Construction of a single-chain interleukin-12-expressing retroviral vector and its application in cytokine gene therapy against experimental coccidioidomycosis.
T-cell-mediated immunity is an important determinant in protection against primary infection with Coccidioides immitis, a dimorphic fungal pathogen that causes the disease coccidioidomycosis. To determine if interleukin-12 (IL-12) gene therapy could potentiate host response against C. immitis, we constructed a single-chain cDNA encoding the p40 and p35 subunits linked by a polylinker and, using a retroviral vector, transfected J774 macrophages with the construct. The transduced J774 cells expressed IL-12 in vitro, with a mean concentration of 28,440 pg from 10(6) cells in 48 h as measured by an IL-12 (p75)-specific enzyme-linked immunosorbent assay. The secreted IL-12 was biologically active, as judged by its ability to induce the production of gamma interferon (IFN-gamma) by spleen cells from BALB/c mice. Treatment of the highly susceptible BALB/c mouse strain with the IL-12-transduced J774 cells inhibited C. immitis growth in tissues from mice challenged by a pulmonary route, as evidenced by 1.37-, 2.59-, and 1.22-log reductions in the number of CFU in the lungs, spleens, and livers, respectively, compared to the fungal load in mice given vector-transduced J774 cells. The protective effect of IL-12 gene therapy was accompanied by increased levels of IFN-gamma in the lungs and sera of mice treated with IL-12-transduced J774 cells and the constitutive production of IFN-gamma by their spleen cells cultured in vitro. These results suggest that IL-12 gene therapy could be used as adjunct therapy for coccidioidomycosis.
Jiang
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Department of Clinical Investigation, Texas Center for Infectious Disease, San Antonio, Texas 78223, USA. chengyong.jiang@tdh.state.tx.us
Magee
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DM
Cox
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D016475:3T3 Cells; D000281:Administration, Intranasal; D000818:Animals; D003047:Coccidioidomycosis / Q000276:immunology / Q000628:therapy*; D016207:Cytokines / Q000235:genetics; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D007371:Interferon-gamma / Q000096:biosynthesis; D018664:Interleukin-12 / Q000235:genetics* / Q000276:immunology / Q000627:therapeutic use; D051379:Mice; D008807:Mice, Inbred BALB C; D012190:Retroviridae; D018417:Th1 Cells / Q000276:immunology
D016207:Cytokines; D018664:Interleukin-12; D007371:Interferon-gamma