多表位恶性疟人工抗原M.RCAg-1的免疫保护效果评价及其免疫机制的研究
发布时间：2018-07-17 20:12
【摘要】： 疟疾是目前世界上对人类危害最严重的蚊媒寄生原虫病。全世界每年有5亿人受到疟原虫感染，约三百万人死于恶性疟疾。其中80％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大难题之一。由于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抗疟疫苗的热点。但是，目前对多抗原表位疫苗的研究仍然是以人工合成多表位肽疫苗或单一固定的串联多表位DNA疫苗为主，前者成本昂贵，后者免疫原性低，难以达到预期的免疫反应多样性和令人满意的保护效果。 本课题组前期工作借鉴了分子育种技术(即DNA改组)的随机重组原理，利用表位改组技术构建了多表位基因库，并用库免疫血清筛选出高抗原性的阳性克隆，发现其中VR312(改名为M．RCAg-1)基因克隆不仅能在小鼠模型诱导交叉免疫保护而且能在家兔模型中诱导抗恶性疟原虫抑制性抗体。 本研究在前期工作的基础上，，开展了M．RCAg-1基因和蛋白质疫苗在不同种属的哺乳动物模型中的免疫原性和保护性研究，以及对M．RCAg-1蛋白质进行相关流行病学分析，并且对利用核酸分子探针检测虫血率的流式细胞术的建立做了相关的探索，取得以下的结果： 1．M．RCAg-1蛋白质抗原能被不同疫区的病人血清识别，提示M．RCAg-1多表位蛋白质疫苗可能能克服虫株之间差异导致的免疫逃逸； 2．非洲喀麦隆疫区居民血清IgG抗体识别M．RCAg-1多表位蛋白质和年龄呈现显著的正相关，提示儿童时期接种M．RCAg-1多表位蛋白质疫苗可能降低恶性疟导致的高病死率； 3．M．RcAg-1多表位蛋白质免疫小鼠、新西兰白兔和恒河猴均能获得高滴度特异性抗体水平和诱发明显的CD4~+T细胞反应，而且产生的特异性抗体能在体外有效抑制恶性疟原虫的生长。提示产生的特异性抗体中抑制性抗体的比例较大，这可能和M．RCAg-1多表位蛋白质的优势构象有关； 4．M．RcAg-1多表位基因疫苗在小鼠和家兔动物模型能产生高滴度抗体水平，但此特异性抗体亲合力不高，体外生长抑制效果不佳。在恒河猴动物模型中，单纯的基因免疫很难诱导高水平的抗体，而采用基因初免-蛋白质加强的方案能有效的诱导出高水平的特异性抗体，然而和小动物模型一样，体外抑制原虫生长的效果不明显。这可能和基因免疫的表达呈递模式有关，从而产生和蛋白质免疫不同比例和不同亲合力的抗体类型； 5．分析M．RcAg-1蛋白质免疫小鼠的细胞免疫实验结果，发现具有相同特异性的细胞可以分化为不同亚群的辅助性T细胞，这主要取决于抗原的构象和周围分泌的细胞因子类型； 6．利用Hydroethidine流式细胞术可以客观快速相对定量疟原虫虫血率，而且能较好地评价原虫同步化的效果。
[Abstract]:Malaria is the most serious mosquito parasitic protozoa in the world at present. 500 million people are infected with malaria parasites every year in the world and about three million people die from malarial malaria. 80% of them are children under five years of age and pregnant women. The prevention and control of malaria has become one of the most difficult problems in the world. Due to the complexity of the life cycle of the Plasmodium, the antigen has the characteristics of stage specificity, strain difference and high variability, it has long been a technical bottleneck for the development of Plasmodium antimalarial vaccine. Therefore, the immunological response to Plasmodium falciparum in different life periods is special. The selection of multiple epitopes with different antigens inducing various types of immune responses has become a hot spot in the study of antimalarial vaccines. However, the study of multiple epitope vaccines is still mainly based on synthetic multi epitope vaccine or single fixed series multi epitopes DNA vaccine, the former is expensive and the latter has low immunogenicity. It is difficult to achieve the desired immune response diversity and satisfactory protective effect.
In the previous work, we used the principle of random recombination of molecular breeding technology (DNA regrouping), and constructed a multi epitope gene pool using epitope modification technique, and screened the positive clones with high antigenicity with the immune sera of the library. It was found that VR312 (renamed M.RCAg-1) gene clones could not only induce cross immunization protection in mice model. It can also induce Plasmodium falciparum inhibitory antibodies in rabbit models.
On the basis of the previous work, the immunogenicity and protection of M.RCAg-1 gene and protein vaccine in the mammalian model of different species were carried out, and the related epidemiological analysis of M.RCAg-1 protein was carried out, and the establishment of the flow cytometry with nucleic acid probe for detecting the blood rate of the insect was related. The following results are obtained:
The 1.M.RCAg-1 protein antigen can be identified by the serum of patients in different epidemic areas, suggesting that the M.RCAg-1 polyepitope protein vaccine can overcome the immune escape caused by the difference between the strains of the insect.
