Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c Mouse
Objective: Polytope DNA vaccines, capable of focusing the cytotoxic T lymphocyte (CTL)response on critical epitopes, represent a promising approach in HCV immunotherapy. Nevertheless,due to controversial rules governing epitope processing and the low level expression/immunogenicity of recombinant po...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Royan Institute (ACECR), Tehran
2009-01-01
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| Series: | Cell Journal |
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| Online Access: | http://celljournal.org/library/upload/article/Article4.pdf |
| _version_ | 1818207357816012800 |
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| author | Arash Memarnejadian Farzin Roohvand Arash Arashkia Farideh Berjisian Mohammad Reza Aghasadeghi |
| author_facet | Arash Memarnejadian Farzin Roohvand Arash Arashkia Farideh Berjisian Mohammad Reza Aghasadeghi |
| author_sort | Arash Memarnejadian |
| collection | DOAJ |
| description | Objective: Polytope DNA vaccines, capable of focusing the cytotoxic T lymphocyte (CTL)response on critical epitopes, represent a promising approach in HCV immunotherapy. Nevertheless,due to controversial rules governing epitope processing and the low level expression/immunogenicity of recombinant polytope peptides, designing and primary expression/immunogenicity analysis of these vaccine types should be the first consideration prior tocostly transgenic animal studies.Materials and Methods: Four HLA-A2 and H-2d restricted CTL epitopes were selected anddesigned in three appropriate sequential tandems based on epitope and proteasomal cleavagepredictions. The related nucleotide sequences were synthesized using SOEing PCRmethod and cloned into a pcDNA3.1 vector, either alone or fused to the small hepatitis B surfaceantigen (HBsAg-S) gene. Following the preparation of polyclonal anti-sera, expression/secretion of polytopes was evaluated in Cos-7 cells by using immunofluorescence, Westernblot,dot blot, ELISA and RT-PCR techniques. The immunogenicity of the plasmids was alsoassessed through the delayed-type hypersensitivity (DTH) assay in BALB/c mice.Results: Due to in silico designs and optimizations, the polytope products of constructedplasmids were efficiently detected in vitro through common techniques and HBsAg-S-basedparticles were shown to be secreted into the culture media (up to 30%). Moreover, all plasmidswere able to efficiently induce a positive DTH response while HBsAg-S fusion constructsindicated a significant immunopotential effect towards the incorporated mouse epitopes.Conclusion: Designed polytope constructs of this study are efficiently expressed and processed.They have the required initial potency for further immunogenicity analysis in transgenicmice. |
| first_indexed | 2024-12-12T04:27:39Z |
| format | Article |
| id | doaj.art-07478ef02800487aba43d4468e29e46f |
| institution | Directory Open Access Journal |
| issn | 2228-5806 2228-5814 |
| language | English |
| last_indexed | 2024-12-12T04:27:39Z |
| publishDate | 2009-01-01 |
| publisher | Royan Institute (ACECR), Tehran |
| record_format | Article |
| series | Cell Journal |
| spelling | doaj.art-07478ef02800487aba43d4468e29e46f2022-12-22T00:38:09ZengRoyan Institute (ACECR), TehranCell Journal2228-58062228-58142009-01-01112122133Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c MouseArash MemarnejadianFarzin RoohvandArash ArashkiaFarideh BerjisianMohammad Reza AghasadeghiObjective: Polytope DNA vaccines, capable of focusing the cytotoxic T lymphocyte (CTL)response on critical epitopes, represent a promising approach in HCV immunotherapy. Nevertheless,due to controversial rules governing epitope processing and the low level expression/immunogenicity of recombinant polytope peptides, designing and primary expression/immunogenicity analysis of these vaccine types should be the first consideration prior tocostly transgenic animal studies.Materials and Methods: Four HLA-A2 and H-2d restricted CTL epitopes were selected anddesigned in three appropriate sequential tandems based on epitope and proteasomal cleavagepredictions. The related nucleotide sequences were synthesized using SOEing PCRmethod and cloned into a pcDNA3.1 vector, either alone or fused to the small hepatitis B surfaceantigen (HBsAg-S) gene. Following the preparation of polyclonal anti-sera, expression/secretion of polytopes was evaluated in Cos-7 cells by using immunofluorescence, Westernblot,dot blot, ELISA and RT-PCR techniques. The immunogenicity of the plasmids was alsoassessed through the delayed-type hypersensitivity (DTH) assay in BALB/c mice.Results: Due to in silico designs and optimizations, the polytope products of constructedplasmids were efficiently detected in vitro through common techniques and HBsAg-S-basedparticles were shown to be secreted into the culture media (up to 30%). Moreover, all plasmidswere able to efficiently induce a positive DTH response while HBsAg-S fusion constructsindicated a significant immunopotential effect towards the incorporated mouse epitopes.Conclusion: Designed polytope constructs of this study are efficiently expressed and processed.They have the required initial potency for further immunogenicity analysis in transgenicmice.http://celljournal.org/library/upload/article/Article4.pdfEpitopeHepatitis C VirusDNA VaccineCytotoxic T-Lymphocytes |
| spellingShingle | Arash Memarnejadian Farzin Roohvand Arash Arashkia Farideh Berjisian Mohammad Reza Aghasadeghi Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c Mouse Cell Journal Epitope Hepatitis C Virus DNA Vaccine Cytotoxic T-Lymphocytes |
| title | Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c Mouse |
| title_full | Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c Mouse |
| title_fullStr | Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c Mouse |
| title_full_unstemmed | Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c Mouse |
| title_short | Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c Mouse |
| title_sort | designing constructing and immunogenic evaluation of polytope dna constructs by the application of hepatitis c virus immunodominant epitopes in balb c mouse |
| topic | Epitope Hepatitis C Virus DNA Vaccine Cytotoxic T-Lymphocytes |
| url | http://celljournal.org/library/upload/article/Article4.pdf |
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