Enhanced bovine herpesvirus type 1 neutralization by multimerized single-chain variable antibody fragments regardless of differential glycosylation.
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.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Yfke
Y
Pasman
Eva
E
Nagy
Azad K
AK
Kaushik
D057134:Antibodies, Neutralizing / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D000939:Epitopes / Q000276:immunology; D015870:Gene Expression; D006031:Glycosylation; D007242:Herpesvirus 1, Bovine / Q000276:immunology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009500:Neutralization Tests; D010843:Pichia / Q000235:genetics; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000939:Epitopes; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies