EXPRESSION OF A BIOLOGICALLY-ACTIVE ANTIVIRAL ANTIBODY USING A SINDBIS VIRUS VECTOR SYSTEM

Monoclonal antibodies to the Sindbis virus E2 envelope glycoprotein pr otect mice against lethal encephalitis and mediate viral clearance fro m neurons. To facilitate structure-function analyses of anti-E2 mAbs, we developed an expression system that can be used for the constructio n of genetically engineered anti-E2 mAbs. We constructed recombinant S indbis/immunoglobulin gene chimeric viruses that express heavy and lig ht chains of an anti-E2 monoclonal antibody, R6. We used a PCR-based s trategy to clone the entire rearranged heavy and light chain genes fro m R6 hybridoma cell cDNA into a double subgenomic Sindbis virus vector . The recombinant viruses, SIN/R6L and SIN/R6H, were generated by tran sfecting BHK-21 cells with in vitro transcribed RNA from Sindbis virus /R6 light chain and Sindbis virus/R6 heavy chain cDNA clones, respecti vely. Twelve hours after co-infection of BHK cells with SIN/R6L and SI N/R6H, the tissue culture supernatant contained up to 1.4 mg/ml of rec ombinant R6 IgG. The heavy and light chains of recombinant R6 were ass ociated as judged by co-purification on protein A/G sepharose and co-e lectrophoresis of non-reduced proteins. The ELISA reactivity to Sindbi s virus antigen was similar for recombinant R6 and R6 purified from as cites fluid. Furthermore, the in vivo biologic activity of recombinant R6 was similar to that of R6 purified from ascites; recombinant R6 tr eatment completely protected Balb/cJ mice from paralysis and death due to infection with neuroadapted Sindbis virus and also resulted in the clearance of infectious virus from the brains of immunodeficient scid mice persistently infected with wild-type Sindbis virus. Thus, the co -infection of BHK cells with SIN/R6L and SIN/RGH leads to the expressi on, assembly, and secretion of a biologically active recombinant antiv iral antibody. Our results suggest that the Sindbis virus vector syste m is a simple and powerful tool for the production of functional, gene tically engineered antibodies. (C) 1998 Elsevier Science Ltd. All righ ts reserved.