结核分枝杆菌融合蛋白疫苗（Ag85B-ESAT6）的构建及免疫学特性研究
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摘要
结核病(Tuberculosis,TB),是由结核分枝杆菌(Mycobacterium tuberculosis, MTB)引起的以呼吸道系统感染为主的慢性传染病,是目前世界死亡人数最多、造成危害性最大的疾病之一。
卡介苗(BCG)是当前预防结核病的唯一疫苗,但它的预防保护效果欠佳,对儿童具有很好的保护力,但对成人无保护作用,主要是由于BCG的不断变异,导致免疫效果不稳定;同时在BCG的制备和传代过程中丢失了部分与保护力相关的基因,使BCG保护性免疫应答减弱;近些年来,伴随着艾滋病的流行和MTB自身耐药性的增强,进一步加剧了结核病对人类的威胁,因此迫切需要研制出更有效的TB疫苗。
研究发现在MTB培养上清滤液(CFP)中存在许多具有保护力的分泌蛋白,其中Ag85B和ESAT6具有很好的免疫原性,Ag85B具有分支杆菌酸转移酶活性,可以介导吞噬细胞对MTB进行吞噬,并且能够诱导很强的Th1免疫反应,ESAT6是MTB短期培养滤液中纯化分离出的一种低分子量分泌性蛋白,不存在于BCG中,在MTB感染早期阶段即可被机体识别,具有较强的细胞免疫活性,它们均能刺激机体产生保护性免疫反应,是机体抗结核感染的主要保护性抗原。研究还表明,Ag85B和EAST6的融合蛋白形式进行免疫可提高机体的免疫应答水平。本研究构建了原核表达载体pET-20b- Ag85B- EAST6,制备了融合蛋白Ag85B- EAST6。实验选用了BALB/c小鼠作为实验模型,重组卡介苗(rBCG)进行初免,Ag85B-ESAT6融合蛋白分别联合三种佐剂DDA/MPL,AD11.sm,ADO-1进行加强,分别检测了抗体效价,Th1/Th2细胞因子含量,流式细胞亚型分选,IFN-r的ELISPOT实验,CTL实验等。结果显示: Ag85B-ESAT6融合蛋白联合佐剂疫苗形式优于rBCG,引起了较强的细胞免疫,但以CD4+细胞反应为主,CD8+细胞反应相较弱;同时也初步评价了三种佐剂在提高Ag85B-ESAT6融合蛋白免疫水平中的效果。
Tuberculosis (TB), a bacterial disease due to Mycobacterium Tuberculosis (M. tb).Mycobacterium bovis, the agent of TB in cattle, has been prevalent since ancient times. Despite being an ancient disease, tuberculosis (TB) remains one of the most devastating causes of morbidity and mortality worldwide. The magnitude of the disease reservoir is immense with about 2 billion people or one-third of the world’s population thought to be infected with M. tuberculosis. In recent years, control of TB has been exacerbated by the deadly intersection of the HIV and TB epidemics and the emergence of multiple drug-resistant tuberculosis.The failure of current TB immunization procedures to adequately protect against M. tuberculosis infections is largely responsible for the unsuccessful global control of this disease. Although the current TB vaccine, M. bovis BCG, a vaccine against TB was developed by Albert Calmette and Camille Gu′erin in 1921, using a live attenuated strain of M. bovis, has widely used for at least 60 years, its efficacy has been shown to be highly variable in well-controlled clinical trials. While BCG is moderately protective against disseminated TB in children, BCG is ineffective in protecting against the most prevalent form of the disease, adult pulmonary TB. The inadequacy of BCG immunization to control the TB epidemic has stimulated a worldwide effort to develop more effective TB vaccination strategies.
