Immunogenetics

, Volume 57, Issue 10, pp 730–738 | Cite as

Analysis of the expressed heavy chain variable-region genes of Macaca fascicularis and isolation of monoclonal antibodies specific for the Ebola virus' soluble glycoprotein

  • Chris Druar
  • Surinder S. Saini
  • Meredith A. Cossitt
  • Fei Yu
  • Xiangguo Qiu
  • Thomas W. Geisbert
  • Steven Jones
  • Peter B. Jahrling
  • Donald I. H. Stewart
  • Erik J. Wiersma
Original Paper

Abstract

The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding it's antibody repertoire is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5′ rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human VH repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the VH3 (41%), VH4 (39%), and VH1 (14%) subgroups used more frequently than the VH5 (3.9%) or VH7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative homolog of JH5β gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding human germline genes, 80–89% for IGHV and 72–92% for IGHJ. M. fascicularis and human VH genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans.

Keywords

Antibody repertoire Cynomolgus monkey VH gene Monoclonal antibody Ebola virus 

References

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefPubMedGoogle Scholar
  2. Amin T, Carter G (2004) Immunogenicity issues with therapeutic proteins. Curr Drug Discov:20–24Google Scholar
  3. Anderson DR, Brams P, Hanna N, Shestowsky WS, Heard C (2000) Human B7.1-specific primatized antibodies and transfectomas expressing said antibodies. US Patent 6113898, IDEC Pharmaceuticals Corporation, San DiegoGoogle Scholar
  4. Andris JS, Miller AB, Abraham SR, Cunningham S, Roubinet F, Blancher A, Capra JD (1997) Variable region gene segment utilization in rhesus monkey hybridomas producing human red blood cell-specific antibodies: predominance of the VH4 family but not VH4-21 (V4-34). Mol Immunol 34:237–253CrossRefPubMedGoogle Scholar
  5. Barbas CF III, Burton DR, Scott JK, Silverman GJ (2001) Phage display. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  6. Barre S, Greenberg AS, Flajnik MF, Chothia C (1994) Structural conservation of hypervariable regions in immunoglobulins evolution. Nat Struct Biol 1:915–920CrossRefPubMedGoogle Scholar
  7. Bible JM, Howard W, Robbins H, Dunn-Walters DK (2003) IGHV1, IGHV5 and IGHV7 subgroup genes in the rhesus macaque. Immunogenetics 54:867–873PubMedGoogle Scholar
  8. Brezinschek HP, Brezinschek RI, Lipsky PE (1995) Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction. J Immunol 155:190–202PubMedGoogle Scholar
  9. Chassagne S, Laffly E, Drouet E, Herodin F, Lefranc MP, Thullier P (2004) A high-affinity macaque antibody Fab with human-like framework regions obtained from a small phage display immune library. Mol Immunol 41:539–546CrossRefPubMedGoogle Scholar
  10. Chothia C, Lesk AM, Tramontano A, Levitt M, Smith-Gill SJ, Air G, Sheriff S, Padlan EA, Davies D, Tulip WR, Colman PM, Spinelli S, Alzari PM, Poljak RJ (1989) Conformations of immunoglobulin hypervariable regions. Nature 342:877–883CrossRefPubMedGoogle Scholar
  11. Cowell LG, Kim HJ, Humaljoki T, Berek C, Kepler TB (1999) Enhanced evolvability in immunoglobulin V genes under somatic hypermutation. J Mol Evol 49:23–26PubMedCrossRefGoogle Scholar
  12. Davidkova G, Petterson S, Holmberg D, Lundkvist I (1997) Selective usage of VH genes in adult human B lymphocyte repertoires. Scand J Immunol 45:62–73CrossRefPubMedGoogle Scholar
  13. Desiderio SV, Yancopoulos GD, Paskind M, Thomas E, Boss MA, Landau N, Alt FW, Baltimore D (1984) Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells. Nature 311:752–755CrossRefPubMedGoogle Scholar
