Host Immune Responses to Antigens of Malaria Parasites

宿主对疟疾寄生虫抗原的免疫反应

• 批准号: 9354823
• 负责人: Carole Long
• 金额: $ 64.18万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9354823
• 关键词: 16 year old; Address; Adult; Affect; African; Alleles; Antibodies; Antibody Formation; Antibody Response; Antigens; Aotus primate; Area; Attention; Biological Assay; Biology; Biometry; Blood; Blood Tests; Cell surface; Characteristics; Child; Clinical; Clinical Trials; Collaborations; Collection; Complex; Culicidae; DNA; Data; Data Analyses; Development; Disease; Drops; Erythrocytes; Evaluation; Female; Fingers; Frequencies; Generations; Genetic Polymorphism; Genetic Transcription; Goals; Gold; Growth; Human; Immune response; Immune system; Immunity; Immunization; Immunology; Individual; Infection; Intervention; Investigation; Laboratories; Life; Life Cycle Stages; Longitudinal Studies; Malaria; Malaria Vaccines; Mali; Manuscripts; Membrane; Messenger RNA; Methodology; Microscopy; Modeling; Molecular; Monkeys; Mosquito Control; Mus; National Institute of Allergy and Infectious Disease; Oocysts; Paper; Parasites; Pathology; Pattern; Phase; Plasmodium falciparum; Population; Preparation; Prevalence; Procedures; Proteins; Publications; Publishing; RNA; RNA analysis; Reporting; Research Personnel; Resistance development; Respiratory Burst; Sampling; Seasons; Senegal; Series; Serum; Specificity; Staging; Surface; Surface Antigens; Techniques; Testing; Time; United States National Institutes of Health; Universities; Vaccines; Variant; Weight; Work; age group; asexual; base; clinical risk; cohort; feeding; genetic signature; humanized monoclonal antibodies; improved; insight; malaria transmission; male; mathematical model; neutrophil; nonhuman primate; novel; prevent; research study; response; successful intervention; transmission process; transmission-blocking vaccine; vaccine candidate; vector mosquito; volunteer

Studies on asexual stage immunity to P. falciparum 1.) Evaluate the merozoite antigen PfRH5 as a vaccine candidate. Our collaborators at Oxford University (Dr. Simon Draper et al.) are pursuing this protein as a vaccine candidate and, as a prelude to that, have conducted an immunization-challenge trial in Aotus monkeys. We have collaborated on evaluating growth inhibition activity (GIA) in sera from the monkeys and these results have now been published; the promising results have supported a human clinical trial of PfRH5 which has recently been completed; we have analyzed results from this trial and a manuscript reporting on this is in preparation. In addition, we are preparing for another clinical trial of PfRH5 which will be initiated later this year. 2.) Investigate other targets of merozoite immunity - AMA1 While AMA1 is a prominent vaccine candidate, it has two issues:1) antigenic polymorphism so that antibody responses tend to be strain-specific and 2) insufficient antibody production to result in protective immunity in humans. Our data supports the use of a mixture of 4-5 different AMA1 alleles to overcome the polymorphism. Further, we have collaborated with LMVR investigators (Dr. Louis Miller) to show that using AMA1 in conjunction with its partner protein RON2 produces antibodies with greater activity in the GIA assay. This complex is required for triggering junction formation between the merozoite and the red cell surface. Evaluation of this combination in an immunization-challenge trial in a non-human primate (Aotus) has been conducted and the results have been submitted for publication by Dr. Miller, supporting a human clinical trial. In addition, we have contributed to a clinical trial of AMA1 in the UK. This trial showed that, even though high levels of antibody were elicited and those antibodies showed GIA activity, they did not affect the rate of parasite growth in the parasite-challenged vaccine recipients. Thus much more has to be done to achieve partial protection in humans with this vaccine. 3.) Studies of immunity to malaria in Kenieroba, Mali. Our 4-year investigation of the acquisition of immunity to malaria in Malian children represents perhaps the most detailed longitudinal study of malaria in African children that has been conducted and the results have now been published. More recently we collaborated with others to broaden the scope of anti-parasite functional activities and have added the neutrophil-dependent antibody-dependent respiratory burst assay. A manuscript on this technique has been published. 