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: 10272127
• 负责人: Carole Long
• 金额: $ 91.17万
• 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/10272127
• 关键词: Address; Adult; Africa; Anti-malarial drug resistance; Antibodies; Antibody Response; Antibody titer measurement; Antigens; Antimalarials; Area; Artemisinins; Attention; B-Lymphocytes; Binding; Biological Assay; Biology; Biometry; Blood; Blood Tests; Bone Marrow; Buffaloes; Child; Clinical Trials; Collaborations; Combined Modality Therapy; Complex; Culicidae; DNA; Data Analyses; Detection; Development; Disease; Drug usage; Epitopes; Erythrocytes; Evaluation; Exposure to; Funding; Generations; Genetic; Genetic Transcription; Goals; Growth; Health; Human; Immune response; Immune system; Immunity; Immunization; Immunoglobulin G; Immunology; In Vitro; Infection; Institutes; Investigation; Japan; Laboratories; Libraries; Malaria; Malaria Vaccines; Measurement; Mefloquine; Membrane; Methods; Modeling; Monitor; Monoclonal Antibodies; Myeloid Cells; National Institute of Allergy and Infectious Disease; New York; Oocysts; Parasites; Pathology; Patients; Pattern; Performance; Pharmaceutical Preparations; Plasmodium falciparum; Plasmodium vivax; Prevalence; Process; Proteins; Publications; Publishing; Recombinant Proteins; Recombinants; Research Personnel; Resistance development; Salivary Glands; Sampling; Series; Serum; Single-Stranded DNA; Source; Specificity; Sporozoites; Standardization; Structure; Surface; Tablets; Tanzania; Techniques; Testing; Time; Universities; Vaccines; Weight; Work; analytical method; analytical tool; aptamer; asexual; base; drug efficacy; drug quality; drug standard; drug testing; experimental study; feeding; field study; healthy volunteer; human monoclonal antibodies; humanized monoclonal antibodies; improved; in vivo; malaria transmission; member; novel; polyclonal antibody; prevent; protein function; response; sensor; sex; small molecule; synergism; tool; transcriptome; transcriptome sequencing; transmission process; transmission-blocking vaccine; vaccine candidate; vaccine development; 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 studies using our standardized growth-inhibition assay (GIA). A clinical trial using recombinant PfRH5 protein showed 20% reduction in parasite growth. This is the first time that positive results have been seen with a blood stage vaccine in a challenge trial. We have completed GIA studies from this trial, showing that the GIA results correlated with the reduced parasite growth rate. For the first time these results provide a marker to show what level of GIA should be necessary to obtain protection in humans. This has been submitted for publication. 2)Two versions of this PfRH5 vaccine are now being tested in field trials conducted by the Institute for Health Initiatives in Tanzania. We are currently evaluating the GIA results from trial samples. 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 identified 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. This has been published. 4) We have initiated a detailed study of immune responses to PfRH5 as well as other associated molecules which are part of the invasion complex with this molecule PfCyRPA and PfRipr. Antibody titers in Malians exposed to these proteins by repeated infection are very low. To determine interaction (synergistic, additive, or antagonistic) between preexisting anti-malaria immunity and vaccine induced anti-RH5 antibodies, we performed GIA with mixtures of purified Malian total IgGs and several different human anti-Rh5 polyclonal and monoclonal Abs provided by Dr. Simon Draper. Overall interactions were additive; however, a proportion of Malian IgGs showed synergistic effects when mixed with anti-RH5 mAbs, and a proportion of other Malian IgGs showed sub-additive interactions with polyclonal antibodies. The importance of these findings is that we did not observe substantial antagonistic effects which were previously seen with AMA1 vaccines. 5) Other targets of merozoite immunity -Pf AMA1. We have collaborated with Dr. Lou Miller to show that using AMA1 in conjunction with its partner protein RON2 elicits antibodies with greater activity in the GIA assay. In addition, we have examined methods for analysis of additive and synergistic effects of antibodies to AMA1-RON2 with antibodies to other parasite antigens including PfRH5, PfRipr, and PfCyRPA. This has been published and it includes an online analytic tool that can be used by other investigators to establish additivity or synergy of antibody combinations. 