Incorporation of recombinant bovine C3d and native leukotoxin from Mannheimia haemolytica into ISCOM-MATRIX using avidin-biotin interaction

MOORE DP; FIRTH MA; MCBEY BA; HODGINS DC; SHEWEN PE

Monona Terrace Convention Center, Madison Wisconsin, USA

Conferencia; The 5th International Veterinary Vaccines and Diagnostics Conference; 2009

Immune stimulating complexes are nanoparticles containing lipids and saponins used to enhance antigen presentation in vaccine formulations. A fragment of complement component C3, called C3d, is found in immune complexes and has been employed as a molecular adjuvant in mice, increasing the immunogenicity of a model antigen up to 10,000 fold. Recombinant bovine C3d (rboC3d) has been successfully expressed in Escherichia coli for evaluation as an adjuvant in cattle. Because improved vaccines for prevention of bovine pneumonic pasteurellosis are needed, the aim of this study was to formulate a vaccine that incorporated rboC3d and protective antigens of M. haemolytica into ISCOM-MATRIX. Log phase culture supernatant of M. haemolytica A1 was used as a source of antigens, including native leukotoxin (Lkt). ISCOM-MATRIX and the antigens were biotinylated using a commercial kit, and rboC3d was expressed as a biotinylated protein with a c-Myc tag. Biotinylated ISCOM-MATRIX was first incubated with neutravidin (Pierce Laboratories) for 90min at room temperature, and then centrifuged in a 10-40% sucrose gradient and visible bands were recovered from the interface. The ISCOM-MATRIX coated with avidin was then incubated with similar volumes of biotinylated rboC3d (4µg/mL) and culture supernatant antigens (450µg/mL). Biotinylated ISCOM-MATRIX without avidin coating was incubated with the biotinylated proteins as negative control. After further centrifugation in a sucrose gradient, formulations were recovered and analyzed by SDS-PAGE and western immunoblot, using monoclonal antibodies: anti c-Myc for rboC3d and MoAb 601 specific for Lkt A. Western blot showed bands at approx. 40 and 100kDa, consistent with rboC3d and Lkt, respectively. Attachment of biotinylated Lkt was also observed in ISCOM-MATRIX without avidin coating. Although rboC3d has not yet been proven as a molecular adjuvant, this formulation has potential as an experimental vaccine for preventing fibrinous pneumonia caused M. haemolytica in cattle.