Production and validation of anti-HCV antibodies for viral neutralization

Abstract: Hepatitis-C Virus (HCV) remains the leading cause of liver transplant in the US and the UK, and the World Health Organization (WHO) estimates that 71 million people are infected worldwide. A vaccine would drastically impact the healthcare-associated burdens that HCV causes globally. The objective of this master’s thesis project is to produce human antibody (IgG) against HCV. This project will focus on the monoclonal antibodies (mAbs) HEPC3, AR3C, HEPC74, and HCV1. These four antibodies have been isolated from patients who have successfully cleared the infection, and their sequences and structures are available in the public domain. These four antibodies have also shown to bind to E2, a glycoprotein on the surface HCV that is crucial for viral binding and entry. This interaction of the mABs with E2 has been implicated in viral neutralization, making them promising choices for this study. Overall, 3 out of 4 mAbs were successfully cloned and produced. The unsuccessful antibody, HEPC74, was discovered to have failed due to an error in the plasmid sequence. Just as the western blot to confirm secretion was ready to be run, the laboratory closed due the Covid-19 outbreak. Therefore, the data can officially declare a ¾ mAb production success, however it is safe to assume that the alternative clone for HEPC74 was also a success due to a perfect sequence match. Since the primary objective of this project was to successfully clone and produce these four antibodies, then this study is considered an overall success. Lastly, this study examined how the same protocol  could be applied the SARS-CoV-2 outbreak, by the cloning and production of anti-RBD IgG and testing them for viral neutralization.