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: 8555943
• 负责人: Carole Long
• 金额: $ 58.78万
• 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/8555943
• 关键词: Address; Adenoviruses; Adult; Affinity; Africa; Age; American; Animals; Antibodies; Antibody Formation; Antigens; Aotus primate; Area; Binding; Biological; Biological Assay; Biometry; Blood; Blood Tests; Blood specimen; Burn injury; CD147 antigen; Cell Line; Cell surface; Child; Chimeric Proteins; Chondroitin Sulfate A; Clinical; Clinical Trials; Collaborations; Complex; Computer Simulation; Culicidae; DNA; Data; Development; Disease; Educational workshop; Enzyme-Linked Immunosorbent Assay; Epitopes; Erythrocytes; Escherichia coli; European; Exposure to; Extramural Activities; Family; Flow Cytometry; Foundations; Gel; Generations; Genotype; Geographic Locations; Germany; Goals; Growth; HIV; Human; Hygiene; Immune Sera; Immune response; Immune system; Immunity; Immunization; Immunoglobulin G; In Vitro; Incidence; India; Infection; Insect Vectors; Intervention; Investigation; Japanese Population; Laboratories; Length; Libraries; Life; Life Cycle Stages; Longitudinal Studies; Malaria; Malaria Vaccines; Mali; Mass Spectrum Analysis; Membrane; Merozoite Surface Protein 1; Methods; Midgut; Monkeys; Monoclonal Antibodies; Mus; Oocysts; Oryctolagus cuniculus; Outcome; Parasitemia; Parasites; Parasitology; Pathology; Pharmaceutical Preparations; Philadelphia; Placenta; Plasmodium falciparum; Play; Population; Preclinical Testing; Pregnancy; Preparation; Procedures; Process; Proteins; Qualifying; Recombinants; Relative (related person); Reproducibility; Research Personnel; Resistance development; Resources; Role; Sampling; Seasons; Serum; Sickle Cell Trait; Societies; Specificity; Sporozoites; Staging; Surface; Surface Antigens; Switzerland; Techniques; Technology; Testing; Transgenic Organisms; Tropical Medicine; Universities; Vaccines; Variant; Woman; Work; World Health Organization; acquired immunity; aptamer; asexual; base; circumsporozoite protein; cohort; feeding; hemoglobin AA; improved; insight; meetings; member; men; next generation; novel; novel strategies; novel vaccines; posters; preclinical study; prevent; response; successful intervention; transmission process; vaccine candidate; vaccine development

During this past year (2011-2012) our laboratory built upon the foundation begun in 2008 to explore the interface between the malaria parasite and the host immune system. We have continued to collaborate with Dr. Rick Fairhurst (LMVR) and Dr. Mahamadou Diakite (MRTC) on a 4-year longitudinal study of 1400 children in 3 villages in Mali. In 2009 we identified a sub-cohort of these children, selecting those with the sickle cell trait (HbAS) and pairing them with age-matched HbAA controls. These children have been followed from 2009 to the present, with blood samples being obtained before and after the transmission season as well as during bouts of clinical malaria infection; these samples provide a unique and valuable resource for studies on the development of humoral and cellular responses to blood-stage antigens of malaria parasites. Analysis has shown that children with HbAS are significantly protected against malaria in this population; this was not true for children with the HbAC genotype. Increasing age, a surrogate of acquired immunity, was also protective. Because long-standing data has shown that antibodies can play a significant role in protection against erythrocytic stages of infection, we have pursued a detailed characterization of the antibody populations present in the Malian children. We have already shown that children with HbAS genotype show lower responses by ELISA to a number of merozoite antigens relative to age-matched controls. We believe that the lower titers in the HbAS children may result from their lower incidence of infection and we are analyzing a study of asymptomatic parasitemia in these children. During the past year we have followed these children into a new transmission season and shown that, surprisingly, their IgG levels to specific merozoite proteins do not diminish during the dry season with little malaria transmission. We are continuing to follow the antibody profiles over several transmission seasons using both ELISA and GIA as readouts and are also conducting more in depth studies of the antibodies themselves. In addition, we have developed a flow cytometry assay to profile the antibodies in these children to antigens present on the surface of parasitized erythrocytes. We have investigated changes in these antibody populations with age, with malaria exposure, and with host genotype. Another aspect of our studies of host responses to proteins on the surface of parasitized erythrocytes, we have continued our collaboration with Dr. Kavita Singh (RTB) on a major surface antigen of infected red cells, viz., VAR2CSA. This protein is a member of the large PfEMP1 family and has been implicated in pregnancy associated malaria through binding to chondroitin sulfate A (CSA) in the placenta. Dr. Singh