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: 10014135
• 负责人: Carole Long
• 金额: $ 98.02万
• 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/10014135
• 关键词: Address; Adult; Antibodies; Antibody Response; Antibody titer measurement; Antigens; Aotus primate; Area; Artemisinins; Attention; B-Lymphocytes; Binding; Biological Assay; Biological Markers; Biology; Biometry; Blood; Blood Tests; Child; Clinical Trials; Collaborations; Collection; Combined Vaccines; Complex; Culicidae; DNA; Data; Data Analyses; Detection; Development; Disease; Epitopes; Erythrocytes; Evaluation; Female; Foundations; Funding; Generations; Genetic; Genetic Transcription; Goals; Growth; Human; Immune response; Immune system; Immunity; Immunization; Immunology; Individual; Infection; Intervention; Japan; Laboratories; Libraries; Life Cycle Stages; Malaria; Malaria Vaccines; Mali; Mass Spectrum Analysis; Measurement; Mefloquine; Membrane; Messenger RNA; Methodology; Modeling; Monkeys; Monoclonal Antibodies; Mosquito Control; National Institute of Allergy and Infectious Disease; Oocysts; Parasites; Pathology; Pattern; Pharmaceutical Preparations; Phase; Plasmodium falciparum; Plasmodium vivax; Population; Prevalence; Procedures; Property; Proteins; Publishing; Quantitative Reverse Transcriptase PCR; RNA; Recombinant Proteins; Recombinants; Research Personnel; Resistance development; Salivary Glands; Series; Serum; Site; Source; Specificity; Sporozoites; Standardization; Surface; System; Tablets; Tanzania; Testing; Time; United States National Institutes of Health; Universities; Vaccines; Weight; Work; aptamer; asexual; base; clinical risk; drug testing; experimental study; feeding; field study; human monoclonal antibodies; humanized monoclonal antibodies; improved; in vivo; insight; malaria transmission; male; member; novel; preclinical study; prevent; protein function; response; sensor; successful intervention; transcriptome sequencing; transmission process; transmission-blocking vaccine; vaccine candidate; vector mosquito

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 we have contributed to various pre-clinical studies using our standardized growth-inhibition assay (GIA). A clinical trial using recombinant PfRH5 protein showed about 20% reduction in parasite growth. This is the first time that positive results have been seen with a blood stage vaccine and we have completed GIA studies from this trial. 2) Quantitation of the human responses to this vaccine combined with the quantitative GIA results have provided a marker which needs to be achieved to see greater protection in humans. The increment is approximately 5-fold and efforts to reach this are under development. 3)Part of this effort has shown that there are different epitopes on the PfRH5 surface and that some are critically important for function of the protein. Characterizing monoclonal antibodies (mabs) to this protein has allowed identification of mabs which themselves do not show GIA but do enhance the activity of other mabs. The functionality of these non-neutralizing antibodies seems to be to slow the invasion of merozoites into red cells. 4) In addition, we have initiated a detailed study of immune responses in field sites to PfRH5 as well as other associated molecules which are part of an invasion complex with this molecule - PfCyRPA, PfRipr, etc. Antibody titers in Malians to these proteins are very low but we are isolating their specific antibodies and are studying their functionality. 5) We have collaborated with Oxford investigators on passive transfer of monoclonal antibodies to PfRH5 into Aotus monkeys then challenged with malaria parasites. We have established a GIA threshold required for protection in vivo in this NHP system. 