Functional analysis and epitope recognition of African swine fever virus pA151R antigen-specific T cells

doi:10.1016/j.jia.2026.01.009

Advanced Online Publication | Current Issue | Archive | Adv Search

Jing Cao¹, Yunfei Tian¹, Longfei Han³, Xiaoping He³, Liming Niu¹, Jiawei Liu¹, Fangyuan Zhang¹, Dongyue Wang², Jiangnan Li³, Changjiang Weng^3, Jiajun Wu^2#, Li Huang^3#, Shaobin Shang^{, 4#}

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

²The Biosafety High-Level Laboratory Management Office, China Animal Disease Control Center, Beijing 102609, China

³Division of Fundamental Immunology, National African Swine Fever Para-reference Laboratory, State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

⁴Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, 225009, China

Highlights

l ASFV non-structural protein pA151R-specific T cells were characterized in immune-protected pigs using advanced immunological techniques suitable for swine.

l T-cell epitopes of pA151R recognized by swine T cells were mapped. Both pA151R-specific and epitope peptide-specific CD4⁺ and CD8⁺ T cells expressed IFN-γ and TNF-α, and are capable of killing epitope peptide-pulsed autologous swine target cells.

l pA151R-specific T cells potentially contribute to protective immunity and pA151R can be used as a component of protective antigen cocktail for vaccine development.

Abstract
References

Download: PDF in ScienceDirect
Export: BibTeX | EndNote (RIS)

摘要

目的：非洲猪瘟（ASF）严重威胁全球养猪业，开发亚单位疫苗首先需要鉴定关键保护性抗原。本研究旨在系统地评价非洲猪瘟病毒（ASFV）非结构蛋白 pA151R 的抗原性、pA151R特异性T细胞的功能及其识别的T细胞表位。

方法：原核表达并纯化重组 pA151R 蛋白（rpA151R），制备抗rpA151R多克隆抗体（pAb），通过Western blot和间接免疫荧光试验验证其与真核转染和病毒感染表达的天然pA151R的反应性。利用ASFV减毒候选疫苗免疫猪，采用流式细胞术、细胞内因子染色分析pA151R特异性T细胞的动态变化与功能。采用IFN-γ ELISPOT试验和重叠多肽鉴定pA151R蛋白中的猪T细胞表位及表位特异性T细胞功能。通过乳酸脱氢酶（LDH）释放试验评估pA151R特异性T细胞对自体靶细胞的杀伤活性。

结果：抗rpA151R pAb能特异性识别真核表达和ASFV感染PAM细胞表达的天然pA151R蛋白。pA151R特异的T细胞应答和抗体水平分别在免疫猪后14和21天达到高峰。pA151R特异性T细胞可同时分泌 IFN-γ、TNF-α、IL-2和穿孔素，具有多功能T细胞的特征。T表位鉴定获得7个肽段：P2、P4、P5 仅被CD4⁺ T细胞识别，含有SLA-II类限制性表位；P6、P10、P12、P13特异性被CD8⁺ T细胞识别，含有SLA-I类限制性表位；P1、P7、P9可同时被CD4⁺ 和CD8⁺ T 细胞识别，含有SLA-I和II类限制性表位。这些多肽特异性CD4⁺或 CD8⁺ T细胞能够以剂量依赖的方式杀伤多肽冲击的自体靶细胞, 具有细胞毒活性。

结论与创新性：本研究系统评估了重组pA151R蛋白被ASFV减毒候选疫苗免疫猪外周血中特异性T细胞和抗体识别的抗原性，鉴定了pA151R特异性T细胞的功能和T细胞表位，证实pA151R是一个潜在的保护性抗原，可作为ASFV多组分亚单位疫苗的候选抗原。本研究的创新性在于：以ASFV免疫保护猪为模型，利用适用于猪的细胞免疫学研究方法，深入分析了pA151R特异性T细胞的多功能性和杀伤活性，绘制了pA151R蛋白的猪T细胞表位图谱，为筛选和评估其它ASFV编码蛋白的保护潜力提供了方法学依据。

