Original Article
Copyright ©2010 Baishideng. All rights reserved.
World J Gastroenterol. Apr 28, 2010; 16(16): 1953-1969
Published online Apr 28, 2010. doi: 10.3748/wjg.v16.i16.1953
Natural epitope variants of the hepatitis C virus impair cytotoxic T lymphocyte activity
Shuping Wang, Rico Buchli, Jennifer Schiller, Jianen Gao, Rodney S VanGundy, William H Hildebrand, David D Eckels
Shuping Wang, David D Eckels, Department of Pathology, University of Utah, Salt Lake City, UT 84112, United States
Rico Buchli, Rodney S VanGundy, Pure Protein L.L.C., Oklahoma City, OK 73104, United States
Jennifer Schiller, Jianen Gao, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, United States
William H Hildebrand, Department of Microbiology and Immunology, Health Sciences Center, University of Oklahoma, Oklahoma City, OK 73104, United States
Author contributions: Wang S, Buchli R, VanGundy RS and Hildebrand WH performed the majority of experiments; Schiller J and Gao J assisted some parts of experiments; Eckels DD, Wang S and Buchli R wrote the manuscript.
Supported by The National Institutes of Health, No. NIH-DK-57732
Correspondence to: David D Eckels, Professor, Department of Pathology, University of Utah, Salt Lake City, UT 84112, United States. david.eckels@path.utah.edu
Telephone: +1-801-2132800  Fax: +1-801-5853670
Received: November 9, 2009
Revised: January 6, 2010
Accepted: January 13, 2010
Published online: April 28, 2010

AIM: To understand how interactions between hepatitis C virus (HCV) and the host’s immune system might lead to viral persistence or effective elimination of HCV.

METHODS: Nucleotides 3519-3935 of the non-structural 3 (NS3) region were amplified by using reverse transcription polymerase chain reaction (PCR). PCR products of the HCV NS3 regions were integrated into a PCR® T7TOPO® TA vector and then sequenced in both directions using an automated DNA sequencer. Relative major histocompatibility complex binding levels of wild-type and variant peptides were performed by fluorescence polarization-based peptide competition assays. Peptides with wild type and variant sequences of NS3 were synthesized locally using F-moc chemistry and purified by high-performance liquid chromatography. Specific cytotoxic T lymphocytes (CTLs) clones toward HCV NS3 wild-type peptides were generated through limiting dilution cloning. The CTL clones specifically recognizing HCV NS3 wild-type peptides were tested by tetramer staining and flow cytometry. Cytolytic activity of CTL clones was measured using target cells labeled with the fluorescence enhancing ligand, DELFIA EuTDA.

RESULTS: The pattern of natural variants within three human leukocyte antigen (HLA)-A2-restricted NS3 epitopes has been examined in one patient with chronic HCV infection at 12, 28 and 63 mo post-infection. Results obtained may provide convincing evidence of immune selection pressure for all epitopes investigated. Statistical analysis of the extensive sequence variation found within these NS3 epitopes favors a Darwinian selection model of variant viruses. Mutations within the epitopes coincided with the decline of CTL responses, and peptide-binding studies suggested a significant impact of the mutation on T cell recognition rather than peptide presentation by HLA molecules. While most variants were either not recognized or elicited low responses, such could antagonize CTL responses to target cells pulsed with wild-type peptides.

CONCLUSION: Cross-recognition of CTL epitopes from wild-type and naturally-occurring HCV variants may lead to impaired immune responses and ultimately contribute to viral persistence.

Keywords: Epitopes, Human, T Cells, Cytotoxic, Anergy, Viral