6) PfRH5 vaccines are now being tested in Tanzania and we are conducting GIA analysis of the human responses. 7) Other targets of merozoite immunity - AMA1. We have collaborated with Dr. Lou Miller to show that using AMA1 in conjunction with its partner protein RON2 produces antibodies with greater activity in the GIA assay. 8) We are also collaborating with Oxford University (Dr. Adrian Hill) on a major EU-funded project to develop a 3-stage multi-component vaccine for P. falciparum. 9) We have started a collaboration with Drs. Peter Crompton/Joshua Tan, and Robert Seder to do high throughput identification of human monoclonal antibodies to whole parasites and recombinant proteins of malaria using B cells from Malians as the source of the antibodies. 10) We are also collaborating with Dr. Richard Eastman (NCATS)on additional monoclonal antibody selection to PfRH5 and other parasite molecules using selected human and camelid libraries. 11) We have identified DNA aptamers which can specifically identify piperaquine and mefloquine, two of the partner drugs for artemisinin used for treatment of malaria around the world. These sensors now provide a platform for the development of a kit to detect these drugs in human blood and to test drug tablets for potency. 12) We have initiated studies of Plasmodium vivax to identify a biomarker of infection to improve detection kits. Studies on parasite sexual stages and transmission blocking vaccine candidates: 1)We have developed quantitative methodology for analysis of the standard mosquito membrane feeding assay (SMFA) to evaluate transmission blocking activity. The 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 in order to have confidence in assessing potential transmission blocking vaccine (TBV) candidates. We have worked with the Biostatistics group at NIAID to develop a model of the assay and more recently we have shown 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. 2) Search for and evaluate new possible transmission blocking vaccine candidates. Using SMFA we have evaluated a number of potential transmission blocking vaccine candidates. We have tested antibodies to significant numbers of sexual stage and mosquito vaccine candidates to compare their activity using quantitative measurements of antibody concentration and SMFA. 3) We have compared the number of oocysts with the number of sporozoites in the mosquito salivary gland. We have shown that there is a strong concordance of oocysts and salivary gland sporozoites in the SMFA. 4) Construction and evaluation of human/humanized monoclonal antibodies (mabs) to several sexual stage parasite proteins. We are currently comparing several mabs to Pfs25 and other candidates as a foundation for human clinical trials. Passive administration of these antibodies will allow evaluation of transmission blocking in vivo and might eventually be used in elimination campaigns. We are comparing them in SMFA, mapping their epitopes, and determining their binding properties. 5) We are continuing studies to examine the transcriptional pattern of P. falciparum gametocytes during differentiation in culture using RNASeq. We have obtained data on RNA transcription from gametocytes in culture and we are currently analyzing the data to identify markers which will correlate with the number of oocysts in the mosquito. In addition as part of this work we are seeking genetic targets which determine whether the gametocyte develops as a male or female. 