Antibody Development to HCV Alternate Reading Frame Protein in Liver Transplant Candidate and its Computational Analysis

Background: HCV Alternate Reading Frame Protein (ARFP) is a frameshift product of HCV-core encoding. Here, we characterized specific anti-ARFP antibodies in Liver Transplant Candidate (LTC) and chronic HCV-infected patients. Methods: The ARFP gene was cloned and the recombinant protein was purified using Nickel chromatography and confirmed by western blotting. ELISA was developed using recombinant core-1a, core- 1b, ARFP-1a protein, and 99-residue synthetic ARFP 1b peptide. By several Bioinformatics tools, general properties, immunogenic epitopes, and structures of these proteins were obtained. Results: The seroprevalence of anti-core and anti-ARFP antibodies was 100% in LTC patients, but only 75.2% and 94.3% of chronic patients had evidence of anti-ARFP and anti-core antibodies, respectively. In-silico results demonstrated physicochemical features, antigen properties and potential interactors that could describe progression toward advanced liver disease. Conclusion: As the first report, the prevalence of anti-ARFP antibodies in LTC patients is of the order of 100% and titer of anti-ARFP antibody was significantly higher in LTC patients compared to chronic individuals, suggesting the possible role of ARFP in the progression toward advanced liver disease. In addition, docking analysis determined several interactor proteins such as prefoldin 2, cathepsin B, vitronectin, and angiotensinogen that have an important role in progression to chronic infection and liver disease development.