Construction of a single-chain interleukin-12-expressing retroviral vector and its application in cytokine gene therapy against experimental coccidioidomycosis

Abstract

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.

FIG. 1

Schematic representation of the retroviral construct that carries both the p40 and p35 genes and the neomycin resistance gene selectable marker. The p40 and p35 cDNAs were linked by a (Gly₄Ser)₃ polylinker and were regulated by the long terminal repeat (LTR) promoter. This plasmid vector, designated pLXSN/mIL-12, was capable of coordinately expressing the p40, p35, and neomycin resistance genes. CMV, cytomegalovirus; SV40, simian virus 40.

FIG. 2

Expression of mRNA transcripts for β-actin, the neomycin resistance gene, and mIL-12 in pLXSN/mIL-12-J774 cells. Lanes 1, 2, and 3 depict the results obtained with cellular RNA obtained from nontransduced J774 cells, J774 cells transduced with the pLXSN plasmid vector alone, and J774 cells transduced with the pLXSN/mIL-12 construct, respectively.

FIG. 3

Secretion of bioactive IL-12 from pLXSN/mIL-12-transduced J774 cells. Supernatants were collected at 48 h from in vitro cultures of 10⁶ pLXSN/mIL-12-transduced J774 cells and, for negative controls, nontransduced and vector-transduced J774 cells. The supernatants were assayed for IL-12 by ELISA (A) and for bioactive IL-12 (B) as measured by the induction of IFN-γ production in (2 × 10⁶) spleen cells from normal BALB/c mice. Results are representative of those obtained in at least two separate experiments.

FIG. 4

Protection in BALB/c mice treated with pLXSN/mIL-12-transfected J774 cells. Mice were infected with 60 arthroconidia via a pulmonary route and then treated with 2 × 10⁶ IL-12-transduced or vector-transduced J774 cells via the i.p. route. A third group of mice received saline alone. Treatments were begun 6 h after pulmonary challenge and repeated on days 1, 4, and 7 after challenge. Twelve days postchallenge, the mice were sacrificed and evaluated for C. immitis CFU in tissues. Bars depict means ± standard errors obtained in two experiments involving a total of 22 mice given saline alone, 23 mice treated with vector-transduced J774 cells, and 21 mice given IL-12-transduced J774 cells.