Construction of single-chain Fv with two possible CDR3H conformations but similar inter-molecular forces that neutralize bovine herpesvirus 1.
Bovine herpesvirus 1 (BoHV-1) causes respiratory and genital diseases in cattle for which available vaccines do not confer adequate protection. Since passive immunization with antibodies permits disease prevention, single-chain fragment variable (scFv), originating from a monoclonal bovine IgG1 antibody against BoHV-1, were constructed and expressed in Pichia pastoris in V(lambda)-V(H) orientation via a flexible seven-amino acid linker. Similar to the intact IgG, the purified recombinant scFv neutralized BoHV-1 in vitro and recognized viral antigens in BoHV-1 infected MDBK cells by immunofluorescence. Homology modeling of the Fv predicts two distinct conformations for CDR3H. Firstly, a long protruding CDR3H conformation where no disulfide linkage occurred between two "non-canonical" Cys residues resulted in a large binding cavity between V(lambda) and V(H). Secondly, a smaller potential antigen-binding cavity is predicted with a disulfide linkage between the two Cys residues of CDR3H creating a six-membered loop in the ascending polypeptide, which fitted into the space between V(lambda) and V(H). Despite such potential configurational diversity of the antigen-binding site, the electrostatic surface potentials that would interact with the BoHV-1 epitope are largely similar for both the topographies where salt-bridge type electrostatic interactions likely occur at the edges of the binding site. Given that IgG1 antibody against BoHV-1 is clonally selected, it is likely that disulfide-stabilized broader and flatter surface topography is specifically generated to accommodate the predicted carbohydrate neutralizing B-epitope on BoHV-1. The specificity and neutralizing capacity for BoHV-1 of the scFv should make this bovine antibody fragment a useful diagnostic and potential therapeutic candidate for an important viral pathogen in cattle.
Koti
Madhuri
M
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Farrugia
William
W
Nagy
Eva
E
Ramsland
Paul A
PA
Kaushik
Azad K
AK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D057134:Antibodies, Neutralizing / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D002417:Cattle; D002460:Cell Line; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D007242:Herpesvirus 1, Bovine / Q000276:immunology*; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D007241:Infectious Bovine Rhinotracheitis / Q000276:immunology* / Q000517:prevention & control; D051379:Mice; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G; D011994:Recombinant Proteins