Abstract

African swine fever (ASF) is a highly lethal hemorrhagic disease of swine caused by African swine fever virus (ASFV). Development of safe and effective ASFV subunit vaccine relies on the identification of protective antigens. In this study, we systematically evaluated the antigenicity of ASFV non-structural protein pA151R recognized by T cells from immune-protected pigs. Recombinant pA151R (rpA151R) was expressed in E. coli and used to generate anti-rpA151R polyclonal antibodies (pAb). This pAb bound both eukaryotically-expressed and native viral pA151R, confirming that rpA151R retains its native antigenicity. Using ASFV attenuated vaccine-immunized pigs, we further analysed the kinetics and functions of pA151R-specific T cells as well as their epitope recognition. The results showed that pA151R-specific T cell responses peaked at 14 days post-immunization in pigs, and secreted IFN-γ, TNF-α, IL-2, and perforin simultaneously, with multifunctional characteristics. T-cell epitope mapping identified seven peptides recognized by these pA151R-specific T cells. Among them, three peptides (P2, P4, and P5) were exclusively recognized by CD4⁺ T cells, four peptides (P6, P10, P12, and P13) were specific for CD8⁺ T cells whereas P1, P7, and P9 were recognized by both CD4⁺ and CD8⁺ T cells. These peptide-specific CD4⁺ or CD8⁺ T cells showed cytotoxicity, killing peptide-pulsed autologous target cells in a dose-dependent manner. These findings demonstrated that pA151R-specific swine T cells are able to contribute to protective immunity against ASFV and pA151R is a potential protective antigen for vaccine development. This study established a benchmark for screening and defining more ASFV protective antigens.

Keywords: African swine fever virus pA151R T-cell epitope cytotoxic T lymphocyte activity

Online: 07 January 2026

Fund: This study was funded by the National Key Research and Development Program of China (2021YFD1800105), the National Natural Science Foundation of China (32322081), the State Key Laboratory for Animal Disease Control and Prevention Foundation (SKLADCPKF202401) and by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

About author: #Correspondence Shaobin Shang, shaobinshang@yzu.edu.cn; Jiajun Wu, wujiajun82@126.com; Li Huang: huangli02@caas.cn

	Service
	E-mail this article
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors

Cite this article:

Jing Cao, Yunfei Tian, Longfei Han, Xiaoping He, Liming Niu, Jiawei Liu, Fangyuan Zhang, Dongyue Wang, Jiangnan Li, Changjiang Weng, Jiajun Wu, Li Huang, Shaobin Shang. 2026. Functional analysis and epitope recognition of African swine fever virus pA151R antigen-specific T cells. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2026.01.009

Blome S, Gabriel C, Beer M. 2014. Modern adjuvants do not enhance the efficacy of an inactivated African swine fever virus vaccine preparation. Vaccine, 32, 3879-3882.

Bosch-Camos L, Alonso U, Esteve-Codina A, Chang C Y, Martin-Mur B, Accensi F, Munoz M, Navas M J, Dabad M, Vidal E, Pina-Pedrero S, Pleguezuelos P, Caratu G, Salas M L, Liu L, Bataklieva S, Gavrilov B, Rodriguez F, Argilaguet J. 2022. Cross-protection against African swine fever virus upon intranasal vaccination is associated with an adaptive-innate immune crosstalk. PLoS Pathog, 18, e1010931.

Bosch-Camos L, Lopez E, Navas M J, Pina-Pedrero S, Accensi F, Correa-Fiz F, Park C, Carrascal M, Dominguez J, Salas M L, Nikolin V, Collado J, Rodriguez F. 2021. Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach. Vaccines (Basel), 9,

Burmakina G, Malogolovkin A, Tulman E R, Xu W, Delhon G, Kolbasov D, Rock D L. 2019. Identification of T-cell epitopes in African swine fever virus CD2v and C-type lectin proteins. J Gen Virol, 100, 259-265.

Cadenas-Fernandez E, Sanchez-Vizcaino J M, Kosowska A, Rivera B, Mayoral-Alegre F, Rodriguez-Bertos A, Yao J, Bray J, Lokhandwala S, Mwangi W, Barasona J A. 2020. Adenovirus-vectored African Swine Fever Virus Antigens Cocktail Is Not Protective against Virulent Arm07 Isolate in Eurasian Wild Boar. Pathogens, 9,

Chen W, Zhao D, He X, Liu R, Wang Z, Zhang X, Li F, Shan D, Chen H, Zhang J, Wang L, Wen Z, Wang X, Guan Y, Liu J, Bu Z. 2020. A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs. Sci China Life Sci, 63, 623-634.

