A single point mutation in framework region 3 of heavy chain affects viral neutralization dynamics of single-chain Fv against bovine herpes virus type 1

Abstract

We constructed functional recombinant single chain Fv (scFv) against bovine herpes virus type 1 (BoHV-1), aetiological agent of respiratory and genital diseases in cattle for which available vaccines do not provide adequate protection. The scFv against BoHV-1 with 18 amino acid long flexible linker (scFv3-18L; monomeric form) recognized target antigen and, also, neutralized BoHV-1 in vitro. A comparison with recombinant scFv with 7 amino acid linker against BoHV-1 (scFv1-7L), capable of forming diabodies, indicated that a relatively higher concentration (two-fold) of monomer scFv3-18L is needed for virus neutralization as compared to scFv1-7L. A single point replacement mutation (Asp98 to Gly98) in the framework-3 (FR3) variable-region of scFv with 18 amino acid linker (scFv4m-18L), however, affected the viral neutralization in a dose-dependent manner where 2.7 fold higher mutant scFv concentration was required to achieve virus neutralization. Despite differences in dose-dependent viral neutralization of the mutant scFv-18L, it detected viral antigen in an immunofluorescent assay. The outlined experiments demonstrate that recombinant scFv against BoHV-1, whether expressed as scFv or diabody, provide an effective antibody based therapeutic and immunodiagnostic protein. Further, single point substitution mutation in the FR3 can affect viral neutralization dynamics without affecting qualitative viral antigen recognition.