Applied Microbiology and Biotechnology

, Volume 100, Issue 24, pp 10531–10542 | Cite as

Identification and characterization of a common B-cell epitope on EIAV capsid proteins

  • Zhe Hu
  • Hao Chang
  • Xiaoyu Chu
  • Shuang Li
  • Meiyue Wang
  • Xiaojun WangEmail author
Applied genetics and molecular biotechnology


The equine infectious anemia virus (EIAV) capsid protein (p26) is one of the major immunogenic proteins during EIAV infection and is widely used for the detection of EIAV antibodies in horses. However, few reports have described the use of EIAV-specific monoclonal antibodies (MAbs) in etiological and immunological detection. Previously, we developed an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) for the quantification of the EIAV p26 protein level. However, the epitopes recognized by the MAbs were not identified, and the utilization of the MAbs needs to be evaluated. In this study, we characterized two monoclonal antibodies (9H8 and 1G11 MAbs) against EIAV p26. Two B-cell epitopes are located in amino acid residues, 73NLDKIAEE81 (HE) and 199KNAMRHLRPEDTLEEKMYAC218 (GE) for the 9H8 and 1G11 MAbs, respectively. The 1G11 epitope (GE) varied among viruses isolated worldwide but can be recognized by anti-EIAV sera from different regions, including China, the USA, and Argentina. Meanwhile, 1G11 MAb could react with the mutants of almost all the EIAV strains. Furthermore, we found that the histidine at position 204 (H204), leucine at position 205 (L205), and aspartic acid at position 209 (D209) of EIAV p26 individually played pivotal roles in binding with the 1G11 MAb. Our results revealed that the GE peptide might be a common B-cell binding epitope of EIAV antibodies. This is also the first report to identify a broad-spectrum monoclonal antibody (1G11) against p26 of EIAV. These findings may provide a useful basis for the development of new diagnostic assays for EIAV.


EIAV Monoclonal antibody Capsid protein p26 Epitope mapping Common B-cell epitope Broad-spectrum monoclonal antibody 



This study was funded by grants from the National Natural Science Foundation of China (grant number 31222054 to Xiaojun Wang and 31300713 to Zhe Hu).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.


