| Pulmonary tuberculosis (TB) is one of the leading causes of deaths by a singleinfectious agent. It's been a serious problem to fight against tuberculosis because ofthe power of resistence, Drug Resistance, and mechanism of dormancy as well. Theonly licensed vaccine against M. tuberculosis, bacillle Calmette-Guerin(BCG), whichis an attenuated strain of Mycobacterium bovis, is typically administeredintradermally as a single dose to newborn infants in developing countries. Recentstudies suggests that BCG vaccination is protective against childhood meningealtuberculosis and systemic forms of the disease. However, protective efficacy isvariable(ranging from0-80%)against adult pulmonary disease. In order to replaceBCG, novel constructed vaccines includs live attenuated vaccine,viral vectors, subunitvaccine. They take advantage of the widespread use of BCG by boosting on top ofBCG or BCG as a prime. It's a great challenge for either DNA or subunit vaccine toinduce strong cell-mediated response to infection involving both CD4+and CD8+Tcells and the ability ro respond with Th1-type cytokines, particularly IFN-γ.A fusionprotein, comprising two immunodominant secreted proteins from M.tuberculosis(Ag85B-ESAT6), has been proven highy efficacious in animal models.The Ag85B is a major secreted protein, belonging to the mycolyl transferase familyfrom M. tuberculosis comprising Ag85A,85B,85C.85A,85B have been shownhighly immunogenetic. Futhermore, Ag85B induces protective immune responsesagainst tuberculosis in mice as well as guinea pigs. To overcome the cross-reaction ofBCG vaccine and M.tuberculosis, great effort has been made in identifying diagnosticantigens that are missing in BCG such as those in the region of differences(RD), butare present in MTB. RD1antigen,6-Kd early secretory antigenic target ESAT6has been discovered and have been widely used in T-cell based IFN-γreleasing diagnosticassays.The fusion of these two genes, broaden the range of immunogenicity,whichare main protective antigens against tuberculosis.The aim of this study is to complete genetic stability research of rAD, rMVA,rBCG to meet the clinical requirments and saftyregards. Also because of the lowexpression and immunogenicity of rMVA. Heterologous eukaryotic tissueplasminogen activator(tPA) was used as signal sequence in order to enhanceexpression and immunogenicity and even protection.In this study, the genetic ability of two viral vectors vaccinerMVA-Ag85B-ESAT6and rAD-Ag85B-ESAT6, as well as rBCG-Ag85B were beingevaluated. They were passaged for20times in BHK-TK-cell line,293cell line, andsuton culture, protein expression and gene are being assayed. Data proved that thegenetic stability of three vaccines above is good enough to meet Clinical requirements.Only in MVA the expression of protein cannot reach our requirements. Accordingly,we design six sequence including Ag85B-ESAT6(eucaryon),tPA-Ag85B-ESAT6(procaryon), tPA-Ag85B-ESAT6(eucaryon),Ag85B-ESAT6-Ha(eucaryon), tPA-Ag85B-ESAT6-Ha(procaryon),tPA-Ag85B-ESAT6-Ha (eucaryon) and put them into pcDNA3.1(-), to transfect293Tcell line as well as BHK-TK-cell line. And then examine protein expression usingwesternblot. Data shows that tPA leading protein is highly expressed both in cell andculture surpernant, also resulted in N-glycosylation increase in expression.Recombinant MVA was constructed by homologous recombination with vacciniavextor, the result proved that signal peptide tPA is able to enhance expression ofprotein also in MVA system. So, rMVA-tPA-Ag85B-ESAT6was constructed toexpress immunodominant secreted protein Ag85B-ESAT6. rMVA was screened andselected, plaque was amplificated, and purified. Virus stock was tittered byimmunostaining.MVA-tPA-Ag85B-ESAT6as well as MVA-Ag85B-ESAT6was inoculatedsubcutaneously in BALB/c. After1week and2week, CTL, ELISPOT, FCM, ELISAwas used to evaluate cellular immune response and humoral immunologic response as well. The result shows that, tPA signal peptide modified MVA-85B-E6, enhanced theIFN-γ significant when stimulated by MHCI-restrited epitode, and also enhanced theIgG level a little which indicates a Th1biased response. However the specificresponse to the stimulation of MVA was pretty limited, and the cytokine secreted afterstimulation of antigen protein does not show great enhancement. These results suggestthat the tuberculosis MVA-tPA-Ag85B-ESAT6was able to elicit substantial immuneresponses in suitably vaccinated mice, but further studies to the constructs or the useof alternative immunization strategies will be needed to improve the efficacy of thisvaccine candidate. |
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