Developing a barcoded malaria parasite panel to assess broadly neutralizing antibodies

项目摘要 疟疾仍然是对全球公共卫生的一个严重威胁，令人关切的是， 我们所掌握的战略正在变得不那么有效。最近的进展和对新疫苗的新兴趣 针对病原体，包括恶性疟原虫疟疾寄生虫， 我们对抗疾病的工具包。一种针对疟疾和其他真核寄生虫的高效疫苗， 一直难以捉摸。已经投入了大量的努力来开发针对 寄生虫的子孢子形式，导致世界卫生组织最近建议使用 在流行地区。在疟疾的临床血液阶段，许多提供免疫应答的功能性抗原， 寄生虫侵入红细胞的途径是已知的，一些天然存在的抗体， 保护性的除其他因素外，抗原多样性和变异对广泛的免疫系统构成了重大挑战。 中和血液阶段疫苗，因为许多用于宿主细胞进入的免疫靶向分子在功能上是 冗余和序列多态性。随着抗原的免疫原性测试的不断进行， 高度优先确定那些最有可能广泛保护免受广泛抗原性 世界各地发现的各种疟疾寄生虫。我们建议在这里开发一个平台， 抗寄生虫抗体的广泛中和潜力。在第一步中，我们将鉴定培养适应的P。 代表全球寄生虫所有主要等位基因的恶性疟原虫克隆系; CRISPR-Cas9方法引入短的、稳定表达的条形码序列以区分品系。 在步骤2中，我们将汇集90个条形码化的细胞系，并验证基于Illumina的扩增子测序的使用， 解卷积池并测量寄生物线的相对水平。在步骤3中，我们将使用合并检测试剂盒 测定单克隆抗体的侵袭抑制和广泛中和潜力的菌株特异性 针对血液阶段疫苗的两种感兴趣的抗原。 群体基因组学、寄生虫的反向遗传修饰和定量高- 通量测序，将使我们能够分析抗原多样性的功能结果，在前所未有的 规模和复杂性;并将改变努力，以确定最有前途的抗原，为一个强大的，广泛的， 中和疟疾疫苗。我们进一步预计，该方法也将有助于非疫苗相关的 恶性疟原虫的研究，包括寄生虫对抗疟药的耐药性分析。

PROJECT SUMMARY Malaria remains a grave threat to global public health with concern that the limited drugs and vector control strategies at our disposal are becoming less effective. Recent progress and renewed interest in new vaccines targeting pathogens, including the Plasmodium falciparum malaria parasite, provide an opportunity to expand our toolkit to combat the disease. A highly efficacious vaccine for malaria, as for other eukaryotic parasites, has been elusive. Substantial effort has been invested in development of a pre-erythrocytic vaccine targeting the sporozoite form of the parasite, resulting in the recent recommendation by the World Health Organization for use in endemic areas. During the clinical blood-stage of malaria, many of the functional antigens that provide the parasite pathways for invasion into erythrocytes are known, and some naturally occurring antibodies are protective. Antigenic diversity and variation, among other factors, poses a major challenge for a broadly- neutralizing blood-stage vaccine, because many immune-targeted molecules for host cell entry are functionally redundant and sequence polymorphic. As antigens continue to be tested for immunogenicity, it will become a high-priority to identify those with the best potential for widespread protection against the breadth of antigenically diverse malaria parasites found across the world. We propose here to develop a platform for facile assessment of the broadly-neutralizing potential of anti-parasite antibodies. In Step 1, we will identify culture-adapted P. falciparum clonal lines that represent all major alleles of the parasite globally; and implement state-of-the-art CRISPR-Cas9 methodologies to introduce short, stably expressed barcode sequences to distinguish the lines. In Step 2, we will pool 90 barcoded lines and validate the use of Illumina-based amplicon-sequencing to deconvolute pools and measure the relative levels of the parasite lines. In Step 3, we will use the pooled assay to measure the strain-specificity of invasion inhibition and broadly-neutralizing potential of monoclonal antibodies targeting two antigens of interest for a blood-stage vaccine. The combination of population genomics, reverse genetic modification of parasites, and quantitative high- throughput sequencing, will permit us to analyze the functional outcomes of antigenic diversity at unprecedented scale and complexity; and will transform efforts to identify the most promising antigens for a potent, broadly- neutralizing malaria vaccine. We further anticipate that the approach will also be useful for non-vaccine related studies for P. falciparum, including analysis of parasite resistance to antimalarials.