  1. Alvarez I, Gutierrez G, Ostlund E, Barrandeguy M, Trono K (2007) Western blot assay using recombinant p26 antigen for detection of equine infectious anemia virus-specific antibodies. Clin Vaccine Immunol 14(12):1646–1648. doi: 10.1128/CVI.00293-07 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Alvarez I, Cipolini F, Wigdorovitz A, Trono K, Barrandeguy ME (2015) The efficacy of ELISA commercial kits for the screening of equine infectious anemia virus infection. Rev Argent Microbiol 47(1):25–28. doi: 10.1016/j.ram.2014.12.001 PubMedGoogle Scholar
  3. Chong YH, Ball JM, Issel CJ, Montelaro RC, Rushlow KE (1991a) Analysis of equine humoral immune responses to the transmembrane envelope glycoprotein (gp45) of equine infectious anemia virus. J Virol 65(2):1013–1018PubMedPubMedCentralGoogle Scholar
  4. Chong YH, Payne SL, Issel CJ, Montelaro RC, Rushlow KE (1991b) Characterization of the antigenic domains of the major core protein (p26) of equine infectious anemia virus. J Virol 65(2):1007–1012PubMedPubMedCentralGoogle Scholar
  5. Chung C, Mealey RH, McGuire TC (2004) CTL from EIAV carrier horses with diverse MHC class I alleles recognize epitope clusters in gag matrix and capsid proteins. Virology 327(1):144–154. doi: 10.1016/j.virol.2004.06.035 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cook RF, Leroux C, Issel CJ (2013) Equine infectious anemia and equine infectious anemia virus in 2013: a review. Vet Microbiol 167(1–2):181–204. doi: 10.1016/j.vetmic.2013.09.031 CrossRefPubMedGoogle Scholar
  7. Fraser DG, Oaks JL, Brown WC, McGuire TC (2002) Identification of broadly recognized, T helper 1 lymphocyte epitopes in an equine lentivirus. Immunology 105(3):295–305CrossRefPubMedPubMedCentralGoogle Scholar
  8. Hammond SA, Cook SJ, Lichtenstein DL, Issel CJ, Montelaro RC (1997) Maturation of the cellular and humoral immune responses to persistent infection in horses by equine infectious anemia virus is a complex and lengthy process. J Virol 71(5):3840–3852PubMedPubMedCentralGoogle Scholar
  9. Hu Z, Chang H, Ge M, Lin Y, Wang X, Guo W (2014) Development of antigen capture ELISA for the quantification of EIAV p26 protein. Appl Microbiol Biotechnol 98(21):9073–9081. doi: 10.1007/s00253-014-6078-8 CrossRefPubMedGoogle Scholar
  10. Hua RH, Liu LK, Chen ZS, Li YN, Bu ZG (2013) Comprehensive mapping antigenic epitopes of NS1 protein of Japanese encephalitis virus with monoclonal antibodies. PLoS One 8(6):e67553. doi: 10.1371/journal.pone.0067553 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Hussain KA, Issel CJ, Rwambo PM, Arnizaut AB, Ball JM, Schnorr KL, Montelaro RC (1988) Identification of gag precursor of equine infectious anaemia virus with monoclonal antibodies to the major viral core protein, p26. J Gen Virol 69(Pt 7):1719–1724. doi: 10.1099/0022-1317-69-7-1719 CrossRefPubMedGoogle Scholar
  12. Issel CJ, Coggins L (1979) Equine infectious anemia: current knowledge. J Am Vet Med Assoc 174(7):727–733PubMedGoogle Scholar
  13. Jin Z, Jin L, Peterson DL, Lawson CL (1999) Model for lentivirus capsid core assembly based on crystal dimers of EIAV p26. J Mol Biol 286(1):83–93. doi: 10.1006/jmbi.1998.2443 CrossRefPubMedGoogle Scholar
  14. Langemeier JL, Cook SJ, Cook RF, Rushlow KE, Montelaro RC, Issel CJ (1996) Detection of equine infectious anemia viral RNA in plasma samples from recently infected and long-term inapparent carrier animals by PCR. J Clin Microbiol 34(6):1481–1487PubMedPubMedCentralGoogle Scholar
  15. Macdonald RA, Hosking CS, Jones CL (1988) The measurement of relative antibody affinity by ELISA using thiocyanate elution. J Immunol Methods 106(2):191–194CrossRefPubMedGoogle Scholar
  16. McDermott J, Farrell L, Ross R, Barklis E (1996) Structural analysis of human immunodeficiency virus type 1 gag protein interactions, using cysteine-specific reagents. J Virol 70(8):5106–5114PubMedPubMedCentralGoogle Scholar
  17. McGuire TC, Leib SR, Lonning SM, Zhang W, Byrne KM, Mealey RH (2000) Equine infectious anaemia virus proteins with epitopes most frequently recognized by cytotoxic T lymphocytes from infected horses. J Gen Virol 81(Pt 11):2735–2739. doi: 10.1099/0022-1317-81-11-2735 CrossRefPubMedGoogle Scholar
