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: 8157019
• 负责人: Carole Long
• 金额: $ 80.75万
• 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/8157019
• 关键词: Antigens; Immune response; Malaria; Parasites

In 2009-2010 the Long laboratory built upon the foundation established in the previous 2 years to explore the interface between the malaria parasite and the hosts immune system. To accomplish this we have employed both clinical research studies in Mali and different rodent models of malaria infection. We have collaborated with Dr. Rick Fairhurst and Dr. Mahamadou Diakite of the MRTC in Mali to continue a 5-year longitudinal study of 1400 children of various ages in 3 villages. 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 through 2009 into 2010 for development of humoral and cellular responses to blood-stage antigens of malaria parasites. From the first year of our study it is already apparent that children with HbAS are significantly protected against malaria in this population. We have tested the hypothesis that the HbAS children develop accelerated antibody responses to blood-stage malaria antigens as a basis for their protection; however, we have shown that in fact their humoral responses are lower than their matched controls. Thus, this cannot be responsible for their increased resistance to infection. In addition to the quantitation of humoral responses by ELISA, we have purified IgG from the same children before and after the rainy season and are testing the ability of these preparations to inhibit parasite growth in vitro. Moreover, we have determined the plasma concentration of a series of cytokines before and after the rainy season. We are currently integrating all these humoral analyses and comparing the responses of children with the sickle cell trait to the control children. These results in children are also being compared with those of adults from three different ethnic groups in Mali. In addition, we are initiating a series of studies on cellular responses of the same children. We have also made significant progress in 2 other areas begun in 2009. The first is the immunologic analysis of antibody responses to the DBL3X domain of the VAR2CSA protein. This PfEMP1 protein has been implicated in pregnancy associated malaria through binding to chondroitin sulfate A (CSA) in the placenta. In collaboration with Dr. Kavita Singh, who determined the crystal structure of the DBL3X domain, we have produced monoclonal and polyclonal antibodies to this domain and shown that some of these antibodies can inhibit binding of parasitized erythrocytes expressing VAR2CSA to CSA. Thus the antibodies have functional activity. More recently, we have shown that this DBL3X domain is recognized by sera from Malian women and we mapped the CSA binding site to the S3 sub-domain of DBL3x (Singh et al, 2010). The second new area is the use of DNA aptamers to identify novel conserved antigenic determinants on the surface of malaria infected erythrocytes. While most blood-stae vaccine candidates have been directed to antigens of merozoites, we believe that antigens present on the surface of the infected red cell have significant advantages as potential vaccine candidates because of their exposure to the serum for long periods. However, the antigenic complexity and diversity of the surface molecules (e.g., PfEMP1) identified to date have proven daunting in terms of vaccine development. Consequently we have taken a novel approach to the identification of strain-conserved targets on the infected red cell membrane. We have prepared several complex DNA aptamer libraries and conducted a series of selections on various targets. To analyze the selected aptamer populations we have used next-generation sequencing technologies primarily Illumina Solexa sequencing. Using this technology we have identified over 100 aptamers with specificity for infected red cells and we have confirmed this by FACS analysis comparing uninfected and infected red cells; some of these aptamers inhibit parasite growth. Once the characterization is completed, we will proceeding to identify the molecular targets of the selected aptamers. An area which we have expanded this year is transmission blocking immunity. In collaboration with PATH/MVI, we are performing mosquito membrane feeding assays and working to standardize this assay. In addition, using a large amount of historical data, we have established the amount of anti-Pfs25 antibody (Pfs25 is an antigen of mosquito stage P. falciparum parasites) required to neutralize 50% of oocysts (IC50) in various species including humans (Cheru et al). This provides a marker for vaccine development with this antigen. Using our standardized blood-stage parasite growth inhibition assay (GIA), we have also collaborated with LMIV and others in analysis of 4 different human trials of various blood-stage vaccine candidates (Sagara et al.; Pierce et al.; Ellis et al.; El Sahly et al). With collaborators (Bruder et al.), we have also shown that recombinant adenoviruses expressing different P. falciparum blood stage antigens can elicit significant levels of antibodies with functional activity in animal studies. Finally we have continued our studies of rodent models of malaria. We are replicating our previous studies of multi-functional CD4+ T cells specific for P. yoelii MSP-8. We had identified such cells and had described their appearance in different tissues after different numbers of injections and after challenge with viable malaria parasites. We have also collaborated on a study of the importance of early interferon-gamma in a mouse model of cerebral malaria performed with P. berghei. These results have been presented at the American Society for Tropical Medicine and Hygiene (9 talks and posters)(Washington, DC, November, 2009) the Multilateral Initiative on Malaria Meeting, Nairobi, Kenya, November 2009); the American Association of Immunologists (AAI, Baltimore MD, May 2010); the Malaria Vaccines for the World, Washington, DC, Sept. 2010). In addition, 9 abstracts/talks are being presented at the 2010 meeting of the American Society for Tropical Medicine and Hygiene in Atlanta, GA.

