Paul Harrigan

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Standard and enhanced genotypic assessment of HIV drug resistance and tropism in subtype-B and non-subtype-B HIV-1 infections (2015)

Sequencing human immunodeficiency virus type 1 (HIV-1) for drug resistance mutations and viral tropism is crucial to the current HIV/AIDS personalized treatment approach. This thesis addresses particular areas where clinical utility data is lacking: First, current methods were validated as clinically useful with plasma viral RNA primarily from patients infected with subtype B HIV-1, the dominant strain in developed countries; however, globally most patients are infected with non-subtype B variants. Secondly, there is insufficient data available with respect to the newest classes of antiretroviral drugs (i.e. CCR5-antagonist and integrase inhibitors). Finally, modern antiretroviral therapies often lead to “undetectable plasma viremia” (i.e. successful ongoing treatment) which make plasma-based genotypic testing impossible; the utility of examining alternative sample types is being actively explored. The overall objective of this thesis is to evaluate genotypic assessment of HIV-1 using standard and “second generation” DNA sequencing methods for guiding clinical decisions in nonconventional sample types. Specifically, it is hypothesized that genotypic assessment of subtype A, C and D HIV-1, plasma viral RNA collected pre-therapy, and viral DNA archived in blood cells are useful predictors of in vitro phenotype and/or clinical outcomes. Chapter 1 and 2 examine the clinical utility of current genotyping approaches when applied to non-subtype-B HIV-1. Results suggest that (1) transmitted genotypic drug resistance predicted small but negative treatment outcomes in non-B infections, and (2) current genotypic tools for predicting viral tropism had poor sensitivities and/or specificities in subtypes A and D, but not C HIV-1. Chapter 3 and 4 examine the clinical utility of current genotypic approaches coupled with alternative sample types. Results suggest that (3) pre-therapy plasma sample tropism results predicted post-therapy post-suppression tropism in 90% of subjects, and (4) viral DNA archived in blood and plasma RNA had similar integrase inhibitor-associated mutations, but mutations in DNA were detected substantially later and were substantially less prevalent. In conclusion, genotypic assessment of HIV-1 using nonconventional sample types is clinically relevant, but has specific limitations. Further methodological research and clinical validation studies are needed to ensure proper interpretation of results.

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The use of genetic sequencing technologies to determine HIV-1 viral tropism and to evaluate the effects of maraviroc on patient viral populations (2015)

HIV-1 infection is reliant on the ability of the virus to enter target cells characterized by the expression of either the CCR5 or CXCR4 co-receptor at the cell surface. It is now well established that the V3 loop of the HIV-1 envelope is the primary determinant of co-receptor use, and that genetic analysis of the V3 loop can be used to infer co-receptor use, or “tropism”. This became clinically relevant with the development of the entry inhibitor, maraviroc (MVC), an anti-HIV compound designed to inhibit HIV-1 cell entry exclusively at the CCR5 co-receptor. As such, the more pathogenic CXCR4-using variants are likely to thrive during MVC therapy. Anti-HIV treatment guidelines now strongly suggest a tropism test be performed when considering the clinical use of MVC.There are two primary objectives discussed in this thesis, 1) the validation of a population-based sequencing tropism assay designed to predict virological response to MVC; 2) to apply a next generation sequencing tropism assay to investigate the selective pressures exerted by MVC on mixed tropic HIV-1 populations. The validation studies presented in this thesis demonstrate the reliability of a population-based sequencing assay to infer virological response to MVC in two large, multinational cohorts. The results of these studies have promoted the worldwide expansion of this genotypic assay as a practical and affordable option for tropism inference. In addition, a more intensive investigation into the selective pressures exerted by MVC on mixed tropic HIV-1 populations demonstrated the reproducible outgrowth of preexisting variants most genetically characteristic of CXCR4-using virus. The results of these studies suggest MVC may be effective against a broader range of variants than previously thought. In general, the four studies described in this thesis demonstrate the clinical utility of genetic sequencing when considering the use of MVC.

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Tools and applications for DNA sequencing in the clinical management of viral infectious diseases : examples from HIV-1 and HCV (2015)

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.

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Deep sequencing of HIV-1 envelope determines coreceptor usage and predicts virologic responses to antiretroviral treatment with CCR5 antagonist medication (2014)

