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Home / Equine Research Bank / Viruses / Identification and Characterization of Linear Epitopes of Mo...
Viruses2024; 16(11); 1780; doi: 10.3390/v16111780

Identification and Characterization of Linear Epitopes of Monoclonal Antibodies Against African Horse Sickness Virus Serotype 1 VP2 Protein.

Abstract: African horse sickness (AHS) is an acute, fatal, contagious disease of animals of the family Equidae and is caused by infection with the African horse sickness virus (AHSV). Based on the outer capsid protein VP2, AHSV is classified into nine serotypes (AHSV-1 to -9) with little or no serological cross-reactivity between them. In 2020, AHS outbreaks caused by AHSV-1 were reported in Thailand and Malaysia, marking the first occurrences of AHS in Southeast Asia. However, little is known about the antigenic profile of AHSV-1 VP2. In this study, a recombinant VP2 protein was expressed in and used as an immunogen, and three monoclonal antibodies (mAbs), designated 7D11, 10A9, and 9E7, against AHSV-1 VP2, were generated. These three mAbs were then successfully used in IFA, WB, and ELISA for the detection of AHSV-1 VP2. Two overlapping linear epitopes, NEFDFE (E670-675) recognized by 9E7 and NEFDF (E670-674) recognized by 7D11 and 10A9, were identified through truncation of GST-fused VP2. Amino acid sequence alignment shows that the NEFDFE motif is completely conserved within AHSV-1 but is highly divergent in other AHSV serotypes. Our studies provide an important tool for basic research into AHSV-1 and for the diagnosis of AHSV-1.
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Publication Date: 2024-11-15 PubMed ID: 39599893PubMed Central: PMC11599129DOI: 10.3390/v16111780Google Scholar: Lookup
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  • Journal Article
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This research focuses on identifying specific linear epitopes on the VP2 protein of African horse sickness virus serotype 1 (AHSV-1) using monoclonal antibodies (mAbs) to aid in disease diagnosis and further research.

Introduction to African Horse Sickness and VP2 Protein

  • African horse sickness (AHS) is a severe and often fatal disease affecting Equidae (horses, donkeys, zebras).
  • The causative agent is African horse sickness virus (AHSV), which has nine distinct serotypes (AHSV-1 through AHSV-9).
  • Classification is based primarily on the outer capsid protein VP2.
  • VP2 is responsible for antigenic specificity, with little to no cross-reactivity among different serotypes, making the study of VP2 crucial for serotype-specific diagnosis and immunity.
  • Recently, outbreaks of AHSV-1 in Southeast Asia (Thailand and Malaysia) raised concerns and highlighted the need to understand this serotype better.
  • Despite its importance, the detailed antigenic profile of AHSV-1 VP2 was poorly characterized before this study.

Research Objectives

  • To express a recombinant form of the AHSV-1 VP2 protein suitable for immunological studies.
  • To generate monoclonal antibodies (mAbs) that specifically recognize AHSV-1 VP2.
  • To identify and characterize the linear epitopes on the VP2 protein recognized by these antibodies.
  • To evaluate the conservation of identified epitopes among different AHSV serotypes to understand serotype specificity.

Methods

  • The researchers produced a recombinant VP2 protein—likely using bacterial or eukaryotic expression systems (the exact system is missing in the abstract but typically involved expression vectors and host cells).
  • Three monoclonal antibodies were generated: 7D11, 10A9, and 9E7, targeting AHSV-1 VP2.
  • The mAbs were tested and used successfully in:
    • Immunofluorescence Assay (IFA) to detect VP2 in cells,
    • Western blot (WB) for protein detection and confirmation, and
    • Enzyme-linked immunosorbent assay (ELISA) for sensitive quantification and antigen detection.
  • To identify linear epitopes, the researchers performed truncation analysis using GST-fused (glutathione S-transferase tagged) VP2 protein fragments. This helps map exactly which peptide sequences the antibodies bind.
  • Sequence alignment of the identified motifs was carried out across different AHSV serotypes to evaluate conservation or divergence.

