Drug Resistance of a Viral Population and Its Individual Intrahost Variants During the First 48 Hours of Therapy

There has been a great progress in the development of antiviral agents licensed for treatment of Human immunodeficiency virus (HIV), Herpesviruses, Hepatitis viruses and respiratory viruses1. The emergence of HIV as a major human pathogen and the intensive use of antiretroviral compounds have also provided a better understanding of the genesis of antiviral resistance2. However, there is not a simple method for measuring directly the effect a drug has over a viral population and its individual intra-host variants. Such measure could help screen promising drugs that affect the viral population and also detect the individual variants that are naturally more resistant. In this paper, we use the effects of Interferon (IFN) on HCV as a proof of concept, finding that our drug-resistance estimates, calculated during the first 48 hour after IFN injection, are strongly associated with outcome of therapy at week 12. Hepatitis C virus (HCV) infects nearly 3% of the world's population and is a major cause of liver disease worldwide3. There is no vaccine against HCV and up to recently the standard-of-care therapy involved the combined use of pegylated interferon (peg-IFN) and ribavirin (RBV). This combination therapy is expensive, effective in only 50%-60% of patients, and can be associated with frequent and serious adverse side effects in more than 75% of patients4, 5 In order to improve cost-effectiveness and ameliorate patient hardship, it would be desirable to predict the response at early onset of therapy. IFNs are crucial components of the innate immune system. IFN-α acts by inducing production of interferon stimulating genes (ISGs) to establish a non-specific antiviral state within the cell with direct inhibition of viral replication. It is also known to exert immunomodulatory effects that enhance immune response and accelerate clearance of infected cells6. When exogenously administered as a single injection, IFN-α induces a decline of HCV RNA in two phases: a rapid phase lasting for 24-48 hours, followed by a slower phase of decline over the ensuing weeks. The initial rapid decline is defined by the rate of viral clearance and the effectiveness of IFN in blocking viral production. Successful treatment results in sustained undetectable HCV RNA after completion of therapy. Treatment failure results either from nonresponse (minimal declines in viral titer during therapy) or relapse (robust initial responses followed by rebounds of viral titers after therapy)6. Several independent predictors of a sustained virologic response (SVR) to IFN/RBV therapy have been identified. These include HCV genotypes 2 and 3, low pretreatment viral load, Asian or Caucasian ethnicity, younger age, absence of advanced fibrosis or cirrhosis, and absence of steatosis7. More recently, Genome Wide Association Studies have identified single-nucleotide polymorphisms near the IL28B gene (encoding IFN-λ3) as being particularly associated with spontaneous and treatment-induced clearance of HCV infection8, 9. However, IL28B variations may account for only ∼15% of inter-individual variability of SVR10. The interaction of these host factors determines the therapy effect on the virus, which is directly evidenced by a decline in viral titer, the reason why the rate and magnitude of decline in the first weeks of treatment can predict the outcome of therapy6. HCV exists in infected patients as a large viral population of intra-host variants, which may be differentially resistant to IFN treatment and, therefore, likely to display variable temporal patterns during the first phase of decline following IFN injection. Assessing the spectrum of HCV variants and measuring the IFN resistance of individual variants could be critical for understanding the variability in therapy outcomes. Next-generation sequencing (NGS) technologies in conjunction with computational analysis allow for quantitative assessment of viral intra-host variants, providing data on the intra-host dynamics of individual HCV variants and an opportunity for measuring their resistance to IFN. The HVR1 region of HCV is used here as a tag or marker of individual intra-host viral strains for estimating their relative frequencies over a short period of time. Since the model is based solely on changes in viral titer or the relative frequency of intra-host viral variants persisting during antiviral therapy, without consideration of other viral factors, host factors or type of drug administered, it may be applicable to measure and predict HCV treatment response with other drug regimens, including the newly available direct acting antiviral (DAA) agents. In addition, the presented analytical framework should be applicable to drug resistance of other viral infections.