Use of recombinant single-chain antibodies for prevention or treatment of diseases has been addressed in various studies including neutralization of viruses1,2 and toxins3, cancer immunotherapy4,5, and recently also in the form of DNA vaccines encoding anti-idiotype single-chain antibodies6. At cell

1177 Use of recombinant single-chain antibodies for prevention or treatment of diseases has been addressed in various studies including neutralization of viruses1,2 and toxins3, cancer immunotherapy4,5, and recently also in the form of DNA vaccines encoding anti-idiotype single-chain antibodies6. At cell culture level, expression of virus-specific single-chain antibodies inside host cells can interfere with virus multiplication within the cell7,8. The present study aimed to establish protection against an infectious pathogen in vivo by administration of antibody genes. The experimental model involved the fish rhabdovirus viral hemorrhagic septicemia virus (VHSV), with rainbow trout (Oncorhynchus mykiss) as the host species9,10. As with the morphologically related mammalian rabies virus, studies have identified the viral glycoprotein G as the target of neutralizing antibodies11, and adoptive transfer of immunity can be obtained by intraperitoneal injection of neutralizing monoclonal antibodies (MAbs) before exposure to VHSV12. Gene constructs encoding single chain antibodies, with the human Ig kappa constant domain fused to the 3′ end (ScAbs) were prepared using cDNA libraries from hybridoma cell lines producing neutralizing antibodies to the VHSV G protein. The variable parts of the immunoglobulin (Ig) heavy and light chain genes were cloned13 and connected by a sequence encoding a 14–amino acid flexible spacer. A constant-domain gene of the human Ig κ-chain was added as a tag at the 3′ end14. Upon expression in bacteria, the resulting ScAbs were able to neutralize VHSV (P. M. C., N. L. et al. unpublished data). Recombinant singlechain neutralizing antibodies have also been reported for several important mammalian viruses, including HIV-1 (ref. 15), rabies virus1, and vesicular stomatitis virus (VSV)16. In the case of VSV, protective activity could furthermore be demonstrated in vivo, but because of the short half-life of the single-chain antibody molecules in circulation, protection was obtained only when the antibody was allowed to interact with the virus in vitro before inoculation of the mice16. Short half-life of neutralizing singlechain antibodies administered to mice before infection was correspondingly used to explain the lack of protection against exposure to a lethal coronavirus17. Here, we aimed at maintaining a stable level of circulating ScAb within the host over a longer period of time by inducing expression in vivo through administration of a plasmid-borne recombinant antibody gene, rather than delivery of the antibody itself. Such a strategy has earlier been suggested for cancer immunotherapy4 but has to our knowledge not been reported as a prophylactic measure.