Chung C J, Cha S H, Grimm A L, Ajithdoss D, Rzepka J, Chung G, Yu J, Davis W C, Ho C S. 2018. Pigs that recover from porcine reproduction and respiratory syndrome virus infection develop cytotoxic CD4+CD8+ and CD4+CD8- T-cells that kill virus infected cells. Plos One, 13, e0203482.

Deutschmann P, Forth J H, Sehl-Ewert J, Carrau T, Viaplana E, Mancera J C, Urniza A, Beer M, Blome S. 2023. Assessment of African swine fever vaccine candidate ASFV-G-∆MGF in a reversion to virulence study. NPJ Vaccines, 8, 78.

Gallardo C, Sanchez E G, Perez-Nunez D, Nogal M, De Leon P, Carrascosa A L, Nieto R, Soler A, Arias M L, Revilla Y. 2018. African swine fever virus (ASFV) protection mediated by NH/P68 and NH/P68 recombinant live-attenuated viruses. Vaccine, 36, 2694-2704.

Ge S, Li J, Fan X, Liu F, Li L, Wang Q, Ren W, Bao J, Liu C, Wang H, Liu Y, Zhang Y, Xu T, Wu X, Wang Z. 2018. Molecular Characterization of African Swine Fever Virus, China, 2018. Emerg Infect Dis, 24, 2131-2133.

Goatley L C, Nash R H, Andrews C, Hargreaves Z, Tng P, Reis A L, Graham S P, Netherton C L. 2022. Cellular and Humoral Immune Responses after Immunisation with Low Virulent African Swine Fever Virus in the Large White Inbred Babraham Line and Outbred Domestic Pigs. Viruses, 14,

Goatley L C, Reis A L, Portugal R, Goldswain H, Shimmon G L, Hargreaves Z, Ho C S, Montoya M, Sánchez-Cordón P J, Taylor G, Dixon L K, Netherton C L. 2020. A Pool of Eight Virally Vectored African Swine Fever Antigens Protect Pigs Against Fatal Disease. Vaccines (Basel), 8,

Goatley L C, Tng P, Al-Adwani L, Hargreaves Z, Levin S, Lambe T, Netherton C L. 2023. ASFV antigens selected from genotype I immunised pigs are immunogenic, but do not protect against genotype II challenge. Veterinary Vaccine, 2, 100042.

Hao X, Zhang F, Yang Y, Shang S. 2021. The Evaluation of Cellular Immunity to Avian Viral Diseases: Methods, Applications, and Challenges. Front Microbiol, 12, 794514.

Hu X, Liu C, Rcheulishvili N, Wang Y, Xiong T, Xie F, Wang X, Chen R, Wang P G, He Y. 2025. Development and efficacy of a novel mRNA cocktail for the delivery of African swine fever virus antigens and induction of immune responses. Microbiol Spectr, 13, e0290924.

Huang J W, Niu D, Liu K, Wang Q, Ma L, Chen C C, Zhang L, Liu W, Zhou S, Min J, Wu S, Yang Y, Guo R T. 2020. Structure basis of non-structural protein pA151R from African Swine Fever Virus. Biochem Biophys Res Commun, 532, 108-113.

Jancovich J K, Chapman D, Hansen D T, Robida M D, Loskutov A, Craciunescu F, Borovkov A, Kibler K, Goatley L, King K, Netherton C L, Taylor G, Jacobs B, Sykes K, Dixon L K. 2018. Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins. J Virol, 92,

Janetzki S, Price L, Schroeder H, Britten C M, Welters M J, Hoos A. 2015. Guidelines for the automated evaluation of Elispot assays. Nat Protoc, 10, 1098-1115.

Li J, Hu L, Liu Y, Huang L, Mu Y, Cai X, Weng C. 2015. DDX19A Senses Viral RNA and Mediates NLRP3-Dependent Inflammasome Activation. J Immunol, 195, 5732-5749.

