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: 8946421
• 负责人: Carole Long
• 金额: $ 63.67万
• 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/8946421
• 关键词: Address; Adult; African; Age; Alleles; American; Anemia; Animals; Antibodies; Antibody Formation; Antigen Presentation; Antigens; Aotus primate; Area; Attention; Binding; Biological Assay; Biology; Biometry; Blood; Blood Tests; Cell surface; Cells; Cessation of life; Characteristics; Child; Chondroitin Sulfate A; Clinical; Clinical Trials; Collaborations; Complex; Computer Simulation; Culicidae; DNA; Data; Development; Disease; Enzyme-Linked Immunosorbent Assay; Epitopes; Erythrocytes; Evaluation; Family; Fingers; Flow Cytometry; Frequencies; Generations; Genes; Genetic Polymorphism; Glutathione S-Transferase; Goals; Gold; Growth; Heme; Human; Hypertension; Immune response; Immune system; Immunity; Immunization; Individual; Infection; Inflammatory Response; Intervention; Invaded; Investigation; Laboratories; Life; Life Cycle Stages; Longitudinal Studies; Low Birth Weight Infant; Malaria; Malaria Vaccines; Mali; Measures; Membrane; Methodology; Modeling; Monoclonal Antibodies; Mus; National Institute of Allergy and Infectious Disease; Oocysts; Paper; Parasites; Pathology; Phase; Placenta; Plasmodium falciparum; Pregnancy; Premature Birth; Procedures; Process; Protein Family; Proteins; RNA; Reading; Recombinants; Research Personnel; Resistance development; Respiratory Burst; Reticulocytes; Reverse Transcriptase Polymerase Chain Reaction; Salivary Glands; Sampling; Senegal; Serum; Sickle Cell Trait; Specificity; Sporozoites; Staging; Surface; Techniques; Testing; Time; United States National Institutes of Health; Universities; Vaccines; Variant; Venipunctures; Weight; Woman; Work; artesunate; asexual; base; density; feeding; improved; insight; men; monocyte; neutrophil; nonhuman primate; novel; novel vaccines; pathogen; prevent; response; successful intervention; tool; transmission process; unborn child; vaccine candidate; vaccine development; vector mosquito; volunteer

Studies on asexual stage immunity to P. falciparum 1. Search for conserved, parasite-encoded epitopes on the surface of malaria IEs Pregnancy associated malaria (PAM) remains a major threat to women and their unborn children in endemic areas. IEs accumulate in the placenta and the ensuing inflammatory response can increase the likelihood of anemia, hypertension, premature delivery and possible death of low birth weight infants. Clinical immunity to PAM in multigravid women has been attributed to antibodies that recognize VAR2CSA, a large multi-domain variant of the PfEMP-1 family, which binds to chondroitin sulfate A (CSA), on the surface of red cells. Opsonization of infected erythrocytes by cytophilic antibodies that recognize VAR2CSA epitopes represents an understudied host effector mechanism in PAM. We have established a flow cytometry-based opsonization assay and we have shown that, in contrast to malaria-nave American adults and malaria-exposed Malian men, purified IgGs from multigravid Malian women showed higher 1) reactivity to recombinant DBL domains by ELISA, 2) binding to red cells expressing VAR2CSA, and 3) opsonization of these infected erythrocytes by human monocytic cells. Importantly, preincubation of IgGs from multigravid women with selected VAR2CSA domains significantly diminished opsonization of VAR2CSA-expressing IEs by monocytes. Antigen reversal of opsonization provides the first evidence that domains DBL3x, DBL5ε, and DBL2x, are the primary targets of these opsonizing antibodies. Our study focuses attention on these domains for PAM vaccine development and introduces a new tool to identify targets of host effector responses to pathogens. 2. Investigate targets of merozoite immunity a. 