Epitope mapping of the E2 glycoprotein, including the hypervariable region 1, of the hepatitis C virus genotype 3a, in the context of humoral immune pressure
Walsh, Nicole Ellen
University College Cork
The hepatitis C virus (HCV) is an enveloped +ssRNA virus, belonging to the family Flaviviridae. HCV is notable for displaying extraordinary genetic diversity and variability, having seven recognised genotypes and over sixty subtypes. HCV is responsible for the disease known as hepatitis C, which is associated with cirrhosis and hepatocellular carcinoma (HCC). The Global Hepatitis Report released by the World Health Organisation (WHO) in 2017 estimated that viral hepatitis was responsible for 1,340,000 deaths in 2015. The report also estimated that 71,000,000 people have ongoing HCV infections. HCV is largely transmitted via exposure to infected blood, with intravenous drug use accounting for approximately 55% of cases. HCV infections can be categorised as acute or chronic. During chronic HCV infections, antibodies (Abs) are produced against HCV - however, the host Abs are unable to neutralise HCV and only accelerate the evolution of circulating HCV variants. HCV variants resistant to the current generation of host Abs become the dominant variant through selective pressure. The variants of HCV within a host are known as quasispecies. Although the host Ab response is not able to resolve the chronic HCV infection, some Abs can bind to particular HCV variants. These Abs form complexes with virus particles and are known as AAVs (antibody-associated virus). AAVs are detectable in the blood of patients with chronic HCV infections and examination of these AAVs could reveal conserved viral structures and vulnerable HCV epitopes. Twenty genotype 3a serum and plasma samples from patients with chronic HCV infections were obtained from the National Virus Registry Laboratory (NVRL) and from the Molecular Virology Research and Diagnostic Laboratory (MVDRL). HCV genotype 3a was chosen for this research project given its prevalence (estimated to account for 17.9% of chronic HCV infections), resistance to treatment, and increased risk of causing severe steatosis and HCC when compared to other genotypes. Building on previous research carried out by the MVDRL, the patient samples were screened for the presence of AAVs. Initially, AAV+ samples were going to be processed and used to generate HCV pseudoparticles (HCVpp). The HCVpp system is a model system that incorporates the E1E2 glycoprotein from HCV into a plasmid. The E1E2 glycoprotein is responsible for HCV entry and infection, meaning the HCVpp can be used to infectivity and Ab neutralisation assays. However, the E1E2 glycoproteins could not be extracted from the AAV+ patient samples. Instead, the IgG from the AAV+ samples was extracted and used for a series of neutralisation experiments on HCV pseudoparticles generated using the HCV H77 isolate. H77 (GenBank: AAB67037.1) is an infectious genotype 1a isolate that has undergone complete genome sequencing. The Abs that showed the greatest neutralisation potential against the H77 pseudoparticles were selected for epitope mapping. The epitope mapping procedure tested the selected Ab samples against a synthesised H77 E2 glycoprotein structure, and characterised the sites where the patient Abs bound to the synthesised E2. This revealed vulnerable epitopes on the HCV E2 glycoprotein. The epitope mapping also revealed a large number of glycosylation sites around the vulnerable epitopes – a phenomenon known as glycan shielding. Glycan shielding is used by a number of viruses (including HCV and HIV) to protect conserved and vulnerable epitopes from Abs. However, strategies are being developed to counter viral glycosylation, including modifications to glycosylation sites and the use of polysaccharides derived from non-mammalian sources as therapeutic agents against glycosylated viruses.
HCV , Hepatitis C , Virology , Pseudoparticles , Antibodies , Epitope mapping , Humoral immunity
Walsh, N. E. 2020. Epitope mapping of the E2 glycoprotein, including the hypervariable region 1, of the hepatitis C virus genotype 3a, in the context of humoral immune pressure. MRes Thesis, University College Cork.