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Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus.

ABSTRACT: AIM:Quasispecies of hepatitis C virus (HCV) are the foundation for rapid sequence evolution of HCV to evade immune surveillance of hosts. The consensus sequence evolution of a segment of HCV NS3 region, which encompasses putative cytotoxic T cell epitopes, was evaluated. METHODS:Three male patients, infected with HCV through multiple transfusions, were identified from clinical symptoms and monitored by aminotransferase for 60 months. Blood samples taken at months 0, 32, and 60 were used for viral RNA extraction. A segment of HCV NS3 region was amplified from the RNA extraction by RT-PCR and subjected to subcloning and sequencing. HLA types of these three patients were determined using complement-dependent microlymphocytotoxic assay. CTL epitopes were predicted using MHC binding motifs. RESULTS:No patient had clinical symptoms or elevation of aspartate/alanine aminotransferase. Two patients showed positive HCV PCR results at all 3 time points. The other one showed a positive HCV PCR result only at month 0. A reported HLA-A2-restricted CTL epitope had no alteration in the HLA-A2-negative carrier over 60 months. In the HLA-A2-positive individuals, all the sequences from 0 month 0 showed an amber mutation on the initial codon of the epitope. Most changes of consensus sequences in the same patient occurred on predicted cytotoxic T cell epitopes. CONCLUSION:Amber mutation and changes of consensus sequence in HCV NS3 region may be related to viral immune escape.

SUBMITTER: Guo HZ

PROVIDER: S-EPMC4726999 | biostudies-literature | 2004 Mar

REPOSITORIES: biostudies-literature

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Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus.

Guo Hua-Zhang HZ Yin Ying Y Wang Wen-Liang WL Zhang Chuan-Shan CS Wang Tao T Wang Zhe Z Zhang Jing J Cheng Hong H Wang Hai-Tao HT

World journal of gastroenterology 20040301 6

<h4>Aim</h4>Quasispecies of hepatitis C virus (HCV) are the foundation for rapid sequence evolution of HCV to evade immune surveillance of hosts. The consensus sequence evolution of a segment of HCV NS3 region, which encompasses putative cytotoxic T cell epitopes, was evaluated.<h4>Methods</h4>Three male patients, infected with HCV through multiple transfusions, were identified from clinical symptoms and monitored by aminotransferase for 60 months. Blood samples taken at months 0, 32, and 60 wer ...[more]

PMID: 15040030

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Project description:CD8(+) cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present at lower frequencies than the amino acid(s) that they replaced. Furthermore, epitopes that underwent escape contained amino acid sites of high variability, whereas epitopes persisting at high frequencies lacked highly variable sites. We therefore infer that escape mutations are likely to be associated with weak functional constraints on the viral protein. This was supported by an extensive analysis of one subject for whom all escape mutations within defined CTL epitopes were studied and by an analysis of all reported escape mutations of defined CTL epitopes in the HIV Immunology Database. In one of these defined epitopes, escape mutations involving the substitution of amino acids with lower database frequencies occurred, and the epitope soon reverted back to the sensitive form. We further show that this escape mutation substantially diminished viral fitness in in vitro competition assays. Coincident with the reversion in vivo, we observed the fixation of a mutation 3 amino acids C terminal to the epitope, coincident with the ablation of the corresponding CTL response. The C-terminal mutation did not restore replication fitness reduced by the escape mutation in the epitope and by itself had little effect on replication fitness. Therefore, this C-terminal mutation presumably impaired the processing and presentation of the epitope. Finally, for one persistent epitope, CTL cross-reactivity to a mutant form may have suppressed the mutant to undetected levels, whereas for two other persistent epitopes, each of two mutants showed poor cross-reactivity and appeared in the subject at later time points. Thus, a viral dynamic exists between the advantage of immune escape, peptide cross-reactivity, and the disadvantage of lost replication fitness, with the balance playing an important role in determining whether a CTL epitope will persist or decline during infection.

Project description:In treating hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections, the rapid reselection of resistance-associated variants (RAVs) is well known in patients with repeated exposure to the same class of antiviral agents. For chronic hepatitis C patients who have experienced virologic failure with direct-acting antiviral drugs, the potential for the reselection of persistent RAVs is unknown. Nine patients who received 14 days of telaprevir monotherapy were retreated with telaprevir-based triple therapy 4.3 to 5.7 years later. In four patients with virologic failure with both telaprevir-containing regimens, population-based and deep sequencing (454 GS-FLX) of the NS3 protease gene were performed before and at treatment failure (median coverage, 4,651 reads). Using deep sequencing, with a threshold of 1.0% for variant calling, no isolates were found harboring RAVs at the baseline time points. While population-based sequencing uncovered similar resistance patterns (V36M plus R155K for subtype 1a and V36A for subtype 1b) in all four patients after the first and second telaprevir treatments, deep sequencing analysis revealed a median of 7 (range, 4 to 23) nucleotide substitutions on the NS3 backbone of the resistant strains, together with large phylogenetic differences between viral quasispecies, making the survival of resistant isolates highly unlikely. In contrast, in a comparison of the two baseline time points, the median number of nucleotide exchanges in the wild-type isolates was only 3 (range, 2 to 8), reflecting the natural evolution of the NS3 gene. In patients with repeated direct antiviral treatment, a continuous evolution of HCV quasispecies was observed, with no clear evidence of persistence and reselection but strong signs of independent de novo generation of resistance. Antiviral therapy for chronic viral infections, like HIV, hepatitis B virus (HBV), or hepatitis C virus (HCV), faces several challenges. These viruses have evolved survival strategies and proliferate by escaping the host's immune system. The development of direct-acting antiviral agents is an important achievement in fighting these infections. Viral variants conferring resistance to direct antiviral drugs lead to treatment failure. For HIV/HBV, it is well known that viral variants associated with treatment failure will be archived and reselected rapidly during retreatment with the same drug/class of drugs. We explored the mechanisms and rules of how resistant variants are selected and potentially reselected during repeated direct antiviral therapies in chronically HCV-infected patients. Interestingly, in contrast to HIV and HBV, we could not prove long-term persistence and reselection of resistant variants in HCV patients who failed protease inhibitor-based therapy. This may have important implications for the potential to reuse direct-acting antivirals in patients who failed the initial direct antiviral treatment. (The phase IIIb study described in this paper is registered at ClinicalTrials.gov under registration number NCT01054573.).

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Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus.

Publications

Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus.

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