Antigenic Mapping of the Hemagglutinin of an H9N2 Avian Influenza Virus Reveals Novel Critical Amino Acid Positions in Antigenic Sites Zhimin Wan, Jianqiang Ye, Liangliang Xu, Hongxia Shao, Wenjie Jin, Kun Qian, Hongquan Wan, Aijian Qin College of Veterinary Medicine, Yangzhou University, Yangzhou, China, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China
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.
H
9N2 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 (TCID50) of virus (X1) were used. All of the selected antibodies inhibited X1 virus with
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TABLE 1 Biological properties of H9-specific MAbs generated in this study MAba
Isotype
HI titer (log2)
MN titer
1C3 2G4 3B10 5B4 6A5 6A10 6B6 6E6
IgG2a IgG1 IgG2a IgG1 IgG1 IgG2a IgG1 IgG1
15 14 11 19 13 18 14 14
327,680 655,360 10,240 655,360 5,120 655,360 327,680 655,360
a
Untreated mouse ascitic fluid of each hybridoma was used in HI (see also Tables 2 and 3) and MN assays. Titers shown are the reciprocals of the highest dilutions showing HI or MN activity against X1 virus.
high titers in both the HI and MN assay (Table 1), suggesting that these MAbs are against the globular head region of H9. To identify amino acids in H9 that are critical for the MAb-HA interaction, we selected MAb escape mutants of X1 virus in embryonated chicken eggs (22). Mutants were obtained for all but 1 of the 8 MAbs used. As shown by the results in Table 2, mutants were either poorly inhibited (in the case of mutants m1C3 and m6A5, selected with MAbs 1C3 and 6A5, respectively) or not inhibited at all (the remaining mutants) by the selecting antibodies in the HI assay. When examined against MAbs other than that used for its selection, each mutant was inhibited by most if not all of the other MAbs at titers close to those of the selecting MAbs (Table 3), suggesting that the epitopes recognized are largely not identical. The exception was mutant m5B4, selected with MAb 5B4, which was efficiently inhibited by MAbs 1C3 and 3B10 but resisted inhibition by MAbs 6A5, 6A10, 6B6, and 6E6. Consistent with these results, MAb 5B4 failed to inhibit mutants m6A10, m6B6, and
Received 21 November 2013 Accepted 11 January 2014 Published ahead of print 15 January 2014 Editor: D. S. Lyles Address correspondence to Hongquan Wan,
[email protected], or Aijian Qin,
[email protected]. Z.W. and J.Y. contributed equally. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.03440-13
p. 3898 –3901
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Novel Amino Acid Positions in H9 Antigenic Sites
TABLE 2 Amino acid mutations in the HA of escape mutants selected with H9-specific MAbs Mutants
HI titer (log2)a
Mutation(s)
m1C3 m3B10 m5B4 m6A5 m6A10 m6B6 m6E6
5 — — 6 — — —
T147Kb D153N T200I, N201S A168D Q164K D196V, D207N N167K
a b
Shown are the titers obtained with each selecting MAb. —, no inhibition in HI assay. H9 numbering.
m6E6 as efficiently as it inhibited wild-type X1 virus (Table 3). These data demonstrate that MAb 5B4 recognizes an epitope that probably overlaps those recognized by MAbs 6A10, 6B6, and 6E6. The amino acid mutations in the HA of mutant viruses were identified through sequencing the HA gene (23). Five mutants, selected with MAbs 1C3, 3B10, 6A5, 6A10, and 6E6, respectively, each bore a single amino acid mutation in its HA, at position 147, 153, 168, 164, and 167, respectively (Table 2). The remaining 2 mutants each had a double mutation in the HA, T200I/N201S in mutant m5B4 and D196V/D207N in mutant m6B6 (selected with MAb m6B6). Taken together, we identified 9 critical amino acids in H9 antigenic sites. As all of the selecting MAbs exhibited efficient HI function against X1 virus (Table 1), it is not surprising that the mutations identified in our study were all distributed on the top of the HA globular head region (Fig. 1). In reference to the antigenic sites described in an earlier report (17), 4 of these mutations, T147K, Q164K, N167K, and A168D, were within antigenic site I in H9, while the remaining 5 mutations, D153N, D196V, T200I, N201S, and D207N, were located in antigenic site II. Among the 9 amino acid mutations identified, those at positions 164, 167, 168, 196, and 207 were not detected in similar studies by other groups (17, 18). Notably, these 5 novel positions are comparable to those in antigenic site B in H3 virus (24–26). 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 (29–31). We examined 1,036 full-length H9 sequences available in GenBank (as of 19 July 2013) by using multiple sequence comparison by log
TABLE 3 Cross-reactions of H9N2 escape mutants with H9-specific MAbs in HI assay a
Inhibition of virus : MAb
Wild-type X1 m1C3 m3B10 m5B4 m6A5 m6A10 m6B6 m6E6
