —/0, 8-fold lower HI titer than that with the parent virus. 0, at least 16-fold lower HI titer than that with the parent virus.
NT, not tested.
Antigenic sites revealed by reactivities of MAbs with escape mutants in HI tests.
In Fig. 1, the 24 positions are shown on the primary sequence of A/Narita/1/2009 HA1 and compared with the corresponding positions within 1918 Spanish influenza virus HA, seasonal H1N1 virus HA, and A/PR/8/34-Mt. Sinai strain HA (PR8-Mt.Sinai HA) (10).
FIG 1.
Comparison of the antigenic sites of HA1 among H1N1 viruses. Alignments of the amino acid sequences of the HA1 region of A/Narita/1/2009 (GenBank accession no. ACR09395), A/California/4/2009 (FJ966082), A/California/7/2009 (FJ966974), A/South Carolina/1/1918 (AF117241), A/Solomon Islands/3/2006 (EU124177), A/Brisbane/59/2007 (CY030230), and A/Puerto Rico/8/1934 (Mt. Sinai) (AF389118) are shown using H1 numbering from the first methionine in the signal peptide. Each antigenic site is shown with the corresponding symbol: ◼, Sa; ◆, Sb; ▶, Ca1; ◀, Ca2; ●, Cb; □, Pa. Symbols for A/Narita/1/2009 and A/Puerto Rico/8/1934 are shown above and below the sequence, respectively.
The reactivity of each MAb with the panel of escape mutants was examined using an HI test (Table 2). Based on their reactivities, the MAbs were assembled into four complementary groups: Sa (n3 to n13), Sb (n15 and n16), Ca2 (n17 and n18), and n2.
(i) Site Sa.
Single amino acid changes at positions 141, 142, 171, 172, and 180 in the Sa site affected the reactions of the mutants with MAbs n3 to n8, whereas the reactivities of MAbs n9 to n13 with the mutants was affected by amino acid substitutions at positions 141, 142, 171, 172, and 177 (except for n13) at site Sa. Further mutations at positions 143 and 174 in the Sa site and position 173 in the Sb site affected the reactivity with n4 to n6, n4 to n10, and n9 to n13, respectively. MAbs n9 and n10 lost reactivity when mutations were present at position 173, whereas n11 to n13 reacted with N173I but not with N173D. These results indicate that (i) the Sa site of Narita HA is composed of the residues at positions 141, 142, 143, 171, 172, 173, 174, 177, and 180, and (ii) there are two distinct regions in the Sa site that are recognized differently by MAbs n3 to n8 and MAbs n9 to n13 (Tables 2 and 3).
TABLE 3.
Amino acid substitutions of escape mutants HAs which affect reactivity with each MAb
| MAb | Amino acid substitution(s)a in each antigenic site |
|||
|---|---|---|---|---|
| Sa site | Sb site | Ca2 site | Pa site | |
| n3 | P141L; N142D; K171E; K171N; K171T; G172E; G172V; K180E; K180N | |||
| n4 | P141T; P141L; N142D; H143Y; K171E; K171N; K171T; K171Q; G172E; G172V; S174L; K180E; K180N | |||
| n5 | P141L; N142D; H143Y; K171E; K171N; K171T; K171Q; G172E; G172V; S174L; K180E; K180N | |||
| n6 | P141L; N142D; H143Y; K171E; K171N; K171T; K171Q; G172E; G172V; S174L; K180E; K180N | |||
| n7 | P141L; N142D; K171E; K171N; K171T; K171Q; G172E; G172V; S174L; K180E; K180N | |||
| n8 | P141L; N142D; K171E; K171N; K171T; K171Q; G172E; G172V; S174L; K180E; K180N | |||
| n9 | P141T; P141L; N142D; K171E; K171N; K171T; K171Q; G172E; G172V; S174L; K177N; K177T | N173I; N173D | ||
| n10 | P141T; P141L; N142D; K171E; K171N; K171T; K171Q; G172E; G172V; S174L; K177N; K177T | N173I; N173D | ||
| n11 | P141T; P141L; N142D; K171E; K171N; K171T; K171Q; G172E; G172V; K177T; K177E | N173I; N173D | ||
| n12 | P141T; P141L; N142D; K171E; K171N; K171T; K171Q; G172E; G172V; K177N; K177T; K177E | N173I | ||
| n13 | P141T; P141L; N142D; K171E; K171N; K171T; K171Q; G172E; G172V | N173I | ||
| n15 | G172V | K170E; K170Q; K170T; N173I; N173D; S202N; Q206E; Q210L; Q210E; N211D; A212E | ||
| n16 | K170E; K170Q; K170T; N173I; N173D; S202N; Q206E; Q210L; N211D; A212E | |||
| n17 | A151V; A151G; P154S; A156V; A156T; A156D; G157E; A158E; K159N; S200P | |||
| n18 | A151G; P154S; P154T; A156D; G157E; G157R; A158E; A158V; K159N; S200P; R238K | K147N; K147Q | ||
| n2 | G172E | K170E | K159N | K147N; K147Q |
All mutations that decreased the HI titer of each MAb at least 8-fold compared to the titer with the parent virus.