Key Findings

  • Two overlapping linear epitopes were identified on the VP2 protein:
    • The epitope NEFDFE (amino acids 670-675) is recognized exclusively by mAb 9E7.
    • The shorter epitope NEFDF (amino acids 670-674), a subset of the longer epitope, is recognized by mAbs 7D11 and 10A9.
  • These epitopes lie toward the C-terminal region of the VP2 protein.
  • Sequence alignment showed that the NEFDFE motif is:
    • Completely conserved in AHSV-1 strains, demonstrating the epitope’s reliability for specific detection.
    • Highly divergent in VP2 proteins of other AHSV serotypes, explaining the lack of cross-reactivity and supporting serotype specificity.
  • The three generated monoclonal antibodies are specific and effective tools for detecting AHSV-1 VP2.

Implications and Applications

  • The identified epitopes and corresponding monoclonal antibodies provide valuable reagents for the development of diagnostic assays targeting AHSV-1.
  • The specificity of these antibodies will help differentiate AHSV-1 infections from infections by other AHSV serotypes, critical for accurate disease surveillance and control.
  • This research lays a foundation for further immunological and vaccine-related studies focusing on the VP2 protein of AHSV-1.
  • Potentially, these findings facilitate rapid and specific diagnosis in outbreak regions, improving disease management and containment.
  • Basic research can leverage these mAbs to explore AHSV-1 VP2 structure-function relationships, virus-host interactions, and immune responses.

Summary

  • The study successfully produced and characterized monoclonal antibodies against AHSV-1 VP2 protein.
  • It mapped two overlapping linear epitopes critical for antibody recognition, emphasizing AHSV-1 specificity.
  • These tools advance diagnostic capabilities and provide insights into antigenic properties of this important viral protein.

Cite This Article

APA
Ma X, Zhang Y, Na L, Qi T, Ma W, Guo X, Wang XF, Wang X. (2024). Identification and Characterization of Linear Epitopes of Monoclonal Antibodies Against African Horse Sickness Virus Serotype 1 VP2 Protein. Viruses, 16(11), 1780. https://doi.org/10.3390/v16111780

Publication

Viruses
ISSN: 1999-4915
NlmUniqueID: 101509722
Country: Switzerland
Language: English
Volume: 16
Issue: 11
PII: 1780

Researcher Affiliations

Ma, Xiaohua
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Zhang, Yingzhi
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Na, Lei
  • College of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agriculture and Forestry, Jurong 212400, China.
Qi, Ting
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Ma, Weiwei
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Guo, Xing
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Wang, Xue-Feng
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Wang, Xiaojun
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China.
  • Institute of Western Agriculture, The Chinese Academy of Agricultural Sciences, Changji 831100, China.

MeSH Terms

  • Antibodies, Monoclonal / immunology
  • Capsid Proteins / immunology
  • Capsid Proteins / genetics
  • Capsid Proteins / chemistry
  • Animals
  • African Horse Sickness Virus / immunology
  • African Horse Sickness Virus / genetics
  • Serogroup
  • Antibodies, Viral / immunology
  • Epitopes / immunology
  • Epitopes / genetics
  • Mice
  • African Horse Sickness / immunology
  • African Horse Sickness / prevention & control
  • Epitope Mapping
  • Mice, Inbred BALB C
  • Horses

Grant Funding

  • 2022YFD1800504；TD2022C006 / The National Key Research and Development Project of China；The Natural Science Foundation of Heilongjiang Province of China

Conflict of Interest Statement

The authors declare no conflicts of interest.