  14. Frazer JK, Capra JD (1999) Immunoglobulins: structure and function. In: Paul WE (ed) Fundamental immunology, 4th edn. Lippincott-Raven, Philadelphia, pp 37–74Google Scholar
  15. Garbutt M, Liebscher R, Wahl-Jensen V, Jones S, Möller P, Wagner R, Volchkov V, Klenk HD, Feldmann H, Ströher U (2004) Properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses. J Virol 78:5458–5465CrossRefPubMedGoogle Scholar
  16. Helmuth EF, Letvin NL, Margolin DH (2000) Germline repertoire of the immunoglobulin V(H)3 family in rhesus monkeys. Immunogenetics 51:519–527CrossRefPubMedGoogle Scholar
  17. Huang C, Stewart AK, Schwartz R, Stollar BD (1992) Immunoglobulin heavy chain gene expression in peripheral blood B lymphocytes. J Clin Invest 89:1331–1343PubMedCrossRefGoogle Scholar
  18. Knappik A, Ge L, Honegger A, Pack P, Fischer M, Wellnhofer G, Hoess A, Wolle J, Pluckthun A, Virnekas B (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296:57–86CrossRefPubMedGoogle Scholar
  19. Lefranc MP, Giudicelli V, Ginestoux C, Bodmer J, Muller W, Bontrop R, Lemaitre M, Malik A, Barbie V, Chaume D (1999) IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 27:209–212CrossRefPubMedGoogle Scholar
  20. Lewis AP, Barber KA, Cooper HJ, Sims MJ, Worden J, Crowe JS (1993) Cloning and sequence analysis of kappa and gamma cynomolgus monkey immunoglobulin cDNAs. Dev Comp Immunol 17:549–560PubMedCrossRefGoogle Scholar
  21. Lin MM, Zhu M, Scharff MD (1997) Sequence dependent hypermutation of the immunoglobulin heavy chain in cultured B cells. Proc Natl Acad Sci U S A 94:5284–5289CrossRefPubMedGoogle Scholar
  22. Link JM, Hellinger MA, Schroeder HW Jr (2002) The rhesus monkey immunoglobulin IGHD and IGHJ germline repertoire. Immunogenetics 54:240–250CrossRefPubMedGoogle Scholar
  23. Logdberg L, Kaplan E, Drelich M, Harfeldt E, Gunn H, Ehrlich P, Dottavio D, Lake P, Ostberg L (1994) Primate antibodies to components of the human immune system. J Med Primatol 23:285–297PubMedGoogle Scholar
  24. Margolin DH, Reimann KA, Sodroski J, Karlsson GB, Tenner-Racz K, Racz P, Letvin NL (1997) Immunoglobulin V(H) usage during primary infection of rhesus monkeys with chimeric simian–human immunodeficiency viruses. J Virol 71:8582–8591PubMedGoogle Scholar
  25. Meek K, Eversole T, Capra JD (1991) Conservation of the most JH proximal Ig VH gene segment (VHVI) throughout primate evolution. J Immunol 146:2434–2438Google Scholar
  26. Newman R, Alberts J, Anderson D, Carner K, Heard C, Norton F, Raab R, Reff M, Shuey S, Hanna N (1992) “Primatization” of recombinant antibodies for immunotherapy of human diseases: a macaque/human chimeric antibody against human CD4. Biotechnology (N Y) 10:1455–1460CrossRefGoogle Scholar
  27. Newman RA, Hanna N, Raab RW (1997) Recombinant antibodies for human therapy. US Patent 5,658,570, IDEC Pharmaceuticals Corporation, San DiegoGoogle Scholar
  28. O'Brien PM, Aitken R (2002) Antibody phage display. Methods in molecular biology. Humana, TotowaGoogle Scholar
  29. Padlan EA (1997) Does base composition help predispose the complementarity-determining regions of antibodies to hypermutation? Mol Immunol 34:765–770CrossRefPubMedGoogle Scholar
  30. Rassenti LZ, Kohsaka H, Kipps TJ (1995) Analysis of immunoglobulin VH gene repertoire by an anchored PCR-ELISA. Ann N Y Acad Sci 764:463–473PubMedCrossRefGoogle Scholar
  31. Rosner K, Winter DB, Tarone RE, Skovgaard GL, Bohr VA, Gearhart PJ (2001) Third complementarity-determining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than non-mutated genes. Immunology 103:179–187CrossRefPubMedGoogle Scholar
