Comprehensive evaluation of HA epitope modifications in H9N2 subtype avian influenza vaccines for broad cross-protection

doi:10.1016/j.jia.2025.08.018

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Nan Zhang¹^*, Keji Quan¹^*, Mengqi Lin¹, Zijun Lu¹, Zhifan Li¹, Yiming Yang¹, Nuo Xu¹, Hui Yang^1,^2,³^{, 4}, Jie Zhu⁵, George Fei Zhang⁶, Tao Qin^1,^2,³^{, 4}, Sujuan Chen^1,^2,³^{, 4#}, Daxin Peng^1,^2,³^{, 4#}, Xiufan Liu^1,^2,³^{, 4}

¹College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China

²Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou 225009, China

³Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China

⁴Jiangsu Research Centre of Engineering and Technology for Prevention and Control of Poultry Disease, Yangzhou 225009, China

⁵Shandong Binzhou Wohua Bioengineering Co., Ltd., Binzhou, Shandong, 256600, China

⁶Laboratory of Microbiology and Immunology, Shandong Binzhou Wohua Bioengineering Co., Ltd., Binzhou, Shandong, 256600, China

Highlights:

1. Mutations in HA antigenic sites of H9N2 AIV were systematically analyzed using three-dimensional antigenic cartography and monoclonal antibody profiling.

2. R118-A was identified as a vaccine candidate with cross-protection against diverse H9N2 antigenic lineages.

Abstract
References

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摘要

（目的）H9N2亚型禽流感病毒（Avian influenza virus，AIV）因其血凝素（Hemagglutinin，HA）蛋白频繁发生抗原漂移，导致灭活疫苗的保护效果减弱，持续对全球家禽养殖业构成威胁。前期研究发现，H9N2 AIV HA蛋白中的12个抗原残基在其两个主要抗原群之间的抗原漂移中起关键作用。为进一步阐明这些抗原残基对病毒抗原性与交叉保护力的影响。（方法）本研究以H9N2 group 2（B4.7）毒株A/chicken/Jiangyin/JY120118/2018（R118）为母本，构建了替换group 1毒株（B4.4和B4.6）HA蛋白抗原区域（A、B1、B2和E）的单一表位或联合表位突变株，通过三维抗原制图及单抗排谱分析各突变对病毒抗原性的改变；随后，选择变突株制备免疫血清，进行交叉血凝抑制（Hemagglutinin inhibition，HI）与微量中和（Microneutralization，MN）试验；最终选择候选疫苗株免疫SPF鸡，采用不同H9N2毒株进行攻毒试验，评估其交叉保护效果。（结果）单抗排谱显示突变株R118-A、R118-AE、R118-B1和R118-AB1E能同时被group 1和group 2的单抗识别。在HI和MN试验中R118-A的免疫血清对group 1和group 2抗原均具有良好的覆盖能力，个体间免疫反应一致性较高。动物试验表明，R118-A对group 2 毒株W120118攻毒的保护效果与母本相当，而针对group 1毒株W100318和group 2异源毒株W05091攻毒时，R118-A显著减少了排毒量和排毒动物的个体数量。（结论）因此，基于抗原区域A（D145S、T149K和G153D）改造的疫苗候选株R118-A对不同分支的H9N2亚型AIV的感染具有良好的交叉保护作用，为广谱H9N2疫苗的设计与优化提供了理论支持和实践指导。

创新性：

1. 通过三维抗原制图与单克隆抗体排谱技术，系统评估了HA蛋白不同抗原区域突变对H9N2禽流感病毒抗原性的影响。

2. 通过上述技术筛选获得了疫苗候选株R118-A，其对不同抗原分支的H9N2禽流感病毒感染具有交叉保护能力。

Abstract

The hemagglutinin (HA) protein of the H9N2 subtype avian influenza virus (AIV) undergoes frequent antigenic drift, which compromises the efficacy of existing inactivated vaccines. We have identified 12 key HA residues responsible for antigenic differences between the 2 major H9N2 antigenic groups; however, their role in eliciting broad cross-reactive immunity remains undefined. In this study, we systematically evaluated the impact of single- and multi-residue mutations in HA antigenic regions A, B1, B2, and E on viral antigenicity using antigenic cartography and monoclonal antibody profiling. 4 recombinant viruses—R118-A, R118-AE, R118-B1, and R118-AB1E—demonstrated broadened antigenic reactivity and were selected for further analysis. Among them, R118-A elicited immune sera with high hemagglutination inhibition and microneutralization titers against a diverse panel of H9N2 strains and exhibited broad antigenic coverage on antigenic cartography. In chicken challenge experiments, immunization with R118-A conferred cross-protection against group 1 (B4.4+B4.6) and group 2 (B4.7) H9N2 viruses, underscoring the critical role of site A modifications in broadening vaccine protection. These findings offer theoretical support and practical strategies for the rational design of next-generation H9N2 vaccines with improved cross-protective efficacy.

Keywords: H9N2 avian influenza virus Hemagglutinin Monoclonal antibody profiling Antigenic cartography Broad-spectrum protection

Online: 21 August 2025

Fund:

This research was funded by the National Key R&D Project of China (2021YFD1800202), the Jiangsu Provincial Key R&D Project, China (BE2022329), the Jiangsu Provincial Natural Science Fund for Distinguished Young Scholars, China (BK20240045), the “Jie Bang Gua Shuai” Project at Yangzhou University, China (YZUXK202316), the Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province, China (CX(23)3071), the Outstanding Technological Innovation Team of College and University in Jiangsu Province, China ([2021] No.1), 111 Project (D18007), and the Priority Academic Program Development of Jiangsu Higher Education, China (PAPD).

About author: #Correspondence Daxin Peng, E-mail: pengdx@yzu.edu.cn; Sujuan Chen. E-mail: chensj@yzu.edu.cn, Tel: +86-514-87975893. * These authors have contributed equally to this work.

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Nan Zhang, Keji Quan, Mengqi Lin, Zijun Lu, Zhifan Li, Yiming Yang, Nuo Xu, Hui Yang, Jie Zhu, George Fei Zhang, Tao Qin, Sujuan Chen, Daxin Peng, Xiufan Liu. 2025. Comprehensive evaluation of HA epitope modifications in H9N2 subtype avian influenza vaccines for broad cross-protection. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.08.018

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