title: Exceptionally long CDR3H of bovine scFv antigenized with BoHV-1 B-epitope generates specific immune response against the targeted epitope.
abstract: We discovered that some bovine antibodies are amongst the largest known to exist due to the presence of an exceptionally long CDR3H (≥49 amino acids) with multiple cysteines that provide a unique knob and stalk structure to the antigen binding site. The large CDR3H size, unlike mouse and human, provides a suitable platform for antigenization with large configurational B-epitopes. Here we report the identification of a B-epitope on the gC envelope protein of bovine herpes virus type-1 (BoHV-1) recognized by a bovine IgG1 antibody. The identified 156 amino acid long gC fragment (gC156) was expressed as a recombinant protein. Subsequently, a functional scFv fragment with a 61 amino-acid long CDR3H (scFv1H12) was expressed such that gC156 was grafted into the CDR3H, replacing the "knob" region (gC156scFv1H12 or Ag-scFv). Importantly, the Ag-scFv could be recognized by a neutralizing antibody fragment (scFv3-18L), which suggests that the engraftment of gC156 into the CDR3H of 1H12 maintained the native conformation of the BoHV-1 B-epitope. A 3D model of gC156 was generated using fold-recognition approaches and this was grafted onto the CDR3H stalk of the 1H12 Fab crystal structure to predict the 3D structure of the Ag-scFv. The grafted antigen in Ag-scFv is predicted to have a compact conformation with the ability to protrude into the solvent. Upon immunization of bovine calves, the antigenized scFv (gC156scFv1H12) induced a higher antibody response as compared to free recombinant gC156. These observations suggest that antigenization of bovine scFv with an exceptionally long CDR3H provides a novel approach to developing the next generation of vaccines against infectious agents that require induction of protective humoral immunity.
authors: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors: Yfke
authors: Y
authors: Pasman
authors: School of Science, RMIT University, Bundoora, VIC 3083, Australia.
authors: Caroline
authors: C
authors: Soliman
authors: School of Science, RMIT University, Bundoora, VIC 3083, Australia; Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 3004, Australia; Department of Immunology, Alfred Medical Research and Education Precinct, Monash University, Melbourne, VIC 3004, Australia; Department of Surgery Austin Health, University of Melbourne, Heidelberg, VIC 3084, Australia.
authors: Paul A
authors: PA
authors: Ramsland
authors: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Electronic address: akaushik@uoguelph.ca.
authors: Azad K
authors: AK
authors: Kaushik
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D015153:Blotting, Western; D002417:Cattle; D022801:Complementarity Determining Regions / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D007242:Herpesvirus 1, Bovine / Q000276:immunology*; D013058:Mass Spectrometry; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology; D057127:Single-Chain Antibodies / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D022801:Complementarity Determining Regions; D018985:Epitopes, B-Lymphocyte; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D014759:Viral Envelope Proteins
keywords: Antibody; Antigenized scFv; B epitope; BoHV-1; CDR3; Exceptionally long CDR3H; H graft; scFv