6) We are continuing a series of experiments to determine what serum-derived factors are necessary for successful differentiation of gametocytes so that robust numbers of oocysts can be obtained after mosquito infection. Mass spectrometry of "good" and "bad" sera have shown consistent differences between them (Dr. M Llinas collaboration). 7) We have initiated studies on PfHAP2 as a novel transmission blocking vaccine candidate and we are starting new studies to identify other transmission blocking vaccine candidates with the group at Ehime University in Japan (Dr. Taka Tsuboi). 8) We are part of a consortium of investigators led by Oxford University (S. Biswas) and funded by the EU to develop a transmission blocking vaccine. Field studies of malaria transmission in Mali 1) We have assessed 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.This work has been completed and published 2)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. 3) We added a new barcoding procedure 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. 4) Using RNA collections from the same study, we used a qRT-PCR procedure to identify Pfs25 mRNA specific for gametocytes. More than 80% of people with parasites also have detectable gametocytes so that no one group can be uniquely targeted for interventions.

对恶性疟原虫的无性阶段免疫的研究 1）评估Merozoite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）正在追求该蛋白作为疫苗候选者，我们使用我们的标准化生长抑制测定法（GIA）为各种临床前研究做出了贡献。 使用重组PFRH5蛋白的临床试验显示，寄生虫生长降低了约20％。这是第一次通过血液级疫苗看到阳性结果，我们已经完成了该试验的GIA研究。 2）定量对这种疫苗的反应与定量GIA结果相结合，已经提供了一个标记，需要获得更大的保护。增量大约是5倍，而实现这一目标的努力正在开发中。 3）这项工作的一部分表明，PFRH5表面上有不同的表位，有些对蛋白质的功能至关重要。表征该蛋白质的单克隆抗体（mAb）允许鉴定mAb，而mAb本身并未显示GIA但确实增强了其他mAb的活性。这些非中和抗体的功能似乎是为了减慢梅罗寄生虫入侵红细胞的侵袭。 4）此外，我们已经详细研究了对PFRH5的现场位点的免疫反应以及其他相关分子，这些分子是该分子的入侵复合物的一部分-PFCYRPA，PFRIPR等。MALIANSMALIANS中的抗体滴定器对这些蛋白质非常低，但我们隔离了其特定的抗体和研究其功能。 5）我们已经与牛津研究人员合作，将单克隆抗体向PFRH5的被动转移到Aotus猴子中，然后对疟疾寄生虫提出了挑战。我们已经在此NHP系统中建立了在体内保护所需的GIA阈值。 6）PFRH5疫苗现在正在坦桑尼亚进行测试，我们正在对人类反应进行GIA分析。 7）其他梅罗洛伊特免疫力的靶标-AMA1。我们已经与Lou Miller博士合作，以表明将AMA1与其伴侣蛋白RON2结合使用，可产生GIA分析中活性更大的抗体。 8）我们还与牛津大学（Adrian Hill博士）合作进行了一个由欧盟资助的重大项目，以开发用于恶性疟原虫的三阶段多组分疫苗。 9）我们已经与Drs合作。 Peter Crompton/Joshua Tan和Robert Seder使用Malians的B细胞作为对整个寄生虫的人类单克隆抗体和对疟疾的重组蛋白的高吞吐量鉴定，作为抗体的来源。 10）我们还使用选定的人和骆驼图库与Richard Eastman博士（NCAT）（NCATS）进行了针对PFRH5和其他寄生虫分子的其他单克隆抗体选择。 11）我们已经确定了DNA适体，可以特异性地识别Piperaquine和Mefloquine，这是青蒿素用于治疗世界各地疟疾的两种伴侣药物。这些传感器现在为开发套件提供了一个平台，以在人类血液中检测这些药物并测试药物片的效力。 12）我们启动了对疟原虫的研究，以鉴定感染的生物标志物，以改善检测试剂盒。 寄生虫性阶段和传播阻塞疫苗候选者的研究： 1）我们开发了用于分析标准蚊子摄食测定法（SMFA）的定量方法，以评估传输阻滞活性。 SMFA是评估抗体阻止传播到蚊子的能力的标准测定法，我们对该测定法进行了深入研究，以便有信心评估潜在的传输阻断疫苗（TBV）候选者。我们已经与Niaid的Biostatistics组合作，开发了一种测定模型，最近我们首次表明，我们可以预测特定抗体对蚊子寄生虫患病率降低的影响，基于SMFA的卵囊减少和摩西群落中的卵母细胞的减少。通过抗体减少蚊子中疟疾患病率是减少田间传播的关键。 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。我们已经测试了大量性阶段和蚊子疫苗候选物的抗体，以使用抗体浓度和SMFA的定量测量进行比较其活性。 3）我们已经将卵囊的数量与蚊子唾液腺中的孢子虫数量进行了比较。我们已经表明，SMFA中有强烈的卵囊和唾液腺孢子菌的一致性。 4）对几种性阶段寄生虫蛋白的人/人源化单克隆抗体（mAb）的结构和评估。我们目前正在将几个mAB与PFS25和其他候选者进行比较，作为人类临床试验的基础。这些抗体的被动施用将允许评估体内传输阻塞，并最终可能用于消除运动。我们在SMFA中比较它们，映射其表位并确定其结合特性。 5）我们正在继续研究，以检查使用RNASEQ在培养中分化过程中恶性疟原虫的转录模式。我们已经获得了来自培养物配子细胞RNA转录的数据，目前我们正在分析数据以识别与蚊子中卵囊数量相关的标记。此外，作为这项工作的一部分，我们正在寻求遗传靶标，这些遗传靶标决定了配子细胞是男性还是女性。 6）我们正在继续进行一系列实验，以确定成功分化配子细胞所需的血清衍生因素，以便在蚊子感染后可以获得稳健的卵囊数量。 “好”和“坏”血清的质谱显示它们之间的差异一致（M Llinas协作博士）。 7）我们已经开始对PFHAP2作为一种新型传输阻断疫苗候选者的研究，我们正在开始新的研究，以识别日本Ehime University的小组的其他传播阻断疫苗候选者（Taka Tsuboi博士）。 8）我们是牛津大学（S. Biswas）领导的调查人员联盟的一部分，并由欧盟资助以开发传输阻断疫苗。 马里疟疾传播的现场研究 1）我们全年都评估了马里肯尼奥巴省居民的无性和性阶段寄生虫的存在。在2013年春季，我们启动了一项新研究（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。这项工作已完成并发布了 2）我们首次表明，全年的纵向纵向纵向患病率增加与临床疟疾风险降低有关。这表明那些持续的寄生虫运输的人获得了更强的保护性免疫。 3）我们添加了一种基于哈佛大学开发的方法，以确定一个人携带的寄生虫的不同克隆。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。 4）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。超过80％的寄生虫患者也具有可检测的配子细胞，因此没有一个人可以独特地针对干预措施。