  32. Ruiz M, Lefranc MP (2002) IMGT gene identification and Colliers de Perles of human immunoglobulins with known 3D structures. Immunogenetics 53:857–883CrossRefPubMedGoogle Scholar
  33. Saini SS, Allore B, Jacobs RM, Kaushik A (1999) Exceptionally long CDR3H region with multiple cysteine residues in functional bovine IgM antibodies. Eur J Immunol 29:2420–2426PubMedCrossRefGoogle Scholar
  34. Samuelsson A, Choidi F, Ohman P, Putkonen P, Norrby E, Persson MA (1995) Chimeric macaque/human Fab molecules neutralize simian immunodeficiency virus. Virology 207:495–502CrossRefPubMedGoogle Scholar
  35. Sanz I, Dang H, Takei M, Talal N, Capra JD (1989) VH sequence of a human anti-Sm autoantibody. Evidence that autoantibodies can be unmutated copies of germline genes. J Immunol 142:883–887PubMedGoogle Scholar
  36. Soltes G, Barker H, Marmai K, Pun E, Yuen A, Wiersma EJ (2003) A new helper phage and phagemid vector system improves viral display of antibody Fab fragments and avoids propagation of insert-less virions. J Immunol Methods 274:233–244CrossRefPubMedGoogle Scholar
  37. Storb U (2001) DNA polymerases in immunity: profiting from errors. Nat Immunol 2:484–485CrossRefPubMedGoogle Scholar
  38. Tomlinson IM, Walter G, Jones PT, Dear PH, Sonnhammer EL, Winter G (1996) The imprint of somatic hypermutation on the repertoire of human germline V genes. J Mol Biol 256:813–817CrossRefPubMedGoogle Scholar
  39. Tonegawa S (1983) Somatic generation of antibody diversity. Nature 302:575–581PubMedCrossRefGoogle Scholar
  40. Tordsson J, Lavasani S, Ohlsson L, Karlstrom P, Svedberg H, Abrahmsson L, Brodin T (2000) A3—a novel colon and pancreatic cancer reactive antibody from a primate phage library selected using intact tumour cells. Int J Cancer 87:559–568CrossRefPubMedGoogle Scholar
  41. Tsurushita N, Vasquez M (2004) Humanization of monoclonal antibodies. In: Honjo T, Alt F, Neuberger M (eds) Molecular biology of B-cells. Elsevier/Academic, San Diego, pp 533–545Google Scholar
  42. VanDyk L, Meek K (1992) Assembly of IgH CDR3: mechanism, regulation, and influence on antibody diversity. Int Rev Immunol 8:123–133PubMedCrossRefGoogle Scholar
  43. Vargas-Madrazo E, Lara-Ochoa F, Ramirez-Benites MC, Almagro JC (1997) Evolution of the structural repertoire of the human V(H) and Vkappa germline genes. Int Immunol 9:1801–1815CrossRefPubMedGoogle Scholar
  44. Via M (2004) Advances. Life Science reports. New directions in monoclonal antibodies. Cambridge Healthtech Advisors, Cambridge, MAGoogle Scholar
  45. Wahl-Jensen V, Kurz SK, Hazelton PR, Schnittler HJ, Stroher U, Burton DR, Feldmann H (2005) Role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages. J Virol 79:2413–2419CrossRefPubMedGoogle Scholar
  46. Wiersma EJ, Stewart DIH (2003) Phagemid display system. Cangene Corporation WO 03/031611 A2 (patent pending)Google Scholar
  47. Yamada M, Wasserman R, Reichard BA, Shane S, Caton A, Rovera G (1991) Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes. J Exp Med 173:395–407CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Chris Druar
    • 1
  • Surinder S. Saini
    • 1
    • 2
  • Meredith A. Cossitt
    • 1
  • Fei Yu
    • 1
  • Xiangguo Qiu
    • 3
  • Thomas W. Geisbert
    • 4
  • Steven Jones
    • 3
  • Peter B. Jahrling
    • 4
  • Donald I. H. Stewart
    • 1
  • Erik J. Wiersma
    • 1
  1. 1.Cangene CorporationMississaugaCanada
  2. 2.Canadian Food Inspection AgencyBeamsvilleCanada
  3. 3.Health Canada, National Microbiology LaboratoryWinnipegCanada
  4. 4.US Army Medical Research Institute of Infectious DiseasesFort DetrickUSA

Personalised recommendations