Li J, Song J, Zhou S, Li S, Liu J, Li T, Zhang Z, Zhang X, He X, Chen W, Zheng J, Zhao D, Bu Z, Huang L, Weng C. 2023. Development of a new effective African swine fever virus vaccine candidate by deletion of the H240R and MGF505-7R genes results in protective immunity against the Eurasia strain. J Virol, 97, e0070423.

Li S, Liu J, Meng L, Yin S, Wu H, Zou J, Yuan D, He H, Yin G, Jia X, Hao X, Shang S. 2025. Cellular immune signatures and differences of four porcine circovirus type 2 vaccines to heterologous PCV2d infection. NPJ Vaccines, 10, 92.

Li Y, Huang L, Li H, Zhu Y, Yu Z, Zheng X, Weng C, Feng W H. 2024. ASFV pA151R negatively regulates type I IFN production via degrading E3 ligase TRAF6. Front Immunol, 15, 1339510.

Liu W, Li H, Liu B, Lv T, Yang C, Chen S, Feng L, Lai L, Duan Z, Chen X, Li P, Guan S, Chen L. 2023. A new vaccination regimen using adenovirus-vectored vaccine confers effective protection against African swine fever virus in swine. Emerg Microbes Infect, 12, 2233643.

Mu S, Chen L, Dong H, Li S, Zhang Y, Yin S, Tian Y, Ding Y, Sun S, Shang S, Guo H. 2024. Enhanced antigen-specific CD8 T cells contribute to early protection against FMDV through swine DC vaccination. J Virol, 98, e0200223.

Neilan J G, Zsak L, Lu Z, Burrage T G, Kutish G F, Rock D L. 2004. Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection. Virology, 319, 337-342.

Netherton C L, Goatley L C, Reis A L, Portugal R, Nash R H, Morgan S B, Gault L, Nieto R, Norlin V, Gallardo C, Ho C S, Sánchez-Cordón P J, Taylor G, Dixon L K. 2019. Identification and Immunogenicity of African Swine Fever Virus Antigens. Front Immunol, 10, 1318.

Oura C a L, Denyer M S, Takamatsu H, Parkhouse R M E. 2005. In vivo depletion of CD8+ T lymphocytes abrogates protective immunity to African swine fever virus. J Gen Virol, 86, 2445-2450.

Pikalo J, Porfiri L, Akimkin V, Roszyk H, Pannhorst K, Kangethe R T, Wijewardana V, Sehl-Ewert J, Beer M, Cattoli G, Blome S. 2022. Vaccination With a Gamma Irradiation-Inactivated African Swine Fever Virus Is Safe But Does Not Protect Against a Challenge. Front Immunol, 13, 832264.

Portugal R, Goldswain H, Moore R, Tully M, Harris K, Corla A, Flannery J, Dixon L K, Netherton C L. 2024. Six adenoviral vectored African swine fever virus genes protect against fatal disease caused by genotype I challenge. J Virol, 98, e0062224.

Ramirez-Medina E, Vuono E, Pruitt S, Rai A, Espinoza N, Valladares A, Spinard E, Silva E, Velazquez-Salinas L, Gladue D P, Borca M V. 2022. ASFV Gene A151R Is Involved in the Process of Virulence in Domestic Swine. Viruses, 14,

Rodríguez I, Redrejo-Rodríguez M, Rodríguez J M, Alejo A, Salas J, Salas M L. 2006. African swine fever virus pB119L protein is a flavin adenine dinucleotide-linked sulfhydryl oxidase. J Virol, 80, 3157-3166.

Sanchez-Cordon P J, Chapman D, Jabbar T, Reis A L, Goatley L, Netherton C L, Taylor G, Montoya M, Dixon L. 2017. Different routes and doses influence protection in pigs immunised with the naturally attenuated African swine fever virus isolate OURT88/3. Antiviral Res, 138, 1-8.

Schafer A, Franzoni G, Netherton C L, Hartmann L, Blome S, Blohm U. 2022. Adaptive Cellular Immunity against African Swine Fever Virus Infections. Pathogens, 11,

Shang S B, Li Y F, Guo J Q, Wang Z T, Chen Q X, Shen H G, Zhou J Y. 2008. Development and validation of a recombinant capsid protein-based ELISA for detection of antibody to porcine circovirus type 2. Res Vet Sci, 84, 150-157.