(ii) Site Sb.
Mutations at positions 170, 173, 202, 206, 210, 211, and 212 in site Sb affected reactivity with n15 and n16. Interestingly, n15 showed decreased reactivity with G172V (Sa site) but reacted with G172E. Similar to residue 173, residue 172 also belonged to epitopes in sites Sa and Sb, depending on the amino acid residue (Tables 2 and 3).
In this study, single-mutation mutants containing H143Y (Sa site) or Q210E or N211D (Sb site) did not react with MAbs n4 to n6, n15, or n15/n16, respectively, as shown in the HI test. However, they reacted well with MAbs n7, n11, and n10/n11, respectively, even these MAbs were used for the selection of those mutants (Table 2).
(iii) Site Ca2.
In this study, two epitopes of n17 and n18 were identified in the Ca2 site. The HI reactivity of n17 and n18 with each escape mutant indicated that their epitopes are composed of residues at positions 151, 154, 156, 157, 158, 159, 200, and 238; however, the reactivity of each MAb differed based on the substituted amino acid residue, which implies that the amino acid sequences are different for the paratope of each MAb (Tables 2 and 3).
(iv) Site Pa.
Three escape mutants, each possessing a single mutation at position 147, 170, or 172, were selected by MAb n2. Moreover, n2 showed decreased reactivity with the other mutant, carrying a K159N change, which was selected by n17 (Table 2). The n2 epitope presumably consisted of the residues at positions 147, 159, 170, and 172, and the last three residues were located in the Ca2, Sb, and Sa sites, respectively (Table 3). A similar epitope consisting of residues K136, D144, K147, G148, K171, and G172 has been identified for the human monoclonal antibody EM4C04, which was derived from a patient infected with the pdm09 virus (13, 15). We designated this novel antigenic site Pa.
The reactivity of ferret antiserum against A/California/7/2009 with the representative escape mutants related to each antigenic site was also examined by the HI test. No mutants showed reduced reactivity with the polyclonal ferret antiserum compared to that of the parent virus (Table 2).
Characteristics of the antigenic structure of pdm09 HA.
Figure 2 shows the antigenic sites of A/Narita/1/2009 HA (Fig. 2). The epitopes for the investigated MAbs, except for epitope n2, were localized within the Sa, Sb, and Ca2 antigenic sites. In this study, no MAbs against Ca1 or Cb were identified. The unique positions of the amino acid substitutions in the escape mutants of A/Narita/1/2009 were 147 and 200. In the three-dimensional (3-D) structure, residue 200 of site Ca2 was located next to the residues of site Sb but was far from the other components of site Ca2 in the 3-D structure (Fig. 2). Although the S200P mutation affected the reactivity with n17 and n18, this influence may be attributed to a conformational change at topologically distant sites rather than to a direct interaction between the epitope and the paratope.
FIG 2.
Antigenic structure of HA of A/Narita/1/2009. Positions of amino acid changes in the escape mutants are located on the globular head of HA of A/Narita/1/2009, shown with numbering. The three-dimensional structure of A/Narita/1/2009 HA was generated by the protein structure homology-modeling server “SWISS MODEL” using the coordinates of A/California/4/2009 (PDB ID 3al4A). The image was created using the software program PyMOL. The residues at each antigenic site are colored as pink for the Sa site, sky blue for the Sb site, green for the Ca2 site, and orange for residue 147. The residues discussed in the text are colored yellow.