2. the M.RCAg-1 polyepitope protein identified by the serum IgG antibody in the Cameroon epidemic area of Africa showed a significant positive correlation, suggesting that the vaccination of M.RCAg-1 polyepitopes in children may reduce the high mortality caused by falciparum malaria.
3.M.RcAg-1 multi epitopes protein immunized mice, New Zealand white rabbits and Ganges RIver monkeys can obtain high titer specific antibody level and induce obvious CD4~+T cell response, and the specific antibodies can effectively inhibit the growth of Plasmodium falciparum in vitro. This suggests that the proportion of the specific antibodies in the specific antibodies is larger. It may be related to the dominant conformation of M.RCAg-1 multi epitope protein.
The 4.M.RcAg-1 multi epitope gene vaccine can produce high titer antibody level in mice and rabbit models, but the specificity of the specific antibody is not high and the inhibition effect is not good in vitro. In the Ganges RIver monkey animal model, the simple gene immunity is difficult to induce the high level of antibody, and the scheme of the genetic primer protein strengthening can be effective. High level specific antibodies were induced, but like small animal models, the effect of inhibition of protozoa growth in vitro was not obvious. This may be related to the expression pattern of gene immunization, thus producing different proportion of protein immunization and the type of antibody with different affinity.
5. the results of cellular immunity test in M.RcAg-1 protein immunized mice showed that the cells with the same specificity could differentiate into the auxiliary T cells of different subgroups, which mainly depends on the conformation of the antigen and the type of cytokines secreted around.
6. Hydroethidine flow cytometry can objectively and rapidly quantify the blood flow rate of malaria parasites, and can better evaluate the synchronization effect of protozoa.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
本文编号：2130805
[Abstract]:Malaria is the most serious mosquito parasitic protozoa in the world at present. 500 million people are infected with malaria parasites every year in the world and about three million people die from malarial malaria. 80% of them are children under five years of age and pregnant women. The prevention and control of malaria has become one of the most difficult problems in the world. Due to the complexity of the life cycle of the Plasmodium, the antigen has the characteristics of stage specificity, strain difference and high variability, it has long been a technical bottleneck for the development of Plasmodium antimalarial vaccine. Therefore, the immunological response to Plasmodium falciparum in different life periods is special. The selection of multiple epitopes with different antigens inducing various types of immune responses has become a hot spot in the study of antimalarial vaccines. However, the study of multiple epitope vaccines is still mainly based on synthetic multi epitope vaccine or single fixed series multi epitopes DNA vaccine, the former is expensive and the latter has low immunogenicity. It is difficult to achieve the desired immune response diversity and satisfactory protective effect.
In the previous work, we used the principle of random recombination of molecular breeding technology (DNA regrouping), and constructed a multi epitope gene pool using epitope modification technique, and screened the positive clones with high antigenicity with the immune sera of the library. It was found that VR312 (renamed M.RCAg-1) gene clones could not only induce cross immunization protection in mice model. It can also induce Plasmodium falciparum inhibitory antibodies in rabbit models.
On the basis of the previous work, the immunogenicity and protection of M.RCAg-1 gene and protein vaccine in the mammalian model of different species were carried out, and the related epidemiological analysis of M.RCAg-1 protein was carried out, and the establishment of the flow cytometry with nucleic acid probe for detecting the blood rate of the insect was related. The following results are obtained:
The 1.M.RCAg-1 protein antigen can be identified by the serum of patients in different epidemic areas, suggesting that the M.RCAg-1 polyepitope protein vaccine can overcome the immune escape caused by the difference between the strains of the insect.
2. the M.RCAg-1 polyepitope protein identified by the serum IgG antibody in the Cameroon epidemic area of Africa showed a significant positive correlation, suggesting that the vaccination of M.RCAg-1 polyepitopes in children may reduce the high mortality caused by falciparum malaria.
3.M.RcAg-1 multi epitopes protein immunized mice, New Zealand white rabbits and Ganges RIver monkeys can obtain high titer specific antibody level and induce obvious CD4~+T cell response, and the specific antibodies can effectively inhibit the growth of Plasmodium falciparum in vitro. This suggests that the proportion of the specific antibodies in the specific antibodies is larger. It may be related to the dominant conformation of M.RCAg-1 multi epitope protein.
The 4.M.RcAg-1 multi epitope gene vaccine can produce high titer antibody level in mice and rabbit models, but the specificity of the specific antibody is not high and the inhibition effect is not good in vitro. In the Ganges RIver monkey animal model, the simple gene immunity is difficult to induce the high level of antibody, and the scheme of the genetic primer protein strengthening can be effective. High level specific antibodies were induced, but like small animal models, the effect of inhibition of protozoa growth in vitro was not obvious. This may be related to the expression pattern of gene immunization, thus producing different proportion of protein immunization and the type of antibody with different affinity.
5. the results of cellular immunity test in M.RcAg-1 protein immunized mice showed that the cells with the same specificity could differentiate into the auxiliary T cells of different subgroups, which mainly depends on the conformation of the antigen and the type of cytokines secreted around.
6. Hydroethidine flow cytometry can objectively and rapidly quantify the blood flow rate of malaria parasites, and can better evaluate the synchronization effect of protozoa.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
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