The hepatitis C virus and immune escape : relation between sequence variations and the in vitro and in vivo functionality of the non-structural 3/4A complex

Abstract

The hepatitis C virus (HCV) is a blood-borne virus responsible for a major part of liver transplants is the US, and 3% of the population world-wide is infected globally. HCV belongs to the Hepacivirus genus of the Flaviviridae family and is transmitted through infectious blood or blood products. In 85% of those infected the virus establishes a chronic infection. The reason for the high rate of viral persistence is largely unknown, although the high genetic variability is believed play a major role.

Today no vaccine is available to prevent or cure the infection, however interferon-a and ribavirin combination therapy can cure around 50% of those chronically infected. Unfortunately are the side-effects severe and hard to cope with, whereby development of new more effective and less expensive treatments are urgently needed.

HCV can be divided into six genotypes of which HCV genotype 1, which represents approximately 50 percent of all HCV infections globally, is the most difficult to treat. One of the proteins with the lowest genetic variability is the non-structural (NS) 3 protein and, in addition, previous studies have shown that NS3-specific CD4+ T-helper-cell (TH) and CD8+ cytotoxic T lymphocyte (CTL) responses are crucial for resolving the viral infection.

Previous studies have shown NS3 to be poorly immunogenic when used as a DNA vaccine. In vivo, NS3 forms a stable complex with NS4A, thereby targeting NS3 to the endoplasmic reticulum and prolonging the NS3 half life. By inclusion of NS4A to the NS3 DNA vaccine, we observed stronger humoral and cellular responses with a TH1-skewed response, as compared to using NS3 alone. The enhanced immunogenicity was most probably a result of both higher expression levels and a prolonged half-life of NS3.

HCV lacks proof-reading during replication, thereby creating a swarm of closely related genetic variants of the virus within the same host, termed quasi-species. This ability to rapidly change makes HCV able to quickly generate the fittest variant for each new environmental pressure. Thus, HCV can positively select escape mutants when under immune pressure or antiviral treatment. We therefore investigated the natural variation of HLA-A2 epitopes within NS3 in HLA-A2 positive and negative patients chronically infected with genotype 1. In agreement with previous studies did we find that NS3 was highly conserved.

Genetic immunizations with NS3/4A in HLA-A2 transgenic mice showed that the 1073-1081 epitope was immunodominant in both genotype (gt) 1 and 3 strains. Fine mapping of the 1073-1081 epitope of gt 1 revealed that positions 2, 3, 5, 7 and 9 were essential for immune recognition. Mutations at these positions would therefore mediate immune escape, however, almost no mutations were identified at these positions. Importantly, mutations at these positions disrupted both the protease activity and viral replication. Thus, a positive selection of such escape mutations in nature is prevented by the negative effects on viral fitness.

To investigate the true plasticity of the NS3 protease we next generated a complete fitness map of the NS3 protease by introducing alanine or glycine substitutions at each of the 181 residues. Surprisingly only 23 (13%) of the 181 mutants resulted in destroyed protease activity, indicating a much higher plasticity than predicted.

Finally, to study the in vivo effects of one of the immune-escape mutations that prevented HLA-A2 binding and destroyed the protease, IIe1074Ala, we generated a transgenic mouse expressing this mutant NS3/4A protein in the liver. This revealed that the protease activity of NS3 was responsible for the previously observed resistance to TNF-α seen in Tg mice expressing a functional NS3 protease. In conclusion, the present studies have increased our understanding of the NS3/4A complex, which hopefully will aid in the development of new preventive and therapeutic regimens for chronic HCV infections.