Antiviral activity of leflunomide against respiratory syncytial virus

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Respiratory synctial virus (RSV) is a major cause of often serious, life-threatening lung disease in infants and young children, immunosuppressed organ transplant recipients, patients suffering from cystic fibrosis, congenital heart disease, and the elderly. No effective vaccine is currently available for prevention of RSV disease. Ribavirin, the only approved antiviral therapy, which must be administered by aerosol for 12 to 18 hours/day for 3 to 7 days, is only marginally effective and has not been shown to significantly reduce mortality associated with RSV disease. While passive immunoprophylaxis with hyperimmune globulin or monoclonal antibody has been shown to reduce disease severity in some cases, satisfactory antiviral therapy has yet to be achieved. Leflunomide, an inhibitor of pyrimidine synthesis and protein kinase activity, is an anti-inflammatory agent approved for treatment of rheumatoid arthritis and is currently in clinical trials as an immunosuppressant in transplant recipients. This agent has been shown to exert powerful antiviral activity against cytomegalovirus in vitro and in vivo, and in preliminary in vitro and in vivo studies against RSV. The specific aims of this investigation are to test the hypothesis that leflunomide reduces RSV viral load and associated bronchiolitis in vivo, and to elucidate its antiviral mechanisms at the moleclular level. Cotton rats inoculated with RSV and treated with leflunomide, ribavirin, Palivizumab, or combinations of theses agents will be euthanized at various intervals. Viral loads will be quantitated by plaque assay of lung homogenates, and tissue injury and pulmonary inflammation will be assessed by histologic and immunohistochemical analysis of lung tissue. Immune status of RSV-infected animals will be assessed by proliferation assays of splenocyte responses to RSV antigen. Molecular antiviral mechanisms will be elucidated by quantitative real time rtPCR assays of viral genomic RNA synthesis and transcription, immunoprecipitation and western blot analysis of viral protein synthesis and phosphorylation, and immunogold electron microscopy. Data generated by these studies are anticipated to demonstrate the bifunctional utility of leflunomide in both the reduction in viral load and the concurrent attenuation of the severe inflammatory reaction primarily responsible for RSV disease manifestations. Furthermore, elucidation of the molecular antiviral mechanisms of this agent may identify novel targets for future antiviral drug development.