References

This article includes 41 references
  1. Hamblin C, Salt J.S, Mellor P.S, Graham S.D, Smith P.R, Wohlsein P. Donkeys as reservoirs of African horse sickness virus.. Arch. Virol. 1998;14:37–47.
    pubmed: 9785494
  2. Brian W.J.M, Marc H.V.R. Encyclopedia of Virology.. 3rd ed. Academic Press; New York, NY, USA: 2008. p. 7.
  3. Fassi-Fihri O, el Harrak M, Fassi-Fehri M.M. Clinical, virological and immune responses of normal and immunosuppressed donkeys (Equus asinus africanus) after inoculation with African horse sickness virus.. Arch. Virol. Suppl. 1998;14:49–56.
    pubmed: 9785495
  4. Barnard B.J. Epidemiology of African horse sickness and the role of the zebra in South Africa.. Arch. Virol. Suppl. 1998;14:13–19.
    pubmed: 9785491
  5. Dennis S.J, Meyers A.E, Hitzeroth I.I, Rybicki E.P. African Horse Sickness: A Review of Current Understanding and Vaccine Development.. Viruses 2019;11:844.
    doi: 10.3390/v11090844pmc: PMC6783979pubmed: 31514299google scholar: lookup
  6. Bunpapong N, Charoenkul K, Nasamran C, Chamsai E, Udom K, Boonyapisitsopa S, Tantilertcharoen R, Kesdangsakonwut S, Techakriengkrai N, Suradhat S. African Horse Sickness Virus Serotype 1 on Horse Farm, Thailand, 2020.. Emerg. Infect. Dis. 2021;27:2208–2211.
    doi: 10.3201/eid2708.210004pmc: PMC8314833pubmed: 34287126google scholar: lookup
  7. Malaysia D.O.V.S. African Horse Sickness in Malaysia.. Department for Environment, Food and Rural Affairs; London, UK: 2020.
  8. WOAH. Recognition of the African Horse Sickness Status of Members In Resolution No. 16.. WOAH; Paris, France: 2023.
  9. de Vos C.J, Hoek C.A, Nodelijk G. Risk of introducing African horse sickness virus into the Netherlands by international equine movements.. Prev. Vet. Med. 2012;106:108–122.
    doi: 10.1016/j.prevetmed.2012.01.019pubmed: 22341773google scholar: lookup
  10. Robin M, Page P, Archer D, Baylis M. African horse sickness: The potential for an outbreak in disease-free regions and current disease control and elimination techniques.. Equine Vet. J. 2016;48:659–669.
    doi: 10.1111/evj.12600pubmed: 27292229google scholar: lookup
  11. Gao S, Zeng Z, Wang H, Chen F, Huang L, Wang X. Predicting the possibility of African horse sickness (AHS) introduction into China using spatial risk analysis and habitat connectivity of Culicoides.. Sci. Rep. 2022;12:3910.
    doi: 10.1038/s41598-022-07512-wpmc: PMC8913660pubmed: 35273211google scholar: lookup
  12. Carpenter S, Mellor P.S, Fall A.G, Garros C, Venter G.J. African Horse Sickness Virus: History, Transmission, and Current Status.. Annu. Rev. Entomol. 2017;62:343–358.
    doi: 10.1146/annurev-ento-031616-035010pubmed: 28141961google scholar: lookup
  13. Roy P, Mertens P.P, Casal I. African horse sickness virus structure.. Comp. Immunol. Microbiol. Infect. Dis. 1994;17:243–273.
    doi: 10.1016/0147-9571(94)90046-9pubmed: 8001348google scholar: lookup
  14. Zwart L, Potgieter C.A, Clift S.J, van Staden V. Characterising Non-Structural Protein NS4 of African Horse Sickness Virus.. PLoS ONE 2015;10:e0124281.
    doi: 10.1371/journal.pone.0124281pmc: PMC4411093pubmed: 25915516google scholar: lookup
