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. 2022 Nov 15;14(11):2530.
doi: 10.3390/v14112530.

Characterization of Neutralizing Monoclonal Antibodies and Identification of a Novel Conserved C-Terminal Linear Epitope on the Hemagglutinin Protein of the H9N2 Avian Influenza Virus

Yanan Wang  1   2 , Xueyang Li  2 , Qianru Xu  2   3 , Xiangxiang Niu  2 , Shenli Zhang  2 , Xiaotian Qu  2 , Hongyan Chu  2 , Jinxuan Chen  2 , Qianqian Shi  2 , Erqin Zhang  2   4 , Gaiping Zhang  1   2   4   5   6   7
Affiliations

Characterization of Neutralizing Monoclonal Antibodies and Identification of a Novel Conserved C-Terminal Linear Epitope on the Hemagglutinin Protein of the H9N2 Avian Influenza Virus

Yanan Wang et al. Viruses. .

Abstract

The H9N2 avian influenza virus (AIV) remains a serious threat to the global poultry industry and public health. The hemagglutinin (HA) protein is an essential protective antigen of AIVs and a major target of neutralizing antibodies and vaccines. Therefore, in this study, we used rice-derived HA protein as an immunogen to generate monoclonal antibodies (mAbs) and screened them using an immunoperoxidase monolayer assay and indirect enzyme-linked immunosorbent assay. Eight mAbs reacted well with the recombinant H9N2 AIV and HA protein, four of which exhibited potent inhibitory activity against hemagglutination, while three showed remarkable neutralization capacities. Western blotting confirmed that two mAbs bound to the HA protein. Linear epitopes were identified using the mAbs; a novel linear epitope, 480HKCDDQCM487, was identified. Structural analysis revealed that the novel linear epitope is located at the C-terminus of HA2 near the disulfide bond-linked HA1 and HA2. Alignment of the amino acid sequences showed that the epitope was highly conserved among multiple H9N2 AIV strains. The results of this study provide novel insights for refining vaccine and diagnostic strategies and expand our understanding of the immune response against AIV.

Keywords: H9N2 avian influenza virus; HA2; hemagglutinin protein; linear B cell epitope; neutralizing monoclonal antibodies.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
H9N2 HA immunization and process of generate mAbs. (A) Schedule of generate mAbs by hybridoma technology. BALB/c mice were immunized with HA antigen and generate mAbs. The spleen cells were fused with the myeloma cells and were screened, and finally hybridoma cells were propagated to produce mAbs. (B) Details of immunizing dose, adjuvant, frequency and routes. S.C represents subcutaneous injection. (C) HA-specific antibody of serum detected by indirect ELISA. N represents OD450nm of mice sera immunized with TP309. P stands for OD450nm of mice sera immunized with recombinant HA protein; P/N ≥ 2.1 is considered as positive. All the data are repeated three times and the average values are calculated.
Figure 2
Figure 2
Screening of mAbs against the H9N2 AIV and HA protein. (A) Screening mAbs by IPMA. Eight mAbs, 3A5, 4E8, 5A5, 6H2, 8G8, 11B9, 11E3, and 12E5 were identified. (B) ELISA results for reaction of mAbs with the HA protein. PC, H9N2 AIV-positive chicken serum. NC, SP2/0 cell culture supernatants.
Figure 3
Figure 3
Identification of mAbs. (A) Neutralizing activity of mAbs. PC, H9N2 AIV-positive chicken serum. NC, H9N2 AIV-negative mice serum. (B) Hemagglutination inhibitory activity of mAbs. PC, H9N2 AIV-positive chicken serum. NC, H9N2 AIV-negative mice serum. (C) Evaluation of the response between the mAbs and HA protein by Western blotting. M, protein marker. Lane 1, HA protein. Lane 2, TP309 soluble protein used as negative control. (D) Specificity detection of the mAbs react with different virus by dot blotting. Lane 1, H9N2 AIV virus. Lane 2, H5N1 AIV virus. Lane 3, NDV virus. Line 4, TP309 soluble protein. All the data were presented as means with error bars.
Figure 4
Figure 4
Identification of the B cell linear epitope on HA protein. (A) A schematic diagram of 41 overlapping peptides containing 20–23 amino acids (containing an offset of 7 aa) of HA protein (residues 19–521). (B) Reactivity of 41 overlapping peptides with mAb 6H2 detected by ELISA. (C) Dot blotting analysis of 41 overlapping peptides reacted with mAb 6H2. (D) Dot blotting results of five truncated peptides reacted with mAb 6H2. (E) ELISA results of five truncated peptides recognized by mAb 6H2. PC, recombinant HA protein diluted in peptide diluent. NC, peptide diluent.
Figure 5
Figure 5
Spatial location of the identified epitope P39-5. (A) The location of the identified epitope using the hemagglutinin structure (PDB: 1JSD) as template by PyMOL. (B) Spatial distribution of the identified epitope in HA protein. 480HKCDDQCM487 (P39-5) is indicated in red, previously published epitopes are orange, HA1 is indicated in green, and HA2 is blue.
Figure 6
Figure 6
The conservation analysis of the identified epitope. Multiple sequence alignment of the identified epitope in 11 different strains of H9N2 AIV and H1-H18 AIV subtypes. The amino acid sequences of the novel identified epitope in different strains were highlighted with a red line.
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Grants and funding

This work was supported by the Key Scientific and Technological Project of Henan Province, China (grant No. 222102110210) and Major Science and Technology Project of Henan Province (grant No. 221100110600), the Major Project to Breed New Genetically Modified Organisms (grant No. 2016ZX08001006-10).