随机重组多表位恶性疟疾疫苗M.RCAg-1的制备及免疫保护性差异的机理研究
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摘要
蚊媒传播的疟疾是全球最严重的三大传染病之一。目前,全球约40%的世界人口处于罹患疟疾的危险之中,而每年有5亿人受到疟原虫感染,约100万人死于恶性疟疾,其中大部分是五岁以下的儿童和孕妇。由于恶性疟原虫对现有药物的抗药性不断出现和蔓延,蚊媒对杀虫剂抗药性的产生等因素影响,疟疾的防治成为世界性的难题之一。疟原虫生活周期复杂,抗原具有阶段特异性、虫株差异性和高度变异性等特点,长期以来形成研制抗疟原虫疫苗的技术瓶颈。因此,针对恶性疟原虫不同生活时期的免疫反应特点,选取诱导产生多种免疫反应类型的不同抗原构建多表位疫苗已经成为研究疟疾疫苗的热点。
在本课题组前期工作中,针对参与抗疟原虫红内期感染的免疫类型,选择了来自8个恶性疟原虫抗原的11个B细胞和Th细胞表位,利用epitope shuffling (表位改组)技术成功构建了随机组合多表位恶性疟疾疫苗M. RCAg-1。通过在BALB/c小鼠、新西兰兔、恒河猴的免疫原性和体内外保护性实验观察,证实其具有良好的免疫原性和免疫保护效果,是一个极具潜力的疟疾候选疫苗。
本研究首先按照国家生物制品研制标准,筛选出表达多表位疫苗M. RCAg-1重组蛋白的工程菌,建立了高效表达蛋白的纯化工艺,评价了三种已用于人临床试验的佐剂与M. RCAg-1抗原配伍后,对小鼠和新西兰白兔体液免疫反应,CD4+T细胞反应及免疫兔血清抗体在体外对恶性疟原虫生长影响的作用。并且针对不同载体表达的M. RCAg-1重组蛋白在免疫动物后,其兔免疫血清在体外对恶性疟原虫生长的不同抑制作用,深入分析了其可能的原因,取得了以下结果：
1.经pDS-ex-EKase载体系统表达的重组抗原蛋白的免疫原性和免疫保护性最好,因此确定将BL21(DE3)-M. RCAg-1/pDS-ex-EKase蛋白表达菌株作为工程菌株,并且确定了M. RCAg-1蛋白最佳的表达、纯化方式,能够获得稳定的纯度大于97%的重组蛋白,提供进一步的疫苗相关临床前评价实验使用；
2.分别在pDS-ex-EKase和pDS-ex-PPase载体系统,以及pBV220温控启动子系统中表达了三种M. RCAg-1重组蛋白,发现尽管这些蛋白的氨基酸序列基本相同,在动物模型体内的免疫原性相同,但是其体外免疫保护性却出现明显差异,其中通过pDS-ex-EKase载体系统制备的抗原,免疫新西兰兔后,血清抗体对恶性疟原虫的体外生长抑制率近100%,而通过其它两类载体系统制备的同一抗原,其兔血清抗体对恶性疟原虫的抑制率下降至50%或以下；
3.利用生物信息学和色谱技术分析,发现从不同表达载体制备的重组蛋白在二级和三级结构上出现极大差异,证明蛋白质分子的空间构象发生了改变,而且一些表位在分子中的位置也有明显的改变；
4.经全面测定包含在三个基本相同序列抗原分子中的每个表位肽所诱导产生的抗体滴度,经统计学相关性方法分析,证明在针对体液免疫反应和辅助性T细胞免疫反应的多表位人工抗原中,影响抗原免疫保护性的关键因素是诱导产生高滴度抗体反应的表位百分率和平均抗体滴度；
本研究确定了制备和纯化M. RCAg-1的稳定高效的原核表达系统,并摸索出符合国家生物制品审批管理规范,能满足工业化生产规模的中试制备条件。通过构建不同表达系统,发现蛋白质分子的空间构象对人工多表位抗原的免疫保护效应至关重要,影响其空间构象的因素不仅涉及读码框架内的肽链序列,还涉及表达载体与插入片段间结合部位的肽段序列,它们都能通过影响人工抗原分子的蛋白折叠形式,从而改变肽链中α-螺旋的数量、表位在分子中的位置和蛋白质分子的稳定性。本文还证实,在针对体液免疫反应和辅助性T细胞免疫反应的多表位人工抗原中,影响抗原免疫保护性的关键因素是诱导产生高抗体水平的表位百分率和平均抗体滴度。这些实验结果对设计出新一代更优化的红细胞内期多表位疟疾疫苗具有重要的指导意义。
Malaria is one of the most infectious diseases to human health in the world. There are an estimated 500 million cases and up to 1 million deaths from malaria each year,which mainly are under 5 years of age and pregnant women. The emergence and spread of drug-resistant parasites and insecticide-resistant Anopheles mosquito vectors make prevention and treatment of malaria a big problem in the world. Plasmodium's complex life cycles, antigenic stage-specificity, diversity and variation, have been the unique obstacles to develop antimalarial vaccines for a long time. Thus, the development of a multiple antigens and epitopes vaccine against Plasmodium falciparum to elicit relative immune responses at different stages has become a major hotspot in the development of malaria vaccines.
