恶性疟疾多表位蛋白疫苗M.RCAg-1和M.RCAg-3不同佐剂配伍的研究

发布时间：2018-03-10 10:06

本文选题：恶性疟原虫　切入点：多表位疫苗　出处：《北京协和医学院》2011年硕士论文　论文类型：学位论文

【摘要】：疟疾是当今世界三大传染性疾病之一。根据WHO最新统计数据显示,2009年全球2.25亿人感染疟疾,有78.1万人因其死亡,而且大多数是5岁以下儿童和孕妇[1]。在所有疟原虫中,恶性疟是危害最严重、致死率最高的一种。在疟疾控制的过程中不断出现恶性疟原虫及按蚊抗药性,这就加剧了人们对于预防性和治疗性疟疾疫苗的需求。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,目前针对单个抗原或表位的疫苗在相关实验中未取得较好的效果,因此多表位嵌合疫苗就成为研究抗疟疫苗的热点[2]。本课题组前期工作中,我们利用表位随机重组技术构建了多表位基因库[3],并用库免疫血清筛选出免疫原性最佳、稳定性最好的人工随机组合抗原M.RCAg-1和M.RCAg-3.用弗氏佐剂在小鼠和新西兰兔动物模型中证实二者具有较强的免疫原性并可在体外抑制恶性疟原虫的生长(Growth Inhibi tion Assay.GIA).换用佐剂Montanide ISA51(Seppic,France)免疫恒河猴后发现局部刺激性较大。因此,本研究目的是选择新的抗原佐剂使M.RCAg-1和M.RCAg-3能顺利用于临床观察.本研究在前期工作的基础上,根据第二代多表位嵌合抗原疫苗M.RCAg-3的理化性质,利用阴离子交换柱、疏水作用层析柱和分子筛,摸索总结出了第二代多表位嵌合抗原疫苗M.RCAg-3的纯化步骤。并且选用了有临床应用前景或正在临床应用的佐剂(CpG.Al.AD.ADL)与M.RCAg-1和M.RCAg-3配伍,以提高其免疫原性。还用纳米粒包裹蛋白或蛋白加佐剂来免疫实验动物,以探索新的抗原递呈途径,增加抗原的免疫原性。通过对不同组别免疫效果的评价来分析其相应的佐剂效应。最终选择ADL和AD分别与M.RCAg-1和M.RCAg-3配伍免疫大白兔,验证其抗体的体外保护效果。主要取得以下进展： 1.摸索出纯化第二代多表位嵌合抗原疫苗M.RCAg-3的纯化步骤,最终纯化结果可达到98.8%。 2.筛选与M.RCAg-1和M.RCAg-3相配伍的佐剂。佐剂ADL和AD分别能显著增强M.RCAg-1和M.RCAg-3免疫反应。而佐剂CpG和CpGA1增强抗原免疫反应的能力相对较弱。初步确定用纳米包裹递呈蛋白的方法不适合M.RCAg-1和M.RCAg-3。 3. M. RCAg-1(小试)、M.RCAg-1(中试)和M.RCAg-3分别与佐剂ADL AD1和AD配伍免疫大白兔后,总IgG和特异性IgG的GIA水平接近WHO规定的阳性对照抗体的体外生长抑制试验(GIA)水平。
[Abstract]:Malaria is one of the three most infectious diseases in the world today. According to the latest WHO statistics, in 2009, 225 million people worldwide were infected with malaria, 781,000 people died from malaria, and most of them were children under the age of five and pregnant women. [1] of all malaria parasites, Falciparum malaria is one of the most harmful and lethal species. Plasmodium falciparum and Anopheles mosquito resistance continue to occur during malaria control. This has increased the demand for preventive and therapeutic malaria vaccines. Due to the complexity of the life cycle of Plasmodium, the antigens are phase specific, the strain diversity and high variability, and so on. At present, the single antigen or epitope vaccine has not achieved good results in related experiments, so multi-epitope chimeric vaccine has become a hot spot in the research of antimalarial vaccine [2]. In our earlier work, we constructed a multiepitope gene bank by using epitope random recombination technique, and screened out the best immunogenicity by using library immune serum. M. RCAg-1 and M.RCAg-3. the best artificial random combinations of antigens M.RCAg-1 and M.RCAg-3. Freund's adjuvants were used in mice and New Zealand rabbits to confirm their strong immunogenicity and to inhibit the growth of Plasmodium falciparum Inhibi tion Assay.GIAA in vitro. The rhesus monkey was immunized with Montanide ISA51 and Seppica France. The aim of this study was to select a new antigen adjuvant so that M.RCAg-1 and M.RCAg-3 could be used in clinical observation successfully. Based on the previous work, based on the physicochemical properties of the second generation multiepitope chimeric antigen vaccine M.RCAg-3, the anion exchange column was used. The purification steps of the second generation multiepitope chimeric antigen vaccine M.RCAg-3 were summarized by hydrophobic interaction chromatography column and molecular sieve, and the adjuvant CpG.Al.AD.ADL, which has clinical application prospect or is in clinical use, was selected to be compatible with M.RCAg-1 and M.RCAg-3. In order to improve its immunogenicity, the experimental animals were immunized with nanoparticles encapsulated protein or protein and adjuvant to explore new ways of presenting antigen. The immunogenicity of antigens was increased. The adjuvant effect was analyzed by evaluating the immune effects of different groups. Finally, ADL and AD were selected to match M.RCAg-1 and M.RCAg-3 to immunize rabbits in vitro to verify the protective effect of their antibodies in vitro. The following progress has been made:. 1. The purification steps of the second generation multiepitope chimeric antigen vaccine M.RCAg-3 were found, and the final purification result was 98.8%. 2. Screening adjuvants compatible with M. RCAg-1 and M.RCAg-3. Adjuvant ADL and AD could significantly enhance the immunoreaction of M.RCAg-1 and M.RCAg-3, respectively. The ability of adjuvant CpG and CpGA1 to enhance the immunoreaction of antigen was relatively weak. The method is not suitable for M.RCAg-1 and M.RCAg-3. 3. After inoculating rabbits with adjuvant ADL AD1 and AD respectively, the GIA levels of total IgG and specific IgG of M. RCAg-1 (pilot-scale test) and M.RCAg-3 were close to the in vitro growth inhibition test (GIA) of the positive control antibody specified by WHO.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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