H9亚型禽流感病毒HA蛋白广谱性B细胞和T细胞表位的筛选与鉴定

doi:10.1016/j.jia.2024.07.005

Journal of Integrative Agriculture ›› 2026, Vol. 25 ›› Issue (4): 1636-1646.DOI: 10.1016/j.jia.2024.07.005

H9亚型禽流感病毒HA蛋白广谱性B细胞和T细胞表位的筛选与鉴定

收稿日期:2024-02-27 修回日期:2024-07-04 接受日期:2025-05-22 出版日期:2026-04-20 发布日期:2026-03-11

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

Keji Quan^1*, Nan Zhang^1*, Mengqi Lin¹, Yuan Liu¹, Yue Li¹, Qun Hu¹, Maoshun Nie¹, Tao Qin^{1, 2, 3}, Jingzhi Li⁴, Hongwei Ma⁴, Sujuan Chen^{1, 2, 3#}, Daxin Peng^{1, 2, 3#}, Xiufan Liu^{1, 2, 3}

¹College of Veterinary Medicine, Yangzhou University/Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China
²Joint International Research Laboratory of Agriculture and Agri-Product Safety, Ministry of Education, Yangzhou 225009, China
³Jiangsu Research Centre of Engineering and Technology for Prevention and Control of Poultry Disease, Yangzhou 225009, China
⁴Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215000, China

Received:2024-02-27 Revised:2024-07-04 Accepted:2025-05-22 Online:2026-04-20 Published:2026-03-11
About author:Keji Quan, E-mail: DZ120190004@yzu.edu.cn; Nan Zhang, E-mail: DZ120210023@stu.yzu.edu.cn; #Correspondence Sujuan Chen, E-mail: chensj@yzu.edu.cn; Daxin Peng, E-mail: pengdx@yzu.edu.cn * These authors contributed equally to this work.
Supported by:
This work was supported by the National Key Research and Development Program of China (2021YFD1800202), the National Natural Science Foundation of China (3237042 and 32172942), the “Jie Bang Gua Shuai” Project at Yangzhou University, China (YZUXK202316), the Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province, China (SCX[22]3547), the Outstanding Technological Innovation Team of College and University at Jiangsu Province, China ([2021] NO.1) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education, China (PAPD).

摘要/Abstract

摘要：

H9N2亚型禽流感病毒（Avian influenza virus，AIV）在我国持续存在，对养鸡业造成了严重的经济损失。疫苗免疫接种是目前主要的防控手段，但由于病毒的不断变异，现有疫苗的免疫效果并不理想。H9N2亚型AIV的血凝素蛋白（HA）是主要免疫原，其中HA1易发生变异，而HA2相对比较保守，本研究目的是筛选和鉴定HA1蛋白上的广谱性抗原表位。通过对华东地区分离的H9N2亚型AIV HA氨基酸序列进行系统发育分析和血清学试验筛选不同抗原群的代表株，以其HA1氨基酸序列为模板，合成了一个重叠多肽库。利用不同来源H9N2亚型AIV免疫血清对重叠多肽库进行扫描，以免疫抗体产生和动物攻毒保护试验鉴定B细胞表位，以淋巴细胞增殖和ELISpot鉴定T细胞表位。结果显示H9N2亚型AIV分离株在遗传进化上均属于h9.4.2.5分支，并进一步分化成两个不同的亚分支。交叉血凝抑制和微量中和试验结果显示Group 1血清与Group 2抗原HI平均效价差值为2^2.86，Group 2血清与Group 1抗原HI平均效价差值为2^3.2；Group 1血清针对Group 1和Group 2毒株的平均中和效价分别为2^13.89和2^10.58；Group 2血清针对Group 1和Group 2毒株的平均中和效价分别为2^6.70和2^12.33。血清和病毒的反应性呈现明显的分群现象，且与遗传进化分析的分群具有一致性。在此基础上，选取了两个分支中的3个代表性毒株A/chicken/Jiangsu/JY040218C/2019、A/pigeon/Jiangsu/JY020616/2019和A/chicken/Jiangsu/WX090312/2018的HA1作为合成模板，我们鉴定出4个区域（H9-2/3、H9-20/21、H9-26、H9-29/30/31）的肽段显示出广谱反应性。多肽免疫试验结果显示，肽段H9-21 (²¹⁹RIFKPLIGPRPLVNGLMGRI²³⁹)、H9-26 (²⁶⁹SGESHGRILKTDLKMGSCTV²⁸⁹) 和 H9-30 (³⁰⁹YAFGNCPKYIGVKSLKLAVG³²⁹) 能有效诱导抗体产生，并对亲本病毒JY040218C提供部分保护效力。淋巴细胞增殖和ELISpot分析结果表明，肽段H9-15 (¹⁵⁹MRWLTQKNNAYPTQDAQYTN¹⁷⁹)、H9-22 (²²⁹PLVNGLMGRINYYWSVLKPG²⁴⁹) 和 H9-23 (²³⁹NYYWSVLKPGQTLRIKSDGN²⁵⁹) 能有效刺激不同H9N2 AIV免疫后的鸡外周血淋巴细胞增殖，并诱导INF-γ的表达。因此，本研究鉴定出了5个新的H9 HA1细胞表位：H9-15、H9-22、H9-23、H9-26和H9-30，其中H9-26被认为是最佳的B细胞表位，而H9-22则被认为是最佳的T细胞表位。这些表位具有广谱的免疫反应性，通过靶向这些表位进行疫苗设计和检测方法研发，有望提高H9N2亚型禽流感的防控效果。