Sun E, Huang L, Zhang X, Zhang J, Shen D, Zhang Z, Wang Z, Huo H, Wang W, Huangfu H, Wang W, Li F, Liu R, Sun J, Tian Z, Xia W, Guan Y, He X, Zhu Y, Zhao D, et al. 2021. Genotype I African swine fever viruses emerged in domestic pigs in China and caused chronic infection. Emerg Microbes Infect, 10, 2183-2193.

Sunwoo S Y, Perez-Nunez D, Morozov I, Sanchez E G, Gaudreault N N, Trujillo J D, Mur L, Nogal M, Madden D, Urbaniak K, Kim I J, Ma W, Revilla Y, Richt J A. 2019. DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain. Vaccines (Basel), 7,

Takamatsu H H, Denyer M S, Lacasta A, Stirling C M, Argilaguet J M, Netherton C L, Oura C A, Martins C, Rodríguez F. 2013. Cellular immunity in ASFV responses. Virus Res, 173, 110-121.

Tian Y, Wang D, He S, Cao Z, Li W, Jiang F, Shi Y, Hao Y, Wei X, Wang Q, Qie S, Wang J, Li T, Hao X, Zhu J, Wu J, Shang S, Zhai X. 2024. Immune cell early activation, apoptotic kinetic, and T-cell functional impairment in domestic pigs after ASFV CADC_HN09 strain infection. Front Microbiol, 15, 1328177.

Tran X H, Phuong L T T, Huy N Q, Thuy D T, Nguyen V D, Quang P H, Ngôn Q V, Rai A, Gay C G, Gladue D P, Borca M V. 2022. Evaluation of the Safety Profile of the ASFV Vaccine Candidate ASFV-G-ΔI177L. Viruses, 14,

Urbano A C, Ferreira F. 2022. African swine fever control and prevention: an update on vaccine development. Emerg Microbes Infect, 11, 2021-2033.

Van Den Born E, Olasz F, Meszaros I, Goltl E, Olah B, Joshi J, Van Kilsdonk E, Segers R, Zadori Z. 2025. African swine fever virus vaccine strain Asfv-G-∆I177l reverts to virulence and negatively affects reproductive performance. NPJ Vaccines, 10, 46.

Xiong W, Chen H, Chen Y, Wang K, Lian T, Zhang W, Yu Q, Gao X, Su J, He Q, Wang X, Yu J, Cui M. 2024. Diverse immune cell profiles in ASFV-associated lymphopenia. Animal Diseases, 4,

Yang J, Shao Z, Zhao X, Zhang W, Zhang Y, Li L, Gao Y, Shao Q, Cao C, Li H, Cui R, Liu H, Gan J. 2024. Structures of African swine fever virus topoisomerase complex and their implications. Nat Commun, 15, 6484.

Yuan F, Cui J, Wang T, Qin J, Jeon J H, Ding H, Whittaker C A, Xu R, Cao H, Chen J. 2025. Selection, Design, and Immunogenicity Studies of ASFV Antigens for Subunit mRNA Cocktail Vaccines with Specific Immune Response Profiles. ACS Infect Dis, 11, 1907-1921.

Zajac M D, Sangewar N, Lokhandwala S, Bray J, Sang H, Mccall J, Bishop R P, Waghela S D, Kumar R, Kim T, Mwangi W. 2022. Adenovirus-Vectored African Swine Fever Virus pp220 Induces Robust Antibody, IFN-γ, and CTL Responses in Pigs. Front Vet Sci, 9, 921481.

Zajac M D, Trujillo J D, Yao J, Kumar R, Sangewar N, Lokhandwala S, Sang H, Mallen K, Mccall J, Burton L, Kumar D, Heitmann E, Burnum T, Waghela S D, Almes K, Richt J, Kim T, Mwangi W. 2023. Immunization of pigs with replication-incompetent adenovirus-vectored African swine fever virus multi-antigens induced humoral immune responses but no protection following contact challenge. Front Vet Sci, 10, 1208275.

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.

No related articles found!

No Suggested Reading articles found!

Viewed

Full text

Abstract

Cited

Shared

Discussed

Cite this article:

About JIA

Editorial board

For authors