RESUMEN
Introduction: Hepatitis C virus [HCV] is a ?blood-borne pathogen, resulting in liver cirrhosis and liver cancer. Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was ?to design and prepare a specific mRNA, without 5' cap and poly [A] tail transcribed in vitro capable of coding core protein and also to determine its functionality
Methods: Candidate mRNA was prepared by in vitro transcription of the designed construct consisting of ??5' and 3' untranslated regions of heat shock proteins 70 [hsp70] mRNA, T7 promoter, internal ribosome entry site [IRES] sequences of eIF4G related to human dendritic cells [DCs], and the ?Core gene of HCV. To design the modified mRNA, the ??5' cap and poly [A] tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA [IVT-mRNA] and the expressions of green fluorescent protein [GFP], and Coregenes were determined by microscopic examination and Western blotting assay
Results: Cell transfection results showed that despite the absence of ??5' cap and poly [A] tail, the structure of the mRNA ?was stable. Moreover, the successful expressions of GFP and Coregenes were achieved
Conclusion: Our findings indicated the effectiveness of a designed IVT-mRNA harboring the Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV
RESUMEN
The obligate intracellular bacterium Chlamydia trachomatis causes sexually transmissible diseases in human. Timely and sensitive detection of this pathogen is very important. There are many cross-reactions in bacteriological and serological methods in detection of this type of pathogens. The aim of this study was to achieve a more specific antigen for serological tests. Blood samples were taken from 192 women with suspected chlamydial infection and sera were isolated. ELISA plate wells were coated with recombinant C. trachomatis OMP2 as antigen. Cut-off system was determined with 40 negative sera. The final results of this research were compared with Euroimmun commercial kit. The ELISA system cut-off was calculated at 0.27 using negative sera samples. ODs of positive samples were higher than 0.27 and negative samples were lower than it. We obtained 30 samples [15.62%] as positive and 162 cases [84.37%] as negative. Sensitivity and specificity of the recombinant antigen were 90% and 86%, respectively. This antigen showed no cross-reactivity with sera of patients infected with Hydatid cyst, HCV, Epstein barr virus, HBV, Helicobacter pylori, Toxoplasma gondii, Cytomegalovirus, Mycoplasma, Measles and Varicella zoster virus. The sensitivity and specificity of rOMP2 in ELISA for detection of C. trachomatis were 90% and 86%, respectively. Though the sensitivity was higher than results of Euroimmun commercial kit, its specificity was calculated lower than reference kit
RESUMEN
There are more than 350 million individuals with hepatitis C in the world. One of the important problems in vaccine project is development of effective and suitable adjuvant in human vaccines. At present research we applied human BHsp90 protein as an adjuvant in recombinant HCV vaccine design. The thermal vector of pGP1-2 was used for human heat shock protein 90 expression. This protein injected to BalbC mice as an adjuvant together with recombinant protein of HCV core. The combination of these proteins was used and we evaluated the humoral and cellular immunity and the cytokine secretion of inguinal and popliteal lymph nodes lymphocytes were analyzed in vitro and ex vivo conditions. So the combination of Core protein together with hsp90 induced total IgG and IgG2a secretion. The spleen lymphocytes proliferation were increased equal to serum IgG2a level that was constant in second time bleeding with significant different to complexes with freund's adjuvant. At first IL-4 and IL-5 cytokines were increased, after one week it decreased. Production of IL-4 showed there was no hypersensitivity reaction after vaccine injection