In the previous work, we have successfully constructed polyepitope Plasmodium falciparum malaria vaccines M.RCAg-1 by random tandem with epitope shuffling technology platform,11 B cell and Th cell epitopes seclected from 8 Plasmodium falciparum antigens as the study subject according to immune type involved in blood-stage infection against Plasmodium. At present, the experiments of immunogenicity and protection in vitro and vivo in immunized mice, rabbits and rhesus monkeys have been observed, which confirmed its good immunogenicity and protective effect in vivo, and the vaccine is an potential malaria candidate vaccine.
Based on the national standard of biological products, the present work study on the screen of engineering bacteria expressing polyeitope vaccine M.RCAg-1 recombination protein, the establishment of the technique for highly effective protein expression and purification, the evaluation of the effect of three adjuvants acceptable in human clinical experiment formulation with purified M.RCAg-1 protein on the humoral immune response and CD4+ T cell response of inbred strain mice and New Zealand white rabbits, and the effect of antibody on Plasmodium falciparum growth in vitro. The potential reason of different inhibition effect of recombinant protein expressing in different victors immune rabbits sera on Plasmodium falciparum growth in vitro were also analyzed. The results are as follows:
1. the imunogenicity and protection of recombinant antigen protein through pDS-ex-Ekase system expression is best, therefore, the bacterial strain of BL21 (DE3)-M.RCAg-1/pDS-ex-Ekase protein expression is determined as engineering bacterial strain. Also the optimal pattern of the expression and purification of M.RCAg-1 protein is determined. The recombinant protein could be obtained stably and over 97% purity which could the need of following experiment of vaccine related preclinical evaluation;
2. Three kind of recombinant proteins were expressed in pDS-ex-Ekase and pDS-ex-PPase carrier system and pBV220 temperature controlling promoter system, the three proteins have almost the same amino acid sequence, the same immunogenicity in animal models, however, the obviously different immunoprotections. When New Zealand white rabbit immunized by antigen of pDS-ex-Ekase carrier system, the sera antibody inhibited the growth of Plasmodium falciparum in vitro almost 100%, However below 50% when immunized by antigens of the other two carrier systems.
3. The significant differences of secondary and tertiary structures were shown in recombinant proteins from different expression vectors, analyzed by bioinformatics and chromatographic technique, which demonstrated the change of protein molecule spaces conformation, and the obviously change of some epitope locations.
4. The statistics analysis of antibody titer induced by every epitope peptid which was contained in three antigens with almost the same sequences showed the key factors of antigen immunoprotection in Humoral immune reaction and adjuvanticity T cell immune reaction. The key factors were epitope percentage and average antibody titer.
