【摘要】：肺結(jié)核是世界范圍內(nèi)導(dǎo)致人類死亡的主要感染性疾病之一。牛分枝桿菌(Bacilli Calmette Guerin,BCG)是目前唯一的抗結(jié)核病疫苗。BCG是來源于上個(gè)世紀(jì)牛分枝桿菌的減毒株,歷時(shí)13年體外傳代所得。幾十年來,BCG疫苗在世界范圍廣泛應(yīng)用,然而在不同的地域,其有效性有巨大的差異,尤其對(duì)成人肺結(jié)核幾乎沒有保護(hù)效應(yīng)。因此,迫切需要研制安全、有效的抗結(jié)核疫苗。在眾多新疫苗中,亞單位疫苗具有成分明確和易于被接受的優(yōu)點(diǎn),并且具有強(qiáng)化BCG保護(hù)效率的作用,是最有前景的疫苗之一。目前的疫苗免疫策略主要使用BCG或者其他疫苗初免,然后選擇亞單位疫苗加強(qiáng)免疫。 我們此前的研究表明,融合蛋白Ag85B-Mpt64190-198-Mtb8.4 (AMM)和佐劑二甲基三十六烷基銨(DDA)、卡介苗多糖核酸(BCG PSN)構(gòu)建的亞單位疫苗能夠誘導(dǎo)較強(qiáng)體液免疫和細(xì)胞免疫。本課題探討了佐劑DDA和卡介苗多糖核酸(BCG PSN)、海藻糖二霉菌酸脂(TDM)、聚肌胞(PolyI:C)不同的免疫活化劑混合,聯(lián)合作為融合蛋白AMM的佐劑,比較不同佐劑輔助誘導(dǎo)的免疫效應(yīng)差異和加強(qiáng)BCG免疫保護(hù)效率的差異。 1.二甲基三十六烷基銨及卡介苗多糖核酸佐劑在結(jié)核亞單位疫苗強(qiáng)化BCG免疫中的效應(yīng)研究 目的：研究DDA和DDA-BCG PSN佐劑在結(jié)核融合蛋白Ag85B-Mpt64190-198-Mtb8.4疫苗強(qiáng)化卡介苗(BCG)免疫中的不同免疫輔助效應(yīng)。 方法：選擇DDA、BCG PSN作為融合蛋白AMM的佐劑,在BCG初免基礎(chǔ)上應(yīng)用AMM疫苗加強(qiáng)免疫小鼠兩次。其中一組聯(lián)合使用兩種佐劑(DDA/BCG PSN),另一組以DDA單獨(dú)作為佐劑,同時(shí)設(shè)立BCG和磷酸緩沖液(phosphate buffered saline,PBS)免疫組為對(duì)照。末次免疫4周后,應(yīng)用ELISA及ELISPOT檢測(cè)免疫小鼠的體液與細(xì)胞免疫反應(yīng)。12周后,經(jīng)尾靜脈H37Rv毒株攻擊被免疫小鼠。感染6周后,檢測(cè)小鼠體內(nèi)結(jié)核菌載量,并分析肺組織病理切片,評(píng)價(jià)不同佐劑疫苗的保護(hù)效果。 結(jié)果：在BCG初免基礎(chǔ)上,AMM亞單位疫苗聯(lián)合佐劑組(AMM/DDA/BCGPSN)和單獨(dú)佐劑組(AMM/DDA)加強(qiáng)免疫兩次后,脾臟淋巴細(xì)胞經(jīng)抗原Ag85B和純蛋白衍生物(purified protein derivative,PPD)刺激后,皆可分泌較BCG組強(qiáng)的IFN-γ。毒力株攻擊后,菌落形成單位(colony-forming units, CFU)計(jì)數(shù)顯示,AMM疫苗單一DDA佐劑組(AMM/DDA)強(qiáng)化BCG免疫后,其肺部荷菌量少于BCG和PBS組(P0.05),而AMM疫苗聯(lián)合佐劑組(AMM/DDA/BCG PSN)脾臟荷菌量少于PBS組和BCG組(P0.05)。組織病理分析結(jié)果表明AMM/DDA/BCG PSN組肺組織病理?yè)p傷較輕,而AMM/DDA組病理?yè)p傷個(gè)體差異較大。 結(jié)論：DDA是較為理想的結(jié)核亞單位疫苗佐劑,能誘導(dǎo)較強(qiáng)的細(xì)胞免疫和較強(qiáng)的免疫保護(hù)作用；BCG PSN可能具有免疫調(diào)節(jié)作用,可以減輕免疫病理?yè)p傷。 2.二甲基三十六烷基銨為基礎(chǔ)的不同免疫佐劑在結(jié)核亞單位疫苗強(qiáng)化BCG免疫中的佐劑效應(yīng)研究 目的：研究免疫佐劑TDM、Poly I:C和BCG PSN聯(lián)合DDA作為融合蛋白AMM的佐劑,在BCG初免基礎(chǔ)上AMM疫苗加強(qiáng)免疫兩次,比較不同的聯(lián)合佐劑組的佐劑效應(yīng)。 