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Updated: Mar 16, 2026
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
Exceptionally long CDR3H of bovine scFv antigenized with BoHV-1 B-epitope generates specific immune response against
Yfke Pasman1, Caroline Soliman2, Paul A Ramsland3
1Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Insights
Bovine antibodies possess exceptionally long CDR3H regions, enabling antigenization for enhanced vaccine development. Grafting a bovine herpesvirus epitope onto these antibodies boosted immune response in calves.
Area of Science:
- Immunology
- Structural Biology
- Vaccine Development
Background:
- Bovine antibodies exhibit unique structural features, including exceptionally long CDR3H regions (≥49 amino acids) with multiple cysteines.
- This unique structure forms a knob and stalk conformation, providing a versatile platform for antigenization, unlike in mice or humans.
Purpose of the Study:
- To identify and characterize a B-epitope on the bovine herpesvirus type-1 (BoHV-1) gC protein.
- To engineer an antigenized single-chain variable fragment (Ag-scFv) by grafting the identified epitope onto a bovine antibody's CDR3H region.
- To evaluate the immunogenicity of the engineered Ag-scFv for potential vaccine applications.
Main Methods:
- Recombinant expression of a 156 amino acid gC fragment (gC156) from BoHV-1.
- Construction and expression of a functional scFv fragment (scFv1H12) with a 61 amino acid CDR3H, into which gC156 was grafted (Ag-scFv).
- Structural modeling of the Ag-scFv and validation using a neutralizing antibody fragment (scFv3-18L).
- Immunization of bovine calves with Ag-scFv and recombinant gC156 to assess antibody response.
Main Results:
- A BoHV-1 B-epitope within gC156 was identified and successfully grafted into the CDR3H of a bovine scFv, creating Ag-scFv.
- The engineered Ag-scFv retained the native conformation of the B-epitope, as confirmed by recognition with a neutralizing antibody fragment.
- Structural prediction indicated a compact, protruding antigen conformation within the Ag-scFv.
- Immunization with the antigenized scFv elicited a significantly higher antibody response in calves compared to the free recombinant antigen.
Conclusions:
- Antigenization of bovine scFv, leveraging their exceptionally long CDR3H regions, offers a novel strategy for vaccine design.
- This approach enhances the induction of protective humoral immunity against infectious agents.
- The engineered Ag-scFv holds promise for developing next-generation vaccines against bovine herpesvirus and potentially other pathogens.
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.