4.) We continue to work with Drs. Amy Bei and Dyann Wirth (Harvard University), who are studying changes in frequency of different P.falciparum clones over time in Senegal. We have worked with them to examine the influence of human immune responses on the changes in clonal parasite patterns, particularly related to the observation of parasites with common genetic signatures. The first results of these studies using GIA and the variant surface antigen assay were published and an expanded study has recently been completed and a manuscript submitted. Studies on parasite sexual stages and malaria transmission: 1.) Develop quantitative methodology for analysis of the standard mosquito membrane feeding assay (SMFA) to evaluate transmission blocking activity. The gold standard assay to evaluate the ability of antibodies to block transmission to mosquitoes is the SMFA, and we have performed an in depth study of this assay to define its characteristics in order to have confidence in assessing potential transmission blocking vaccine (TBV) candidates. We have shown that the SMFA is quite reproducible at high concentrations of antibody but highly variable at low concentrations, and we have worked with the Biostatistics group at NIAID to develop a mathematical model of the assay. Recently we have tested a modified model of the assay to show for the first time that we can predict the impact of a specific antibody on the reduction of P. falciparum parasite prevalence in mosquitoes based on the reduction in oocysts in an SMFA and the number of oocysts in control mosquitoes. Reduction in malaria prevalence in mosquitoes by an antibody is key to reducing transmission in the field. These results have recently been published and a detailed mathematical treatment of this work by our statistical colleagues is in press. 2.) Search for and evaluate new possible transmission blocking vaccine candidates. Using SMFA we have evaluated a number of potential transmission blocking vaccine candidates. In collaboration with colleagues at Oxford University we have produced and tested a number of known and unknown sexual stage proteins from P. falciparum. Sera from immunized mice have been evaluated by SMFA but none have shown high levels of oocyst inhibition. Further we have tested antibodies produced by collaborators to a number of other sexual stage and mosquito vaccine candidates to compare their activity. 3.) Assess the presence of asexual and sexual stage parasites in residents of Kenieroba, Mali throughout the year. In the spring of 2013 we initiated a new study (NIH 13-I-N107) to address the limited information on transmission in malaria endemic areas, and in 2014 we completed the field aspect of this study. Volunteers representing all age groups were finger pricked twice per month for 1 year to collect DNA and RNA. During the 2014-2016 time frame we completed analysis of over 10,000 samples of parasite DNA on filter paper. In November, 2013 (wet season), P. falciparum prevalence in the cohort was 37.0%, and in May of 2014 (dry season), the prevalence dropped to 10.0%. We also analyzed the longitudinal prevalence of the cohort; we observed that the 9-16 years old age group had the highest median longitudinal prevalence compared to the other age groups, and males had a higher median than females (22.0%). 4.)We showed for the first time that increasing P.falciparum longitudinal prevalence throughout the year was associated with decreasing risk of clinical malaria. This suggests that those with persistent parasite carriage acquire stronger protective immunity against clinical malaria. 5.) We added a new barcoding procedure this year based on a methodology developed at Harvard University to determine how many different clones of parasites an individual was carrying. This allowed us to show that at least 70% of infections were polygenomic, so that most people in the population are carrying more than one parasite clone at any given time. 6.) Using the RNA collections from the same study, we used a qRT-PCR procedure to identify Pfs25 mRNA specific for gametocytes. In 2015-2016 we completed RNA analysis to determine who is carrying gametocytes. Interestingly, even though you might not see them on microscopy, more than 80% of people with parasites also have detectable gametocytes by molecular criteria. This shows that no one group can be uniquely targeted for interventions to reduce transmission. 7.) With support from PATH/MVI, we have begun to evaluate humanized monoclonal antibodies to 3 different sexual/mosquito stage antigens. 8.) We have initiated studies to examine the differentiation pattern of P. falciparum gametocytes in culture for 3 weeks using RNASeq techniques. We have obtained data on RNA transcription during differentiation and we are analyzing the data to identify better markers of early and late stage gametocyte development. In addition, we are conducting a series of experiments to determine what factors are necessary for successful differentiation of gametocytes so that robust numbers of oocysts can be obtained after mosquito infection.