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 as well as that of our collaborators supports the use of a mixture of 4-5 different AMA1 alleles to overcome the polymorphism and elicit broadly reactive antibodies. Further, we have collaborated with LMVR investigators to show that using AMA1 in conjunction with its partner protein RON2 produces antibodies with greater activity in a standardized parasite growth inhibition assay (GIA). 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) is ongoing to obtain supportive data for a human clinical trial. b. We continue to work with other investigators on two different merozoite protein families - the reticulocyte binding-like family (RBL) and the erythrocyte binding-like (EBL) proteins as novel candidates either as single proteins or as mixtures. We have collaborated with others from LIG to show that antibodies to RH5 isolated from those living in Mali do have GIA activity, lending support to this protein as a new vaccine candidate. Also we have evaluated the GIA activity of antibodies to both RH5 and the RIPr protein (RH5 interacting protein) which appear together on the surface of the invading merozoite. Each protein elicits strong GIA activity. 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, and it reveals the impact of the sickle cell trait (HbAS) in protection against malaria. We have evaluated antibody responses of these children in several different assays including ELISA and GIA, and in 2014 we are adding other functional measures such as the neutrophil-dependent antibody-dependent respiratory burst assay and opsonization assays to obtain a broad scope of anti-parasite functional activities. 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 and extend this to parasites in the field. 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. 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 computer model of the assay. In 2014 we have worked with others at LMVR and at FDA to develop improved read-outs for this assay. 2. In 2014 we used the SMFA assay to assess whether children immunized with the RTS,S vaccine (the most advanced malaria vaccine candidat) could develop antibodies that would block transmission. We were not able to show an impact of the polyclonal human or monoclonal antibodies on transmission to mosquitoes whether measured by reductions in oocyst density or sporozoites in salivary glands. 3. Search for and evaluate new possible transmission blocking vaccine candidates. Using SMFA in 2014 we have evaluated a number of potential transmission blocking vaccine candidates as well as different presentations of these antigens. In collaboration with colleagues at Oxford University we have produced and tested a number of known and unknown sexual stage proteins from P. falciparum. These proteins have been produced in HEK293 cells and the products were used to immunize mice. Sera from these animals are being evaluated in the SMFA. 4. Assess the presence of asexual and sexual stage parasites in Kenieroba residents throughout the year. In the spring of 2013 we initiated a new study (NIH 13-I-N107) of malaria transmission in Kenieroba to address the limited information on actual transmission in malaria endemic areas, and in 2014 we completed the field aspect of this study. Volunteers were finger pricked twice per month for 1 year to collect DNA and RNA. During this year we have worked out the methodology for the PCR to detect parasite DNA on filter papers and also the RT-PCR procedure to identify RNA specific for gametocytes. Using these techniques, we are evaluating the carriage of parasite DNA as well as gametocytes in 500 villagers of all ages. This will identify those individuals capable of malaria transmission who might be targeted for intervention. 5. Determine whether Kenieroba, Mali residents develop antibodies which have blocking activity in SMFA. The Kenieroba study of transmission includes a venipuncture sample from all the volunteers 3 times during the year. These larger volume serum samples will be used in SMFA to determine whether antibodies have transmission blocking activity. 