These immunization strategies have included live attenuated strains, viral vectored vaccine constructs and subunit formulations. These new approaches have potential advantages relative to BCG immunization such as increased safety, improved stability, and decreased interference by exposure to environmental mycobacteria. However, developing a vaccination strategy based on genetic or subunit vaccines that induces strong and sustainable anti-tuberculosis cellular immunity is a significant scientific challenge. Although immunizations with single antigen preparations have usually been inadequate for controlling intracellular infections, recent studies have shown that levels of pathogen specific cellular immunity can be substantially enhanced by combining different vaccines in a heterologous prime/boost. These heterologous prime/boost vaccination strategies often improve the magnitude and quality of T cell responses and can broaden epitope coverage. The subunit approach holds a number of advantages, such as increased safety and stability as well as the demonstrated ability to boost prior BCG. In addition, as subunit vaccines appear not to be influenced by environmental mycobacteria this type of vaccine. However, progress in this field has been delayed by the lack of adjuvant that induces a strong cell-mediated immune (CMI) response. These recent progresses can be partly attributed to the significant advances achieved within the field of adjuvant research.
In this paper, ESAT6 gene and Ag85B gene were obtained by the basic operation of molecular cloning. Two genes connected through the linker as Ag85B-EAST6 fusion gene. Constructed pET-20b-Ag85B-EAST6 prokaryotic expression vector, transformed into BL21, expression of the success of the target protein with selected strain. Using His tag affinity chromatography method, the target protein was purified and the purified protein purity of more than 90%. The protein was identified by Western blot experiment. Used glycerol recovery solution on protein refolding, refolding rate was up to 90%.
Experiment selected BALB/c mice as experimental models for the initial immunization use of rBCG, and Ag85B-EAST6 fusion protein were combined three adjuvant DDA/MPL, AD11.sm, ADO-1 to strengthen the immunity. Experimental results were evaluated by antibody titers, Th1/Th2 cytokines, IFN-r ELISPOT, CTL and other experiments. In the adjuvant DDA / MPL combined antigen Ag85B-ESAT6, found that DDA / MPL combined antigen Ag85B-ESAT6 immunization antibody titers of 1:25600, higher than the protein alone antibody titers after immunization to 1:12800, indicating DDA / MPL to enhance the intensity of the humoral immune protein promoted. More importantly, the DDA/MPL combined antigen Ag85B-ESAT6 of immunization, with Ag85B protein, ESAT6 protein and 9-2 peptide, 18-2 peptide, have emerged in response to stimulation by IFN-γin the ELISPOT experiments. While use of the protein alone and adjuvant alone had no spots, indicating the protein combined adjuvant had a significant immune response after immunization. During the prime-boost test, antibody titer of rBCG was 1:1600, rBCG + Ag85B-ESAT6 + DDA / MPL group is 1:25600, and the same as Ag85B-ESAT6 + DDA / MPL group after immunization. Moreover, other experiment groups of antibody titers were higher than rBCG. The rBCG group of IFN-γcontent 2000pg/ml was 4 times higher than the PBS group. Ag85B-ESAT6 + DDA / MPL group, rBCG + Ag85B-ESAT6 + AD11.sm group and rBCG + Ag85B-ESAT6 + ADO-1 group of IFN-γlevels compared with the PBS group increased by 11 times, 19 times and 7 times. IL-2 levels in supernatants of spleen cells with lower levels overall. Compared with the PBS group, rBCG group, rBCG + Ag85B-ESAT6 + DDA / MPL group and rBCG + Ag85B-ESAT6 + AD11.sm Group of IL-2 contents have increased in more than 2 times, 4 times and 5 times. ELISPOT results showed that, the number of spots of rBCG +Ag85B-ESAT6 + DDA / MPL group, rBCG + Ag85B-ESAT6 + AD11.sm group and rBCG + Ag85B-ESAT6 + ADO-1 group were 5.6 times, 6 times and 3 times higher than rBCG group. After 20 peptide stimulation, the three groups produced 97,100 and 10 spots respectively. After 9-1 peptide and 9-2 peptide stimulation, rBCG +Ag85B-ESAT6 + AD11.sm group produced 33 spots and 43 spots. After stimulation with the Ag85B protein, activated CD4 + cells of rBCG +Ag85B-ESAT6 + DDA / MPL group and rBCG + Ag85B-ESAT6 + AD11.sm group were doubled than stimulation before. CTL results showed that each group of target cell for specific killing rate of less than 10%, indicating that specific CD8 + induced killing is not prominent.