Abstract:

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.

Key words: H9N2 subtype avian influenza virus , HA protein , Epitope , Microarray , Peptide

Keji Quan, Nan Zhang, Mengqi Lin, Yuan Liu, Yue Li, Qun Hu, Maoshun Nie, Tao Qin, Jingzhi Li, Hongwei Ma, Sujuan Chen, Daxin Peng, Xiufan Liu. H9亚型禽流感病毒HA蛋白广谱性B细胞和T细胞表位的筛选与鉴定[J]. Journal of Integrative Agriculture, 2026, 25(4): 1636-1646.

Keji Quan, Nan Zhang, Mengqi Lin, Yuan Liu, Yue Li, Qun Hu, Maoshun Nie, Tao Qin, Jingzhi Li, Hongwei Ma, Sujuan Chen, Daxin Peng, Xiufan Liu.

Identification of broad-spectrum B-cell and T-cell epitopes of H9 subtype avian influenza virus HA protein using polypeptide scanning [J]. Journal of Integrative Agriculture, 2026, 25(4): 1636-1646.

参考文献

Arpaia N, Green J A, Moltedo B, Arvey A, Hemmers S, Yuan S, Treuting P M, Rudensky A Y. 2015. A distinct function of regulatory T cells in tissue protection. Cell, 162, 1078–1089.

Belser J A, Sun X, Brock N, Pappas C, Pulit-Penaloza J A, Zeng H, Jang Y, Jones J, Carney P J, Chang J, Long N V, Diep N T, Thor S, Di H, Yang G, Cook P W, Creager H M, Wang D, Mcfarland J, Dong P V, et al. 2020. Genetically and antigenically divergent influenza a(h9n2) viruses exhibit differential replication and transmission phenotypes in mammalian models. Journal of Virology, 94, e00451–20.

Bi Y, Li J, Li S, Fu G, Jin T, Zhang C, Yang Y, Ma Z, Tian W, Li J, Xiao S, Li L, Yin R, Zhang Y, Wang L, Qin Y, Yao Z, Meng F, Hu D, Li D, et al. 2020. Dominant subtype switch in avian influenza viruses during 2016–2019 in China. Nature Communications, 11, 5909.

Cao Y, Liu H, Liu D, Liu W, Luo T, Li J. 2022. Hemagglutinin gene variation rate of H9N2 avian influenza virus by vaccine intervention in China. Viruses, 14, 1043.

Carnaccini S, Perez D R. 2020. H9 influenza viruses: An emerging challenge. Cold Spring Harbor Perspectives in Medicine, 10, a038588.

Charostad J, Rezaei Zadeh Rukerd M, Mahmoudvand S, Bashash D, Hashemi S M A, Nakhaie M, Zandi K. 2023. A comprehensive review of highly pathogenic avian influenza (HPAI) H5N1: An imminent threat at doorstep. Travel Medicine and Infectious Disease, 55, 102638.