Residue 147 is located near the right edge of the receptor-binding site and is near the other members of epitope n2, including residues 159, 170, and 172, in the 3-D structure. This epitope was not detected in the antigenic site of PR8-Mt. Sinai HA or several seasonal influenza viruses (H1N1) that circulated during and after 1994/1995 because of the lack of residue 147. However, because of the presence of residue 147, the Pa site may exist in the Spanish influenza virus, PR8-Cambridge strain, and seasonal H1N1 viruses that were isolated before 1994.
Acquisition of a glycosylation site is known to shield epitopes from antibody recognition. In the seasonal influenza H1N1 virus, two highly conserved glycosylation sites, at positions 142 and 177, were identified in the Sa site, but both sites were absent in pdm09 HA. In this study, a new oligosaccharide attachment site, K177N, was generated in an escape mutant that was selected by MAbs n10 and n11. However, this mutant and others with K177T or K177E all reacted with the MAbs targeting site Sa. Thus, to determine whether the HA mutant with K177N was modified with N-linked glycans, we compared the electrophoretic mobilities of the mutant HAs, i.e., those with K177N and K177T, in SDS-polyacrylamide gels. As shown in Fig. 3, in the absence of TM, the parental virus and both mutant HAs showed slower electrophoretic mobility than in the presence of TM, but the mobility of these HAs was similar, suggesting that an additional glycosylation did not occur at position 177 in the mutant carrying N177.
FIG 3.
Immunoprecipitation analysis of the HA molecules of escape mutants carrying amino acid substitutions at position 177. MDCK cells infected with parental virus (lanes 1 and 4) and mutants with the K177T (lanes 2 and 5) and K177N (lanes 3 and 6) substitutions were labeled with [35S]methionine for 30 min in the absence of tunicamycin (lanes 1 to 3) or for 60 min in the presence of tunicamycin (lanes 4 to 6) at 7 h postinfection. Cells were immunoprecipitated with MAb n17 to site Ca2, and the resulting precipitates were analyzed by SDS-PAGE.
Antigenic characterization of pdm09 HA viruses from 2009 to 2013.
To detect signs of antigenic drift, genetic and antigenic analyses have been extensively performed for pdm09 isolates obtained from 2009 to 2013 (6–8). Compared with the pdm09 virus, various mutations were found at the antigenic sites of these natural isolates; however, significant antigenic differences from the A/California/7/2009 vaccine strain were not found when using ferret antiserum against A/California/7/2009 (6, 7).
To confirm that the mutations in the antigenic sites of the natural isolates affected the reactivities of the corresponding MAbs, cross-reactions of the MAbs were investigated using natural isolates obtained in Yamagata Prefecture, Japan, from 2009 to 2013. Table 4 shows the reactivities of the representative MAbs with seven representative pdm09 isolates. Among the isolates from 2009/2010, A/Yamagata/232/2009 and A/Yamagata/143/2010 reacted well with all of the investigated MAbs, whereas A/Yamagata/752/2009 failed to react with MAbs to site Sa and showed an 8-fold-lower reactivity with n2 than did A/Narita/1/2009, possibly because of the G172E mutation (sites Sa and Pa). Two other isolates from 2010-2011, A/Yamagata/203/2011 and A/Yamagata/206/2011, exhibited no or decreased reactivity with n17/n18 and n16, respectively, possibly because of the A151T/A158S/S200P (site Ca2) and S202T (site Sb) mutations. Two isolates obtained in 2012-2013, A/Yamagata/264/2012 and A/Yamagata/87/2013, showed low reactivity with n16 and n3/n7/n16, presumably because of S202T (site Sb) and K180I/S202T (site Sa/Sb), respectively.
TABLE 4.
