Identification of Unique and Conserved Neutralizing Epitopes of Vestigial Esterase Domain in HA Protein of the H9N2 Subtype of Avian Influenza Virus

Xiangyu Huang; Guihu Yin; Yiqin Cai; Jianing Hu; Jingwen Huang; Qingtao Liu; Xiuli Feng

doi:10.3390/v14122739

Identification of Unique and Conserved Neutralizing Epitopes of Vestigial Esterase Domain in HA Protein of the H9N2 Subtype of Avian Influenza Virus

Viruses. 2022 Dec 8;14(12):2739. doi: 10.3390/v14122739.

Authors

Xiangyu Huang^{1

2

3}, Guihu Yin^{1

3}, Yiqin Cai^{1

3}, Jianing Hu^{1

3}, Jingwen Huang^{1

3}, Qingtao Liu², Xiuli Feng^{1

3}

Affiliations

¹ Key Laboratory of Animal Microbiology of China's Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
² Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
³ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.

Abstract

The H9N2 subtype of avian influenza virus (AIV) has been reported to infect not only birds, but also humans. The hemagglutinin (HA) protein is the main surface antigen of AIV and plays an important role in the viral infection. For treatment strategies and vaccine development, HA protein has been an important target for the development of broadly neutralizing antibodies against influenza A virus. To investigate the vital target determinant cluster in HA protein in this work, HA gene was cloned and expressed in the prokaryotic expression vector pET28a. The spleen lymphocytes from BALC/c mice immunized with the purified recombinant HA protein were fused with SP2/0 cells. After Hypoxanthine-Aminopterin-Thymidine (HAT) medium screening and indirect ELISA detection, six hybridoma cell lines producing anti-HA monoclonal antibodies were screened. The gradually truncated HA gene expression and western blotting were used to identify their major locations in epitopes specific to these monoclonal antibodies. It was found that the epitopes were located in three areas: ¹¹²NVENLEEL¹¹⁹, ¹¹⁷EELRSLFS¹²⁴, and ¹⁷⁰PIQDAQ¹⁷⁵. Epitope ¹¹²NVENLEEL¹¹⁹ has a partial amino acid crossover with ¹¹⁷EELRSLFS¹²⁴, which is located in the vestigial esterase domain "110-helix" of HA, and the monoclonal antibody recognizing these epitopes showed the neutralizing activity, suggesting that the region ¹¹²NVENLEELRSLFS¹²⁴ might be a novel neutralizing epitope. The results of the homology analysis showed that these three epitopes were generally conserved in H9N2 subtype AIV, and will provide valuable insights into H9N2 vaccine design and improvement, as well as antibody-based therapies for treatment of H9N2 AIV infection.

Keywords: H9N2 avian influenza virus; HA protein; antigen epitope; monoclonal antibody; neutralization.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Antibodies, Monoclonal
Antibodies, Viral
Chickens
Epitopes
Esterases
Hemagglutinin Glycoproteins, Influenza Virus / genetics
Hemagglutinins
Humans
Influenza A Virus, H9N2 Subtype* / genetics
Influenza in Birds*
Mice

Substances

Epitopes
Hemagglutinins
Esterases
Antibodies, Monoclonal
Hemagglutinin Glycoproteins, Influenza Virus
Antibodies, Viral

Grants and funding

This research was funded by Key Laboratory for prevention and control of Avian Influenza and Other Major Poultry Diseases, Ministry of Agriculture and Rural Affairs, P.R. China (grant number: YDWS202211), National Natural Science Foundation (grant number: 31872458) and National Key R&D Program of China (grant number: 2017YFD0500706) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The funding sources did not influence the work.