Chen H, Yuan H, Gao R, Zhang J, Wang D, Xiong Y, Fan G, Yang F, Li X, Zhou J, Zou S, Yang L, Chen T, Dong L, Bo H, Zhao X, Zhang Y, Lan Y, Bai T, Dong J, et al. 2014. Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: A descriptive study. Lancet, 383, 714–721.

Chen J M, Sun Y X, Chen J W, Liu S, Yu J M, Shen C J, Sun X D, Peng D. 2009. Panorama phylogenetic diversity and distribution of type A influenza viruses based on their six internal gene sequences. Virology Journal, 6, 137.

Clemens E B, Van De Sandt C, Wong S S, Wakim L M, Valkenburg S A. 2018. Harnessing the power of T cells: The promising hope for a universal influenza vaccine. Vaccines (Basel), 6, 18.

Cummings J F, Guerrero M L, Moon J E, Waterman P, Nielsen R K, Jefferson S, Gross F L, Hancock K, Katz J M, Yusibov V. 2014. Safety and immunogenicity of a plant-produced recombinant monomer hemagglutinin-based influenza vaccine derived from influenza A (H1N1)pdm09 virus: A Phase 1 dose-escalation study in healthy adults. Vaccine, 32, 2251–2259.

Du L, Leung V H, Zhang X, Zhou J, Chen M, He W, Zhang H Y, Chan C C, Poon V K, Zhao G, Sun S, Cai L, Zhou Y, Zheng B J, Jiang S. 2011. A recombinant vaccine of H5N1 HA1 fused with foldon and human IgG Fc induced complete cross-clade protection against divergent H5N1 viruses. PLoS ONE, 6, e16555.

Egarnes B, Gosselin J. 2018. Contribution of regulatory T cells in nucleotide-binding oligomerization domain 2 response to influenza virus infection. Frontiers in Immunology, 9, 132.

Ekkens M J, Shedlock D J, Jung E, Troy A, Pearce E L, Shen H, Pearce E J. 2007. Th1 and Th2 cells help CD8 T-cell responses. Infection and Immunity, 75, 2291–2296.

Frey S S, Versage E, Van Twuijver E, Hohenboken M. 2023. Antibody responses against heterologous H5N1 strains for an MF59-adjuvanted cell culture-derived H5N1 (aH5n1c) influenza vaccine in adults and older adults. Human Vaccines & Immunotherapeutics, 19, 2193119.

Graham C M, Smith C A, Thomas D B. 1998. Novel diversity in Th1, Th2 type differentiation of hemagglutinin-specific T cell clones elicited by natural influenza virus infection in three major haplotypes (H–2b,d,k). The Journal of Immunology, 161, 1094–1103.

Guo Y J, Krauss S, Senne D A, Mo I P, Lo K S, Xiong X P, Norwood M, Shortridge K F, Webster R G, Guan Y. 2000. Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia. Virology, 267, 279–288.

Kaverin N V, Rudneva I A, Ilyushina N A, Lipatov A S, Krauss S, Webster R G. 2004. Structural differences among hemagglutinins of influenza A virus subtypes are reflected in their antigenic architecture: Analysis of H9 escape mutants. Journal of Virology, 78, 240–249.

Knowlden Z A G, Richards K A, Moritzky S A, Sant A J. 2019. Peptide epitope hot spots of CD4 T cell recognition within influenza hemagglutinin during the primary response to infection. Pathogens, 8, 220.

Kreijtz J H C M, Fouchier R A M, Rimmelzwaan G F. 2011. Immune responses to influenza virus infection. Virus Research, 162, 19–30.

Letunic I, Bork P. 2021. Interactive Tree of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Research, 49, W293–W296.

Li Y, Ma M L, Lei Q, Wang F, Hong W, Lai D Y, Hou H, Xu Z W, Zhang B, Chen H, Yu C, Xue J B, Zheng Y X, Wang X N, Jiang H W, Zhang H N, Qi H, Guo S J, Zhang Y, Lin X, et al. 2021. Linear epitope landscape of the SARS-CoV-2 Spike protein constructed from 1,051 COVID-19 patients. Cell Reports, 34, 108915.