Antigenic analysis of pdm09 natural isolates in Yamagata Prefecture and seasonal H1N1 viruses using representative MAbs
| Virus strain | Amino acid change(s)a | HI titerb |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| MAbs of respective antigenic site |
Postinfection ferret antiserum against A/California/7/2009 | ||||||||
| Sa |
Sb |
Ca2 |
Pa |
||||||
| n3 | n7 | n10 | n16 | n17 | n18 | n2 | |||
| pdm09 natural isolates | |||||||||
| A/Narita/1/2009 | 12,800 | 640 | 6,400 | 25,600 | 25,600 | 25,600 | 640 | 1,280 | |
| 2009-2010 season | |||||||||
| A/Yamagata/232/2009 | S220T | 6,400 | 320 | 3,200 | 25,600 | 12,800 | 12,800 | 640 | 1,280 |
| A/Yamagata/752/2009 | G172E, K188R, S220T, A214T | <c | < | < | 6,400 | 6,400 | 3,200 | 80 | 640 |
| A/Yamagata/143/2010 | V36I, D52G, A203T, S220T | 12,800 | 320 | 3,200 | 12,800 | 12,800 | 25,600 | 320 | 640 |
| 2010-2011 season | |||||||||
| A/Yamagata/203/2011 | V64I, A151T, A158S, S200P, S220T, I312V | 6,400 | 640 | 6,400 | 12,800 | < | 160 | 640 | 1,280 |
| A/Yamagata/206/2011 | K136N, S160G, S202T, S220T, A214T | 6,400 | 640 | 6,400 | 1,600 | 2,560 | 2,560 | 640 | 1,280 |
| 2012-2013 season | |||||||||
| A/Yamagata/264/2012 | D114N, S202T, S220T, F227S, V251I, K300E | 6,400 | 320 | 1,600 | 320 | 12,800 | 12,800 | 320 | 640 |
| A/Yamagata/87/2013 | S101G, S160G, K180I, S202T, A214T, S220T | 20 | 80 | 1,600 | 320 | 12,800 | 12,800 | 320 | 640 |
| Seasonal H1N1 virusesd | |||||||||
| A/Solomon Islands/3/2006 | < | < | < | < | 40 | 20 | < | NTe | |
| A/Brisbane/59/2007 | < | < | < | < | < | < | < | NT | |
Amino acid differences in HA1 between pdm09 natural isolates and A/Narita/1/2009 are shown.
The HI test was performed with 0.5% turkey erythrocytes. The HI titer is expressed as the reciprocal of the highest antibody dilution which completely inhibited hemagglutination.
<, less than 20.
Amino acid sequences of seasonal H1N1 viruses are shown in Fig. 1.
NT, not tested.
To determine whether any MAbs exhibit cross-reactivity with seasonal H1N1 viruses, we also examined the reactivities of 16 MAbs with A/Solomon Island/3/2006 and A/Brisbane/59/2007 HAs. No MAbs reacted well with these seasonal H1N1 virus HAs (Table 4).
During 2009 to 2013, amino acid changes at positions 202 and 220 were found in the main stream of the evolutionary pathway of these natural isolates of HA1 in Yamagata Prefecture (Fig. 4). According to the antigenic analysis of the viruses shown in Table 4, n16 lost reactivity with a S202T mutant, whereas the other mutation, S220T, which is located in the Ca1 site of PR8 HA, did not affect the reactivities of the viruses with the MAbs that were used in this study. These results show that the mutations in the antigenic site of the natural variants predictably affected reactivity with their corresponding MAbs. However, no variant lost reactivity with the ferret postinfection antiserum against A/California/7/2009, as observed for the several escape mutants in this study (Table 2). The antibodies produced in the naive ferret infected with A/California/7/2009 therefore presumably recognize more than one antigenic site of HA.
FIG 4.
Phylogenetic tree of the HA1 polypeptide of the natural isolates from 2009 to 2013 that were used in this study. A phylogenetic tree was constructed using the neighbor-joining method in the CLUSTAL W software program, version 2.1. Numbers refer to the mainstream amino acid changes that were fixed in most of the subsequent strains.
