Human cytomegalovirus (HCMV) causes considerable morbidity and mortality in patients with AIDS, cancer, and organ transplants. Prophylactic and maintenance therapies for HCMV disease involving ganciclovir, foscarnet, or cidofovir lead to the emergence of drug resistant HCMV clinical isolates. Both genotypic and phenotypic methods are available for measuring drug susceptibility of HCMV clinical isolates. However, genotypic assays are too specific to detect all possible mutations that lead to resistance and the currently used phenotypic assays are too time consuming and labor intensive to have an impact on clinical decision making. A flow cytometry assay that takes days instead of weeks was developed for the phenotypic determination of drug susceptibility of HCMV clinical isolates. The assay involves the use of reagents containing fluorochrome labeled monoclonal antibodies to HCMV antigens and flow cytometry to automatically detect and quantitate the number of cells expressing these antigens. The reduction in the percentage of antigen positive cells in the presence of inhibitory concentrations of antiviral drugs is used to determine inhibitory concentration 50 (IC50) values. This assay was compared to the plaque reduction assay (PRA) to determine the drug susceptibility to ganciclovir, foscarnet and cidofovir of over 30 HCMV clinical isolates. The IC50 values obtained with the flow cytometry assay are qualitatively, but not quantitatively, identical to those obtained from the time consuming and labor intensive plaque reduction assay. The Phase I portion of this grant application proposes to validate the flow Cytometry drug susceptibility assay for HCMV clinical isolates. Completion of this Phase I project will result in a validated detection system for the phenotypic determination of the drug susceptibility of HCMV clinical isolates. The long term goal is to develop a line of reagents that can be used in flow cytometry to determine drug susceptibility of a wide variety of viruses, including the human immunodeficiency virus (HIV), to clinically useful antiviral drugs.
A line of monoclonal antibody reagents that can be used with flow cytometry for determining drug susceptibility of HCMV clinical isolates will be developed. Over time this line of monoclonal antibody reagents will be developed for determining drug susceptibilities of a number of different viruses for clinically useful antiviral agents and for screening novel antiviral agents for antiviral activity.
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