Development and characterization of monoclonal antibodies against p72 protein of African swine fever virus reveals a novel conserved B-cell epitope

doi:10.1016/j.jia.2025.10.024

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Hua Cao^{1, 2 ,3}, Mengjia Zhang^{1, 2, 3}, Junhua Dong ^{1, 3}, Pengfei Li^{1, 2, 3}, Ahmed H Ghonaim^{1, 2, 3}, Xuexiang Yu^{1, 2}, Yongtao Li⁷^,⁸, Suphot Wattanaphansak⁹, Wenjuan Du^7,8, Anan Jongkaewwattana¹⁰, Chao Kang¹, Pan Tao^{1, 3}, Qigai He^{1, 2}^#, Wentao Li^{1, 2, 3,4}^{,5, 6#}

National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China

²Key Laboratory of Prevention & Control for African Swine Fever and Other Major Pig Diseases, Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture and Rural Affairs, Wuhan 430070, China

³ Hubei Hongshan Laboratory, Wuhan 430070, China

⁴ Frontiers Science Center for Animal Breeding and Sustainable Production, Wuhan 430070, China

⁵ Hubei Jiangxia Laboratory, Wuhan 430200, China

⁶ International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China

⁷College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China

⁸ International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China

⁹ Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand

¹⁰National Center for Genetic Engineering and Biotechnology, Pathum Thani 12120, Thailand

Highlights

l Discovery of a novel and conserved linear B-cell epitope (aa 130-152) on the major capsid protein p72 of ASFV.

l Structural insights confirm the epitope's surface accessibility on the p72 trimer, making it a prime target for antibodies.

l Validated utility for serodiagnosis through strong reactivity with sera from infected pigs.

Abstract
References

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摘要

目的： 非洲猪瘟（African Swine Fever, ASF）是由非洲猪瘟病毒（African Swine Fever Virus, ASFV）引起的一种烈性传染病，对全球养猪业构成严重威胁。p72是ASFV的主要衣壳蛋白，是血清学诊断和亚单位疫苗开发的关键靶标。本研究旨在基于灭活的非洲猪瘟病毒作为免疫原，制备针对p72蛋白的特异性单克隆抗体（monoclonal Antibodies, mAbs），鉴定新型B细胞线性表位，并评估其临床应用中的潜力。

方法与结果：使用灭活的ASFV基因II型毒株免疫BALB/c小鼠，通过杂交瘤技术制备抗p72的mAbs。成功获得5株能稳定分泌抗p72 mAbs的杂交瘤细胞株（11F6、21B10、28H1、32G5、38C9）。经过间接免疫荧光试验（IFA）验证，所有mAbs均能特异性识别HEK-293T细胞中表达的重组p72蛋白以及ASFV感染的细胞。利用mAb 32G5进行亚细胞定位研究发现，在感染早期（2小时），p72即可被检测到；感染中后期（8-18小时），p72主要聚集于病毒工厂，并随后向细胞膜迁移。通过噬菌体展示技术与截短肽段分析，将32G5识别的表位精细定位至p72蛋白第130–152位氨基酸（¹³⁰AHGQLQTFPRNGYDWDNQTPLEG¹⁵²）。原核系统表达的该表位GST融合蛋白能被32G5高效识别（抗体效价达1:3.2 × 10⁵），并能被ASFV感染后的阳性血清识别。使用ESPript 3软件保守性分析表明，该表位在13个不同基因型的ASFV参考毒株中高度保守。利用PyMOL软件空间结构分析显示，该线性表位位于p72三聚体螺旋帽区（由DEN、HIN和DEC环构成）顶端的暴露区域。

结论： 本研究成功制备了5株具有良好反应性的抗ASFV p72蛋白mAbs，并鉴定出一个位于p72蛋白130–152位氨基酸、高度保守、且表面暴露的新型B细胞表位。该表位在自然感染中具有良好的免疫原性。

创新性： 1）首次系统报道了位于p72蛋白ER1区螺旋帽顶端的一个新型、高度保守的线性B细胞表位；2）该B细胞表位可以被自然感染中产生的抗体识别；3）针对该表位的mAb 32G5可用于追踪病毒感染过程中p72蛋白的动态定位，为研究ASFV的复制与组装过程提供了有力工具。

Abstract

African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious disease that has spread globally, posing a significant threat to swine production and international trade. As rapid diagnosis is crucial for controlling ASF, its major capsid protein, p72, has become a key target for diagnostic and vaccine development. In this study, we generated five monoclonal antibodies (mAbs) against the p72 protein by immunizing mice with inactivated virus. Using phage display technology, we identified the epitope for one mAb as a novel linear B-cell epitope within amino acids 130-152 of the p72 protein. Structural and homology analyses revealed that this epitope is highly conserved across diverse ASFV genotypes and is exposed on the surface of the p72 trimer. Importantly, the epitope showed strong reactivity with sera from ASFV-positive swine. These findings offer a foundation for creating improved serological diagnostics and designing epitope-based vaccines against ASFV.

