The goal of this project is the production of a cross-protective DEN anti-Id vaccine, based on the murine monoclonal antibody (Mab) 4G2, which is directed against a cross-protective neutralizing epitope present on all flaviviruses. The project involves the following steps: (1) purification of Mab 4G2 (the idiotype); (2) immunization of mice with 4G2; (3) """"""""capture"""""""" of the cells producing anti-4G2 antibodies (anti-Id) as hybridomas by cell fusion; and (4) use of purified anti-Id antibodies as immunogens, followed by virus challenge of immunized animals. Phase U of the project has been accomplished, with the production of over 150 mg of 4G2 antibody from ascites fluid collected from pristane-primed mice injected with the 4G2 hybridoma cell line. Because the hybridoma cell produces both a non-specific IgG1 in addition to the anti-DEN IgG2a, Protein A (which binds IgG1 poorly) was used as the first step in the purification scheme. Some of the antibody was treated with papain to produce Fab fragment, which was then purified by protein-G affinity chromatography. Both Fab and whole Ig were then coupled to the carrier protein KLH by glutaraldehyde treatment; additionally, Ig was coupled to BSA. The above materials were used as immunogens and in screening tests. Separate groups of mice were immunized using the conjugate materials: initial priming injections were given in which the antigen was mixed with complete adjuvant, followed by booster injections in which incomplete adjuvant was used, and a final (pre-fusion) injection in which the antigen alone was administered. Twenty-one fusions were performed using mice immunized with the KLH conjugates, and over 450 resultant hybridomas were analyzed for the production of anti-4G2 antibodies using both competition and direct-binding ELISA. NO stable reactive clones of cells were obtained. However, fusions using spleens from mice receiving the Ig-KLH conjugate gave rise to larger numbers of hybrid colonies than the Fab-KLH conjugate, so immunizations now underway are utilizing an Ig-BSA conjugate, along with modifications in the immunization schedule in an attempt to maximize the response.