DISCUSSION
The prototypes of the pdm09 virus are A/California/4/2009 (MDCK isolate) and A/California/7/2009 (egg isolate). The primary sequence of A/Narita/1/2009 HA1 is similar to that of the prototype viruses (Fig. 1). HA1 from A/Narita/1/2009 differs from A/California/4/2009 and A/California/7/2009 by two amino acids and one amino acid, respectively. These differences were not observed in the antigenic region, and the reactivity of the ferret antiserum against A/California/7/2009 is similar among those viruses (data not shown). Therefore, we have analyzed the antigenic structure of HA of A/Narita/1/2009, as a representative pdm09 virus, using 16 anti-HA MAbs and their escape mutants. Most epitopes of the MAbs were located in the antigenic sites Sa, Sb, and Ca2. In addition, epitope n2 was defined in a novel antigenic site (Pa) and was proximal to the receptor-binding site (Fig. 2).
Based on the antigenic analysis of PR8-Mt. Sinai HA, sites Sa and Sb are defined as operationally distinct areas that are separated by residues 170/173 of the Sb site and 172/174 of the Sa site, which lie in the same region of the polypeptide chain (10). Although sites Sa and Sb are contiguous and have been suggested to share a close linkage, simultaneous binding of antibodies to each site has not been identified (10, 11, 20). However, a study of the crystal structure of human MAb 2DI in complex with the 1918 pandemic HA demonstrated that the epitope of MAb 2DI, which is derived from survivors of the 1918 pandemic, contained the residues at positions 142 (in the Sa site) and 171 to 183 (in the Sa and Sb sites). Similarly, in this study, epitopes of mouse MAbs n9 to n13 and n15 were composed of residues in the Sa and Sb sites (16).
Based on the residues of epitopes n2 and EM4C04 (13), we have determined that the novel antigenic site (Pa) of pdm09 HA is between sites Sa, Sb, and Ca2 and residue 147. In a previous study using mouse MAbs, this epitope in the Pa site was not identified in PR8-Mt. Sinai HA (20). We therefore considered that the Pa antigenic site may be specific to pdm09 HA. Recently, epitopes of broadly neutralizing human MAbs, i.e., 5J8 and CH65, were found around the receptor-binding site (15, 16). MAb 5J8 was derived from a healthy, middle-aged woman and had HI activity against A/South Carolina/1/1918 and A/California/4/2009. Epitope 5J8 is composed of residues 147, 151, and 236, which are near epitope n2. Because residue 151 is near residue 159, epitope 5J8 may partially overlap epitope n2 and comprise the Pa site. In contrast, MAb CH65 was obtained by rearranging the heavy- and light-chain genes derived from a subject immunized with the 2007 trivalent vaccine, including the seasonal H1N1 virus lacking residue 147 of HA. The crystal structure of the complex between CH65 and A/Solomon Islands/3/2006 HA, which had a deletion of residue 147, suggested that residues 150, 151, and 240, which were located on the right edge of the receptor-binding site, were involved in the interaction. A neutralization assay showed that among the historical H1N1 viruses, the viruses with an insertion at position 147 were resistant to CH65. Taking these results into consideration, the region around position 147 is presumably a component of epitopes for human antibodies against H1HA. Considering the location of residue 147, a mutation at this position may significantly affect the receptor-binding ability. However, in this study, two escape mutants of Narita/1/2009, carrying the mutations K147N and K147Q, were isolated using MAb n2. In another study, a similar escape mutant of A/California/4/2009, with K147Q, was selected by human MAb 5J8 (15). During the circulation of the H1N1 seasonal influenza viruses in 1947 to 1957 and 1977 to 1995, only mutations K to R and R to K were recognized, and residue 147 was deleted after 1995. Similarly, residue 144, which is located near position 147 of older H1N1 viruses, was also deleted immediately before the disappearance of these viruses in 1957 (21). Deletions of residues in close proximity to the receptor-binding site of HA seem preferable for the escape of H1N1 viruses from immune selection in the human population. It is well known that the glycosylation of HA prevents its neutralization by antibodies. A previous study implied that pdm09 HA modified with additional glycosylation sites at positions 142 and 177 was resistant to neutralization by antibodies to wild-type HA (22). Therefore, glycosylation at these positions has implications for the antigenic drift of pdm09 viruses. However, as indicated in this study, the asparagine at position 177 was not glycosylated, even though the mutation affected reactivity with the MAb.