has produced 6 domains of the VAR2CSA protein in recombinant E. coli and we have tested their recognition by antisera from Malian adults. Antibodies from multiparous Malian women but not men recognize some of these domains by ELISA. We have also standardized an opsonization assay using these IgGs and shown that FCR3 parasitized red cells expressing VAR2CSA can be efficiently opsonized by a human monocytic cell line. We have developed a novel procedure testing the ability of various domains to inhibit opsonization by these antibodies. We have thus identified several domains that are important for recognition by anti-VAR2CSA antibodies in this functional assay. Our second major area of investigation relates to the identification of malaria parasite-encoded antigens which could be the targets of new vaccines or drugs. We have hypothesized that there are conserved epitopes present on the infected red cell which could represent such targets. While most blood-stage vaccine candidates are from merozoites, antigens present on the surface of the infected red cell have significant advantages as targets because of their exposure to the serum for long periods. However, the antigenic complexity and diversity of the known surface molecules (e.g., PfEMP1) have proven daunting for vaccine development. To address this problem, we have pursued a new strategy using DNA aptamers. We had previously prepared several DNA aptamer libraries and performed repetitive selections on various targets, and we analyzed the selected aptamer populations using next-generation sequencing technology. We have focused on a small subset which binds to infected but not uninfected red cells; some of these aptamers inhibit parasite growth in vitro. This year we have completed broader studies on the reactivity profiles of this subset and shown that some recognize all the parasite isolates tested. More recently we have developed methods to affinity purify the parasite targets of the aptamers. This is a complex process, involving preparation of extracts from parasitized erythrocytes, binding to immobilized aptamers, and identification of target molecules by mass spectrometry. However, we now have bands on SDS-PAGE gels and are confident we will have identification of target molecules. Using our standardized blood-stage parasite growth inhibition assay (GIA), we have collaborated with others in analysis of several different human trials of various blood-stage vaccine candidates and in testing of preclinical animal sera. An important step forward has been made in collaboration with Dr. Simon Draper and colleagues (Oxford University), who are using recombinant adenoviruses encoding P. falciparum blood stage antigens. A new potential blood-stage vaccine candidate - PfRH5 - has been identified that elicits antibodies with very high levels of GIA activity. Binding of PfRH5 to the red cell protein basigin has been shown to be essential for merozoite invasion. We have tested antibodies to PfRH5 against parasites from different geographic locations and are evaluating antibodies from an immunization-challenge study in Aotus monkeys for functional activity. We are continuing to collaborate with Dr. James Burns (Drexel University) on his novel fusion protein of P. falciparum MSP1 and MSP8. We have shown that it elicits extremely high levels of antibodies with GIA acitivty in rabbits; this construct is being extended to preclinical studies in Aotus monkeys. We have continued to expand our studies on transmission blocking immunity and in collaboration with PATH/MVI, we are performing mosquito membrane feeding assays (MFA). This involves culturing P. falciparum sexual stages in vitro, feeding these parasites to laboratory-reared mosquitoes in the presence or absence of specific antibodies, and later counting oocysts in the mosquito midgut. To accelerate the development of transmission blocking vaccine, this year we have qualified the MFA to test its reproducibility. Using these results we have worked with Dr. Michael Fay of the Biostatistics group to develop a computerized model of the MFA which allows us to ask questions about assay variables and predict outcomes. In addition, it allows us to test sexual stage vaccine candidates for transmission blocking activity and obtain confidence limits. We have constructed and characterized a new set of mouse monoclonal antibodies to the full-length P. falciparum circumsporozoite protein prepared by Dr. Sanjay Singh at Gennova, India. Seven of these monoclonals have been produced in quantity, and we are working with extramural collaborators (Drs. Fidel Zavala and Chris Ockenhouse) to test these for passive protective activity using transgenic P. berghei parasites. Results have been presented at the American Society for Tropical Medicine and Hygiene (10 talks and posters-Philadelphia, PA)(2011). Results have been presented at the European BioMalPar meeting in Germany (2011), the Japanese Society for Parasitology (2012), a World Health Organization workshop in Geneva, Switzerland (2012), and at Gennova, India.

