Poster Presentation International Veterinary Immunology Symposium 2016

Exceptionally long CDR3H of bovine scFv provides a suitable scaffold for antigenization with conformational B- epitope capable of inducing specific immune response (#126)

Azad Kaushik 1 , Yfke Pasman 1
  1. University of Guelph, Guelph, ON, Canada

Earlier, our laboratory discovered that some bovine antibodies are among the largest known to exist in a species with an exceptionally long CDR3H (>48 amino acids) with multiple cysteine amino acids. Such CDR3H generates a unique antigen-binding site with a "stalk-knob" structure capable of providing configurational diversity via variable intra-CDR3H disulfide bridging. Given the large CDR3H size, unlike mouse and human immunoglobulins where CDR3H is relatively shorter, these provide suitable platform for antigenization with large configurational B-epitopes. First, we determined whether VH+VL pair, expressed as scFv (single chain fragment variable; scFv1H12) in Pichia pastoris, from a polyspecific bovine IgM, with an exceptionally long CDR3H (61 amino acids) sustained required functional conformation. The scFv1H12 showed polyspecific antigen recognition similar to parent IgM antibody with minor differences. Thus, these observations suggested that atypical CDR3H conformation was retained in scFv format, similar to parent antibody. Then, we identified a B-epitope on gC envelope protein of bovine herpes virus type-1 (BoHV-1), an economically significant cattle pathogen. A 156 amino acid long gC fragment (gC156) containing BoHV-1 B-epitope was expressed as a recombinant antigen in P. pastoris. This was followed by grafting of gC156 containing B epitope into the CDR3H of scFv1H12 together with flexible linkers (gC156scFv1H12). The gC156scFv1H12 was expressed in P. pastoris. The secreted recombinant proteins were characterized and purified. Interestingly, B-epitope in gC156scFv1H12 sustained configuration similar to native glycoprotein on BoHV-1 envelope. Free recombinant gC156 and its antigenized form, gC156scFv1H12, were used to immunize bovine calves. The antigenized scFv, gC156scFv1H12, induced higher antibody response as compared to free recombinant gC156 fragment in the calves. These observations provide evidence that bovine antibodies with exceptionally long CDR3H provide a suitable scaffold capable of sustaining B epitope conformation and inducing targeted humoral immunity upon immunization. [Supported by NSERC Canada Discovery Grant]