Amino acid changes at positions 170 to 174 have been identified after the cell culture adaptation of pdm09 viruses (9, 23, 24). In this study, the variants with G172E comprised one-third of all of the mutants. However, each variant lost reactivity with the MAbs that were used for the selection of the variant, suggesting that variants with G172E were not selected during adaptation to MDCK cells.
Since the emergence of the pdm09 virus as a pandemic virus in 2009, stringent surveillance of pdm09 viruses has been applied to detect antigenic variants. From 2009 to 2013, amino acid changes at positions 220 (Ca1 site) and 202 (Sb site) were found in the mainstream of the evolutionary pathway of pdm09 HA (Fig. 4). However, as shown in Table 4, hemagglutination abilities of natural isolates with the mutations S202T and S220T were efficiently inhibited by postinfection ferret sera against the A(H1N1)pdm09 virus. No antigenic differences from the vaccine strain, A/California/7/2009, were reported for the natural isolates in the various region of the world in 2013 (25, 26). In the case of the reappearance of H1N1 viruses in 1977, several antigenic variants, A/Lackland/3/78 and A/Brazil/11/78, showed 4- to 8-fold decreases in reactivity with the postinfection ferret sera against A/USSR/90/77. These variants also failed to react with MAb 264 against A/USSR/90/77 HA (27, 28). Using binding assays of MAb 264 with mutant HA, we have identified E233K as a mutation responsible for the antigenic drift of A/USSR/90/77 (29). In fact, this mutation was found in the mainstream of the evolutionary pathway (30). It is difficult to identify such a crucial mutation in antigenic variants, which usually possess extra mutations unrelated to antigenicity. In the present study, we identified the residues that composed the epitopes of 16 MAbs against A/Narita/1/2009 HA. The use of such MAbs should be helpful in determining critical amino acid substitutions for the antigenic drift of A(H1N1)pdm09 viruses, once such drift occurs. Consequently, these findings may help the WHO to make recommendations for the next vaccine virus among pdm09 H1N1 viruses.
ACKNOWLEDGMENTS
This study was supported in part by a Health Labor Sciences Research Grant, Research on Emerging and Re-emerging Infectious Diseases (to E.N.; no. H22-//Shinko-Ippan-001//) from the Ministry of Health, Labor and Welfare of Japan.
We are greatly indebted to Katsuhisa Nakajima for helpful suggestions and valuable discussions. We thank Yasushi Suzuki for preparing the image of 3-D structure and Yoko Kadowaki for her assistance with sequence analysis.
We declare that we have no potential conflicts of interests related to this article.
Footnotes
Published ahead of print 13 August 2014
REFERENCES
- 1.Smith GJ, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus OG, Ma SK, Cheung CL, Raghwani J, Bhatt S, Peiris JS, Guan Y, Rambaut A. 2009. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459:1122–1125. 10.1038/nature08182. [DOI] [PubMed] [Google Scholar]
- 2.World Health Organization. 2010. Recommended viruses for influenza vaccines for use in the 2010-2011 northern hemisphere influenza season. Wkly. Epidemiol. Rec. 85:81–92. [PubMed] [Google Scholar]
- 3.World Health Organization. 2011. Influenza A(H1N1) 2009 virus: current situation and post-pandemic recommendations. Wkly. Epidemiol. Rec. 86:61–72. [PubMed] [Google Scholar]
- 4.Nakajima K, Desselberger U, Palese P. 1978. Recent human influenza A (H1N1) viruses are closely related genetically to strains isolated in 1950. Nature 274:334–339. 10.1038/274334a0. [DOI] [PubMed] [Google Scholar]