Li Y T, Linster M, Mendenhall I H, Su Y C F, Smith G J D. 2019. Avian influenza viruses in humans: Lessons from past outbreaks. British Medical Bulletin, 132, 81–95.

Liang L, Bai Y, Huang W, Ren P, Li X, Wang D, Yang Y, Gao Z, Tang J, Wu X, Gao S, Guo Y, Hu M, Wang Z, Wang Z, Ma H, Li J. 2024. Genetic and biological properties of H9N2 avian influenza viruses isolated in central China from 2020 to 2022. Journal of Integrative Agriculture, 23, 2778-2791.

Lim C M L, Komarasamy T V, Adnan N, Radhakrishnan A K, Balasubramaniam V. 2024. Recent advances, approaches and challenges in the development of universal influenza vaccines. Influenza and Other Respiratory Viruses, 18, e13276.

Liu D, Shi W, Gao G F. 2014. Poultry carrying H9N2 act as incubators for novel human avian influenza viruses. Lancet, 383, 869.

Liu G, Zhang F, Shi J, Tian G, Chen H, Yu K, Meng Q. 2013. A subunit vaccine candidate derived from a classic H5N1 avian influenza virus in China protects fowls and BALB/c mice from lethal challenge. Vaccine, 31, 5398–5404.

Liu Q, Zhao L, Guo Y, Zhao Y, Li Y, Chen N, Lu Y, Yu M, Deng L, Ping J. 2022. Antigenic evolution characteristics and immunological evaluation of H9N2 avian influenza viruses from 1994–2019 in China. Viruses, 14, 726.

Liu S, Ji K, Chen J, Tai D, Jiang W, Hou G, Chen J, Li J, Huang B. 2009. Panorama phylogenetic diversity and distribution of Type A influenza virus. PLoS ONE, 4, e5022.

Lu Y, Li Z, Teng H, Xu H, Qi S, He J, Gu D, Chen Q, Ma H. 2015. Chimeric peptide constructs comprising linear B-cell epitopes: Application to the serodiagnosis of infectious diseases. Scientific Reports, 5, 13364.

Peacock T, Reddy K, James J, Adamiak B, Barclay W, Shelton H, Iqbal M. 2016. Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape. Scientific Reports, 6, 18745.

Peacock T H P, James J, Sealy J E, Iqbal M. 2019. A global perspective on H9N2 avian influenza virus. Viruses, 11, 620.

Price G E, Huang L, Ou R, Zhang M, Moskophidis D. 2005. Perforin and Fas cytolytic pathways coordinately shape the selection and diversity of CD8+ T-cell escape variants of influenza virus. Journal of Virology, 79, 8545–8559.

Pu J, Wang S, Yin Y, Zhang G, Carter R A, Wang J, Xu G, Sun H, Wang M, Wen C, Wei Y, Wang D, Zhu B, Lemmon G, Jiao Y, Duan S, Wang Q, Du Q, Sun M, Bao J, et al. 2015. Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus. Proceedings of the National Academy of Sciences of the United States of America, 112, 548–553.

Rattan A, Richards K A, Knowlden Z A G, Sant A J. 2019. Protein vaccination directs the CD4+ T cell response toward shared protective epitopes that can be recalled after influenza virus infection. Journal of Virology, 93, e00947–19.

Rhee J W, Kim D, Park B K, Kwon S, Cho S, Lee I, Park M S, Seo J N, Kim Y S, Choi H S, Lee Y, Kwon H J. 2012. Immunization with a hemagglutinin-derived synthetic peptide formulated with a CpG-DNA-liposome complex induced protection against lethal influenza virus infection in mice. PLoS ONE, 7, e48750.

Richards K A, Chaves F A, Krafcik F R, Topham D J, Lazarski C A, Sant A J. 2007. Direct ex vivo analyses of HLA-DR1 transgenic mice reveal an exceptionally broad pattern of immunodominance in the primary HLA-DR1-restricted CD4 T-cell response to influenza virus hemagglutinin. Journal of Virology, 81, 7608–7619.

