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Degree

Doctor of Philosophy, Ohio State University, Integrated Biomedical Science Graduate Program, 2010.

Abstract

Respiratory syncytial virus (RSV) is the primary cause of bronchiolitis and pneumonia in infants and young children worldwide. RSV is an opportunistic pathogen that is often unsuspected as the cause of infections in solid organ and bone marrow transplant recipients, patients suffering cystic fibrosis or congenital heart disease, and the elderly. Ribavirin, the only currently approved antiviral therapy, is only marginally effective against RSV and has not been shown to reduce mortality due to infection. In addition, ribavirin must be administered by small-particle aerosol for 12 to 18 hours per day for 3 to 7 days, thus requiring hospitalization. The only option for prevention in at-risk infants and transplant patients is passive immunoprophylaxis with monoclonal antibody specific for the fusion protein of RSV. Leflunomide, an anti-inflammatory drug approved for treatment of rheumatoid arthritis, is an inhibitor of protein kinase activity and pyrimidine synthesis. It has been shown to be effective against acute and chronic transplant rejection in animal models and is currently showing promise as an immunosuppressant in clinical trials in human transplant recipients. Studies have also shown it to exert powerful antiviral activity against cytomegalovirus, herpes simplex virus, and polyomavirus BK. In the current study, we have tested the hypothesis that leflunomide possesses antiviral activity against RSV in vitro and in vivo. Phase contrast microscopy and immunohistochemical staining studies of RSV-infected cultured HEp2 cells and human small airway epithelial cells showed profound attenuation of virally-induced cytopathic change and syncytia formation in cultures incubated in the presence of A77 1726, the active metabolite of leflunomide. Data generated by quantitative plaque assay experiments demonstrated dramatic reduction in the production of infectious virus in the presences of pharmacologically relevant levels A77 1726, with an EC50 of ~15-20 μM. Data generated by identical experiments conducted in the presence of exogenous uridine, which restores intracellular pyrimidines to normal levels, suggested that the antiviral activity may be partially accountable to pyrimidine depletion at lower doses of A77, but not at higher, therapeutic doses. To determine whether leflunomide exerts antiviral activity in vivo, cotton rats were inoculated intranasally with each of 2 low-passage clinical isolates of RSV and treated daily with leflunomide or vehicle alone by gavage, either beginning on the day of inoculation or on day 3 post-inoculation (to more accurately simulate a clinical scenario). On day 5 post-inoculation animals were euthanized and lungs were harvested and homogenized for plaque assay. Viral loads in leflunomide-treated animals were >3 logs lower than those measured in vehicle-treated controls even when treatment was delayed, and were not restored by administration of exogenous uridine. To identify specific antiviral mechanisms of leflunomide, real time rt-PCR was used to quantitate viral genomic RNA and viral transcripts in RNA isolated from infected cultured cells incubated in the presence or absence of A77 1726 ± uridine. Data generated by these experiments demonstrated nearly complete inhibition of viral genomic RNA synthesis and gene transcription by A77 1726, which was only modestly restored by exogenous uridine. Western blot analysis of protein extracts from cells treated as above and probed with polyclonal RSV antibody likewise demonstrated attenuation of synthesis of several viral proteins in A77-treated infected cells, with viral protein synthesis partially restored by exogenous uridine. Collectively, these findings suggest promise for leflunomide, which can be administered orally, as a convenient addition to the growing arsenal of antiviral therapeutics.

Subject Headings

Biomedical research

Keywords

Respiratory Syncytial Virus; Leflunomide; antiviral therapy

Committee / Advisors

W. James Waldman, PhD (Advisor)
Joan Durbin, MD/PhD (Committee Member)
Larry Schlesinger, MD/PhD (Committee Member)
Marshall Williams, PhD (Committee Member)

Pages

129p.

Document number: osu1269449453
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=osu1269449453

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