| Summary: | Combination antiretroviral therapy has transformed Human Immunodeficiency Virus (HIV) infection from what was once a fatal diagnosis to a manageable chronic condition. Similarly, new direct-acting antivirals offer a potential cure for individuals infected with Hepatitis C Virus (HCV). Treatment of these two infectious diseases is now routinely guided by genotypic drug resistance testing: portions of the viral genome are sequenced and analyzed for mutations in order to select drug combinations best suited to treat each individual’s unique viral population. The primary aim of this thesis is to develop new methods to personalize therapies for HIV and HCV using a variety of DNA sequencing technologies. First, manual review of Sanger sequences is highly subjective, leading to potential bias in the detection of resistance mutations in diverse viral populations. Automated sequence analysis software that provides standardization between users and laboratories is presented. Second, HIV treatment in resource-limited settings is compromised by insufficient access to resistance testing. To facilitate individual-level monitoring, a low-cost resistance test, whereby hundreds of samples are simultaneously sequenced on a next-generation instrument, is proposed and validated. Third, novel screening and drug resistance tests are required to assess the efficacy of new antivirals. For example, certain regimens containing the protease inhibitor simeprevir are less effective in treating individuals infected with HCV harboring a common polymorphism. Two independent sequencing assays that test for this polymorphism are described and validated. A secondary aim is to measure HIV evolution under immune and drug selection pressures using these same sequencing methods. The hypothesis that effective treatment suppresses viral replication and retards viral evolution is supported by evidence from longitudinal sequences from patients on antiretroviral therapy. A second study demonstrates limited selection of drug resistance mutations in patients with low-level viremia, supporting the hypothesis that many results from an approved clinical test are false positives. Finally, next-generation sequencing is used to quantify HIV variants in cultured virus in order to measure their relative replicative fitness.
This thesis provides evidence that new and existing assays and bioinformatic tools will remain invaluable in the clinical management of HIV and HCV as DNA sequencing technologies continue to evolve. === Medicine, Faculty of === Medicine, Department of === Experimental Medicine, Division of === Graduate
|
|---|
