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Application of a high-resolution melting technique for the rapid detection of partial replacement of HCV-1b by HCV-1a after PEG-IFNα/RBV therapy.

ABSTRACT: A modified method which can be used for the rapid screening of mutations in the protein kinase R-binding domain (PKR-BD) region and the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) is described. This method is based on a high-resolution melting (HRM) technique used for genotyping single nucleotide polymorphisms and allows the detection of single nucleotide substitutions in the DNA sequence by measuring its Tm. The modified method, in addition to precisely measuring the Tm, allows the recording of the melting curve of the investigated cDNA fragment, which can provide provisional information about the number of different quasi-species present in the sample. The HRM analysis of the amplified cDNAs encoding the PKR-BD and HVR1 allowed the detection of partial replacement of HCV-1b by HCV-1a subspecies in one of our patients, as well as evaluation of the effectiveness of pegylated interferon α/ribavirin (PEG-IFNα/RBV) therapy. The HRM technique has never been used for the rapid screening of sequence variations in these regions and may be used for a similar purpose in any viral genome.

SUBMITTER: Holysz M

PROVIDER: S-EPMC4412693 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Application of a high-resolution melting technique for the rapid detection of partial replacement of HCV-1b by HCV-1a after PEG-IFNα/RBV therapy.

Holysz M M Bialas K K Migdalski P P Kmieciak D D Trzeciak W H WH

Journal of applied genetics 20141108 2

A modified method which can be used for the rapid screening of mutations in the protein kinase R-binding domain (PKR-BD) region and the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) is described. This method is based on a high-resolution melting (HRM) technique used for genotyping single nucleotide polymorphisms and allows the detection of single nucleotide substitutions in the DNA sequence by measuring its Tm. The modified method, in addition to precisely measuring the Tm, allows the ...[more]

PMID: 25380691

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Project description:BackgroundThe hepatitis C virus (HCV) infection has been identified as a leading cause of progressive liver diseases worldwide. Despite new treatment strategies, pegylated interferon alfa-2a (Peg-IFNα-2a), in combination with ribavirin (RBV), still represents the gold standard of therapy for hepatitis C in developing countries.ObjectivesThe aim of this study was to investigate the association of substitutions in the HCV subtype 1b (HCV-1b) core protein and the rs12979860 polymorphism in the interleukin 28B gene (IL28B) with the response to Peg-IFNα-2a/RBV combination therapy in Azerbaijani patients.Patients and methodsA total of fifty-one chronically HCV-1b-infected Azerbaijani patients were enrolled in this cross-sectional study from March 2010 to June 2015. After RNA extraction from pre-treatment plasma, the core region of the HCV genome was amplified using the nested reverse transcription (RT) polymerase chain reaction (PCR) method, followed by standard sequencing. In addition, genomic DNA was extracted from peripheral blood mononuclear cell (PBMC) specimens, and the rs12979860 single nucleotide polymorphism (SNP) was identified using a PCR-restriction fragment length polymorphism (PCR-RFLP) assay.ResultsIn this study, a significant association was observed between the non-responders and relapsers to antiviral therapy and substitutions in the HCV-1b core region at positions 43 (R43K, P = 0.047), 70 (R70Q, P < 0.001), 91 (M91L, P = 0.037), and 106 (S106N, P = 0.018). Concerning the IL28B polymorphism, the results showed that sustained virological response was significantly associated with homozygous CC patients (P = 0.009) as compared with other genotypes, while homozygous TT subjects were associated with HCV relapse after therapy (P = 0.006).ConclusionsThe data of the present study suggest that amino acid substitutions at position 43, 70, 91, and 106 in the HCV-1b core protein are correlated with the response to the Peg-IFNα-2a/RBV treatment in Azerbaijani patients with chronic hepatitis C. Moreover, host genetic polymorphisms, such as those of the IL28B locus, might be useful for predicting the responsiveness to Peg-IFNα-2a/RBV combination therapy against HCV.

Project description:Chronic HCV is a surreptitious disease currently affecting approximately 3% of the world's population that can lead to liver failure and cancer decades following initial infection. However, there are currently no vaccines available for the prevention of chronic HCV. From patients who acutely resolve HCV infection, it is apparent that a strong and broad cytotoxic T lymphocyte (CTL) response is important in HCV clearance. DNA vaccines are naked plasmid DNA molecules that encode pathogen antigens to induce a pathogen-specific immune response. They are inexpensive to produce and have an excellent safety profile in animals and humans. Additionally, DNA vaccines are able to induce strong CTL responses, making them well-suited for an HCV vaccine. We aimed to maximize vaccine recipients' opportunity to induce a broad T cell response with a novel antigenic sequence, multi-antigen vaccine strategy. We have generated DNA plasmids encoding consensus sequences of HCV genotypes 1a and 1b non-structural proteins NS3/4a, NS4b, NS5a, and NS5b. Rhesus macaques were used to study the immunogenicity of these constructs. Four animals were immunized 3 times, 6 weeks apart, at a dose of 1.0mg per antigen construct, as an intramuscular injection followed by in vivo electroporation, which greatly increases DNA uptake by local cells. Immune responses were measured 2 weeks post-immunization regimen (PIR) in immunized rhesus macaques and showed a broad response to multiple HCV nonstructural antigens, with up to 4680 spot-forming units per million peripheral blood mononuclear cells (PBMCs) as measured by Interferon-? ELISpot. In addition, multiparametric flow cytometry detected HCV-specific CD4+ and CD8+ T cell responses by intracellular cytokine staining and detected HCV-specific CD107a+/GrzB+ CD8+ T cells indicating an antigen specific cytolytic response 2 weeks PIR compared with baseline measurements. At the final study time point, 6 weeks PIR, HCV-specific CD45RA- memory-like T cells remained detectable in peripheral blood. Data presented in this manuscript support the notion that vaccine immunogenicity studies using a macaque model can be used to depict key anti-HCV nonstructural antigenic cellular immune responses and support the development of DNA-based prophylactic HCV vaccines.

Dataset Information

Application of a high-resolution melting technique for the rapid detection of partial replacement of HCV-1b by HCV-1a after PEG-IFNα/RBV therapy.

Publications

Application of a high-resolution melting technique for the rapid detection of partial replacement of HCV-1b by HCV-1a after PEG-IFNα/RBV therapy.

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