The present study determined the stable and highly effective preparation and purification of M.RCAg-1 in prokaryotic expression systems, and explored the condition of semi-works production according to national biological product examination and management standard which can meet industrialization production. The study of construction in different expression systems showed the space conformation of protein molecule is crucial to immunoprotection effect of artificial polyepitope antigen. Factors influencing space conformation involved not only the peptide sequence of reading frame but also the peptide sequence in binding site between expression carrier and insertion element, these factors changed the amount ofα-helix in peptide, the epitope location in molecule, and the protein stability by influence of protein fold style in artificial antigen. The present study also demonstrated, to polyepitope artificial antigen which induced humoral immune reaction and adjuvanticity T cell immune reaction, the key factors influencing antigen immunoprotection were epitope percentage and average antibody titer which induced highly antibody level. The results could be important to the design of new and optimal erythrocytic stage polyepitope malaria vaccine.
在本课题组前期工作中,针对参与抗疟原虫红内期感染的免疫类型,选择了来自8个恶性疟原虫抗原的11个B细胞和Th细胞表位,利用epitope shuffling (表位改组)技术成功构建了随机组合多表位恶性疟疾疫苗M. RCAg-1。通过在BALB/c小鼠、新西兰兔、恒河猴的免疫原性和体内外保护性实验观察,证实其具有良好的免疫原性和免疫保护效果,是一个极具潜力的疟疾候选疫苗。
本研究首先按照国家生物制品研制标准,筛选出表达多表位疫苗M. RCAg-1重组蛋白的工程菌,建立了高效表达蛋白的纯化工艺,评价了三种已用于人临床试验的佐剂与M. RCAg-1抗原配伍后,对小鼠和新西兰白兔体液免疫反应,CD4+T细胞反应及免疫兔血清抗体在体外对恶性疟原虫生长影响的作用。并且针对不同载体表达的M. RCAg-1重组蛋白在免疫动物后,其兔免疫血清在体外对恶性疟原虫生长的不同抑制作用,深入分析了其可能的原因,取得了以下结果：
1.经pDS-ex-EKase载体系统表达的重组抗原蛋白的免疫原性和免疫保护性最好,因此确定将BL21(DE3)-M. RCAg-1/pDS-ex-EKase蛋白表达菌株作为工程菌株,并且确定了M. RCAg-1蛋白最佳的表达、纯化方式,能够获得稳定的纯度大于97%的重组蛋白,提供进一步的疫苗相关临床前评价实验使用；
2.分别在pDS-ex-EKase和pDS-ex-PPase载体系统,以及pBV220温控启动子系统中表达了三种M. RCAg-1重组蛋白,发现尽管这些蛋白的氨基酸序列基本相同,在动物模型体内的免疫原性相同,但是其体外免疫保护性却出现明显差异,其中通过pDS-ex-EKase载体系统制备的抗原,免疫新西兰兔后,血清抗体对恶性疟原虫的体外生长抑制率近100%,而通过其它两类载体系统制备的同一抗原,其兔血清抗体对恶性疟原虫的抑制率下降至50%或以下；
3.利用生物信息学和色谱技术分析,发现从不同表达载体制备的重组蛋白在二级和三级结构上出现极大差异,证明蛋白质分子的空间构象发生了改变,而且一些表位在分子中的位置也有明显的改变；
4.经全面测定包含在三个基本相同序列抗原分子中的每个表位肽所诱导产生的抗体滴度,经统计学相关性方法分析,证明在针对体液免疫反应和辅助性T细胞免疫反应的多表位人工抗原中,影响抗原免疫保护性的关键因素是诱导产生高滴度抗体反应的表位百分率和平均抗体滴度；
本研究确定了制备和纯化M. RCAg-1的稳定高效的原核表达系统,并摸索出符合国家生物制品审批管理规范,能满足工业化生产规模的中试制备条件。通过构建不同表达系统,发现蛋白质分子的空间构象对人工多表位抗原的免疫保护效应至关重要,影响其空间构象的因素不仅涉及读码框架内的肽链序列,还涉及表达载体与插入片段间结合部位的肽段序列,它们都能通过影响人工抗原分子的蛋白折叠形式,从而改变肽链中α-螺旋的数量、表位在分子中的位置和蛋白质分子的稳定性。本文还证实,在针对体液免疫反应和辅助性T细胞免疫反应的多表位人工抗原中,影响抗原免疫保护性的关键因素是诱导产生高抗体水平的表位百分率和平均抗体滴度。这些实验结果对设计出新一代更优化的红细胞内期多表位疟疾疫苗具有重要的指导意义。
Malaria is one of the most infectious diseases to human health in the world. There are an estimated 500 million cases and up to 1 million deaths from malaria each year,which mainly are under 5 years of age and pregnant women. The emergence and spread of drug-resistant parasites and insecticide-resistant Anopheles mosquito vectors make prevention and treatment of malaria a big problem in the world. Plasmodium's complex life cycles, antigenic stage-specificity, diversity and variation, have been the unique obstacles to develop antimalarial vaccines for a long time. Thus, the development of a multiple antigens and epitopes vaccine against Plasmodium falciparum to elicit relative immune responses at different stages has become a major hotspot in the development of malaria vaccines.
