Hepatitis C virus (HCV) infects millions of people worldwide, causing chronic liver disease that can lead to cirrhosis, hepatocellular carcinoma, and liver transplant. In the last several years, the advent of direct-acting antivirals, including NS3/4A protease inhibitors (PIs), has remarkably improved treatment outcomes of HCV-infected patients. However, selection of resistance-associated substitutions and polymorphisms among genotypes can lead to drug resistance and in some cases treatment failure. A proactive strategy to combat resistance is to constrain PIs within evolutionarily conserved regions in the protease active site. Designing PIs using the substrate envelope is a rational strategy to decrease the susceptibility to resistance by using the constraints of substrate recognition. We successfully designed two series of HCV NS3/4A PIs to leverage unexploited areas in the substrate envelope to improve potency, specifically against resistance-associated substitutions at D168. Our design strategy achieved better resistance profiles over both the FDA-approved NS3/4A PI grazoprevir and the parent compound against the clinically relevant D168A substitution. Crystallographic structural analysis and inhibition assays confirmed that optimally filling the substrate envelope is critical to improve inhibitor potency while avoiding resistance. Specifically, inhibitors that enhanced hydrophobic packing in the S4 pocket and avoided an energetically frustrated pocket performed the best. Thus, the HCV substrate envelope proved to be a powerful tool to design robust PIs, offering a strategy that can be translated to other targets for rational design of inhibitors with improved potency and resistance profiles.IMPORTANCE Despite significant progress, hepatitis C virus (HCV) continues to be a major health problem with millions of people infected worldwide and thousands dying annually due to resulting complications. Recent antiviral combinations can achieve > 95% cure, but late diagnosis, low access to treatment, and treatment failure due to drug resistance continue to be roadblocks against eradication of the virus. We report the rational design of two series of HCV NS3/4A protease inhibitors with improved resistance profiles by exploiting evolutionarily constrained regions of the active site using the substrate envelope model. Optimally filling the S4 pocket is critical to avoid resistance and improve potency. Our results provide drug design strategies to avoid resistance that are applicable to other quickly evolving viral drug targets.

Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response; thus, an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412 to 423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, as well as a bivalent immunogen with two copies of the epitope on the E2 surface. We solved the X-ray structure of a cyclic immunogen in complex with the HCV1 antibody and confirmed preservation of the epitope conformation and the HCV1 interface. Mice vaccinated with our designed immunogens produced robust antibody responses to epitope I, and their serum could neutralize HCV. Notably, the cyclic designs induced greater epitope-specific responses and neutralization than the native peptide epitope. Beyond successfully designing several novel HCV immunogens, this study demonstrates the principle that neutralizing anti-HCV antibodies can be induced by epitope-based, engineered vaccines and provides the basis for further efforts in structure-based design of HCV vaccines. IMPORTANCE: Hepatitis C virus is a leading cause of liver disease and liver cancer, with approximately 3% of the world's population infected. To combat this virus, an effective vaccine would have distinct advantages over current therapeutic options, yet experimental vaccines have not been successful to date, due in part to the virus's high sequence variability leading to immune escape. In this study, we rationally designed several vaccine immunogens based on the structure of a conserved epitope that is the target of broadly neutralizing antibodies. In vivo results in mice indicated that these antigens elicited epitope-specific neutralizing antibodies, with various degrees of potency and breadth. These promising results suggest that a rational design approach can be used to generate an effective vaccine for this virus.

Objective: To examine trends in the direct acting antiviral (DAA) uptake in a multi-state Medicaid population with hepatitis C virus (HCV) prior to and after ledipasvir/sofosbuvir (LDV/SOF) approval and changes in prior authorization (PA) requirements. Data sources: Analyses utilized enrollment, medical, and pharmacy claims in four states, December 2013-December 2017. Study design: An interrupted time series examined trends in uptake (1+ claim for a DAA) before and after two events: LDV/SOV approval (October 2014) and lifting of PA requirements for 40% of members (July 2016). Analyses were also performed in subgroups defined by the number and dates of change in PA requirements in members' Medicaid plans. Data collection/extraction methods: Members aged 18-64 years with an ICD code for HCV were included in the sample from diagnosis date until treatment initiation or Medicaid disenrollment. Principal findings: The annual sample size ranged from 38,302 to 45,005 with approximately 30% ages 18-34 years and 40% female. In December 2013, 0.08% was treated, rising to 0.74% in December 2017 (p < 0.001). Uptake increased from 0.34%/month in October 2014 to 0.70%/month after LDV/SOF approval, (p < 0.001), and increased relative to the pre-LDV/SOV trend through June 2016 (p = 0.04). Uptake increased to 1.18%/month after PA change, (p < 0.001) and remained flat through 2017 (p = 0.64). Cumulatively, 20.1% were treated by December 2017. In plans with few/no requirements through 2017, uptake increased to 1.19%/month after LDV/SOF approval (p < 0.001) and remained flat through 2017 (p = 0.11), with 22.2% cumulatively treated. Among plans that lifted PA requirements from three to zero in mid-2016, uptake did not increase after LDV/SOF approval (p = 0.36) but did increase to 1.41%/month (p < 0.001) after PA change, with 18.1% cumulatively treated. Conclusions: HCV Treatment increased through 2017. LDV/SOF approval and lifting PA requirements led to an increase in uptake followed by flat monthly utilization. Cumulative uptake was higher in plans with few/no PA requirements relative to those with three requirements through mid-2016.