Enhanced bovine herpesvirus type 1 neutralization by multimerized single-chain variable antibody fragments regardless of differential glycosylation

Clin Vaccine Immunol. 2012 Aug;19(8):1150-7. doi: 10.1128/CVI.00130-12. Epub 2012 Jun 13.

Abstract

Single-chain variable antibody fragments (scFvs) with a 2-amino-acid linker capable of multimerization as di-, tri-, or tetrabodies that neutralize bovine herpesvirus type 1 (BoHV-1) in vitro were constructed and expressed in Pichia pastoris. In contrast to the monomeric form, multimeric scFvs had a higher virus neutralization potency, as evidenced by a 2-fold increase in their ability to neutralize BoHV-1 due to avidity effects. Mass spectrum (quadrupole time of flight [Q-TOF]) analyses of multimeric scFv demonstrated extensive heterogeneity due to differential cleavage, variable glycosylation (1 to 9 mannose residues), and the incorporation of minor unidentified adducts. Regardless of the differential glycosylation patterns, the scFvs recognized non-gB or -gE target viral epitopes in the BoHV-1 envelope fraction in a Western blot and also neutralized BoHV-1 in infected Madin-Darby kidney (MDBK) cells in vitro. Indirect evidence for the noncovalent multimerization of scFv was the presence of a major peak of multimerized scFv without a His tag (due to differential cleavage) in the Q-TOF profile, unlike monomeric scFv, which copurified with normally His-tagged scFv and recognized the target antigen. Overall, differentially glycosylated recombinant scFvs against BoHV-1 with a short linker (2 amino acids) are capable of assembly into functional multimers that confer high avidity, resulting in increased virus neutralization in vitro compared to that of monovalent scFv with a long (18-amino-acid) flexible linker. Overall, recombinant multimerized scFv5-2L potentially provides a high-potency therapeutic and immunodiagnostic reagent against BoHV-1, which is suitable for passive immunization and topical application.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Neutralizing / immunology*
  • Antibodies, Viral / immunology*
  • Base Sequence
  • Blotting, Western
  • Epitopes / immunology
  • Gene Expression
  • Glycosylation
  • Herpesvirus 1, Bovine / immunology*
  • Mass Spectrometry
  • Molecular Sequence Data
  • Molecular Weight
  • Neutralization Tests
  • Pichia / genetics
  • Protein Multimerization
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • Single-Chain Antibodies / chemistry
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology*
  • Single-Chain Antibodies / metabolism

Substances

  • Antibodies, Neutralizing
  • Antibodies, Viral
  • Epitopes
  • Recombinant Proteins
  • Single-Chain Antibodies