  18. Montelaro RC, Parekh B, Orrego A, Issel CJ (1984) Antigenic variation during persistent infection by equine infectious anemia virus, a retrovirus. J Biol Chem 259(16):10539–10544PubMedGoogle Scholar
  19. Parekh B, Issel CJ, Montelaro RC (1980) Equine infectious anemia virus, a putative lentivirus, contains polypeptides analogous to prototype-C oncornaviruses. Virology 107(2):520–525CrossRefPubMedGoogle Scholar
  20. Payne S, Parekh B, Montelaro RC, Issel CJ (1984) Genomic alterations associated with persistent infections by equine infectious anaemia virus, a retrovirus. J Gen Virol 65(Pt 8):1395–1399. doi: 10.1099/0022-1317-65-8-1395 CrossRefPubMedGoogle Scholar
  21. Rosati S, Profiti M, Lorenzetti R, Bandecchi P, Mannelli A, Ortoffi M, Tolari F, Ciabatti IM (2004) Development of recombinant capsid antigen/transmembrane epitope fusion proteins for serological diagnosis of animal lentivirus infections. J Virol Methods 121(1):73–78. doi: 10.1016/j.jviromet.2004.06.001 CrossRefPubMedGoogle Scholar
  22. Salinovich O, Payne SL, Montelaro RC, Hussain KA, Issel CJ, Schnorr KL (1986) Rapid emergence of novel antigenic and genetic variants of equine infectious anemia virus during persistent infection. J Virol 57(1):71–80PubMedPubMedCentralGoogle Scholar
  23. Schots A, Van der Leede BJ, De Jongh E, Egberts E (1988) A method for the determination of antibody affinity using a direct ELISA. J Immunol Methods 109(2):225–233CrossRefPubMedGoogle Scholar
  24. Scicluna MT, Issel CJ, Cook FR, Manna G, Cersini A, Rosone F, Frontoso R, Caprioli A, Antognetti V, Autorino GL (2013) Is a diagnostic system based exclusively on agar gel immunodiffusion adequate for controlling the spread of equine infectious anaemia? Vet Microbiol 165(1–2):123–134. doi: 10.1016/j.vetmic.2013.02.027 CrossRefPubMedGoogle Scholar
  25. Shane BS, Issel CJ, Montelaro RC (1984) Enzyme-linked immunosorbent assay for detection of equine infectious anemia virus p26 antigen and antibody. J Clin Microbiol 19(3):351–355PubMedPubMedCentralGoogle Scholar
  26. Soutullo A, Garcia MI, Bailat A, Racca A, Tonarelli G, Malan Borel I (2005) Antibodies and PMBC from EIAV infected carrier horses recognize gp45 and p26 synthetic peptides. Vet Immunol Immunopathol 108(3–4):335–343. doi: 10.1016/j.vetimm.2005.06.007 CrossRefPubMedGoogle Scholar
  27. Soutullo A, Santi MN, Perin JC, Beltramini LM, Borel IM, Frank R, Tonarelli GG (2007) Systematic epitope analysis of the p26 EIAV core protein. J Mol Recognit 20(4):227–237. doi: 10.1002/jmr.825 CrossRefPubMedGoogle Scholar
  28. Stephens RM, Casey JW, Rice NR (1986) Equine infectious anemia virus gag and pol genes: relatedness to visna and AIDS virus. Science 231(4738):589–594CrossRefPubMedGoogle Scholar
  29. Sun M, Yu D, Mo H, Cao H, Chen C, Chen F (2012) Epitope mapping of a monoclonal antibody against the Gp85 of avian leukosis virus subgroup J. J Vet Med Sci 74(6):693–697CrossRefPubMedGoogle Scholar
  30. Worthylake DK, Wang H, Yoo S, Sundquist WI, Hill CP (1999) Structures of the HIV-1 capsid protein dimerization domain at 2.6 A resolution. Acta Crystallogr D Biol Crystallogr 55(Pt 1):85–92. doi: 10.1107/S0907444998007689 CrossRefPubMedGoogle Scholar
  31. Yang Y, Cao MJ, Alcocer M, Liu QM, Fei DX, Mao HY, Liu GM (2015) Mapping and characterization of antigenic epitopes of arginine kinase of Scylla paramamosain. Mol Immunol 65(2):310–320. doi: 10.1016/j.molimm.2015.02.010 CrossRefPubMedGoogle Scholar
  32. Zhang W, Auyong DB, Oaks JL, McGuire TC (1999) Natural variation of equine infectious anemia virus gag protein cytotoxic T lymphocyte epitopes. Virology 261(2):242–252. doi: 10.1006/viro.1999.9862 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Zhe Hu
    • 1
  • Hao Chang
    • 1
  • Xiaoyu Chu
    • 1
  • Shuang Li
    • 1
  • Meiyue Wang
    • 1
  • Xiaojun Wang
    • 1
    Email author
  1. 1.State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institutethe Chinese Academy of Agriculture SciencesHarbinPeople’s Republic of China

Personalised recommendations