Objective:
Hepatitis C virus (HCV) is a major global health concern, infecting an estimated 50–70 million people worldwide. Chronic HCV infection can lead to severe liver disease, including cirrhosis and hepatocellular carcinoma. While direct-acting antivirals are highly effective at curing infection, they do not prevent reinfection and are not accessible to all populations. A prophylactic vaccine is urgently needed to support global elimination efforts. mRNA vaccine platforms have shown promise in rapidly generating potent immune responses and offer advantages such as scalable production and precise antigen design. This study investigates mRNA-encoded HCV envelope glycoproteins as vaccine candidates, aiming to improve immunogenicity and presentation of conserved neutralising epitopes.
Methods:
Previously, a modified E2 glycoprotein (E2Δ123), lacking hypervariable regions which typically act as immune decoys, was designed to focus immune responses on conserved neutralising epitopes. In this study, mRNA constructs were designed, including E2Δ123, E2Δ123A7 (seven cysteines mutated to alanine for consistent monomeric structure), and E1E2. Expression was assessed via transient expression, His-tag affinity and size exclusion chromatography. Antigenicity was characterised through ELISA and flow cytometry and immunisation studies in small animals were also conducted to assess the immunogenicity of the mRNA-encoded proteins. Neutralising antibody responses were evaluated using a panel of 14 tier 1-4 E1E2 pseudotyped viruses.
Results:
Transfection of mRNA-encoded E2Δ123 resulted in expression of monomers only, in contrast to the heterogeneity observed with DNA-encoded E2Δ123. All glycoproteins effectively presented neutralising epitopes, as evidenced by the binding of neutralising monoclonal antibodies to these proteins via ELISA and flow cytometry. Immunisation studies showed that the mRNA constructs elicited E2-specific antibody responses, but we were unable to detect AR4A-like antibodies using E1E2 as an immunogen. Neutralising antibody titres were low and the breadth of neutralisation observed with Δ123 immunogens was comparable to that seen with E1E2. However, neutralisation was largely restricted to genotype 1a and tier 1 and 2 viruses.
Conclusion:
mRNA-based HCV glycoproteins exhibit potential as vaccine candidates by eliciting targeted immune responses and presenting key neutralising epitopes. This work represents a critical step toward developing a potent mRNA-based HCV vaccine; however, current candidates demonstrate limited breadth of neutralisation, and mRNA vaccine platforms face challenges including potency, breadth and durability.