方法：本研究共設(shè)立了七個(gè)實(shí)驗(yàn)組,各個(gè)實(shí)驗(yàn)組在AMM融合蛋白基礎(chǔ)上加入不同的佐劑,分別是：DDA(A/D); DDA聯(lián)合TDM(A/D/T); DDA聯(lián)合TDM、Poly I:C(A/D/T/P); DDA聯(lián)合Poly I:C(A/D/P), DDA聯(lián)合BCG PSN(A/D/B),且設(shè)立BCG和磷酸緩沖液(PBS)免疫組為對(duì)照。各實(shí)驗(yàn)組BCG初次免疫后,用不同佐劑的亞單位疫苗AMM分別在第12周和第14周進(jìn)行加強(qiáng)免疫。末次加強(qiáng)免疫4周后,采用ELISA及ELISPOT檢測(cè)免疫小鼠的體液與細(xì)胞免疫應(yīng)答水平；10周后,以H37Rv毒株攻擊被免疫小鼠,并每周觀察記錄每組小鼠的平均體重。疫苗免疫小鼠感染6周后,檢測(cè)小鼠體內(nèi)結(jié)核菌載量,并分析肺組織病理切片。評(píng)價(jià)二甲基三十六烷基銨為基礎(chǔ)的不同免疫佐劑在結(jié)核亞單位疫苗強(qiáng)化BCG免疫中的佐劑效應(yīng)。 結(jié)果：A/D/T和A/D/T/P實(shí)驗(yàn)組加強(qiáng)BCG免疫兩次后,能夠誘發(fā)小鼠產(chǎn)生更高抗原特異性抗體,且ELISPOT分析顯示該兩組小鼠分泌抗原特異性IFN-γ明顯強(qiáng)于對(duì)照BCG；肺部荷菌量試驗(yàn)表明各個(gè)佐劑實(shí)驗(yàn)組加強(qiáng)BCG免疫后,肺部荷菌量都較PBS組低；各組免疫小鼠經(jīng)尾靜脈H37Rv毒株攻擊后,肺組織病理分析顯示聯(lián)合有BCG PSN或Poly I:C的聯(lián)合佐劑的亞單位疫苗強(qiáng)化BCG免疫后,肺組織病理?yè)p傷均較BCG組和DDA為佐劑的亞單位疫苗組輕(P0.05)；在毒株攻擊前后對(duì)不同實(shí)驗(yàn)組小鼠的體重進(jìn)行動(dòng)態(tài)觀察,結(jié)果表明：PBS實(shí)驗(yàn)組體重減少明顯,而聯(lián)合三種佐劑(DDA、TDM和Poly I:C)和聯(lián)合BCG PSN和DDA兩種佐劑組體重增加明顯。 結(jié)論：佐劑Poly I:C和BCG PSN可能在亞單位疫苗加強(qiáng)BCG免疫后,通過某種免疫機(jī)制,最終可以減輕結(jié)核分枝桿菌感染所造成的病理?yè)p傷。
[Abstract]:Tuberculosis is one of the leading infectious diseases leading to human death worldwide. Mycobacterium bovis (BCG) is currently the only anti-tuberculosis vaccine. BCG is an attenuated strain of Mycobacterium bovis from the last century. It has been passed for 13 years in vitro. For decades, BCG vaccine has been widely used worldwide, however. The effectiveness of subunit vaccines varies greatly from region to region, especially in adults with little protection against tuberculosis. Therefore, there is an urgent need to develop safe and effective anti-tuberculosis vaccines. Current vaccination strategies use BCG or other vaccines for primary immunization and then select subunit vaccines to enhance immunity.
