| [Objective]Tuberculosis (TB), one of the highest mortality of infectious diseases, remains a serious threat to public health. Mycobacterium bovis Bacille Calmette-Guerin (BCG), currently the only vaccine available against TB, has effective protection against severe forms of TB in children. Therefore, more effective vaccines against adult TB are urgently needed. BCG priming-heterologous vaccine booster and recombinant BCG technologies have been considered as two promising regimens against TB. Obviously, defining the protective efficacy of the two regimens would benefit more rational design of the future adult TB vaccines.In this study, a recombinant BCG strain (rBCG::685A) expressing the fusion protein of ESAT-6and Ag85A (r685A) was constructed successfully and confirmed its secretion by Western blotting. The immune responses and protective effects in rBCG::685A vaccinated C57BL/6mice were compared with the BCG prime-pcD685A booster regimen.[Methods]1. The fusion gene ESAT-6-Ag85A of M. tuberculosis was isolated from pcD685A by digesting, and then subcloned into the pre-digested E.coli-mycobacterium shuttle vector pMS261vector to yield the recombinant plasmid pM685A. The anticipated sequence of the insert was confirmed by DNA sequencing.2. pM685A was introduced into BCG China strain by electroporation and the recombinant BCG strain (rBCG::685A) was screened on kanamycin resistance plates. The secret expression of r685A protein was detected by Western-blotting.3. C57BL/6mice were vaccinated with rBCG::685A and BCG prime-pcD685A booster regimens, separately. Negative control mice were immunized with PBS, and BCG-vaccinated as positive control. Eight weeks later, splenocytes were stimulated with PPD and r685A, and the concentration of antigen-specific IFN-y was detected by ELISA. The expressions of IFN-y, TNF-a, IL-10and iNOS in the lung were detected by qRT-PCR.4. Eight weeks after immunization, mice were challenged with a dose of1×106CFU M.tb H37Rv strain via tail vein. Four weeks post-infection, mice were sacrificed. Bacterial loads of the lungs and spleens were analyzed. Moreover, the right lobes of the lungs were harvested and sectioned for light microscopy. To analyze the pathological changes, sections were stained with haematoxylin and eosin and Ziehl-Neelsen method separately.[Results]1. The E.coli-mycobacterium shuttle plasmid pM685A which contains ESAT-6-Ag85A was successfully constructed, and confirmed by enzyme-digestion and DNA sequencing.2. rBCG::685A was constructed and the secretory expression of r685A fusion protein, with a molecular size about38kDa, was confirmed only in the culture supernatants of rBCG::685A.3. Eight weeks after immunization, specific IFN-y secreted by splenocytes was detected by ELISA. Upon stimulation with PPD or r685A, all BCG vaccinated groups produced higher levels of IFN-y than PBS control mice (P<0.05). Most importantly, higher levels of r685A specific IFN-y were secreted by splenocytes of mice vaccinated with BCG prime-pcD685A booster than rBCG::685A (P<0.05).4. The mRNA expression levels of molecules in the lung of vaccinated mice were detected by qRT-PCR. When compared with control mice, the expression of these molecules in the different vaccinated groups increased at different degrees. Especially, all BCG based vaccination strategies increased the expression of IFN-y and IL-10compared with BCG vaccination. Notably, the expression of both TNF-a and iNOS in the lung of BCG prime-pcD685A booster vaccination mice increased most significantly than BCG and rBCG::685A groups (P<0.05).5. Four weeks after challenge, bacterial load of organs were analyzed. BCG prime-pcD685A booster vaccinated mice resulted in the most significant reduction of bacterial load in the lung and spleen (P<0.05). In addition, the difference of bacterial load in both organs between rBCG::685A and BCG vaccinated groups had no statistics significance (P>0.05). Lest lung inflammation were observed in alveolar tissue from mice vaccination with BCG plus pcD685A boosting under microscopy. Fewer perivasculitis and alveolitis were observed in BCG group and rBCG::685A group than PBS group.[Conclusion]1. Boosting BCG with pcD685A DNA provided higher immune response and better protection effect against virulent M. tuberculosis, compared with mice vaccinated with rBCG::685A.2. The rational design of adult TB vaccines will need to confirm the protective effect of combination BCG or effective enhanced rBCG strains as prime vaccine and heterogolous boost strategy. Finally, effective protection could be established against TB. [Objective]The current widely used BCG vaccine provides effective protection only for meningitis and military tuberculosis in infants and children, but variable protection against adult TB. Our previous study compared the effect of prime-boost regimen with rBCG regimen, and indicatied that BCG prime-heterologous vaccine booster is an effective regimen against TB. Therefore, new vaccines which are supposed to replace or enhance the effect induced by BCG are urgently needed to improve the protection against adult TB.Subunit vaccines have been established through large amount of animal experiments and clinical studies to confirm its reliability. Pathogenesis researchs on TB showed that specific antigens from different stage were expressed through M.tb growth period, while BCG immunization established weak, even no response to some of these antigens. We chose and fused antigens from different stages to construct a novel subunit vaccine. We hoped this vaccine could effectively target M.tb among different periods to control the primary TB infection and reactivion.Thus, this study designed a fusion protein A1D3R1which contains multi-stage antigens. When delivered with the novel adjuvant MTO, the immunogenicity and protective effect of subunit vaccine were confirmed through animal model.