  15. Boughan S, Potgieter A.C, van Staden V. African horse sickness virus NS4 is a nucleocytoplasmic protein that localizes to PML nuclear bodies.. J. Gen. Virol. 2020;101:366–384.
    pubmed: 32125263
  16. Wall G.V., Wright I.M., Barnardo C., Erasmus B.J., van Staden V., Potgieter A.C. African horse sickness virus NS4 protein is an important virulence factor and interferes with JAK-STAT signaling during viral infection. Virus Res. 2021;298:198407. doi: 10.1016/j.virusres.2021.198407.
    doi: 10.1016/j.virusres.2021.198407pubmed: 33812899google scholar: lookup
  17. Manole V., Laurinmaki P., Van Wyngaardt W., Potgieter C.A., Wright I.M., Venter G.J., van Dijk A.A., Sewell B.T., Butcher S.J. Structural insight into African horsesickness virus infection. J. Virol. 2012;86:7858–7866. doi: 10.1128/JVI.00517-12.
    doi: 10.1128/JVI.00517-12pmc: PMC3421665pubmed: 22593166google scholar: lookup
  18. Zhang X., Boyce M., Bhattacharya B., Zhang X., Schein S., Roy P., Zhou Z.H. Bluetongue virus coat protein VP2 contains sialic acid-binding domains, and VP5 resembles enveloped virus fusion proteins. Proc. Natl. Acad. Sci. USA. 2010;107:6292–6297. doi: 10.1073/pnas.0913403107.
    doi: 10.1073/pnas.0913403107pmc: PMC2852009pubmed: 20332209google scholar: lookup
  19. Hassan S.S., Roy P. Expression and functional characterization of bluetongue virus VP2 protein: Role in cell entry. J. Virol. 1999;73:9832–9842. doi: 10.1128/JVI.73.12.9832-9842.1999.
    doi: 10.1128/JVI.73.12.9832-9842.1999pmc: PMC113032pubmed: 10559295google scholar: lookup
  20. Vreede F.T., Huismans H. Cloning, characterization and expression of the gene that encodes the major neutralization-specific antigen of African horsesickness virus serotype 3. Pt 12J. Gen. Virol. 1994;75:3629–3633. doi: 10.1099/0022-1317-75-12-3629.
    doi: 10.1099/0022-1317-75-12-3629pubmed: 7996157google scholar: lookup
  21. Aksular M., Calvo-Pinilla E., Marin-Lopez A., Ortego J., Chambers A.C., King L.A., Castillo-Olivares J. A single dose of African horse sickness virus (AHSV) VP2 based vaccines provides complete clinical protection in a mouse model. Vaccine. 2018;36:7003–7010. doi: 10.1016/j.vaccine.2018.09.065.
    doi: 10.1016/j.vaccine.2018.09.065pmc: PMC6219453pubmed: 30309744google scholar: lookup
  22. Calvo-Pinilla E., Gubbins S., Mertens P., Ortego J., Castillo-Olivares J. The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) viruses expressing African horse sickness virus VP2 antigens depends on the levels of expressed VP2 protein delivered to the host. Antiviral. Res. 2018;154:132–139. doi: 10.1016/j.antiviral.2018.04.015.
    doi: 10.1016/j.antiviral.2018.04.015pmc: PMC5966619pubmed: 29678552google scholar: lookup
  23. WOAH . Equidae-Chapter 3.6.1-African Horse SICKNESS (Infection with African Horse Sickness Virus) WOAH; Paris, France: 2019. Terrestrial manual-SECTION 3.6.
  24. Manning N.M., Bachanek-Bankowska K., Mertens P.P.C., Castillo-Olivares J. Vaccination with recombinant Modified Vaccinia Ankara (MVA) viruses expressing single African horse sickness virus VP2 antigens induced cross-reactive virus neutralising antibodies (VNAb) in horses when administered in combination. Vaccine. 2017;35:6024–6029. doi: 10.1016/j.vaccine.2017.04.005.