1C3 3B10 5B4 6A5 6A10 6B6 6E6 Neg ctrlb
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺
⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺
⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺
⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺
⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺
⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺
⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺
⫹, HI titer ⱕ8-fold different from that obtained with wild-type X1 virus; ⫺, HI titer ⱖ16-fold different from that obtained with wild-type X1 virus or, for the negative control (Neg ctrl), no inhibition. b Neg ctrl, ascitic fluid containing MAb against Newcastle disease virus. a
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FIG 1 Locations of 9 critical amino acid positions in H9 antigenic sites. The 9 positions were identified by analyzing MAb escape mutants of A/Chicken/ Jiangsu/X1/2004 (H9N2) virus. The images were generated with Pymol software (Delano Scientific). Shown are the side view (A) and top view (B) of the locations of these positions on an H9 monomer (PDB ID 1JSD). The 5 novel positions identified in this study are highlighted in green, while the remaining 4 are in red. Ten conserved and variable residues (Y98, S136, W153, T155, N183, P186, V190, L194, L226, and G228) (H3 numbering) involving in receptor binding (16) are colored yellow in panel A to depict the location of the receptor binding site. The positions in panel B are labeled in H9 numbering.
expectation (MUSCLE) (32) and found variations at each of the 9 amino acid positions (Table 4). Some of these positions, such as 167, 168, 200, and 201, are highly variable, with multiple mutations having occurred naturally. Positions 147 and 207 are comparatively conserved, as evidenced by their having only 0.2% and 0.3% variations among the 1,036 H9 sequences analyzed. Interestingly, the D153N, Q164K, N167K, A168D, T200I, and N201S mutations detected in the HA of escape mutants in our study are already present in some of the natural H9N2 isolates, indicative of antibody selection pressure on H9N2 viruses in the field. H9N2 virus has high genetic compatibility with many other subtypes of influenza viruses. Besides contributing its internal genes to H5N1 and H7N9 viruses, H9N2 virus has been in active genetic exchange with H3N2 and H7N3 viruses (12, 33, 34). Recent studies demonstrate that H9N2 virus can also easily reassort with the 2009 pandemic H1N1 virus, creating reassortant viruses with higher pathogenicity or better aerosol transmissibility than the parental viruses in mammalian models (35–37). These findTABLE 4 Natural mutations at 9 key amino acid positions in the HA of H9N2 viruses Amino acid positiona 147 153 164 167 168 196 200 201 207
Mutations (% of isolates bearing each residue)b T (99.8), I (0.1), A (0.1) D (80.2), N (15.1), G (3.7), S (0.5), E (0.5) Q (83.3), H (15.9), K (0.6), P (0.1), E (0.1) N (73.9), G (23.4), D (16), S (0.6), R (0.3), T (0.1), K (0.1) A (53.8), S (20.5), T (2.2), F (1.4), D (0.5), L (10.7), N (7.0), Q (0.2), V (3.6), G (0.1) D (94), E (5.0), N (0.5), Y (0.5) T (96.6), A (0.2), I (0.6), K (0.6), M (1.6), N (0.4) N (93.1), E (0.7), S (2.4), D (3.6), K (0.1), T (0.1) D (99.7), E (0.3)
a
H9 numbering. A total of 1,036 full-length H9 sequences available in GenBank (as of 19 July 2013) were analyzed. b
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ings repeatedly demonstrate that H9N2 virus contributes to the genesis of influenza viruses with an increased threat for mammalian species. Moreover, with its continuous prevalence in avian and mammalian species, H9N2 virus has been undergoing profound genetic and antigenic changes. Since its introduction into terrestrial poultry in Asia in the late 1980s (38), H9N2 has evolved into multiple genetic lineages (10, 13, 14, 39), and further genetic variations within lineages are continuously occurring (40–42). With the massive vaccination against H9N2 infection in poultry in many countries, novel antigenic groups have emerged (34, 43– 45). There is no doubt that a continuous and close surveillance on the antigenic evolution of H9N2 virus will facilitate better strategies for the control of H9N2 virus infection. The critical amino acid positions in H9 antigenic sites, including those identified in our study, can serve as valuable tools for this purpose. ACKNOWLEDGMENTS This study was made possible by funding from The Priority Academic Program Development of Jiangsu Province Higher Education Institutions and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses. We thank Bing Chen and Bolin Hang for their technical help in the studies.
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