Construction of HCV-mRNA Vaccine, study its antigenicity and cell mediated responses in in vitro settings.

Abstract

Hepatocellular carcinoma (HCC) is a prominent contributor to mortality associated with liver diseases on a global scale. Hepatitis, liver cirrhosis, hepatocellular carcinoma, and the necessity for liver transplantation are all significantly influenced by HCV globally. The Flaviviridae family resulted in around 71 million individuals worldwide being afflicted with chronic HCV infections in 2020. Direct-Acting Antivirals (DAAs) are one type of treatment for the Hepatitis C Virus (HCV), But are costly, yet there is a possibility of recurring infection. HCV is characterized by the presence of 7 distinct genotypes. This study focuses on seven genotypes for the construction of vaccines and suggested a multi-epitope vaccination. The vaccine utilizes mRNA technology to replicate the genetic makeup of the HCV virus, imitating its natural genome. The vaccine was constructed of epitopesthat are connected by linkers and signaling peptides, which are surrounded by 5' and 3' UTRs. The 6xHis-tag will be affixed to the 5' end for immunocytochemistry. Vaccine was developed, and its characteristics were analyzed using an in-silico technique. The vaccine was stabilized using a 5' cap and a 3' poly A tail. The IVT mRNA vaccine was prepared using the mMACHINE T7 Transcription Kit. The HEK293 cell line was transfected and subsequently utilized to produce the desired protein. Immunocytochemistry (ICC) results confirmed the presence of the protein antigen in the HEK293 cells. Our vaccine candidate also produces the antigen to stimulate a cellular immune response in peripheral blood mononuclear cells (PBMCs). The in vitro transcribed mRNA demonstrated an elevation in the mRNA levels of both T helper 1 (TH1) response inducer, interleukin-12, and TH1 cytokine interferon gamma, thereby indicating the production of memory T cells. This study has demonstrated that it holds great potential as a vaccine for further evaluation against HCV.