6) Collaborative projects on anti-malarial monoclonal antibodies. We have started a collaboration with Drs. Peter Crompton/Joshua Tan to aid in characterizing human monoclonal antibodies to whole parasites and recombinant proteins of malaria using B cells from Malians as the source of the antibodies. We are also collaborating with Dr. Richard Eastman (NCATS) on selecting and characterizing additional monoclonal antibodies to PfRH5 and other parasite molecules using various human and camelid libraries. 7) Tracking antimalarial drug use and efficacy is essential for monitoring the current spread of antimalarial drug resistance. However, available methods for assessing patient drug levels and tablet quality are often inaccessible. We have addressed this problem by developing aptamer-based fluorescent sensors for the rapid, specific detection of the antimalarial compounds piperaquine and mefloquine two slow-clearing partner drugs in first-line artemisinin-based combination therapies (ACTs). DNA aptamers were identified that bind piperaquine and mefloquine with high selectivity over similarly structured small molecules. The aptamers were isolated from a library of single-stranded DNA molecules using a capture-SELEX technique then adapted into structure-switching aptamer fluorescent sensors. Sensor performance was optimized for the detection of drug from human serum and crushed tablets.The patient sample platform was validated against an LC-MS standard drug detection method in healthy volunteer and malaria patient samples. This assay provides a rapid and inexpensive method for tracking antimalarial drug use and quality. This work has been submitted for publication. 8) We are collaborating with Dr. Adrian Hill of Oxford University on a major EU-funded project to develop a 3-stage, multi-component vaccine for P. falciparum. Studies on parasite sexual stages and transmission blocking vaccine candidates: 1)The standard assay to evaluate the ability of antibodies to block malaria transmission to mosquitoes is the Standard Membrane Feeding Assay (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 SMFA results. 2)This year we have extended this analysis to Plasmodium vivax and showed that similar analytic methods and models could be applied to SMFA with this parasite. This has been published. 3) Search for and evaluate new possible transmission blocking vaccine candidates. In collaboration with PATH/MVI, we have evaluated a number of potential transmission blocking vaccine candidates and vaccine formulations by SMFA. 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. 4) We have also initiated studies with Dr. Taka Tsuboi at Ehime University in Japan to identify other transmission blocking vaccine candidates. This work is in progress. 5) 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. This work has now been published. 6) We continue a collaboration with Dr. Jonathan Lovell (New York State University at Buffalo) to characterize antibody interaction using various sexual stage antigens and vaccine formulations. 7) Evaluation of human/humanized monoclonal antibodies (mabs) to several sexual stage parasite proteins. We are working with various investigators (including Dr. Patrick Duffy of LMIV) to compare mabs to Pfs25 and other sexual stage vaccine candidates by SMFA. 8) We are continuing studies to examine the transcriptional pattern of P. falciparum gametocytes during differentiation in culture using RNASeq. We are currently analyzing the data to provide a detailed transcriptome of this differentiation process. We are seeking genetic targets which determine sex assignment of the gametocytes. 9)The differentiation of malaria gametocytes often takes place within the bone marrow of the vertebrate host. We have initiated a project to explore the interaction of developing gametocytes with myeloid cells within the bone marrow. 10) We are continuing a series of experiments to determine what serum-derived factors are necessary for successful differentiation of gametocytes. 11) We are part of a consortium of investigators led by Dr. Sumi Biswas of Oxford University and funded by the EU to develop a transmission blocking vaccine.