In a word: priming with rBCG and boosting with Ag85B-ESAT6 fusion protein combine with adjuvant were better than rBCG used alone. The adjuvant enhanced the immune response caused by the CD4 + T cell response, the CD8 + T cell response was weak. AD11.sm combined protein triggered stronger CD8 + T cell response than DDA / MPL and ADO-1. DDA / MPL was a pronounced effect kind of adjuvant, the experimental studies have shown AD11.sm adjuvant slightly better than DDA / MPL. Ag85B-ESAT6 fusion protein combined adjuvant as vaccine was better than rBCG, not only lead to humoral immunity, but also caused cell immunity, but mainly in CD4 + T cell response and CD8 + T cell response was relatively weak.
卡介苗(BCG)是当前预防结核病的唯一疫苗,但它的预防保护效果欠佳,对儿童具有很好的保护力,但对成人无保护作用,主要是由于BCG的不断变异,导致免疫效果不稳定;同时在BCG的制备和传代过程中丢失了部分与保护力相关的基因,使BCG保护性免疫应答减弱;近些年来,伴随着艾滋病的流行和MTB自身耐药性的增强,进一步加剧了结核病对人类的威胁,因此迫切需要研制出更有效的TB疫苗。
研究发现在MTB培养上清滤液(CFP)中存在许多具有保护力的分泌蛋白,其中Ag85B和ESAT6具有很好的免疫原性,Ag85B具有分支杆菌酸转移酶活性,可以介导吞噬细胞对MTB进行吞噬,并且能够诱导很强的Th1免疫反应,ESAT6是MTB短期培养滤液中纯化分离出的一种低分子量分泌性蛋白,不存在于BCG中,在MTB感染早期阶段即可被机体识别,具有较强的细胞免疫活性,它们均能刺激机体产生保护性免疫反应,是机体抗结核感染的主要保护性抗原。研究还表明,Ag85B和EAST6的融合蛋白形式进行免疫可提高机体的免疫应答水平。本研究构建了原核表达载体pET-20b- Ag85B- EAST6,制备了融合蛋白Ag85B- EAST6。实验选用了BALB/c小鼠作为实验模型,重组卡介苗(rBCG)进行初免,Ag85B-ESAT6融合蛋白分别联合三种佐剂DDA/MPL,AD11.sm,ADO-1进行加强,分别检测了抗体效价,Th1/Th2细胞因子含量,流式细胞亚型分选,IFN-r的ELISPOT实验,CTL实验等。结果显示: Ag85B-ESAT6融合蛋白联合佐剂疫苗形式优于rBCG,引起了较强的细胞免疫,但以CD4+细胞反应为主,CD8+细胞反应相较弱;同时也初步评价了三种佐剂在提高Ag85B-ESAT6融合蛋白免疫水平中的效果。
Tuberculosis (TB), a bacterial disease due to Mycobacterium Tuberculosis (M. tb).Mycobacterium bovis, the agent of TB in cattle, has been prevalent since ancient times. Despite being an ancient disease, tuberculosis (TB) remains one of the most devastating causes of morbidity and mortality worldwide. The magnitude of the disease reservoir is immense with about 2 billion people or one-third of the world’s population thought to be infected with M. tuberculosis. In recent years, control of TB has been exacerbated by the deadly intersection of the HIV and TB epidemics and the emergence of multiple drug-resistant tuberculosis.The failure of current TB immunization procedures to adequately protect against M. tuberculosis infections is largely responsible for the unsuccessful global control of this disease. Although the current TB vaccine, M. bovis BCG, a vaccine against TB was developed by Albert Calmette and Camille Gu′erin in 1921, using a live attenuated strain of M. bovis, has widely used for at least 60 years, its efficacy has been shown to be highly variable in well-controlled clinical trials. While BCG is moderately protective against disseminated TB in children, BCG is ineffective in protecting against the most prevalent form of the disease, adult pulmonary TB. The inadequacy of BCG immunization to control the TB epidemic has stimulated a worldwide effort to develop more effective TB vaccination strategies.
