Short interval change in hepatitis C hypervariable region 1 in chronic infection. Are there treatment windows in the envelope?

Abstract

Hepatitis C Virus (HCV), an RNA virus, is one of the leading causes of cirrhosis worldwide and, remains the leading indication for orthoptic liver transplantation in the United States. Dual treatment with pegylated interferon and ribavirin has until 2010 been the mainstay of treatment. The emergence of newer agents with direct activity against specific virus proteins has revolutionised HCV treatment but, the high cost of these medications are likely to prevent universal access, particularly in developing countries and, strategies to optimise response to cheaper combination treatments are required. The Irish Hepatitis C outcomes research network (ICORN) has proposed a target of 2025 for the complete eradication of Hepatitis C from Ireland. HCV replicates in an error prone fashion resulting in mutant progeny known as quasispecies(QS), thought to form an important mechanism of host immune evasion in the establishment and maintenance of chronic infection, which develops in 50-80% of those acutely infected. HCV has three hypervariable regions (sections of the virus genome that appear to tolerate higher substitution rates) and one of these, Hypervariable region 1 (HVR1) has been recognised as a major target of the adaptive immune response. HVR1 quasispecies complexity and diversity have been implicated as predictive of response to dual therapy. Little, however, is known about the natural history of these parameters in chronic infection. We discuss evolutionary concepts and how they apply to quasispecies and hypothesise how viruses might select a setting appropriate mutation rate in order to optimise adaptation, advancing the theory of replicative homeostasis. We prospectively study 23 patients with chronic HCV infections and, differing degrees of liver fibrosis fortnightly for a 16 week period prior to commencement of treatment. Using amplicon sequencing, cloning and next generation sequencing we explore the behaviour of HVR1 QS, establishing the utility of each technique in describing QS change. We identify variable and unpredictable HVR1 change in our cloning data which precludes the use of these metrics in pre treatment prediction models. HVR1 change is far greater in non cirrhotic patients and the transition to cirrhosis appears to be associated with a change from positive to purifying selection. Using molecular clock techniques we illustrate differing substitution rates within HVR1 among cirrhotic and non cirrhotic patients. We identify, by including an additional retrospective sample, that the patterns we describe are sustained over prolonged periods and further clarify the mode and tempo of HVR1 change by estimating the substitution rates. Using next generation sequencing techniques we identify similar patterns of HCV change when compared with our cloning data. However, the sequence depth provided permits the description of time specific network of HVR1 clones, all connected by a single amino acid substitution to a central node. By separating our samples into immunoglobulin bound and free fractions we describe the importance of host immune mediated change driving the changes seen in our pyrosequencing and cloning data. Finally, using known viral and host molecular markers predictive of treatment response we explore unsuccessfully for models predictive of treatment response.