Next-generation sequencing can be used to genotype an array of HIV variants within clinical specimens, in a process referred to as deep sequencing. When directed at the gene for HIV envelope, this approach can be used to generate a high-sensitivity overview of the viral tropism of the HIV quasispecies in an infected individual. Since HIV variants with tropism for the CXCR4 coreceptor are not susceptible to the CCR5 antagonist agent maraviroc, their detection is crucial in order to screen out patients unlikely to achieve viral load declines on maraviroc. There are several assays that test HIV tropism, but it has been unclear as to which are most useful in the clinical setting. The aim of this thesis is to compare next-generation sequencing using massively parallel pyrosequencing against several alternative tropism assays in a total of four large randomized clinical trials of maraviroc. The methodologies are refined and optimized in the early chapters and applied to clinical specimens from a total of 2864 HIV-infected individuals. The concordance of next-generation sequencing with phenotypic tropism assays reached 87%. Relative to both the original Trofile phenotypic assay and standard population-based sequencing, deep sequencing had higher sensitivity to detect minority non-R5 HIV. Where assays gave discordant results, deep sequencing tended to outperform the comparator assay and was able to better discriminate maraviroc responders from non-responders. Next-generation sequencing had excellent performance in populations of both treatment-experienced and treatment-naïve individuals. It was consistently able to determine coreceptor usage, and to predict which patients would respond to maraviroc. It could be performed using either HIV RNA from blood plasma or HIV DNA from peripheral blood mononuclear cells. Additionally, longitudinal deep sequencing was performed on samples taken prior to maraviroc administration and again at treatment failure. Phylogenetic analyses confirmed that the non-R5 variants present at time of maraviroc treatment failure were derived from variants detected by deep sequencing before treatment was initiated. In conclusion, next-generation sequencing was applied to thousands of samples from phase III clinical trials, and was a superior screening tool to those originally used during trial enrollment. This thesis demonstrates the clinical utility of next-generation sequencing.

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Master's Student Supervision (2010 - 2018)
HIV genomics : trends in antiretroviral resistance and future directions for pharmacogenetic testing (2018)

Antiretroviral drugs are fundamental to the treatment of Human Immunodeficiency Virus (HIV), effectively inhibiting viral replication, the emergence of Acquired Immune Deficiency Syndrome (AIDS), and subsequent mortality. Combined antiretroviral therapy (cART) is the cornerstone of HIV treatment, and has been adopted by agencies around the world. An estimated 18.2 million individuals accessed cART worldwide in 2016. However, HIV drug resistance to cART leads to ineffective HIV treatment, is associated with AIDS-related morbidity and mortality, and the potential onward transmission of drug resistant HIV strains. Since cART became available in BC in the mid-1990s, antiretroviral drugs and clinical guidelines for HIV management have evolved to reflect best practices. Over the past two decades, genetic testing for HIV drug resistance has become an important tool for HIV care. Next generation massively parallel sequencing has proven to be a powerful sequencing tool rivaling the gold standard Sanger sequencing method, however it is not yet widely adopted for HIV-related genetic testing in BC. There are three primary objectives discussed in this thesis: 1) identification of long-term trends in transmitted and acquired HIV drug resistance in BC, Canada; 2) determination of sociodemographic covariates of drug resistance development and testing uptake; and 3) validation and application of an HIV-related next generation sequencing (NGS) assay for abacavir hypersensitivity screening. Prevalence data of acquired and transmitted drug resistance over the past two decades are presented. Acquired resistance was examined in further detail in order to assess the effect of therapy duration on drug resistance, as well as temporal effects and other factors. Covariates of acquired drug resistance were also examined over calendar time, with a particular focus on adherence to treatment regimens, including sociodemographic predictive factors, as well as sociodemographic covariates of resistance testing uptake. After characterizing historical trends of drug resistance, a glimpse at the future of HIV-related genetic testing is presented: an NGS assay for abacavir hypersensitivity screening was validated and applied as a proof of principle on the Illumina MiSeq platform. This assay was shown to be highly accurate and reliable, providing higher resolution sequencing compared to currently used methods, and expediting testing.

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Populational studies of HIV-1 drug resistance in British Columbia (2010)

Objective:Highly active antiretroviral therapy (HAART) has led to a dramatic decrease in AIDS-related morbidity and mortality, but can be compromised by the development of HIV drug resistance. The objective of this thesis is to explore issues important to the understanding of HIV drug resistance at the populational level in British Columbia, Canada.Methods:HIV drug resistance was analyzed via retrospective, observational analyses of the population of HIV-infected individuals receiving treatment through the HIV/AIDS Drug Treatment Program in British Columbia, Canada. Analyses were largely based upon viral protease and reverse transcriptase genotypic sequences obtained from archived plasma samples as well as information in the BC Centre for Excellence in HIV/AIDS’ monitoring and evaluation system.Results: One analysis demonstrated a drastic, exponential decrease in the incidence of new cases of HIV-1 drug resistance over time in individuals followed from 1996-2008 that has been concomitant with linear increases in the proportion of individuals with undetectable plasma viral loads. An analysis of the probability of developing resistance, adjusted for several factors, between different initial antiretroviral regimens showed 2.4-fold lower odds of developing HIV drug resistance among individuals initiating HAART with a boosted protease-inhibitor-based regimen versus other common regimens, as well as a decreased likelihood of developing resistance based upon initiating more modern HAART regimens. Finally, a third analysis focussed on specific K65K and K66K silent mutations in HIV reverse transcriptase, which are strongly co-selected with known thymidine analogue mutations, in particular D67N. In steady-state kinetic assays, the presence of these mutations was shown to alleviate replicative pausing and/or dissociation events of HIV-1 reverse transcriptase on RNA reverse transcriptase templates that contained the D67N and K70R mutations.Conclusion:Changes in HIV drug resistance have occurred at the populational level in BC over the period of 1996-2008. The superiority of modern HAART regimens in regard to the development of resistance, and overall drastic exponential decreases in the levels of incident drug resistance over time, provide new benchmarks for the analysis of the efficacy of current and future antiretroviral regimens. Furthermore, the identification of novel silent mutations co-selected with therapy exposure may have clinical implications.

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