These immunization strategies have included live attenuated strains, viral vectored vaccine constructs and subunit formulations. These new approaches have potential advantages relative to BCG immunization such as increased safety, improved stability, and decreased interference by exposure to environmental mycobacteria. However, developing a vaccination strategy based on genetic or subunit vaccines that induces strong and sustainable anti-tuberculosis cellular immunity is a significant scientific challenge. Although immunizations with single antigen preparations have usually been inadequate for controlling intracellular infections, recent studies have shown that levels of pathogen specific cellular immunity can be substantially enhanced by combining different vaccines in a heterologous prime/boost. These heterologous prime/boost vaccination strategies often improve the magnitude and quality of T cell responses and can broaden epitope coverage. The subunit approach holds a number of advantages, such as increased safety and stability as well as the demonstrated ability to boost prior BCG. In addition, as subunit vaccines appear not to be influenced by environmental mycobacteria this type of vaccine. However, progress in this field has been delayed by the lack of adjuvant that induces a strong cell-mediated immune (CMI) response. These recent progresses can be partly attributed to the significant advances achieved within the field of adjuvant research.
In this paper, ESAT6 gene and Ag85B gene were obtained by the basic operation of molecular cloning. Two genes connected through the linker as Ag85B-EAST6 fusion gene. Constructed pET-20b-Ag85B-EAST6 prokaryotic expression vector, transformed into BL21, expression of the success of the target protein with selected strain. Using His tag affinity chromatography method, the target protein was purified and the purified protein purity of more than 90%. The protein was identified by Western blot experiment. Used glycerol recovery solution on protein refolding, refolding rate was up to 90%.
Experiment selected BALB/c mice as experimental models for the initial immunization use of rBCG, and Ag85B-EAST6 fusion protein were combined three adjuvant DDA/MPL, AD11.sm, ADO-1 to strengthen the immunity. Experimental results were evaluated by antibody titers, Th1/Th2 cytokines, IFN-r ELISPOT, CTL and other experiments. In the adjuvant DDA / MPL combined antigen Ag85B-ESAT6, found that DDA / MPL combined antigen Ag85B-ESAT6 immunization antibody titers of 1:25600, higher than the protein alone antibody titers after immunization to 1:12800, indicating DDA / MPL to enhance the intensity of the humoral immune protein promoted. More importantly, the DDA/MPL combined antigen Ag85B-ESAT6 of immunization, with Ag85B protein, ESAT6 protein and 9-2 peptide, 18-2 peptide, have emerged in response to stimulation by IFN-γin the ELISPOT experiments. While use of the protein alone and adjuvant alone had no spots, indicating the protein combined adjuvant had a significant immune response after immunization. During the prime-boost test, antibody titer of rBCG was 1:1600, rBCG + Ag85B-ESAT6 + DDA / MPL group is 1:25600, and the same as Ag85B-ESAT6 + DDA / MPL group after immunization. Moreover, other experiment groups of antibody titers were higher than rBCG. The rBCG group of IFN-γcontent 2000pg/ml was 4 times higher than the PBS group. Ag85B-ESAT6 + DDA / MPL group, rBCG + Ag85B-ESAT6 + AD11.sm group and rBCG + Ag85B-ESAT6 + ADO-1 group of IFN-γlevels compared with the PBS group increased by 11 times, 19 times and 7 times. IL-2 levels in supernatants of spleen cells with lower levels overall. Compared with the PBS group, rBCG group, rBCG + Ag85B-ESAT6 + DDA / MPL group and rBCG + Ag85B-ESAT6 + AD11.sm Group of IL-2 contents have increased in more than 2 times, 4 times and 5 times. ELISPOT results showed that, the number of spots of rBCG +Ag85B-ESAT6 + DDA / MPL group, rBCG + Ag85B-ESAT6 + AD11.sm group and rBCG + Ag85B-ESAT6 + ADO-1 group were 5.6 times, 6 times and 3 times higher than rBCG group. After 20 peptide stimulation, the three groups produced 97,100 and 10 spots respectively. After 9-1 peptide and 9-2 peptide stimulation, rBCG +Ag85B-ESAT6 + AD11.sm group produced 33 spots and 43 spots. After stimulation with the Ag85B protein, activated CD4 + cells of rBCG +Ag85B-ESAT6 + DDA / MPL group and rBCG + Ag85B-ESAT6 + AD11.sm group were doubled than stimulation before. CTL results showed that each group of target cell for specific killing rate of less than 10%, indicating that specific CD8 + induced killing is not prominent.
