Neutralisation and binding of VHS virus by monovalent antibody fragments

We have previously reported the cloning and characterisation of the heavy a nd light chain variable domain genes encoding three monoclonal antibodies ( Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these a ntibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2 appeare d to recognise a broader range of virus isolates. The variable domains of t hese two antibodies differ by only four residues (Lorenzen et al., 2000a. F ish Shellfish Immunol. 10, 129-142). To further study the mechanism of neut ralisation, Fab fragments as well as a series of recombinant bacterial sing le chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 iso late DK-F1. In addition, a series of scAb fragments were produced using the 3F1H10 variable heavy (VH) chain and variable light (V kappa) chain domain s but containing, either alone or in dual combination, each of the four dif ferent residues present in 3F1A2. The dissociation constants of Mabs 3F1H10 and 3F1A2 and their respective Fab and scAb fragments were measured by BIA core analysis and found to correlate with the capacity of each molecule to neutralise DK-F1. These investigations, together with computer assisted mol ecular analysis of the theoretical influence of each mutation on antigen bi nding, led to the identification of a single mutation at position 35a in th e VH domain as having the most marked impact on viral neutralisation. (C) 2 001 Published by Elsevier Science B.V.