Developing a barcoded malaria parasite panel to assess broadly neutralizing antibodies

项目总结 疟疾仍然是对全球公共卫生的严重威胁，人们担心有限的药物和病媒控制 我们可以利用的战略正在变得不那么有效。新疫苗的最新进展和新的兴趣 针对病原体，包括恶性疟疾寄生虫，提供了扩大的机会 我们对抗疾病的工具包。一种高效的疟疾疫苗，就像其他真核寄生虫一样，已经 一直难以捉摸。已经投入了大量的精力来开发针对日本血吸虫病患者的前红细胞疫苗 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。在疟疾的临床血液期，许多提供 寄生虫入侵红细胞的途径是已知的，一些自然产生的抗体是 防护性的。抗原的多样性和变异性，以及其他因素，对广泛的- 中和血液阶段疫苗，因为许多免疫靶向进入宿主细胞的分子在功能上是 冗余和序列多态。随着抗原继续接受免疫原性测试，它将成为一种 高度优先，以确定那些具有最大潜力广泛防御广泛抗原性的人 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简化评估的平台 抗寄生虫抗体的广谱中和潜力。在第一步中，我们将确定适应文化的P. 在全球范围内代表恶性疟原虫所有主要等位基因的克隆系；并实施最先进的 CRISPR-CAS9方法引入短的、稳定表达的条形码序列来区分线条。 在步骤2中，我们将汇集90条条形码线路，并验证基于Illumina的扩增序列的使用 去卷曲池，并测量寄生虫线的相对水平。在步骤3中，我们将使用集合分析 检测单抗的侵袭抑制和广谱中和能力的菌株特异性 将两种感兴趣的抗原作为血液期疫苗的靶标。 群体基因组学，寄生虫的反向遗传修饰，以及数量高的 吞吐量测序，将使我们能够以前所未有的速度分析抗原多样性的功能结果 规模和复杂性；并将改变为一种有效的、广泛的- 中和疟疾疫苗。我们还预计，该方法也将适用于与疫苗无关的 恶性疟原虫的研究，包括寄生虫对抗疟疾药物的抗药性分析。

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.