year 4, Issue 4 (Winter 2011)
Iran J Med Microbiol 2011, 4(4): 30-40 |
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Arashkia A, Rouhvand F, Memarnejadian A, Alizadeh S, Motevalli F, Ebrahimi M. Immunoinformatics modeling, construction of DNA plasmids Carrying CTL epitopes of hepatitis C virus and their preliminary immunological analysis. Iran J Med Microbiol 2011; 4 (4) :30-40
URL: http://ijmm.ir/article-1-25-en.html
URL: http://ijmm.ir/article-1-25-en.html
Arash Arashkia 
, Farzin Rouhvand , Arash Memarnejadian , Shahadeh Alizadeh , Fatemeh Motevalli , Mekania Ebrahimi
, Farzin Rouhvand , Arash Memarnejadian , Shahadeh Alizadeh , Fatemeh Motevalli , Mekania Ebrahimi
Abstract: (18992 Views)
Background and objectives: High mutation rate and immunosuppressive effect of some key proteins of hepatitis C virus (HCV) encouraged researchers to find conserved and small immunogenic units to induce immune responses. Thus epitope-based vaccines were introduced to find out any potential immunogens. However, the relatively weak immune responses against these vaccines require a creation of additional measures. This study aimed to construct plasmids carrying CTL epitope of hepatitis C virus using immunoinformatics modeling method and assessment of their preliminary immunogenicity.
Materials and Methods: One H-2Dd and two HLA-A*0201-restricted CD8+ T-cell epitopes from E2, E1 and core regions of HCV were selected and immunoinformatically analyzed for optimum sequentiality. The multiepitope sequence was constructed by SOEing PCR and cloned in pcDNA3.1+ vector. PADRE, endoplasmic reticulum signal sequence (ERss) and hepatitis B surface antigen (HBsAg) immuno-enhancer sequences were fused to the 5′ end of multiepitope constructs. In vitro, expression of each plasmid was analyzed by RT-PCR, dot-blot, Western-blot and immunofluorescence techniques. Preliminary in vivo immunogenicity of the construct was assessed by delayed-type hypersensitivity (DTH) response in BALB/c mice.
Results: In vitro, analyses confirmed the expression of plasmids. Preliminary, in vivo assessments indicated the processing and presentation of the H-2Dd epitope in BALB/c mice. Moreover, although the immunostimulation effects of ERss and PADRE were shown, fusion of HBsAg did not enhance the DTH response.
Conclusion: This study shows the value of immunoinformatics prediction of hepatitis C virus epitopes as a multiepitope vaccine candidate and DTH assay for preliminary analysis using BALB/c mice. Data obtained, provide enough support for further evaluation of the designed constructs in HLA-A2 transgenic mice.
Materials and Methods: One H-2Dd and two HLA-A*0201-restricted CD8+ T-cell epitopes from E2, E1 and core regions of HCV were selected and immunoinformatically analyzed for optimum sequentiality. The multiepitope sequence was constructed by SOEing PCR and cloned in pcDNA3.1+ vector. PADRE, endoplasmic reticulum signal sequence (ERss) and hepatitis B surface antigen (HBsAg) immuno-enhancer sequences were fused to the 5′ end of multiepitope constructs. In vitro, expression of each plasmid was analyzed by RT-PCR, dot-blot, Western-blot and immunofluorescence techniques. Preliminary in vivo immunogenicity of the construct was assessed by delayed-type hypersensitivity (DTH) response in BALB/c mice.
Results: In vitro, analyses confirmed the expression of plasmids. Preliminary, in vivo assessments indicated the processing and presentation of the H-2Dd epitope in BALB/c mice. Moreover, although the immunostimulation effects of ERss and PADRE were shown, fusion of HBsAg did not enhance the DTH response.
Conclusion: This study shows the value of immunoinformatics prediction of hepatitis C virus epitopes as a multiepitope vaccine candidate and DTH assay for preliminary analysis using BALB/c mice. Data obtained, provide enough support for further evaluation of the designed constructs in HLA-A2 transgenic mice.
Keywords: Hepatitis C Virus, Immunoinformatics, Multiepitope vaccine, In vivo expression, Immunostimulant.
Type of Study: Original Research Article |
Subject:
Medical Virology
Received: 2013/10/25 | Accepted: 2013/11/10 | ePublished: 2013/11/10
Received: 2013/10/25 | Accepted: 2013/11/10 | ePublished: 2013/11/10
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