Smith D J, Lapedes A S, De Jong J C, Bestebroer T M, Rimmelzwaan G F, Osterhaus A D M E, Fouchier R a M. 2004. Mapping the antigenic and genetic evolution of influenza virus. Science, 305, 371–376.

Spackman E. 2020. Animal Influenza Virus: Methods and Protocols. Humana Press, New York. pp. 11–28.

Steel J, Lowen A C, Wang T T, Yondola M, Gao Q, Haye K, García-Sastre A, Palese P. 2010. Influenza virus vaccine based on the conserved hemagglutinin stalk domain. mBio, 1, e00018–10.

Strutt T M, Mckinstry K K, Dibble J P, Winchell C, Kuang Y, Curtis J D, Huston G, Dutton R W, Swain S L. 2010. Memory CD4+ T cells induce innate responses independently of pathogen. Nature Medicine, 16, 558–564.

Sun Z, Wang Q, Li G, Li J, Chen S, Qin T, Ma H, Peng D, Liu X. 2021. Development of an inactivated h7n9 subtype avian influenza serological diva vaccine using the chimeric HA epitope approach. Microbiology Spectrum, 9, e0068721.

Tenbusch M, Grunwald T, Niezold T, Storcksdieck Genannt Bonsmann M, Hannaman D, Norley S, Uberla K. 2010. Codon-optimization of the hemagglutinin gene from the novel swine origin H1N1 influenza virus has differential effects on CD4+ T-cell responses and immune effector mechanisms following DNA electroporation in mice. Vaccine, 28, 3273–3277.

Trifinopoulos J, Nguyen L T, Von Haeseler A, Minh B Q. 2016. W-IQ-TREE: A fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research, 44, W232-W235.

Wang Q, Sun Z, Li J, Qin T, Ma H, Chen S, Peng D, Liu X. 2021. Identification of a universal antigen epitope of influenza A virus using peptide microarray. BMC Veterinary Research, 17, 22.

Wang X, Liu K, Guo Y, Pei Y, Chen X, Lu X, Gao R, Chen Y, Gu M, Hu J, Liu X, Hu S, Jiao X A, Liu X, Wang X. 2023. Emergence of a new designated clade 16 with significant antigenic drift in hemagglutinin gene of H9N2 subtype avian influenza virus in eastern China. Emerging Microbes & Infections, 12, 2249558.

Yan W, Cui H, Engelsma M, Beerens N, Van Oers M M, De Jong M C M, Li X, Liu Q, Yang J, Teng Q, Li Z. 2022. Molecular and antigenic characterization of avian H9N2 viruses in Southern China. Microbiology Spectrum, 10, e0082221.

Zhang N, Quan K, Chen Z, Hu Q, Nie M, Xu N, Gao R, Wang X, Qin T, Chen S, Peng D, Liu X. 2023. The emergence of new antigen branches of H9N2 avian influenza virus in China due to antigenic drift on hemagglutinin through antibody escape at immunodominant sites. Emerging Microbes & Infections, 12, 2246582.

Zhang S, Yu J L, He L, Gong L, Hou S, Zhu M, Wu J B, Su B, Liu J, Wu G, He J. 2021. Molecular characteristics of the H9N2 avian influenza viruses in live poultry markets in Anhui Province, China, 2013 to 2018. Health Science Reports, 4, e230.

Zhang X, Li Y, Jin S, Wang T, Sun W, Zhang Y, Li F, Zhao M, Sun L, Hu X, Feng N, Xie Y, Zhao Y, Yang S, Xia X, Gao Y. 2022. H9N2 influenza virus spillover into wild birds from poultry in China bind to human-type receptors and transmit in mammals via respiratory droplets. Transboundary and Emerging Diseases, 69, 669–684.

Zhao Y R, Zhao Y Z, Liu S D, Xiao Y H, Li N, Liu K H, Meng F L, Zhao J, Liu M D, Li B Q. 2023. Phylogenetic and epidemiological characteristics of H9N2 avian influenza viruses in Shandong Province, China from 2019 to 2021. Journal of Integrative Agriculture, 22, 881–896.

H9亚型禽流感病毒HA蛋白广谱性B细胞和T细胞表位的筛选与鉴定

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

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