在2009 - 2010年，长期实验室建立在过去两年中建立的基础基础上，以探索疟疾寄生虫与宿主免疫系统之间的界面。为了实现这一目标，我们在马里和不同的疟疾感染模型中都采用了临床研究。 我们与马里MRTC的Rick Fairhurst博士和Mahamadou Diakite博士合作，继续对3个村庄的1400名不同年龄的儿童进行了5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。这些儿童一直遵循2009年至2010年，以发展对疟疾寄生虫血液阶段抗原的体液和细胞反应。从我们研究的第一年开始，已经很明显，HBA患者在该人群中受到明显的保护。我们已经检验了HBAS儿童对血液阶段疟疾抗原产生加速抗体反应的假设，作为保护其保护的基础。但是，我们已经表明，实际上它们的体液反应低于其匹配的对照。因此，这不能对他们增加对感染的抵抗力负责。 除了通过ELISA对体液反应进行定量外，我们还从雨季之前和之后纯化了同一儿童的IgG，并正在测试这些制剂在体外抑制寄生虫生长的能力。此外，我们已经确定了雨季前后一系列细胞因子的血浆浓度。我们目前正在整合所有这些体液分析，并将镰状细胞特征儿童与对照儿童的反应进行比较。这些结果也与马里三个不同种族的成年人的成年人相提并论。此外，我们正在对同一儿童的细胞反应进行一系列研究。 我们还在2009年开始的其他两个区域取得了重大进展。第一个是对VAR2CSA蛋白DBL3X域的抗体反应的免疫学分析。该PFEMP1蛋白通过与胎盘中的硫酸软骨素A（CSA）结合，与妊娠相关的疟疾与妊娠有关。通过确定DBL3X结构域的晶体结构的Kavita Singh博士，我们与该结构域生产了单克隆和多克隆抗体，并表明这些抗体中的某些可以抑制向CSA表达VAR2CSA的寄生虫的寄生的红细胞结合。因此，抗体具有功能活性。最近，我们表明该DBL3X结构域被马里妇女的血清认识到，我们将CSA结合位点映射到DBL3X的S3子域（Singh等，2010）。 第二个新区域是使用DNA适体在疟疾感染的红细胞表面鉴定新的保守抗原决定因素。虽然大多数候选血液疫苗候选物都被引导到蛋白酶的抗原，但我们认为，由于长时间暴露于血清，因此受感染红细胞表面存在的抗原具有显着的优势。但是，迄今为止确定的表面分子的抗原复杂性和表面分子的多样性（例如PFEMP1）在疫苗发育方面已被证明令人生畏。因此，我们采取了一种新颖的方法来鉴定受感染的红细胞膜上的应变靶标。我们已经准备了几个复杂的DNA适体库，并对各种目标进行了一系列选择。为了分析选定的适体种群，我们使用了下一代测序技术，主要是光明Solexa测序。使用这项技术，我们已经确定了超过100多个具有特异性感染红细胞的适体，并且通过FACS分析比较未感染和感染的红细胞来证实这一点。这些适体中的一些抑制了寄生虫的生长。表征完成后，我们将继续确定所选适体的分子靶标。 我们今年扩大的区域是传输阻止免疫力。通过与PATH/MVI合作，我们正在执行蚊子膜进食测定法，并致力于标准化该测定法。此外，使用大量的历史数据，我们已经确定了抗PFS25抗体的量（PFS25是蚊子阶段的抗原抗原疟原虫寄生虫），以中和包括人类在内的各种物种（Cheru等人）中和50％的卵囊（IC50）。这为这种抗原提供了疫苗开发的标记。 使用我们标准化的血液阶段寄生虫生长抑制测定法（GIA），我们还与LMIV和其他人合作，分析了4种不同血阶段疫苗候选物的人类试验（Sagara等人； Pierce等； Ellis等； Ellis等； Els Sahly等人）。与合作者（Bruder等人），我们还表明，表达不同恶性疟原虫血液阶段抗原的重组腺病毒可以在动物研究中引起具有功能活性的显着水平的抗体。 最终，我们继续研究了疟疾啮齿动物模型。我们正在复制对P. yoelii MSP-8的多功能CD4+ T细胞的先前研究。我们已经鉴定出这种细胞，并在不同的注射量和挑战后描述了它们在不同组织中的外观。我们还合作研究了早期干扰素伽马在与伯格（P. Berghei）进行的脑疟疾的小鼠模型中的重要性。 这些结果已在美国热带医学与卫生学会（9次谈判和海报）（2009年11月，华盛顿特区）（2009年11月），2009年11月，肯尼亚内罗毕的多边疟疾倡议）； 美国免疫学家协会（AAI，巴尔的摩医学博士，2010年5月）；华盛顿特区的世界疟疾疫苗，2010年9月）。此外，在2010年在佐治亚州亚特兰大的美国热带医学和卫生学会会议上，还提出了9次摘要/谈判。