对恶性疟原虫的无性阶段免疫的研究 1.）评估梅罗罗兹酸盐抗原PFRH5作为疫苗候选者。我们在牛津大学的合作者（Simon Draper等人）正在追求这种蛋白质作为疫苗候选者，并且作为该蛋白的序曲，已经在Aotus Monkeys进行了免疫挑战试验。我们已经合作评估猴子血清中血清中的生长抑制活性（GIA），这些结果现已发布。有希望的结果支持了最近完成的PFRH5的人类临床试验。我们已经分析了该试验的结果，并准备了有关此试验的手稿。此外，我们正在为PFRH5的另一项临床试验做准备，该试验将于今年晚些时候开始。 2.）调查其他梅罗洛伊特免疫力的其他靶标-AMA1 虽然AMA1是候选疫苗的突出疫苗，但它有两个问题：1）抗原性多态性，因此抗体反应倾向于特异性菌株，而2）抗体产生不足，从而导致人类的保护性免疫。我们的数据支持使用4-5种不同AMA1等位基因的混合物来克服多态性。此外，我们还与LMVR研究者（Louis Miller博士）合作，以表明将AMA1与其伴侣蛋白蛋白RON2结合使用，可在GIA测定中产生具有更大活性的抗体。这种复合物是触发merozoite和红细胞表面之间的连接形成所必需的。在非人类灵长类动物（AOTU）的一项免疫挑战试验中对该组合进行了评估，结果已由米勒博士提交，支持人类的临床试验。 此外，我们为英国的AMA1进行了临床试验。该试验表明，即使引起了高水平的抗体，并且这些抗体表现出GIA活性，但它们也不会影响寄生虫挑战者的疫苗受体中寄生虫生长的速率。因此，必须采取更多措施以通过这种疫苗在人类中获得部分保护。 3.）对马里肯尼奥巴的疟疾的免疫力研究。我们对马里儿童中对疟疾的免疫的四年调查可能是对非洲儿童中疟疾最详细的纵向研究，现已发表，结果已发表。最近，我们与其他人合作，以扩大抗寄生虫功能活性的范围，并添加了中性粒细胞依赖性抗体依赖性呼吸爆发测定法。有关此技术的手稿已发表。 4.）我们继续与Drs合作。艾米·贝伊（Amy Bei）和迪恩·沃思（Dyann Wirth）（哈佛大学），他们正在研究塞内加尔随时间的时间频率的变化。我们已经与他们合作，研究了人类免疫反应对克隆寄生虫模式变化的影响，尤其是与具有常见遗传特征的寄生虫观察有关。这些研究使用GIA和变体表面抗原测定法的第一个结果发表了，最近完成了一项扩展的研究并提交了手稿。 寄生虫性阶段和疟疾传播的研究： 1.）开发定量方法来分析标准蚊子膜进食测定法（SMFA）以评估传输阻滞活性。 SMFA是评估抗体阻断向蚊子传播能力的黄金标准测定法，我们对此测定法进行了深入研究以定义其特征，以便有信心评估潜在的传播阻断疫苗（TBV）候选者。我们已经表明，在高浓度的抗体下，SMFA是相当可重现的，但在低浓度下变化很大，并且我们已经与NIAID的生物统计学组合作开发了该测定的数学模型。最近，我们首次测试了一个修改的测定模型，以证明我们可以预测特定抗体对蚊子中蚊子寄生虫患病率的减少的影响，这是基于SMFA中的卵囊减少以及对照蚊子中的卵囊数量的减少。通过抗体减少蚊子中疟疾患病率是减少田间传播的关键。这些结果最近已经发布，我们的统计同事对这项工作进行了详细的数学处理。 2.）搜索并评估新的可能的传输阻断疫苗候选物。使用SMFA，我们评估了许多潜在的传输阻断疫苗候选物。在牛津大学与同事合作，我们生产并测试了恶性疟原虫的许多已知和未知的性阶段蛋白质。已通过SMFA评估了免疫小鼠的血清，但没有显示出高水平的卵囊抑制作用。此外，我们还测试了合作者生产的抗体，以比较许多其他性阶段和蚊子疫苗来比较其活动。 3.）评估全年马里肯尼奥巴居民的无性和性阶段寄生虫的存在。 2013年春季，我们开始了一项新的研究（NIH 13-I-N107），以解决有关疟疾流行地区传播的有限信息，并在2014年完成了这项研究的领域方面。代表所有年龄段的志愿者每月两次手指刺1年，以收集DNA和RNA。在2014 - 2016年期间，我们完成了对滤纸上10,000多个寄生虫DNA样品的分析。 2013年11月（潮湿季节），该队列的恶性疟原虫患病率为37.0％，2014年5月（旱季），患病率下降到10.0％。我们还分析了队列的纵向患病率。我们观察到，与其他年龄组相比，9-16岁年龄段的纵向患病率最高，男性的中位数高于女性（22.0％）。 4.）我们首次表明，全年的纵向纵向纵向患病率的增加与临床疟疾风险降低有关。这表明那些持续的寄生虫托架的人可以对临床疟疾获得更强的保护性免疫。 5.）我们根据哈佛大学开发的方法来确定一个人正在携带的寄生虫有多少种不同的寄生虫，我们今年添加了一种新的条形码程序。这使我们能够表明至少有70％的感染是多基因组，因此在任何给定时间，大多数人口中的大多数人都携带多个寄生虫克隆。 6.）使用同一研究中的RNA收集，我们使用QRT-PCR程序来识别针对配子细胞特异的PFS25 mRNA。在2015 - 2016年，我们完成了RNA分析，以确定谁携带配子细胞。有趣的是，即使您可能看不到显微镜，超过80％的寄生虫患者也通过分子标准可以检测到可检测的配子细胞。这表明，没有一个人可以独特地针对干预措施来减少传输。 7.）在PATH/MVI的支持下，我们开始评估对3种不同性/蚊子阶段抗原的人源化单克隆抗体。 8.）我们已经开始研究使用RNASEQ技术在培养中检查培养物中的恶性疟原虫植物细胞的分化模式。我们已经在分化过程中获得了RNA转录的数据，并且正在分析数据，以确定早期和晚期配子体发展的更好标记。此外，我们正在进行一系列实验，以确定成功分化配子细胞的必要因素，以便在蚊子感染后可以获得稳健数量的卵囊。