6. Determine the relationship between the SMFA assay and the DMFA, which measures the transmission blocking activity of antibodies to sexual stage parasites from infected people in the field. We are evaluating whether models that we have developed that work with SMFA are also applicable to DMFA. Other studies: 1) We have collaborated with Dr. Akhil Vaidya of Drexel University in showing that asexual P. falciparum parasites do not require synthesis of heme, but it is necessary for sexual stage differentiation and infection of mosquitoes; 2) In collaboration with Dr. Olivier Lichtarge at Baylor University we have provided supporting data for a new strategy to identify function of unknown genes(supergenomic network compression) and have aided evaluation of PfEXP1 as a glutathione transferase which can be inhibited by artesunate;3)We continue to work with Drs. Amy Bei and Dyann Wirth to profile the changes in P. falciparum clonal frequency in Senegal and the influence of immune responses on that process.

对恶性疟原虫的无性阶段免疫的研究 1。在疟疾表面搜索保守的寄生虫编码的表位 与妊娠相关的疟疾（PAM）仍然是对地方性地区妇女及其未出生儿童的主要威胁。 IE在胎盘中积聚，随之而来的炎症反应会增加贫血，高血压，过早分娩以及低出生体重婴儿可能死亡的可能性。多格拉维女性对PAM的临床免疫归因于识别var2csA的抗体，抗体是PFEMP-1家族的大型多域变体，该抗体与红细胞表面与硫酸软骨素A（CSA）结合。通过识别VAR2CSA表位的细胞抗体对感染的红细胞的调整化表示PAM中的一种正在研究的宿主效应器机制。 We have established a flow cytometry-based opsonization assay and we have shown that, in contrast to malaria-nave American adults and malaria-exposed Malian men, purified IgGs from multigravid Malian women showed higher 1) reactivity to recombinant DBL domains by ELISA, 2) binding to red cells expressing VAR2CSA, and 3) opsonization of these infected erythrocytes by human monocytic细胞。重要的是，由单核细胞从具有选定的VAR2CSA结构域的多格拉维妇女的IgG进行预孵育，从而显着减少了表达VAR2CSA表达IE的IES。抗原反转提供了第一个证据，表明域DBL3X，DBL5ε和DBL2X是这些调子化抗体的主要靶标。我们的研究将注意力集中在PAM疫苗开发的这些领域上，并引入了一种新工具，以识别宿主效应子对病原体的反应的靶标。 2。研究梅罗罗派免疫的靶标 一个。 AMA1 - 虽然AMA1是一个突出的疫苗候选者，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和2）2）不足的抗体产生以导致人类​​保护性免疫。我们的数据以及我们的合作者支持4-5种不同AMA1等位基因的混合物来克服多态性并引起广泛反应性的抗体。此外，我们已经与LMVR研究人员合作，表明将AMA1与其伴侣蛋白RON2结合使用，在标准化的寄生虫生长抑制测定法（GIA）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）的免疫挑战试验中对此组合的评估正在进行中，以获取人类临床试验的支持性数据。 b。我们将继续与其他研究者一起在两个不同的蛋白蛋白家族上合作 - 网状细胞结合家族（RBL）和红细胞结合样蛋白（EBL）蛋白作为单蛋白或混合物。我们已经与其他从LIG合作，以表明与居住在马里的人分离的RH5的抗体确实具有GIA活动，对这种蛋白质作为新疫苗候选者提供了支持。另外，我们还评估了抗Rh5和RIPR蛋白（RH5相互作用蛋白）的GIA活性，这些蛋白（RH5相互作用蛋白）一起出现在入侵的Merozoite表面上。每种蛋白质都会引起强大的GIA活性。 3。对马里肯尼奥巴的疟疾免疫的研究。我们对马里儿童中对疟疾免疫的四年调查可能是对非洲儿童疟疾最详细的纵向研究，它揭示了镰状细胞性状（HBAS）对疟疾的影响的影响。我们已经评估了这些儿童在包括ELISA和GIA在内的几种不同测定中的抗体反应，并在2014年添加了其他功能措施，例如中性粒细胞依赖性抗体依赖性呼吸爆发测定法和调查测定法，以获得广泛的抗寄生虫功能范围。 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并将其扩展到现场的寄生虫。 SMFA是评估抗体阻止向蚊子传播能力的金标准测定法，我们对此测定法进行了深入研究以定义其特征。 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，我们已经与NIAID的生物统计组合作，开发了一种测定的计算机模型。 2014年，我们与LMVR和FDA的其他人合作，为此测定法进行了改进的读数。 2。在2014年，我们使用SMFA测定法评估了用RTS免疫的儿童S疫苗（最先进的疟疾疫苗念珠菌）可以开发会阻止传播的抗体。我们无法显示多克隆人或单克隆抗体对向蚊子传播的影响，无论是通过唾液腺中的卵囊密度降低还是孢子虫的测量。 3。搜索并评估新的可能的传输阻断疫苗候选物。我们在2014年使用SMFA评估了许多潜在的传输阻断疫苗候选物以及这些抗原的不同表现。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。这些蛋白质已在HEK293细胞中产生，并使用产物免疫小鼠。这些动物的血清正在SMFA中评估。 4.全年评估肯尼奥巴居民中无性和性阶段寄生虫的存在。在2013年春季，我们开始了一项在肯尼奥巴（Kenieroba）进行的新研究（NIH 13-I-N107），以解决有关疟疾流行地区实际传播的有限信息，并在2014年完成了这项研究的领域方面。志愿者每月两次手指刺穿1年，以收集DNA和RNA。在今年中，我们已经确定了PCR检测过滤纸上寄生虫DNA以及RT-PCR程序的方法，以鉴定针对配子细胞的RNA。使用这些技术，我们正在评估所有年龄段的500个村民的寄生虫DNA的运输以及配子细胞。这将确定那些可能针对干预的疟疾传播的人。 5。确定Kenieroba，马里居民是否会产生在SMFA中具有阻塞活性的抗体。 Kenieroba的传播研究包括一年中所有志愿者的静脉穿刺样本。这些较大的体积血清样品将在SMFA中使用，以确定抗体是否具有传输阻滞活性。 6.确定SMFA测定法与DMFA之间的关系，该测定法测量了该领域感染者对性寄生虫抗体的传输阻断活性。我们正在评估我们开发了与SMFA合作的模型也适用于DMFA。 其他研究：1）我们与德雷克塞尔大学的阿基尔·瓦迪亚（Akhil Vaidya）博士合作，表明无性疟原虫寄生虫不需要混合血红素，但对于性阶段的分化和蚊子感染是必要的。 2）与贝勒大学的Olivier Lichtarge博士合作，我们为识别未知基因（超基因组网络压缩）功能的新策略提供了支持数据，并有助于对PFEAXP1作为谷胱甘肽转移酶进行评估，该酶可以被敏捷抑制； 3）我们继续与DRS合作。 Amy Bei和Dyann Wirth介绍了塞内加尔的恶性疟原虫克隆频率的变化以及免疫反应对该过程的影响。