在过去的一年（2011年至2012年）中，我们的实验室建立在2008年始于2008年的基金会基础上，以探索疟疾寄生虫与宿主免疫系统之间的界面。我们继续与Rick Fairhurst博士（LMVR）和Mahamadou Diakite博士（MRTC）合作，对马里3个村庄的1400名儿童进行了为期4年的纵向研究。在2009年，我们确定了这些儿童的子果，选择了具有镰状细胞性状（HBA）的孩子，并将其与年龄匹配的HBAA对照组合。从2009年到现在，这些儿童已受到关注，在传播季节之前和之后以及临床疟疾感染期间获得了血液样本。这些样品为研究对疟疾寄生虫血液阶段抗原的发展和细胞反应的发展提供了独特而宝贵的资源。分析表明，患有HBA的儿童在该人群中受到明显保护的疟疾。对于患有HBAC基因型的儿童而言，事实并非如此。增加的年龄，是获得免疫的代孕，也是保护性的。 由于长期存在的数据表明，抗体可以在防御感染的红细胞阶段发挥重要作用，因此我们对马里儿童中存在的抗体种群进行了详细的特征。我们已经表明，患有HBA基因型的儿童相对于年龄匹配的对照，ELISA对许多Merozoite抗原的反应较低。我们认为，HBAS儿童的较低滴度可能是由于感染的发病率较低而导致的，我们正在分析对这些儿童无症状寄生虫血症的研究。在过去的一年中，我们一直跟随这些儿童进入一个新的传播季节，这表明，在旱季很少传播的情况下，在干旱季节，它们的IgG水平不足，在干旱季节不会减少。我们将继续使用ELISA和GIA作为读数来遵循几个传输季节的抗体曲线，并且还对抗体本身进行了更深入的研究。此外，我们开发了一种流式细胞仪测定法，以介绍这些儿童的抗体与存在于寄生的红细胞表面上的抗原。我们已经调查了随着年龄的增长，暴露疟疾和宿主基因型的这些抗体种群的变化。 我们对寄生虫红细胞表面上宿主对蛋白质反应的研究的另一个方面是，我们继续与Kavita Singh博士（RTB）的合作，在受感染的红细胞的主要表面抗原上，即VAR2CSA。该蛋白是大型PFEMP1家族的成员，通过与胎盘中的硫酸软骨素A（CSA）结合，与妊娠相关的疟疾。 Singh博士在重组大肠杆菌中生产了6个域的VAR2CSA蛋白，我们已经测试了马里人的Antisera识别。来自多妇女的抗体，但男性不认识ELISA的某些领域。我们还使用这些IgG进行了标准化的调子化测定，并表明表达VAR2CSA的FCR3寄生的红细胞可以通过人类单核细胞系有效地调查。我们已经开发了一种新的程序，测试了各种域抑制这些抗体调子化的能力。因此，我们已经确定了该功能测定中抗VAR2CSA抗体识别至关重要的几个域。 我们的第二个主要研究领域与鉴定疟疾寄生虫编码的抗原有关，这可能是新疫苗或药物的靶标。我们假设在被感染的红细胞上存在保守的表位，可以代表这种靶标。虽然大多数候选血液疫苗来自梅罗寄生疫苗，但由于长期暴露于血清，因此被感染红细胞表面存在的抗原具有显着的靶标。然而，已知的表面分子的抗原复杂性和多样性（例如PFEMP1）证明了疫苗发育的艰巨。为了解决这个问题，我们已经使用DNA适体采取了新的策略。我们以前曾准备好几个DNA适体库，并在各种目标上进行了重复选择，我们使用下一代测序技术分析了所选的适体种群。我们专注于一个与感染但未感染的红细胞结合的小子集。这些适体中的一些抑制了体外寄生虫的生长。今年，我们已经完成了有关该子集的反应性谱的更广泛的研究，并表明有些人识别所有测试的寄生虫分离株。最近，我们开发了将适体的寄生虫靶标相关的方法。这是一个复杂的过程，涉及从寄生的红细胞中制备提取物，与固定的适体结合以及通过质谱鉴定靶分子的提取物。但是，我们现在有SDS-PAGE凝胶上的频段，并且有信心我们可以识别目标分子。 使用我们标准化的血液阶段寄生虫生长抑制测定法（GIA），我们与其他人合作，分析了各种血阶段疫苗候选物的几种不同人类试验以及临床前动物血清的测试。与西蒙·德雷珀（Simon Draper）博士（牛津大学）合作迈出了重要的一步，后者使用编码编码恶性疟原虫血液阶段抗原的重组腺病毒。已经确定了一种新的潜在血阶段疫苗候选疫苗-PFRH5-引起GIA活性水平很高的抗体。 PFRH5与红细胞蛋白basigin的结合已被证明对于梅罗祖铁虫侵袭至关重要。我们已经对来自不同地理位置的寄生虫进行了对PFRH5的抗体，并正在评估AOTUS猴子的免疫挑战研究中的抗体用于功能活性。 我们将继续与James Burns博士（Drexel University）合作，他的小说《恶性疟原虫MSP1》和MSP8的融合蛋白。我们已经表明，它引起了极高的兔子抗体抗体。该构建体扩展到Aotus猴子的临床前研究。 我们继续扩大有关传输阻断免疫力的研究，并与PATH/MVI合作，我们正在进行蚊子膜进食测定法（MFA）。这涉及在体外培养恶性疟原虫的性阶段，在存在或不存在特定抗体的情况下将这些寄生虫喂入实验室饲养的蚊子，然后在蚊子中心计数卵囊。为了加快传输阻断疫苗的发展，今年我们已批准了MFA测试其可重复性。使用这些结果，我们与Biostatistics Group的Michael Fay博士一起开发了MFA的计算机模型，该模型使我们能够询问有关测定变量的问题并预测结果。此外，它使我们能够测试候选性疫苗的传输阻断活动并获得置信度限制。 我们已经构建并表征了一组新的小鼠单克隆抗体，该抗体是由印度Gennova的Sanjay Singh博士制备的全长恶性疟原虫蛋白质蛋白。这些单克隆人中有七个是数量生产的，我们正在与壁外合作者（Fidel Zavala博士和Chris Ockenhouse）合作，使用转基因P. Berghei寄生虫测试这些它们的被动保护活性。 结果已在美国热带医学和卫生学会（10次谈判和海报 - 宾夕法尼亚州）（2011年）提出。 在德国的欧洲生物瘤会议（2011年），日本寄生虫学会（2012年），瑞士日内瓦的世界卫生组织研讨会（2012年）和印度的Gennova上。