Relation between viral fitness and immune escape within the hepatitis C virus protease

Abstract

Background: The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non-structural (NS) 3/4A protease to escape cytotoxic T lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally. Aims: We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073-1081 of the NS3 protease was limited by viral fitness. Patients: Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection. Methods: NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA-A2-restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA-A2-transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication. Results: Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073-1081 epitopes that were generally recognised by cross reactive human and murine HLA-A2 restricted CTLs. Introduction of mutations at five positions of the 1073-1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication. Conclusions: Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans.

Membrane markers of the immune cells in swine: an update

“…Depending on their origin and size, antigenic peptides are either presented by MHC-class II and recognised by Th CD4 lymphocytes or presented by MHCclass I and recognised by CD8 Tc lymphocytes. MHC-class I presents antigenic peptides of eight to nine amino-acids following endogenous degradation in the cytosol, whereas MHC-class II present peptides of 12 to 25 amino-acids following exogenous degradation in phagolysosomes [65,200]. Using synthetic pentadecapeptides, classical swine fever virusspecific T cell epitopes of SLAd/d miniswine were identified.…”

Heterologous T Cells Can Help Restore Function in Dysfunctional Hepatitis C Virus Nonstructural 3/4A-Specific T Cells during Therapeutic Vaccination

“…ELISPOT assays were performed as previously described (11,29). In brief, splenocytes and lymphocytes from groups of immunized mice were pooled and immediately tested for presence of HCV NS3-specific or HBcAgspecific T cells with a commercial anti-IFN-g or anti-IL-2 ELISPOT assay (MabTech, Stockholm, Sweden) using known HCV NS3 peptides, rNS3 protein, or rHBcAg to stimulate CTLs for 36 h before detection.…”

“…Proper expression of each protein and successful NS3 protease cleavage were analyzed through in vitro translation using [ 35 S]methionine-labeled (Amhersham Biosciences, Uppsala, Sweden) prokaryotic T7-coupled reticulocyte lysate TNT system (Promega, Madison, WI) as described (11,24).…”

“…Additionally, the specific T cell responses seem to be dysfunctional during the chronic infection (2)(3)(4)(5)(6)(7)(8). This impairment may be caused by escape mutations within T cell epitopes where this is allowed by the viral fitness (9)(10)(11)(12) or by a direct effect of viral proteins (2). Importantly, the dysfunctionality is clearly reversible in that the depletion of CD25 + regulatory T cells (Tregs) or programmed cell death ligand 1 (PD-L1) ligand-expressing cells restores T cell function in vitro (13,14).…”

Natural epitope variants of the hepatitis C virus impair cytotoxic T lymphocyte activity

“…Epitopes such as HLA-A2 restricted NS3 1073-1081 are consistently targeted by CD8 + T cells, but amino acid mutations facilitating immune evasion are rarely observed [16,71] . Since the NS3 protein shares both protease and NTPase-dependent helicase functions, it has been proposed that mutations in these epitopes may be lethal to the virus [72] . Additionally, another study indicates that CTL escape mutations emerging early in infection are not necessarily stable, but are eventually replaced with variants that achieve a balance between immune evasion and fitness for replication [73] .…”