Keywords: ASFV monoclonal antibody B-cell epitope diagnostic development

Online: 01 November 2025

Fund:

This project was funded by the National Key Research and Development Program of China (2023YFF1000901), the Hubei Hongshan Laboratory, China (2022hszd023), the National Key Laboratory of Agricultural Microbiology, China (AML2023A02), the Fundamental Research Funds for the Central Universities (2662023DKPY004 and 2662025DKPY007), the Innovation Fund of International Joint Research Center of National Animal Immunology (2025IJRCNAI09), the Key Program of Science and Technology of Wuhan, China (2023020302020573) and the Hubei Agricultural Research System, China (HBHZD-ZB-2020-005).

About author: #Correspondence Wentao Li, E-mail: wentao@mail.hzau.edu.cn; Qigai He, E-mail: he628@mail.hzau.edu.cn

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Hua Cao, Mengjia Zhang, Junhua Dong, Pengfei Li, Ahmed H Ghonaim, Xuexiang Yu, Yongtao Li, Suphot Wattanaphansak, Wenjuan Du, Anan Jongkaewwattana, Chao Kang, Pan Tao, Qigai He, Wentao. 2025. Development and characterization of monoclonal antibodies against p72 protein of African swine fever virus reveals a novel conserved B-cell epitope. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.10.024

Alejo A, Matamoros T, Guerra M, Andrés G. 2018. A Proteomic Atlas of the African Swine Fever Virus Particle. J Virol, 92.

Cao H, Zhang M, Liao Z, Li D, He X, Ma H, Li P, Yu X, Peng G, Xie S, He Q, Li W. 2024. A porcine kidney-derived clonal cell line with clear genetic annotation is highly susceptible to African swine fever virus. Vet Res, 55, 42.

Chandana M S, Nair S S, Chaturvedi V K, Abhishek, Pal S, Charan M S S, Balaji S, Saini S, Vasavi K, Deepa P. 2024. Recent progress and major gaps in the vaccine development for African swine fever. Braz J Microbiol, 55, 997-1010.

Eustace Montgomery R. 1921. On A Form of Swine Fever Occurring in British East Africa (Kenya Colony). Journal of comparative pathology and therapeutics, 34, 159-191.

García-Escudero R, Andrés G, Almazán F, Viñuela E. 1998. Inducible gene expression from African swine fever virus recombinants: analysis of the major capsid protein p72. J Virol, 72, 3185-3195.

Hu Y, Wang A, Yan W, Li J, Meng X, Chen L, Li S, Tong W, Kong N, Yu L, Yu H, Shan T, Xu J, Tong G, Zheng H. 2023. Identification of Linear Epitopes in the C-Terminal Region of ASFV p72 Protein. Microorganisms, 11.

Li M, Pan Y, Xi Y, Wang M, Zeng Q. 2023. Insights and progress on epidemic characteristics, genotyping, and preventive measures of PEDV in China: A review. Microbial Pathogenesis, 181, 106185.

Li M, Zheng H. 2025. Insights and progress on epidemic characteristics, pathogenesis, and preventive measures of African swine fever virus: A review. Virulence, 16, 2457949.

Liao H C, Shi Z W, Zhou G J, Luo J C, Wang W Y, Feng L, Zhang F, Shi X T, Tian H, Zheng H X. 2024. Epitope mapping and establishment of a blocking ELISA for mAb targeting the p72 protein of African swine fever virus. Appl Microbiol Biotechnol, 108, 350.

Liberti R, Colabella C, Anzalone L, Severi G, Di Paolo A, Casciari C, Casano A B, Giammarioli M, Cagiola M, Feliziani F, De Giuseppe A. 2023. Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies. Open Vet J, 13, 358-364.

Liu Q, Ma B, Qian N, Zhang F, Tan X, Lei J, Xiang Y. 2019. Structure of the African swine fever virus major capsid protein p72. Cell Res, 29, 953-955.

Miao C, Yang S, Shao J, Zhou G, Ma Y, Wen S, Hou Z, Peng D, Guo H, Liu W, Chang H. 2023. Identification of p72 epitopes of African swine fever virus and preliminary application. Front Microbiol, 14, 1126794.

Orosco F L. 2024. African swine fever virus proteins against host antiviral innate immunity and their implications for vaccine development. Open Vet J, 14, 941-951.

