Rapid Antigen Test for Diagnosis of Active HCV Infection

Summary/Abstract Hepatitis C virus (HCV) causes a global epidemic with 71 million people infected. HCV is a blood-borne flavivirus that can cause cirrhosis and liver cancer and is responsible for ~400,000 deaths each year. Direct- acting antivirals (DAAs) can effectively cure HCV infection; however, most infected individuals are unaware of their infection status or only receive treatment after severe liver damage. Moreover, DAAs are cost-prohibitive and do not reach at-risk populations including people living in low- and middle-income countries or injecting drug users. Consequently, the number of yearly new infections (~1.4 million) exceeds the number of people receiving treatment (~650,000), thereby exacerbating the epidemic. Elimination of HCV by 2030 – a goal set by the World Health Organization (WHO) – will require multifaceted, decentralized public health prevention strategies. One such strategy calls for the development of point-of-care assays for active HCV infection, which is expected to accelerate the diagnoses of individuals who are most likely to transmit the virus and link those infected to care. As such, this proposal aims to develop a low-cost, instrument-free rapid antigen test for active HCV infection. We recently immunized mice with the HCV core antigen (HCVcAg), a protein secreted into the blood that can be detected almost simultaneously with HCV RNA during an active infection. Using flow cytometry, we sorted HCVcAg-specific B cells from immunized mice, then sequenced the antibody variable segments, and cloned and recombinantly expressed monoclonal antibodies (mAbs) that target the HCVcAg. In Aim 1, we will characterize the binding kinetics of the HCVcAg-specific mAbs. The mAbs with the most robust binding kinetics will then be used to generate a rapid antigen test. In Aim 2, we will determine the analytical and clinical sensitivities and specificities of the rapid antigen test using recombinant proteins, native viruses, and cryopreserved, de-identified serum/plasma previously collected from HCV viral load positive and negative patients and healthy controls. Our rapid antigen test is expected to have a sample-to-result processing time of <20 minutes and a maximum manufacturing price per test of <$4 USD. The successful completion of this project will lead to one of, if not the first, cost-effective, instrument-free rapid antigen test for the point-of-care diagnosis of active HCV infection.