In the previous work, we have successfully constructed polyepitope Plasmodium falciparum malaria vaccines M.RCAg-1 by random tandem with epitope shuffling technology platform,11 B cell and Th cell epitopes seclected from 8 Plasmodium falciparum antigens as the study subject according to immune type involved in blood-stage infection against Plasmodium. At present, the experiments of immunogenicity and protection in vitro and vivo in immunized mice, rabbits and rhesus monkeys have been observed, which confirmed its good immunogenicity and protective effect in vivo, and the vaccine is an potential malaria candidate vaccine.
Based on the national standard of biological products, the present work study on the screen of engineering bacteria expressing polyeitope vaccine M.RCAg-1 recombination protein, the establishment of the technique for highly effective protein expression and purification, the evaluation of the effect of three adjuvants acceptable in human clinical experiment formulation with purified M.RCAg-1 protein on the humoral immune response and CD4+ T cell response of inbred strain mice and New Zealand white rabbits, and the effect of antibody on Plasmodium falciparum growth in vitro. The potential reason of different inhibition effect of recombinant protein expressing in different victors immune rabbits sera on Plasmodium falciparum growth in vitro were also analyzed. The results are as follows:
1. the imunogenicity and protection of recombinant antigen protein through pDS-ex-Ekase system expression is best, therefore, the bacterial strain of BL21 (DE3)-M.RCAg-1/pDS-ex-Ekase protein expression is determined as engineering bacterial strain. Also the optimal pattern of the expression and purification of M.RCAg-1 protein is determined. The recombinant protein could be obtained stably and over 97% purity which could the need of following experiment of vaccine related preclinical evaluation;
2. Three kind of recombinant proteins were expressed in pDS-ex-Ekase and pDS-ex-PPase carrier system and pBV220 temperature controlling promoter system, the three proteins have almost the same amino acid sequence, the same immunogenicity in animal models, however, the obviously different immunoprotections. When New Zealand white rabbit immunized by antigen of pDS-ex-Ekase carrier system, the sera antibody inhibited the growth of Plasmodium falciparum in vitro almost 100%, However below 50% when immunized by antigens of the other two carrier systems.
3. The significant differences of secondary and tertiary structures were shown in recombinant proteins from different expression vectors, analyzed by bioinformatics and chromatographic technique, which demonstrated the change of protein molecule spaces conformation, and the obviously change of some epitope locations.
4. The statistics analysis of antibody titer induced by every epitope peptid which was contained in three antigens with almost the same sequences showed the key factors of antigen immunoprotection in Humoral immune reaction and adjuvanticity T cell immune reaction. The key factors were epitope percentage and average antibody titer.
The present study determined the stable and highly effective preparation and purification of M.RCAg-1 in prokaryotic expression systems, and explored the condition of semi-works production according to national biological product examination and management standard which can meet industrialization production. The study of construction in different expression systems showed the space conformation of protein molecule is crucial to immunoprotection effect of artificial polyepitope antigen. Factors influencing space conformation involved not only the peptide sequence of reading frame but also the peptide sequence in binding site between expression carrier and insertion element, these factors changed the amount ofα-helix in peptide, the epitope location in molecule, and the protein stability by influence of protein fold style in artificial antigen. The present study also demonstrated, to polyepitope artificial antigen which induced humoral immune reaction and adjuvanticity T cell immune reaction, the key factors influencing antigen immunoprotection were epitope percentage and average antibody titer which induced highly antibody level. The results could be important to the design of new and optimal erythrocytic stage polyepitope malaria vaccine.
引文
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