Developing a barcoded malaria parasite panel to assess broadly neutralizing antibodies

开发带条形码的疟原虫面板来评估广泛中和抗体

• 批准号: 10511052
• 负责人: Manoj T Duraisingh
• 金额: $ 23.93万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-29 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10511052
• 关键词: Affect; Alleles; Antibodies; Antibody-mediated protection; Antigen Targeting; Antigenic Diversity; Antigenic Variation; Antigens; Antimalarials; Area; Bar Codes; Biological; Biological Assay; Blood; COVID-19 pandemic; CRISPR/Cas technology; Cells; Child; Clinical; Development; Disease; Drug Controls; Epitopes; Equilibrium; Erythrocytes; Evaluation; Exhibits; Falciparum Malaria; Formulation; Genetic Polymorphism; Genetic Recombination; Genomics; Growth; High-Throughput Nucleotide Sequencing; Human; Humoral Immunities; Immune Targeting; Immunization; Immunologics; In Vitro; Individual; Infection; Insecticides; Investments; Malaria; Malaria Vaccines; Measurement; Measures; Mediating; Methodology; Modification; Monoclonal Antibodies; Natural Immunity; Parasite resistance; Parasites; Pathogenicity; Pathway interactions; Phenotype; Plasmodium falciparum; Population; Public Health; Recommendation; Research; Resistance; Resources; Sampling; Specificity; Sporozoites; Testing; Vaccine Research; Vaccines; Variant; World Health Organization; base; burden of illness; combat; cross immunity; functional outcomes; immunogenicity; interest; malaria infection; neutralizing antibody; neutralizing vaccine; next generation sequencing; novel vaccines; parasite invasion; pathogen; prevent; reverse genetics; success; tool; vaccine development; vector control

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.

项目概要 疟疾仍然对全球公共卫生构成严重威胁，令人担忧的是有限的药物和病媒控制 我们所掌握的策略正在变得越来越无效。最新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，提供了扩大 我们对抗这种疾病的工具包。与其他真核寄生虫一样，一种针对疟疾的高效疫苗已问世 一直难以捉摸。已投入大量精力开发针对红细胞前疫苗 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。在疟疾的临床血液阶段，许多功能性抗原提供了 寄生虫侵入红细胞的途径是已知的，并且一些天然存在的抗体 保护的。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。 中和血期疫苗，因为许多进入宿主细胞的免疫靶向分子在功能上 冗余和序列多态性。随着抗原的免疫原性测试不断进行，它将成为一种 优先考虑那些最有潜力针对广泛抗原性进行广泛保护的人 世界各地发现了多种疟原虫。我们在这里建议开发一个方便评估的平台 抗寄生虫抗体的广泛中和潜力。在步骤 1 中，我们将识别文化适应的 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；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细胞系。 在第 2 步中，我们将汇集 90 个条形码线并验证基于 Illumina 的扩增子测序的使用 对池进行解卷积并测量寄生虫线的相对水平。在第 3 步中，我们将使用混合分析 测量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、寄生虫反向基因改造和定量高通量的结合 通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；并将改变鉴定最有希望的抗原的努力，以用于有效的、广泛的 中和疟疾疫苗。我们进一步预计该方法也将适用于非疫苗相关的领域 恶性疟原虫的研究，包括分析寄生虫对抗疟药的耐药性。