In a word: priming with rBCG and boosting with Ag85B-ESAT6 fusion protein combine with adjuvant were better than rBCG used alone. The adjuvant enhanced the immune response caused by the CD4 + T cell response, the CD8 + T cell response was weak. AD11.sm combined protein triggered stronger CD8 + T cell response than DDA / MPL and ADO-1. DDA / MPL was a pronounced effect kind of adjuvant, the experimental studies have shown AD11.sm adjuvant slightly better than DDA / MPL. Ag85B-ESAT6 fusion protein combined adjuvant as vaccine was better than rBCG, not only lead to humoral immunity, but also caused cell immunity, but mainly in CD4 + T cell response and CD8 + T cell response was relatively weak.
引文
[1] Ali Nasser Eddine, Sven Baumann, Stefan H.E. Kaufmann.New tuberculosis vaccines approaching clinical trial– An overview.Department of Immunology, Schumannstrasse 21-22, 10117 Berlin, Germany.
[2] BehrMA, WilsonMA, Anderson P, et al. Comparative genenomic of BCG vaccines by whole genome DNA microarray [J]. Science, 1996, 284 (541): 1520 - 1523.
[3] Leitner WW, Ying H, Restifo NP. DNA and RNA2based vaccines :principle,progress and prospects. Vaccine,1999 ,18 :7652767.
[4] Brewer, T. Fand G. A. Colditz. 1995. Bacille Calmette-Guerin vaccination for the prevention of tuberculosis in health care workers. Clin. Infect. Dis.20:136–142.
[5] Andersen, 1994. Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins.Infect.Immun. 62: 2536.
[6] Tascon, R. E., M. J. Colston, S. Ragno, E. Stavropoulos, D. Gregory, and D. B.Lowrie ,1996. Vaccination against tuberculosis by DNA injection. Nat. Med. 2: 888–892.
[7] Hess J , Kaufmann SH. Development of novel tuberculosis vaccines. CR Acad Sci III , 1999 , 322 : 953– 958.
[8] Orme IM, McMurray DN , Belisle JT. Tuberculosis vaccine developement: Recent progress. Trends Microbiol, 2001, 9: 115– 118
[9] Kamath AT, Feng CG, Macdonald M, et al. Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect Immun, 1999, 67:170221707.
[10] Wedlock DN, Skinner MA, Parlane NA, Vordermeier HM, Hewinson RG, de Lisle GW, et al.Vaccination with DNA vaccines encoding MPB70 or MPB83 or a MPB70 DNA prime-protein boost does not protect cattle against bovine tuberculosis. Tuberculosis (Edinb) 2003; 83(6):339–49.
[11] Skinner M, Buddle BM, Wedlock N, et al. A DNA prime–BCG boost vaccination strategy in cattle induces protection against bovine tuberculosis. Infect Immune 2003; 71:4901–7.
[12] Jackson M, Phalen SE, Lagranderie M, et al. Perisrence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine [J]. Infect Immune, 1999, 67(6):2867 - 2873.
[13] Collins DM. New tuberculosis vaccines based on attenuated strains of the Mycobacterium tuberculosis complex. Immunology Cell Biol 2000; 78(4):342–8.
[14] Guleria I, Teitelbaum R, McAdam RA, Kalpana G, Jacobs JrWR, Bloom BR. Auxotrophic vaccines for tuberculosis. Nat Med 1996; 2(3):334–7.