[Methods]1. Gene design and synthesis. Candidate genes included:Rv3407, PhoY2, Ag85A, Rv2626c and RpfB. Analyzed and fused the fusion sequence named A1D3R1by software. The sequence was synthesized and sequenced by company. Specific PCR primers were designed and synthesized.2. Construction of prokaryotic expression strain, and protein expression, purification and quantification. Prokaryotic strain E.coli BL21(DE3)-pET30b-A1D3R1was constructed The protein AlD3R1was purified under denaturing conditions by Ni SepharoseTM6Fast Flow columns, and confirmed by SDS-PAGE gel and Western-blotting. The concentration was detected by BCA method. Residual LPS contamination was eliminated by the Endotoxin Removal Solution.3. WBIA to detect whether candidate angtigens could be recognized by Chinese M.tb infections and non-infections. Gathered blood samples. Samples were classified by clinical diagnosis, and the rCM-WBIA standard which had been established by our previous work. The concentration of IFN-y were detected and analyzed by enzyme-linked immunosorbent method.4. Immunogenicity analysis of subunit vaccine A1D3R1/MTO. C57BL/6mice were immunized with A1D3R1/MTO which combined A1D3R1with new adjuvant MTO (mainly including TDB and MPL). PBS negative control, MTO group and BCG positive control were setted. Nine weeks after immunization, immunological indexes were detected: serum were collected to detect the specific antibody and subclasses; splenocytes were stimulated, and the secretion of cytokines, including:IFN-γ, TNF-a and IL-2were detected; lymphocytes which secret for single or multi cytokines were analyzed by flow cytometry.5. Short-term protective effection of subunit vaccine A1D3R1/MTO. Nine weeks after immunization, mice were challenged with M.tb H37Rv strain. During the infection period, observed and recorded the numbers of mice that still alived to analyze the survival rate. Four weeks post-infection, mice were sacrificed. The bacterial loads of organs were detected. Moreover, the upper left lobes of the lungs were harvested and sectioned for light microscopy. To analyze the pathological changes and detect the acid-fast bacilli, sections were stained with haematoxylin and eosin and Ziehl-Neelsen method separately. [Results]1. Fusion protein Al D3R1was successfully constructed, expressed and pufified:1) The prokaryotic expressing vector pET30b-AlD3R1which contains the AlD3R1was constructed successfully. The size of gene was confirmed by restriction endonucleases digestion and DNA gel electrophoresis.2) A1D3R1protein was successfully expressed and confirmed by SDS-PAGE. The purity and biological activity were confirmed by Western-blotting with the anti-his tag antibody and anti-A1D3R1mouse sera, respectively.2. A1D3R1and each individual protein were recognized by T cells of Chinese M. tb infections, and high level of IFN-γ was secreted. The crowd was classified by clinical diagnosis and rCM-WBIA:1) A1D3R1and individual proteins could be recognized by Chinese M.tb infections.2) Compared with Rv3407, PhoY2, Ag85A and Rv2626c (except for RpfB), A1D3R1could induce significantly higher levels of IFN-y after stimulation, whatever in all subjects or Chinese M.tb infections (P<0.05).3. A1D3R1/MTO could induce effective Thl-type immune response:1) A1D3R1/MTO could induce high level of IgG and subclasses in immunized mice, and skewed toward Thl type immunity.2) Higher level AlD3R1specific IFN-y, IL-2and TNF-a were secreted in A1D3R1/MTO immunized mice.3) PPD or A1D3R1specific total IFN-γ secretion CD4+and CD8+T lymphocytes significantly increased in A1D3R1/MTO immunized mice.4) PPD or A1D3R1specific IFN-γ+IL-2+secretion CD4+T lymphocytes significantly increased in A1D3R1/MTO immunized mice.5) PPD or A1D3R1specific IFN-γ+, IFN-γ+IL-2+and IFN-γ+TNF-a+secretion CD8+T lymphocytes significantly increased in A1D3R1/MTO immunized mice.4. Better protective effection against TB was established in A1D3R1/MTO group. Four weeks post-infection, the bacterial load of A1D3R1/MTO mice less than the PBS group and MTO group in lung and spleen (P<0.05), but still higher than the BCG group (P<0.001). Less severe lung pathology were found in A1D3R1/MTO group, when compared with MTO and PBS groups. Short term survival rate of A1D3R1/MTO immunized mice reached83.33%.5. MTO adjuvant could establish some protective effection against TB. Spelnocytes in MTO immunized mice could produce higher IFN-y and TNF-a than PBS group (P<0.05). IFN-γ+secretion T cells and IFN-y+IL-2+secretion CD4+T cells increased when compared with PBS group (P<0.05). Protective effection against TB was better than PBS group. Short term survival rate reached83.33%.[Conclusion]Subunit vaccine A1D3R1/MTO could establish effective protection against acute TB infection in mouse-model, and the protection effect may be related to the specific CD4+Thl-type immune response. Further analyzed the immune index, vaccine could induce specific IFN-y+IL-2+secretion CD4+T cells, and IFN-y+, IFN-y+IL-2+, IFN-γ+TNF-a+secretion CD8+T cells. However, the novel adjuvant-MTO could induce the Thl-type cell immune response which secreted IFN-y and TNF-a, and would benefit the further research of vaccine. |
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