Predicting naturally acquired humoral immunity against malaria

描述(由申请人提供)：我们开发了一种在全基因组范围内构建和探测蛋白质微阵列的方法，以表征宿主对超过25种医学上重要的感染微生物的抗体反应。该实验室已经制造了3.5万个质粒，将编码的蛋白质打印在2.5万个微阵列上，并用1.2万个人类血清样本进行了探测。印制在这些阵列上的单个蛋白捕获存在于感染者血清中的抗体，捕获的抗体的量可以使用荧光二次抗体和感染后产生的抗体图谱来定量。在这里，我们建议应用这一方法来理解自然获得体液免疫(NAI)在临床疟疾中的基础，识别与预防恶性疟原虫感染相关的抗体生物标记物，并发现新的疟疾疫苗候选抗原。生活在恶性疟原虫高传播区的人们在童年和青春期反复暴露于恶性疟原虫感染后，对疟疾症状产生了抵抗力。用从接触PF的马里儿童和成人收集的血清对含有2,320个PF蛋白的蛋白质芯片进行探测表明，只有在反复感染PF多年后才能获得长期的抗体反应，并针对49种特定的PF抗原鉴定出保护性抗体。(2)我们的目标是通过在马里进行更全面的免疫流行病学分析来扩展这些初步发现。将对已经从马里NIAID支持的三项研究中收集的4000个血浆样本进行探测和分析。这些研究的纵向性质允许在感染PF之前、期间和之后的关键时间点进行抗体概况分析，从而能够对抗体概况与疟疾预防之间的关系进行前瞻性分析。我们还将调查3个月至5岁的婴儿和儿童的恶性疟原虫特异性抗体谱，因为他们通过接触恶性疟原虫而从母源抗体转变为自然获得的抗体。最后，在横断面分析中，我们将调查富拉尼人和多贡人中PF特异性抗体的同型特征和抗原特异性。 马里，不同的族裔群体，对疟疾的易感性有很好的记录。这项建议还利用NIAID基因组测序中心(与J·克雷格·文特尔研究所合作)最近提供的资金，对其中一项队列研究中的700个人的目标基因组区域进行基因分型，以及全基因组转录图谱(通过RNA-seq.)同一个体在感染恶性疟原虫之前、期间和之后。这将提供一个独特的机会，前瞻性地检查遗传多态、白细胞转录图谱和恶性疟原虫特异性抗体反应之间的关系。

DESCRIPTION (provided by applicant): We developed an approach to construct and probe protein microarrays on a genome-wide scale to characterize host antibody responses against more than 25 medically important infectious microorganisms. The lab has made 35,000 plasmids, printed the encoded proteins on 25,000 microarrays and probed them with 12,000 human serum specimens. The individual proteins printed on these arrays capture antibodies present in the serum of infected individuals, the amount of captured antibody can be quantified using fluorescent secondary antibody and antibody profiles that result after infection determined. Here we propose to apply this approach toward understanding the basis of naturally-acquired humoral immunity (NAI) to clinical malaria, to identify antibody biomarkers associated with protection from P. falciparum infection, and to discover novel malaria vaccine antigen candidates. People living in areas of intense Plasmodium falciparum (Pf) transmission develop resistance to the symptoms of malaria after repeated exposures to Pf parasite infection through childhood and adolescence. Probing a protein microarray containing 2,320 Pf proteins with sera collected from Pf-exposed Malian children and adults indicated that long-lived antibody responses are only acquired after years of repeated Pf infections and protective antibodies were identified against 49 specific Pf antigens.(2) Here we aim to extend these preliminary findings with a more comprehensive immuno-epidemiological analysis in Mali. Four thousand plasma samples which have already been collected from three NIAID-supported studies in Mali will be probed and analyzed. The longitudinal nature of these studies allows for antibody profiling at strategic time points before, during and after Pf infection and thus permits a prospective analysis of the relationship between antibody profiles and protection from malaria. We will also investigate the P. falciparum-specific antibody profile of infants and children aged 3 months to 5 years as they transition from maternally-derived antibodies to naturally-acquired antibodies through P. falciparum exposure. Finally, in a cross-sectional analysis we will investigate the isotype profile and antigen specificity of Pf-specific antibodies in the Fulani and Dogon people in Mali, distinct ethnic groups which have well-documented differential susceptibility to malaria. This proposal also takes advantage of recent funding from the NIAID Genome Sequencing Center (in collaboration with the J. Craig Venter Institute) to perform genotyping of targeted genomic regions of 700 individuals in one of the cohort studies as well as genome-wide transcription profiling (by RNA-seq.) of the same individuals before, during and after P. falciparum infection. This will provide a unique opportunity to examine prospectively the relationship between genetic polymorphisms, leukocyte transcription profiles and P. falciparum-specific antibody responses.