Immunoprophylaxis in fish by injection of mouse antibody genes.

Antibodies are a crucial part of the body's specific defense against infectious diseases and have considerable potential as therapeutic and prophylactic agents in humans and animals. The development of recombinant single-chain antibodies allows a genetic application strategy for prevention of infectious diseases. To test this in a fish model, a gene construct encoding a neutralizing single-chain antibody to the fish-pathogenic rhabdovirus VHSV (viral hemorrhagic septicemia virus) was administered to rainbow trout by intramuscular injection of plasmid DNA. Circulating recombinant antibodies could later be detected in the fish, and protective immunity to the viral disease was established.

Neutralisation and binding of VHS virus by monovalent antibody fragments.

We have previously reported the cloning and characterisation of the heavy and light chain variable domain genes encoding three monoclonal antibodies (Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these antibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2 appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial single 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 isolate DK-F1. In addition, a series of scAb fragments were produced using the 3F1H10 variable heavy (VH) chain and variable light (Vkappa) chain domains but containing, either alone or in dual combination, each of the four different residues present in 3F1A2. The dissociation constants of Mabs 3F1H10 and 3F1A2 and their respective Fab and scAb fragments were measured by BIAcore analysis and found to correlate with the capacity of each molecule to neutralise DK-F1. These investigations, together with computer assisted molecular analysis of the theoretical influence of each mutation on antigen binding, led to the identification of a single mutation at position 35a in the VH domain as having the most marked impact on viral neutralisation.

Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments in E. coli.

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments in E. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.

Three monoclonal antibodies to the VHS virus glycoprotein: comparison of reactivity in relation to differences in immunoglobulin variable domain gene sequences.

Three monoclonal antibodies (MAbs) to the VHSV G protein were compared in different immunoassays and the variable domain cDNA sequences from the respective immunoglobulin (Ig) genes were determined. One MAb (IP1H3) was non-neutralising and recognised different virus isolates equally well in ELISA. The other two were neutralising and recognised the same or closely related epitopes. One of these two MAbs (3F1H10) was more restricted in its ability to neutralise heterologous VHSV isolates than the other (3F1A2). A semi-quantitative relationship between binding of the two neutralising MAbs in ELISA and their neutralising activity was evident. Binding kinetic analyses by plasmon resonance identified differences in the dissociation rate constant (kd) as a possible explanation for the different reactivity levels of the MAbs. The Ig variable heavy (VH) and light (V kappa) domain gene sequences of the three hybridomas were compared. The inferred amino acid sequence of the two neutralising antibody VH domains differed by three amino acid residues (97% identity) and only one residue difference was evident in the V kappa domains. In contrast, IP1H3 shared only 38 and 39% identity with the 3F1A2 and 3F1H10 VH domains respectively and 49 and 50% identity with the 3F1A2 and 3F1H10 V kappa domains respectively. The neutralising antibodies were produced by hybridomas originating from the same fusion and the high nucleotide sequence homology of the variable Ig gene regions indicated that the plasma cell partners of the hybridomas originated from the same virgin B lymphocyte. The few differences observed in the VH and V kappa amino acid sequences were probably due to somatic mutations arising during affinity maturation and might explain the observed reactivity differences between the two MAbs.

Expression of monovalent and bivalent antibody fragments in Escherichia coli.

The technology of humanization of rodent antibodies has opened the way for a broad range of therapeutic antibodies with very low immunogenicity, which are, therefore, suitable for repeated dosing. Such intact antibodies have extended serum half-lives and biodistribution profiles very similar to human antibodies. For some applications, however, the ideal therapeutic should have reduced serum half-life and altered biodistribution patterns typical of antibody fragments, such as Fab or single chain Fv. Bispecific antibody fragments offer exciting additional therapeutic possibilities, but their successful manufacture and purification on a large scale require the development of new methods. Antibody fragments often assemble in Escherichia coli as monovalent fragments with reduced affinities. We describe the spontaneous assembly of bivalent antibody fragments in E. coli and methods of purification that yield either bivalent or monovalent molecules as required.

Cloning and expression of single chain antibody fragments in Escherichia coli and Pichia pastoris.

The potential of recombinant antibody fragments is likely to be fulfilled only if they can be produced routinely at high concentrations. We have compared the ability of Escherichia coli and Pichia pastoris to produce functional recombinant single chain antibody (scAb) fragments. Two scAb fragments were expressed, an antihuman type V acid phosphatase (TRAP) and an anti-Pseudomonas aeruginosa lipoprotein I. We report here that, while expression from P. pastoris resulted in a significantly increased level of expression of the anti-TRAP scAb compared to E. coli, neither fragment was able to bind its target antigen as well as the bacterial product.