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

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.

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.

Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection.

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.

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.

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.