Petrovan V, Murgia M V, Wu P, Lowe A D, Jia W, Rowland R R R. 2020. Epitope mapping of African swine fever virus (ASFV) structural protein, p54. Virus Res, 279, 197871.

Ruedas-Torres I, Thi To Nga B, Salguero F J. 2024. Pathogenicity and virulence of African swine fever virus. Virulence, 15, 2375550.

Shrewsbury J V, Vitus E S, Koziol A L, Nenarokova A, Jess T, Elmahdi R. 2024. Comprehensive phage display viral antibody profiling using VirScan: potential applications in chronic immune-mediated disease. J Virol, 98, e0110224.

Shrock E, Fujimura E, Kula T, Timms R T, Lee I H, Leng Y, Robinson M L, Sie B M, Li M Z, Chen Y, Logue J, Zuiani A, McCulloch D, Lelis F J N, Henson S, Monaco D R, Travers M, Habibi S, Clarke W A, Caturegli P, Laeyendecker O, Piechocka-Trocha A, Li J Z, Khatri A, Chu H Y, Collection M C-, Processing T, Villani A C, Kays K, Goldberg M B, Hacohen N, Filbin M R, Yu X G, Walker B D, Wesemann D R, Larman H B, Lederer J A, Elledge S J. 2020. Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity. Science, 370.

Song J, Li J, Li S, Zhao G, Li T, Chen X, Hu B, Liu J, Lai X, Liu S, Zhou Q, Huang L, Weng C. 2025. Autophagy promotes p72 degradation and capsid disassembly during the early phase of African swine fever virus infection. J Virol, 99, e0170124.

Wang A, Jiang M, Liu H, Liu Y, Zhou J, Chen Y, Ding P, Wang Y, Pang W, Qi Y, Zhang G. 2021a. Development and characterization of monoclonal antibodies against the N-terminal domain of African swine fever virus structural protein, p54. Int J Biol Macromol, 180, 203-211.

Wang G, Xie M, Wu W, Chen Z. 2021b. Structures and Functional Diversities of ASFV Proteins. Viruses, 13.

Wang L, Li D, Zeng D, Wang S, Wu J, Liu Y, Peng G, Xu Z, Jia H, Song C. 2024. Development of a fully automated chemiluminescent immunoassay for the quantitative and qualitative detection of antibodies against African swine fever virus p72. Microbiol Spectr, 12, e0080924.

Xin G, Kuang Q, Le S, Wu W, Gao Q, Gao H, Xu Z, Zheng Z, Lu G, Gong L, Wang H, Zhang G, Shi M, Sun Y. 2023. Origin, genomic diversity and evolution of African swine fever virus in East Asia. Virus Evol, 9, vead060.

Xu Q, Li D, Chen X, Liu X, Cao H, Wang H, Wu H, Cheng T, Ren W, Xu F, He Q, Yu X, Li W. 2024. In Vivo Study of Inoculation Approaches and Pathogenicity in African Swine Fever. Vet Sci, 11.

Yu Q, Liang D, Fu W, Zhang L, Wang J, Zhang Z, Sun Y, Zhu D, Zheng B, Zhu L, Xiang Y, Zhao D, Wang X. 2024. p72 antigenic mapping reveals a potential supersite of vulnerability for African swine fever virus. Cell Discovery, 10, 80.

Yu X, Zhu X, Chen X, Li D, Xu Q, Yao L, Sun Q, Ghonaim A H, Ku X, Fan S, Yang H, He Q. 2021. Establishment of a Blocking ELISA Detection Method for Against African Swine Fever Virus p30 Antibody. Front Vet Sci, 8, 781373.

Zhao D, Sun E, Huang L, Ding L, Zhu Y, Zhang J, Shen D, Zhang X, Zhang Z, Ren T, Wang W, Li F, He X, Bu Z. 2023. Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs. Nat Commun, 14, 3096.

Zhou X, Li N, Luo Y, Liu Y, Miao F, Chen T, Zhang S, Cao P, Li X, Tian K, Qiu H J, Hu R. 2018. Emergence of African Swine Fever in China, 2018. Transbound Emerg Dis, 65, 1482-1484.

Zhu J, Liu Q, Li L, Zhang R, Chang Y, Zhao J, Liu S, Zhao X, Chen X, Sun Y, Zhao Q. 2024a. Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies. Virol Sin, 39, 478-489.

Zhu X, Li F, Fan B, Zhao Y, Zhou J, Wang D, Liu R, Zhao D, Fan H, Li B. 2024b. TRIM28 regulates the coagulation cascade inhibited by p72 of African swine fever virus. Vet Res, 55, 149.

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