- 5.Kendal AP, Joseph JM, Kobayashi G, Nelson D, Reyes CR, Ross MR, Sarandria JL, White R, Woodall DF, Noble GR, Dowdle WR. 1979. Laboratory-based surveillance of influenza virus in the United States during the winter of 1977-1978. I. Periods of prevalence of H1N1 and H3N2 influenza A strains, their relative rates of isolation in different age groups, and detection of antigenic variants. Am. J. Epidemiol. 110:449–461. [DOI] [PubMed] [Google Scholar]
- 6.Dapat IC, Dapat C, Baranovich T, Suzuki Y, Kondo H, Shobugawa Y, Saito R, Suzuki H. 2012. Genetic characterization of human influenza viruses in the pandemic (2009-2010) and post-pandemic (2010-2011) periods in Japan. PLoS One 7:e36455. 10.1371/journal.pone.0036455. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Barr IG, Cui L, Komadina N, Lee RT, Lin RT, Deng Y, Caldwell N, Shaw R, Maurer-Stroh S. 2010. A new pandemic influenza A(H1N1) genetic variant predominated in the winter 2010 influenza season in Australia, New Zealand and Singapore. Euro Surveill. 15:19692. [DOI] [PubMed] [Google Scholar]
- 8.Strengell M, Ikonen N, Ziegler T, Julkunen I. 2011. Minor changes in the hemagglutinin of influenza A(H1N1)2009 virus alter its antigenic properties. PLoS One 6:e25848. 10.1371/journal.pone.0025848. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Guarnaccia T, Carolan LA, Maurer-Stroh S, Lee RT, Job E, Reading PC, Petrie S, McCaw JM, McVernon J, Hurt AC, Kelso A, Mosse J, Barr IG, Laurie KL. 2013. Antigenic drift of the pandemic 2009 A(H1N1) influenza virus in A ferret model. PLoS Pathog. 9:e1003354. 10.1371/journal.ppat.1003354. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Caton AJ, Brownlee GG, Yewdell JW, Gerhard W. 1982. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell 31:417–427. 10.1016/0092-8674(82)90135-0. [DOI] [PubMed] [Google Scholar]
- 11.Gerhard W, Yewdell J, Frankel ME, Webster R. 1981. Antigenic structure of influenza virus haemagglutinin defined by hybridoma antibodies. Nature 290:713–717. 10.1038/290713a0. [DOI] [PubMed] [Google Scholar]
- 12.Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, Balish A, Sessions WM, Xu X, Skepner E, Deyde V, Okomo-Adhiambo M, Gubareva L, Barnes J, Smith CB, Emery SL, Hillman MJ, Rivailler P, Smagala J, de Graaf M, Burke DF, Fouchier RA, Pappas C, Alpuche-Aranda CM, Lopez-Gatell H, Olivera H, Lopez I, Myers CA, Faix D, Blair PJ, Yu C, Keene KM, Dotson PD, Jr, Boxrud D, Sambol AR, Abid SH, St George K, Bannerman T, Moore AL, Stringer DJ, Blevins P, Demmler-Harrison GJ, Ginsberg M, Kriner P, Waterman S, Smole S, Guevara HF, Belongia EA, Clark PA, Beatrice ST, Donis R, Katz J, Finelli L, Bridges CB, Shaw M, Jernigan DB, Uyeki TM, Smith DJ, Klimov AI, Cox NJ. 2009. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325:197–201. 10.1126/science.1176225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.O'Donnell CD, Vogel L, Wright A, Das SR, Wrammert J, Li GM, McCausland M, Zheng NY, Yewdell JW, Ahmed R, Wilson PC, Subbarao K. 2012. Antibody pressure by a human monoclonal antibody targeting the 2009 pandemic H1N1 virus hemagglutinin drives the emergence of a virus with increased virulence in mice. mBio 3(3):e00120–00112. 10.1128/mBio.00120-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Rudneva I, Ignatieva A, Timofeeva T, Shilov A, Kushch A, Masalova O, Klimova R, Bovin N, Mochalova L, Kaverin N. 2012. Escape mutants of pandemic influenza A/H1N1 2009 virus: variations in antigenic specificity and receptor affinity of the hemagglutinin. Virus Res. 166:61–67. 10.1016/j.virusres.2012.03.003. [DOI] [PubMed] [Google Scholar]
