DDA为基础的不同免疫佐剂对结核亚单位疫苗AMM免疫保护效果的影响
发布时间：2018-08-28 11:13
【摘要】：肺结核是世界范围内导致人类死亡的主要感染性疾病之一。牛分枝杆菌(Bacilli Calmette Guerin,BCG)是目前唯一的抗结核病疫苗。BCG是来源于上个世纪牛分枝杆菌的减毒株,历时13年体外传代所得。几十年来,BCG疫苗在世界范围广泛应用,然而在不同的地域,其有效性有巨大的差异,尤其对成人肺结核几乎没有保护效应。因此,迫切需要研制安全、有效的抗结核疫苗。在众多新疫苗中,亚单位疫苗具有成分明确和易于被接受的优点,并且具有强化BCG保护效率的作用,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,然后选择亚单位疫苗加强免疫。 我们此前的研究表明,融合蛋白Ag85B-Mpt64190-198-Mtb8.4 (AMM)和佐剂二甲基三十六烷基铵(DDA)、卡介苗多糖核酸(BCG PSN)构建的亚单位疫苗能够诱导较强体液免疫和细胞免疫。本课题探讨了佐剂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)不同的免疫活化剂混合,联合作为融合蛋白AMM的佐剂,比较不同佐剂辅助诱导的免疫效应差异和加强BCG免疫保护效率的差异。 1.二甲基三十六烷基铵及卡介苗多糖核酸佐剂在结核亚单位疫苗强化BCG免疫中的效应研究 目的：研究DDA和DDA-BCG PSN佐剂在结核融合蛋白Ag85B-Mpt64190-198-Mtb8.4疫苗强化卡介苗(BCG)免疫中的不同免疫辅助效应。 方法：选择DDA、BCG PSN作为融合蛋白AMM的佐剂,在BCG初免基础上应用AMM疫苗加强免疫小鼠两次。其中一组联合使用两种佐剂(DDA/BCG PSN),另一组以DDA单独作为佐剂,同时设立BCG和磷酸缓冲液(phosphate buffered saline,PBS)免疫组为对照。末次免疫4周后,应用ELISA及ELISPOT检测免疫小鼠的体液与细胞免疫反应。12周后,经尾静脉H37Rv毒株攻击被免疫小鼠。感染6周后,检测小鼠体内结核菌载量,并分析肺组织病理切片,评价不同佐剂疫苗的保护效果。 结果：在BCG初免基础上,AMM亚单位疫苗联合佐剂组(AMM/DDA/BCGPSN)和单独佐剂组(AMM/DDA)加强免疫两次后,脾脏淋巴细胞经抗原Ag85B和纯蛋白衍生物(purified protein derivative,PPD)刺激后,皆可分泌较BCG组强的IFN-γ。毒力株攻击后,菌落形成单位(colony-forming units, CFU)计数显示,AMM疫苗单一DDA佐剂组(AMM/DDA)强化BCG免疫后,其肺部荷菌量少于BCG和PBS组(P0.05),而AMM疫苗联合佐剂组(AMM/DDA/BCG PSN)脾脏荷菌量少于PBS组和BCG组(P0.05)。组织病理分析结果表明AMM/DDA/BCG PSN组肺组织病理损伤较轻,而AMM/DDA组病理损伤个体差异较大。 结论：DDA是较为理想的结核亚单位疫苗佐剂,能诱导较强的细胞免疫和较强的免疫保护作用；BCG PSN可能具有免疫调节作用,可以减轻免疫病理损伤。 2.二甲基三十六烷基铵为基础的不同免疫佐剂在结核亚单位疫苗强化BCG免疫中的佐剂效应研究 目的：研究免疫佐剂TDM、Poly I:C和BCG PSN联合DDA作为融合蛋白AMM的佐剂,在BCG初免基础上AMM疫苗加强免疫两次,比较不同的联合佐剂组的佐剂效应。 方法：本研究共设立了七个实验组,各个实验组在AMM融合蛋白基础上加入不同的佐剂,分别是：DDA(A/D); DDA联合TDM(A/D/T); DDA联合TDM、Poly I:C(A/D/T/P); DDA联合Poly I:C(A/D/P), DDA联合BCG PSN(A/D/B),且设立BCG和磷酸缓冲液(PBS)免疫组为对照。各实验组BCG初次免疫后,用不同佐剂的亚单位疫苗AMM分别在第12周和第14周进行加强免疫。末次加强免疫4周后,采用ELISA及ELISPOT检测免疫小鼠的体液与细胞免疫应答水平；10周后,以H37Rv毒株攻击被免疫小鼠,并每周观察记录每组小鼠的平均体重。疫苗免疫小鼠感染6周后,检测小鼠体内结核菌载量,并分析肺组织病理切片。评价二甲基三十六烷基铵为基础的不同免疫佐剂在结核亚单位疫苗强化BCG免疫中的佐剂效应。 结果：A/D/T和A/D/T/P实验组加强BCG免疫两次后,能够诱发小鼠产生更高抗原特异性抗体,且ELISPOT分析显示该两组小鼠分泌抗原特异性IFN-γ明显强于对照BCG；肺部荷菌量试验表明各个佐剂实验组加强BCG免疫后,肺部荷菌量都较PBS组低；各组免疫小鼠经尾静脉H37Rv毒株攻击后,肺组织病理分析显示联合有BCG PSN或Poly I:C的联合佐剂的亚单位疫苗强化BCG免疫后,肺组织病理损伤均较BCG组和DDA为佐剂的亚单位疫苗组轻(P0.05)；在毒株攻击前后对不同实验组小鼠的体重进行动态观察,结果表明：PBS实验组体重减少明显,而联合三种佐剂(DDA、TDM和Poly I:C)和联合BCG PSN和DDA两种佐剂组体重增加明显。 结论：佐剂Poly I:C和BCG PSN可能在亚单位疫苗加强BCG免疫后,通过某种免疫机制,最终可以减轻结核分枝杆菌感染所造成的病理损伤。
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
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