Our previous studies have shown that subunit vaccines constructed with fusion protein Ag85B-Mpt64190-198-Mtb8.4 (AMM) and adjuvant dimethylhexadecyl ammonium (DDA) and BCG polysaccharide nucleic acid (BCG PSN) can induce stronger humoral and cellular immunity. TDM, polymyocyte (PolyI:C) were mixed with different immunoactivators and used as adjuvants of fusion protein AMM to compare the difference of immune effects induced by different adjuvants and the efficiency of enhancing BCG immune protection.
1. Effect of dimethyltrialkyl ammonium and BCG polysaccharide nucleic acid adjuvant on BCG immunity enhanced by tuberculosis subunit vaccine
OBJECTIVE: To study the different immuno-adjuvant effects of DDA and DDA-BCG PSN adjuvants on the immunization of tuberculosis fusion protein Ag85B-Mpt64190-198-Mtb8.4 vaccine with BCG.
Methods: DDA and BCG PSN were used as adjuvants of AMM, and AMM vaccine was used twice on the basis of BCG immunization. One group used two adjuvants (DDA/BCG PSN) together, the other group used DDA as adjuvant alone, and BCG and phosphate buffered saline (PBS) as control group. After 12 weeks, the immunized mice were attacked by H37Rv strain of caudal vein. After 6 weeks of infection, the tuberculosis load in mice was detected, and the lung pathological sections were analyzed to evaluate the protective effect of different adjuvant vaccines.
RESULTS: On the basis of BCG primary immunization, the splenic lymphocytes of AMM subunit vaccine combined adjuvant group (AMM/DDA/BCGPSN) and AMM/DDA group were stimulated by Ag85B and purified protein derivative (PPD) after two times of intensive immunization. Both the splenic lymphocytes secreted IFN-gamma, which was stronger than that of BCG group. The counts of lony-forming units (CFU) showed that the lung load of AMM vaccine with single DDA adjuvant (AMM/DDA) was less than that of BCG and PBS (P 0.05), while the spleen load of AMM vaccine with adjuvant (AMM/DDA/BCG PSN) was less than that of PBS and BCG (P 0.05). And the pathological damage of AMM/DDA group was quite different.
CONCLUSION: DDA is an ideal adjuvant of tuberculosis subunit vaccine, which can induce stronger cellular immunity and stronger immunoprotective effect, BCG PSN may have immunomodulatory effect and can alleviate immunopathological injury.
2. Study on adjuvant effect of different immune adjuvants based on dimethyltrialkyl ammonium on BCG immunization of tuberculosis subunit vaccine
AIM: To study the adjuvant effect of TDM, Poly I:C and BCG PSN combined with DDA as the adjuvant of fusion protein AMM.
METHODS: Seven experimental groups were set up in this study. Each group added different adjuvants on the basis of AMM fusion protein, namely: DDA (A/D); DDA combined with TDM (A/D/T); DDA combined with TDM, Poly I: C (A/D/T/P); DDA combined with Poly I: C (A/D/P); DDA combined with BCG PSN (A/D/B), and BCG and phosphate buffer (PBS) immunization group as control. After the first immunization of BCG, AMM vaccine with different adjuvants was used to strengthen the immunization at 12 and 14 weeks respectively. After 4 weeks of the last immunization, the humoral and cellular immune responses were detected by ELISA and ELISPOT. After 10 weeks, the immunized mice were attacked with H37Rv strain, and the average body size of each group was observed and recorded weekly. Weight. After 6 weeks of infection, the tuberculosis load in mice was detected and the lung histopathological sections were analyzed. The adjuvant effects of different adjuvants based on dimethyltrialkyl ammonium (DMTA) on BCG immunization were evaluated.
Results: After two times of BCG immunization, the mice in A/D/T and A/D/T/P groups could produce higher antigen-specific antibodies, and the ELISPOT analysis showed that the antigen-specific IFN-gamma secreted by the two groups of mice was significantly stronger than that of the control group. The lung histopathological analysis showed that the lung injury of the immunized mice was lighter than that of the BCG group and the DDA subunit vaccine group (P 0.05) after being attacked by H37Rv strain of caudal vein, and the weight of the mice in the different experimental groups before and after being attacked by the virus strain was less than that of the BCG group and the DDA subunit vaccine group. The results of dynamic observation showed that the weight loss of PBS experimental group was obvious, while the weight gain of PBS experimental group combined with three adjuvants (DDA, TDM and Poly I:C) and BCG PSN and DDA adjuvants was significant.
CONCLUSION: Adjuvants Poly I:C and BCG PSN may alleviate the pathological damage caused by Mycobacterium tuberculosis infection through some immune mechanism after BCG immunization is strengthened by subunit vaccine.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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