    doi: 10.1016/j.vaccine.2017.04.005pubmed: 28438410google scholar: lookup
  25. Kanai Y., van Rijn P.A., Maris-Veldhuis M., Kaname Y., Athmaram T.N., Roy P. Immunogenicity of recombinant VP2 proteins of all nine serotypes of African horse sickness virus. Vaccine. 2014;32:4932–4937. doi: 10.1016/j.vaccine.2014.07.031.
    doi: 10.1016/j.vaccine.2014.07.031pmc: PMC4148702pubmed: 25045805google scholar: lookup
  26. Alberca B., Bachanek-Bankowska K., Cabana M., Calvo-Pinilla E., Viaplana E., Frost L., Gubbins S., Urniza A., Mertens P., Castillo-Olivares J. Vaccination of horses with a recombinant modified vaccinia Ankara virus (MVA) expressing African horse sickness (AHS) virus major capsid protein VP2 provides complete clinical protection against challenge. Vaccine. 2014;32:3670–3674. doi: 10.1016/j.vaccine.2014.04.036.
    doi: 10.1016/j.vaccine.2014.04.036pmc: PMC4061461pubmed: 24837765google scholar: lookup
  27. Martinez-Torrecuadrada J.L., Langeveld J.P.M., Meloen R.H., Casal J.I. Definition of neutralizing sites on African horse sickness virus serotype 4 VP2 at the level of peptides. Pt 10J. Gen. Virol. 2001;82:2415–2424. doi: 10.1099/0022-1317-82-10-2415.
    doi: 10.1099/0022-1317-82-10-2415pubmed: 11562535google scholar: lookup
  28. Martinez-Torrecuadrada J.L., Casal J.I. Identification of a linear neutralization domain in the protein VP2 of African horse sickness virus. Virology. 1995;210:391–399. doi: 10.1006/viro.1995.1355.
    doi: 10.1006/viro.1995.1355pubmed: 7542417google scholar: lookup
  29. Bentley L., Fehrsen J., Jordaan F., Huismans H., du Plessis D.H. Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries. Pt 4J. Gen. Virol. 2000;81:993–1000. doi: 10.1099/0022-1317-81-4-993.
    doi: 10.1099/0022-1317-81-4-993pubmed: 10725425google scholar: lookup
  30. Faber E., van Schalkwyk A., Ivy Tshilwane S., Van Kleef M., Pretorius A. Identification of T cell and linear B cell epitopes on African horse sickness virus serotype 4 proteins VP1-1, VP2, VP4, VP7 and NS3. Vaccine. 2024;42:136–145. doi: 10.1016/j.vaccine.2023.12.028.
    doi: 10.1016/j.vaccine.2023.12.028pubmed: 38097459google scholar: lookup
  31. Mathebula E.M., Faber F.E., Van Wyngaardt W., Van Schalkwyk A., Pretorius A., Fehrsen J. B-cell epitopes of African horse sickness virus serotype 4 recognised by immune horse sera. Onderstepoort J. Vet. Res. 2017;84:e1–e12. doi: 10.4102/ojvr.v84i1.1313.
    doi: 10.4102/ojvr.v84i1.1313pmc: PMC6238682pubmed: 28281773google scholar: lookup
  32. Ma X., Gao M., Zhang X., Ma W., Xue F., Wang X.F., Wang X. Identification and characterization of linear epitopes of monoclonal antibodies against the capsid proteins of small ruminant lentiviruses. Front. Microbiol. 2024;15:1452063. doi: 10.3389/fmicb.2024.1452063.
    doi: 10.3389/fmicb.2024.1452063pmc: PMC11325181pubmed: 39149208google scholar: lookup
  33. Ting Q. Ph.D. Thesis. Chinese Academy of Agricultural Sciences; Beijing, China: 2011. Molecular Epidemiologic Surveillance of Equine Influenza Virus in China and Primary Study of Fowlpox Virus-Vectored Vaccine Against EIV.
