CONSTRUCTION OF VECTORS EXPRESSING BIOACTIVE HETERODIMERIC AND SINGLE-CHAIN MURINE INTERLEUKIN-12 FOR GENE-THERAPY

It has been well demonstrated that interleukin-12 (IL-12) could be use ful to defend against a variety of pathogens, to suppress tumor growth and metastasis, and even to be employed as an adjuvant of vaccines to enhance beneficial type 1 T helper (Th1) cell response over detriment al type 2 T helper (Th2) cell responses. To apply IL-12 genes in gene therapy such as a DNA vaccine, a pIL-12 vector was constructed that co ntained two cytomegalovirus (CMV) promoters to drive the expression of p35 and p40 subunits, respectively. In addition, a pscIL-12 vector wa s designed with a linker to fuse p35 cDNA with p40 cDNA to produce a s ingle-chain IL-12 protein, ensuring not only that the expression of p3 5 and p40 subunits was equally expressed, but also that no free p40 su bunits interfered with IL-12 activity. The data suggested pIL-12 could produce a rather high level of biologically active IL-12 after transf ection of COS cell lines as well as C2C12 muscle cell lines, as measur ed by both concanavalin A blast proliferation assay and enzyme-linked immunosorbent assay. Interestingly, the pscIL-12 vector could also exp ress a bioactive murine IL-12 fusion protein in vitro. Furthermore, in vivo functional studies also demonstrated that mice co-immunized with a pS vector expressing the major envelope protein of hepatitis B viru s (HBV) and IL-12 vectors encoding native IL-12 or single-chain IL-12 fusion protein elicited higher levels of IgG(2a) anti-HBs antibody and of Th1-related cytokine. Because p35 and p40 genes can be expressed i n a vector by using a single promoter, pscIL-12 should be useful in fu ture applications for nucleic acid vaccination or for gene therapy aga inst diseases.