Identification of broad-spectrum B-cell and T-cell epitopes of H9 subtype avian influenza virus HA protein using polypeptide scanning1

The H9N2 subtype avian influenza virus (AIV) hemagglutinin (HA) protein is a major immunogen in which HA1 is a genetic variant and HA2 is relatively conserved. Identifying broad-spectrum antigen epitopes targeting HA1 is crucial for vaccine design and detection. Based on the phylogenetic and serological analyses, we identified 2 antigenic groups and 3 representative viruses: A/chicken/Jiangsu/JY040218C/2019, A/pigeon/Jiangsu/JY020616/2019, and A/chicken/Jiangsu/WX090312/2018. An overlapping peptide library was synthesized using HA1 amino acid sequences of the viruses as templates. Through peptide scanning of the sera against different strains of H9N2 subtype AIV, we identified peptides from 4 regions (H9-2/3, H9-20/21, H9-26, and H9-29/30/31) that demonstrated broad-spectrum reactivity. Immunological assay results demonstrated that H9-21 (219RIFKPLIGPRPLVNGLMGRI239), H9-26 (269SGESHGRILKTDLKMGSCTV289), and H9-30 (309YAFGNCPKYI GVKSLKLAVG329) effectively induced antibody generation and conferred partial protective efficacy against the parent virus JY040218C. The results of lymphocyte proliferation and ELISpot assays indicated that peptides H9-15 (159MRWLTQKNNAYPTQDAQYTN179), H9-22 (229PLVNGLMGRINYYWSVLKP G249), and H9-23 (239NYYWSVLKPGQTLRIKSDGN259) could effectively stimulate the expression of interferon-gamma in peripheral blood lymphocytes of chickens immunized against different strains of H9N2 AIV. Collectively, 5 novel cell epitopes H9-15, H9-22, H9-23, H9-26, and H9-30, including the best B cell epitope H9-26 and the best T cells epitope H9-22, were identified that could be targeted for vaccine design or detection approaches against H9N2 AIVs.