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，我们使用标准化的生长抑制测定法（GIA）为研究做出了贡献。 使用重组PFRH5蛋白的临床试验显示，寄生虫生长降低了20％。这是在挑战试验中首次看到血液阶段疫苗的阳性结果。我们已经完成了该试验的GIA研究，表明GIA结果与降低的寄生虫生长速率相关。这些结果首次提供了标记，以表明在人类中获得保护应需要的GIA水平。这已提交出版。 2）现在，该PFRH5疫苗的两个版本正在坦桑尼亚卫生计划进行的现场试验中进行测试。我们目前正在评估试验样本的GIA结果。 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）已经鉴定出MABS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。这已经出版了。 4）我们已经开始了对PFRH5的免疫反应以及其他相关分子的详细研究，这些分子与该分子Pfcyrpa和PFRIPR是入侵复合物的一部分。通过反复感染暴露于这些蛋白质的马里人的抗体滴度非常低。为了确定先前存在的抗马拉症免疫力和疫苗诱导的抗RH5抗体之间的相互作用（协同，加性或拮抗），我们用纯化的Malian Total IgG和几种不同的人类抗RH5多克罗纳尔和单colonalal allaL和单网alnal ABS提供了GIA。总体相互作用是加性的；然而，当与抗RH5 mAb混合时，一部分马里IgG显示出协同作用，而其他马里IgG的比例显示出与多克隆抗体的亚添加相互作用。这些发现的重要性是，我们没有观察到以前从AMA1疫苗中看到的实质性拮抗作用。 5）其他梅罗罗兹免疫的靶标-PF 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，在GIA测定中引起了具有更大活性的抗体。此外，我们还研究了分析抗AMA1-RON2抗体的添加剂和协同作用的方法，该抗体具有与PFRH5，PFRIPR和PFCYRPA在内的其他寄生虫抗原的抗体。这已经发表了，其中包括一种在线分析工具，可以由其他研究人员使用，以建立抗体组合的增强性或协同作用。 6）抗疟疾单克隆抗体的合作项目。我们已经开始与Drs合作。 Peter Crompton/Joshua tan有助于将人类单克隆抗体与疟疾的整个寄生虫和重组蛋白进行表征，并使用马里人的B细胞作为抗体的来源。我们还与理查德·伊斯特曼（Richard Eastman）博士（NCATS）合作，使用各种人和骆驼库来选择和表征PFRH5和其他寄生虫分子的其他单克隆抗体。 7）跟踪抗疟药的使用和功效对于监测抗性耐药性的当前扩散至关重要。但是，评估患者药物水平和片剂质量的可用方法通常无法获得。我们通过开发基于适体的荧光传感器来解决这个问题，以快速，特定的检测抗性化合物Piperaquine和Mefloquine在一线基质素组合疗法中（ACT）中的两种慢清除伴侣药物。鉴定出DNA适体与类似结构的小分子相对于类似结构化的小分子结合载管和甲氟喹。使用捕获 - 塞雷克斯技术从单链DNA分子的库中分离出适体，然后改编成结构切换的适体荧光传感器。优化了传感器性能，可用于从人血清和碎片粉碎的药物中检测。患者样品平台通过健康志愿者和疟疾患者样品中的LC-MS标准药物检测方法进行了验证。该测定法提供了一种快速且廉价的方法来跟踪抗疟药的使用和质量。这项工作已提交出版。 8）我们正在与牛津大学的阿德里安·希尔（Adrian Hill）合作，开发了一个由欧盟资助的主要项目，以开发用于恶性疟原虫的三阶段的多组分疫苗。 寄生虫性阶段和传播阻塞疫苗候选者的研究： 1）评估抗体阻止疟疾向蚊子传播的能力的标准测定是标准的膜进食测定法（SMFA），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。我们与NIAID的生物统计学组合作，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基于SMFA结果的蚊子中寄生虫患病率的降低的影响。 2）今年，我们将此分析扩展到了体内疟原虫，并表明可以使用该寄生虫将类似的分析方法和模型应用于SMFA。这已经出版了。 3）搜索并评估新的可能的传输阻塞疫苗候选物。通过与PATH/MVI合作，我们通过SMFA评估了许多潜在的传输阻断疫苗和疫苗制剂。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。 4）我们还向日本埃希姆大学的Taka Tsuboi博士进行了研究，以识别其他传输阻断疫苗候选者。这项工作正在进行中。 5）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。这项工作现已发布。 6）我们继续与乔纳森·洛维尔（Jonathan Lovell）（布法罗纽约州立大学）合作，以使用各种性阶段抗原和疫苗配方来表征抗体相互作用。 7）评估人/人源化单克隆抗体（mAb）对几种性寄生虫蛋白的评估。我们正在与各种调查人员（包括LMIV的Patrick Duffy博士）合作，将MAB与SMFA的PFS25和其他性阶段疫苗候选者进行比较。 8）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。我们目前正在分析数据，以提供此分化过程的详细转录组。我们正在寻求确定配子细胞性别分配的遗传靶标。 9）疟疾配子细胞的分化通常发生在脊椎动物宿主的骨髓内。我们已经启动了一个项目，以探索骨髓中与髓样细胞发展的配子细胞的相互作用。 10）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。 11）我们是牛津大学Sumi Biswas博士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。