Adapting in-cell ELISA to investigate the localization of hepatitis C virus envelope glycoproteins

The development of hepatitis C virus (HCV) vaccines remains challenging due to extensive viral genetic diversity and the complex biology of the envelope glycoproteins E1 and E2, which are major targets of broadly neutralizing antibodies and are associated with spontaneous viral clearance. Assessment of E1E2 expression is complicated by reported intracellular retention, limiting the ability to evaluate antigen localization efficiently. Here, we adapted an in-cell enzyme-linked immunosorbent assay (ELISA) to quantify membrane-associated and intracellular E1E2 expression and explored its utility as an early screening tool for HCV vaccine antigens. HEK293T and Huh-7 cells were transfected with mRNA encoding untruncated E1E2 sequences from two antigenically distinct HCV isolates, C110D0 and C176D0. Cells were analyzed following fixation alone to detect membrane-associated antigen or following fixation and permeabilization to detect total cellular antigen. In-cell ELISA reproducibly detected E1 and E2 above mock-transfected controls under both conditions. For C110D0, E1 and E2 signals were significantly greater following permeabilization than fixation alone, consistent with partial intracellular retention. Similar trends were observed for C176D0, although differences between conditions were less pronounced. Flow cytometry using anti-E2 monoclonal antibodies confirmed surface expression of E2 on a subset of transfected cells and corroborated the localization patterns by in-cell ELISA. These findings establish in-cell ELISA as a scalable and reproducible approach for evaluating the expression and localization of HCV envelope glycoproteins, and support its use in the prioritization of candidate antigens for downstream studies.IMPORTANCEHepatitis C virus remains a major cause of chronic liver disease and liver cancer worldwide. Although highly effective direct-acting antivirals can cure infection, access to treatment remains limited in low- and middle-income countries, with new infections continuing to occur globally. An effective vaccine is therefore likely to be critical for long-term hepatitis C virus (HCV) control. One challenge in vaccine development is determining whether viral proteins are displayed on the surface of cells, where they can be recognized by the immune system or remain hidden inside infected cells. We describe a simple laboratory assay that distinguishes between these two patterns of protein expression using standard equipment and scalable workflow. Applying this approach, we demonstrate surface expression of envelope proteins from two HCV isolates while also identifying differences in their intracellular retention. This method provides a practical tool for early evaluation and prioritization of vaccine antigens before their progression to more detailed downstream studies.