- 15.Krause JC, Tsibane T, Tumpey TM, Huffman CJ, Basler CF, Crowe JE., Jr 2011. A broadly neutralizing human monoclonal antibody that recognizes a conserved, novel epitope on the globular head of the influenza H1N1 virus hemagglutinin. J. Virol. 85:10905–10908. 10.1128/JVI.00700-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Whittle JR, Zhang R, Khurana S, King LR, Manischewitz J, Golding H, Dormitzer PR, Haynes BF, Walter EB, Moody MA, Kepler TB, Liao HX, Harrison SC. 2011. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin. Proc. Natl. Acad. Sci. U. S. A. 108:14216–14221. 10.1073/pnas.1111497108. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Nakajima S, Nakajima K, Nobusawa E, Zhao J, Tanaka S, Fukuzawa K. 2007. 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. Microbiol. Immunol. 51:1179–1187. 10.1111/j.1348-0421.2007.tb04013.x. [DOI] [PubMed] [Google Scholar]
- 18.Nobusawa E, Sato K. 2006. Comparison of the mutation rates of human influenza A and B viruses. J. Virol. 80:3675–3678. 10.1128/JVI.80.7.3675-3678.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Tsuchiya E, Sugawara K, Hongo S, Matsuzaki Y, Muraki Y, Li ZN, Nakamura K. 2001. Antigenic structure of the haemagglutinin of human influenza A/H2N2 virus. J. Gen. Virol. 82:2475–2484. [DOI] [PubMed] [Google Scholar]
- 20.Lubeck MD, Gerhard W. 1981. Topological mapping antigenic sites on the influenza A/PR/8/34 virus hemagglutinin using monoclonal antibodies. Virology 113:64–72. 10.1016/0042-6822(81)90136-7. [DOI] [PubMed] [Google Scholar]
- 21.Nakajima S, Nishikawa F, Nakajima K. 2000. Comparison of the evolution of recent and late phase of old influenza A (H1N1) viruses. Microbiol. Immunol. 44:841–847. 10.1111/j.1348-0421.2000.tb02572.x. [DOI] [PubMed] [Google Scholar]
- 22.Wei CJ, Boyington JC, Dai K, Houser KV, Pearce MB, Kong WP, Yang ZY, Tumpey TM, Nabel GJ. 2010. Cross-neutralization of 1918 and 2009 influenza viruses: role of glycans in viral evolution and vaccine design. Sci. Transl. Med. 2:24ra21. 10.1126/scitranslmed.3000799. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Yang H, Carney P, Stevens J. 2010. Structure and receptor binding properties of a pandemic H1N1 virus hemagglutinin. PLoS Curr. 2:RRN1152. 10.1371/currents.RRN1152. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Chen Z, Wang W, Zhou H, Suguitan AL, Jr, Shambaugh C, Kim L, Zhao J, Kemble G, Jin H. 2010. Generation of live attenuated novel influenza virus A/California/7/09 (H1N1) vaccines with high yield in embryonated chicken eggs. J. Virol. 84:44–51. 10.1128/JVI.02106-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.World Health Organization. 2013. Review of the 2012-2013 winter influenza season, northern hemisphere. Wkly. Epidemiol. Rec. 88:225–232. [Google Scholar]
- 26.World Health Organization. 2013. Review of the 2013 influenza season in the southern hemisphere. Wkly. Epidemiol. Rec. 88:509–520. [PubMed] [Google Scholar]
- 27.Webster RG, Kendal AP, Gerhard W. 1979. Analysis of antigenic drift in recently isolated influenza A (H1N1) viruses using monoclonal antibody preparations. Virology 96:258–264. 10.1016/0042-6822(79)90189-2. [DOI] [PubMed] [Google Scholar]
- 28.Nakajima S, Kendal AP. 1981. Antigenic drift in influenza A/USSR/90/77(H1N1) variants selected in vitro with monoclonal antibodies. Virology 113:656–662. 10.1016/0042-6822(81)90194-X. [DOI] [PubMed] [Google Scholar]
- 29.Nobusawa E, Nakajima K, Nakajima S. 1987. Determination of the epitope 264 on the hemagglutinin molecule of influenza H1N1 virus by site-specific mutagenesis. Virology 159:10–19. 10.1016/0042-6822(87)90342-4. [DOI] [PubMed] [Google Scholar]
- 30.Raymond FL, Caton AJ, Cox NJ, Kendal AP, Brownlee GG. 1983. Antigenicity and evolution amongst recent influenza viruses of H1N1 subtype. Nucleic Acids Res. 11:7191–7203. 10.1093/nar/11.20.7191. [DOI] [PMC free article] [PubMed] [Google Scholar]