COXSACKIEVIRUS AS VECTOR FOR ANTIINFLAMMATORY CYTOKINES

The coxsackie B virus cause acute myocarditis and dilated cardiomyopathy (DCM); DCM often results in a failing heart, the solution to which is generally transplantation to avoid eventual death. The long-term goal of this work is to determine whether genetically engineered cardiotropic CVB3 vectors expressing key cytokines will be useful as beneficial therapeutic, even prophylactic, approaches to diminishing inflammatory heart disease, including the ablation of rejection in heart transplantation. Our objective in this proposal is to evaluate and explore the efficacy of attenuated CVB3 strains, engineered to express the murine cytokines IL-4 of IL-10, in the diminution of symptoms of acute cardiac inflammatory disease. The specific aims are [1] Study attenuated CVB3 strains that express murine IL-4 (mIL-4) or mIL-10 in cell culture systems. Human and murine cell cultures will be used to evaluate the kinetics and extent of viral expression, genetic stability upon multiple passages, extent of export and biological activity of the expressed cytokines. [2] Study the murine immune response against the interleukin-expressing CVB3 strain. Mice will be evaluated for extent of viral replication in heart, extents and types of B and T cell responses, and how these compare temporally following virus inoculation. Cytokines specific for the Th1 and Th2 type T cell responses, in addition to mIL4 or mIL10, in murine sera and hearts following infection will be monitored and quantitated. RT-PCR analysis of cytokine gene expression in the murine host will also be analyzed as a function of the specific infecting virus. [3] Determine how CVB3-expressed mIL-4 or mIL-10 in mice affects progression and extent of CVB3-induced inflammatory heart disease. Extent of inflammation as a function of time will be photo microscopically mapped and quantitated. Extent and types of anti-viral T cell responses and circulating cytokine expression will be evaluated in test mice versus controls, as will types of anti- viral antibodies. Transient, low-level expression of key modulatory cyto- and chemokines by attenuated picornaviral vectors is a novel and potentially valuable new approach to treating serious inflammatory diseases.