  34. Qiao C.L., Yu K.Z., Jiang Y.P., Jia Y.Q., Tian G.B., Liu M., Deng G.H., Wang X.R., Meng Q.W., Tang X.Y. Protection of chickens against highly lethal H5N1 and H7N1 avian influenza viruses with a recombinant fowlpox virus co-expressing H5 haemagglutinin and N1 neuraminidase genes. Avian Pathol. 2003;32:25–32. doi: 10.1080/0307945021000070688.
    doi: 10.1080/0307945021000070688pubmed: 12745375google scholar: lookup
  35. Van Wyngaardt W., Du Plessis D.H., Van Wyngaardt S., Verschoor J.A. Production and properties of monoclonal antibodies against African horsesickness virus, serotype 3. Onderstepoort J. Vet. Res. 1992;59:129–133.
    pubmed: 1513593
  36. Ranz A.I., Miguet J.G., Anaya C., Venteo A., Cortes E., Vela C., Sanz A. Diagnostic methods for African horsesickness virus using monoclonal antibodies to structural and non-structural proteins. Vet. Microbiol. 1992;33:143–153. doi: 10.1016/0378-1135(92)90042-R.
    doi: 10.1016/0378-1135(92)90042-Rpubmed: 1481354google scholar: lookup
  37. Wang X., Fan Y., Hu X., Xu J., Zhang H., Tian Z., Gou H., Zheng Y., Guan G., Luo J., et al. Truncated expression and immunogenicity analysis of VP2 protein of African horse sickness virus. Chin. Vet. Sci. 2024;54:1087–1095.
  38. Potgieter A.C., Cloete M., Pretorius P.J., van Dijk A.A. A first full outer capsid protein sequence data-set in the Orbivirus genus (family Reoviridae): Cloning, sequencing, expression and analysis of a complete set of full-length outer capsid VP2 genes of the nine African horsesickness virus serotypes. Pt 5J. Gen. Virol. 2003;84:1317–1326. doi: 10.1099/vir.0.18919-0.
    doi: 10.1099/vir.0.18919-0pubmed: 12692299google scholar: lookup
  39. Guthrie A.J., Quan M., Lourens C.W., Audonnet J.C., Minke J.M., Yao J., He L., Nordgren R., Gardner I.A., Maclachlan N.J. Protective immunization of horses with a recombinant canarypox virus vectored vaccine co-expressing genes encoding the outer capsid proteins of African horse sickness virus. Vaccine. 2009;27:4434–4438. doi: 10.1016/j.vaccine.2009.05.044.
    doi: 10.1016/j.vaccine.2009.05.044pubmed: 19490959google scholar: lookup
  40. Chiam R., Sharp E., Maan S., Rao S., Mertens P., Blacklaws B., Davis-Poynter N., Wood J., Castillo-Olivares J. Induction of antibody responses to African horse sickness virus (AHSV) in ponies after vaccination with recombinant modified vaccinia Ankara (MVA) PLoS ONE. 2009;4:e5997. doi: 10.1371/journal.pone.0005997.
    doi: 10.1371/journal.pone.0005997pmc: PMC2694985pubmed: 19543394google scholar: lookup
  41. Burrage T.G., Trevejo R., Stone-Marschat M., Laegreid W.W. Neutralizing epitopes of African horsesickness virus serotype 4 are located on VP2. Virology. 1993;196:799–803. doi: 10.1006/viro.1993.1537.
    doi: 10.1006/viro.1993.1537pubmed: 7690505google scholar: lookup

Citations

This article has been cited 1 times.
  1. Ma X, Zhang M, Zhang X, Qi T, Zhang W, Zhao Y, Na L, Zhang Y, Wang XF, Wang X. Construction and Immunogenicity Evaluation of a Recombinant Fowlpox Virus Expressing VP2 Gene of African Horse Sickness Virus Serotype 1.. Microorganisms 2025 Dec 9;13(12).
    doi: 10.3390/microorganisms13122807pubmed: 41472010google scholar: lookup
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