[15] Wedlock DN, Aldwell FE, Collins DM, de Lisle GW, Wilson T, Buddle BM. Immune responses induced in cattle by virulent and attenuated Mycobacterium bovis strains: correlation of delayed-type hypersensitivity with ability of strains to grow in macrophages. Infect Immune 1999; 67(5):2172–7.
[16] Buddle BM, Wards BJ, Aldwell FE, Collins DM, de Lisle GW. Influence of sensitisation to environmental mycobacteria on subsequent vaccination against bovine tuberculosis. Vaccine 2002; 20(7–8):1126–33.
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[33] Schijns VE. Mechanisms of vaccine adjuvant activity : initiation and regulation of immune responses by vaccine adjuvants. Vaccine, 2003, 21 (9-10):829-831.
[34] Powers DC. Summary of a clinical trial with liposomese adjuvanted influenza a virus vaccine in elderly adults. Mechanismof Ageing and Development, 1997, 93:179-188.
[2] BehrMA, WilsonMA, Anderson P, et al. Comparative genenomic of BCG vaccines by whole genome DNA microarray [J]. Science, 1996, 284 (541): 1520 - 1523.
[3] Leitner WW, Ying H, Restifo NP. DNA and RNA2based vaccines :principle,progress and prospects. Vaccine,1999 ,18 :7652767.
[4] Brewer, T. Fand G. A. Colditz. 1995. Bacille Calmette-Guerin vaccination for the prevention of tuberculosis in health care workers. Clin. Infect. Dis.20:136–142.
[5] Andersen, 1994. Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins.Infect.Immun. 62: 2536.
[6] Tascon, R. E., M. J. Colston, S. Ragno, E. Stavropoulos, D. Gregory, and D. B.Lowrie ,1996. Vaccination against tuberculosis by DNA injection. Nat. Med. 2: 888–892.
[7] Hess J , Kaufmann SH. Development of novel tuberculosis vaccines. CR Acad Sci III , 1999 , 322 : 953– 958.
[8] Orme IM, McMurray DN , Belisle JT. Tuberculosis vaccine developement: Recent progress. Trends Microbiol, 2001, 9: 115– 118
[9] Kamath AT, Feng CG, Macdonald M, et al. Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect Immun, 1999, 67:170221707.
[10] Wedlock DN, Skinner MA, Parlane NA, Vordermeier HM, Hewinson RG, de Lisle GW, et al.Vaccination with DNA vaccines encoding MPB70 or MPB83 or a MPB70 DNA prime-protein boost does not protect cattle against bovine tuberculosis. Tuberculosis (Edinb) 2003; 83(6):339–49.
[11] Skinner M, Buddle BM, Wedlock N, et al. A DNA prime–BCG boost vaccination strategy in cattle induces protection against bovine tuberculosis. Infect Immune 2003; 71:4901–7.
[12] Jackson M, Phalen SE, Lagranderie M, et al. Perisrence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine [J]. Infect Immune, 1999, 67(6):2867 - 2873.
[13] Collins DM. New tuberculosis vaccines based on attenuated strains of the Mycobacterium tuberculosis complex. Immunology Cell Biol 2000; 78(4):342–8.
[14] Guleria I, Teitelbaum R, McAdam RA, Kalpana G, Jacobs JrWR, Bloom BR. Auxotrophic vaccines for tuberculosis. Nat Med 1996; 2(3):334–7.
[15] Wedlock DN, Aldwell FE, Collins DM, de Lisle GW, Wilson T, Buddle BM. Immune responses induced in cattle by virulent and attenuated Mycobacterium bovis strains: correlation of delayed-type hypersensitivity with ability of strains to grow in macrophages. Infect Immune 1999; 67(5):2172–7.
[16] Buddle BM, Wards BJ, Aldwell FE, Collins DM, de Lisle GW. Influence of sensitisation to environmental mycobacteria on subsequent vaccination against bovine tuberculosis. Vaccine 2002; 20(7–8):1126–33.
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