Extent, dynamics and mechanisms of Plasmodium vivax immune evasion caused by PvDBP gene amplification

消除间日疟原虫将极大地受益于血液期疫苗。PvDBP 是一种寄生虫配体，通过与其人类受体Duffy的相互作用参与红细胞的入侵 蛋白。这种相互作用对寄生虫的进入至关重要，使PvDBP成为最先进的 正在进行第二阶段临床试验的血液阶段疫苗。最近的研究发现并刻画了人类 允许超越菌株中和寄生虫的单抗(人抗体)，无论其 PvDBP序列多样性。然而，我们已经证明了在柬埔寨收集的PV有多个副本 这些抗体能够克服PvDBP基因的体外中和作用。这些观察结果提供了 PvdBP扩增在光伏种群中广泛存在的进化优势的第一个证据，以及 创造了一种考虑病原体免疫逃避机制的新范式。这些结果提高了 担心PvDBP疫苗的实施可能会选择多个PvDBP复制寄生虫。总目标 这一提议的目的正是为了确定pvdBP扩增是否可能危及pvDBP疫苗 策略。第一个特定目标(SA)是确定多个pvdbp复制寄生虫的遗传距离有多大 来自柬埔寨的分离株对抗PvDBP抗体有反应，并评估pvdBP扩增是否与 人类群体中的Duffy基因多态性。通过评价抗PvDBP抗体的体外中和作用 来自埃塞俄比亚的单个和多个pvdbp拷贝的寄生虫，我们将能够评估免疫逃避的程度。 用柬埔寨PV描述的pvdbp扩增所赋予的表型。通过(I)关联体外侵袭 与入侵红细胞的全长Duffy序列的比率，以及(Ii)前瞻性测试关联性 在纵向队列中登记的参与者的pvdbp拷贝数与人类Duffy序列之间的关系 在柬埔寨和埃塞俄比亚，我们将能够确定pvdbp扩增和达菲之间的关系。 人类的多态。我们的第二个SA将评估宿主内和种群内的动态 随着时间的推移，pvdbp扩增。通过对我们纵向队列的血清学动力学的分析 参与者，测量PV感染和感染寄生虫的pvdBP拷贝数，我们就能够 测试基因扩增是否在体内由人类宿主的免疫状态选择，以及它与 人群中PV患病率的变化。PK系的体外实验进化将提供补充 抗PvDBP抗体选择pvdBP扩增的证据。我们的第三个SA将是破译 多拷贝寄生虫逃避抗PvDBP抗体中和的分子机制。我们会 具体测试多拷贝寄生虫产生的蛋白质数量增加是否导致免疫逃避 和/或通过多个不同的等位基因和变体同时存在于给定的 寄生虫。通过表型和基因组方法的结合，我们的结果将提供无价的 在疫苗开发的背景下，为克服这种免疫逃避的战略提供信息的数据。 。

Elimination of Plasmodium vivax (Pv) malaria parasites would greatly benefit from a blood-stage vaccine. PvDBP is a parasite ligand involved in erythrocyte invasion through the interaction with its human receptor, the Duffy protein. This interaction is critical for the parasite’s entry making PvDBP the most advanced candidate for a blood-stage vaccine with Phase II clinical trials undergoing. Recent work has identified and characterized human monoclonal antibodies (humabs) that allow strain-transcending neutralization of parasites regardless of their PvDBP sequence diversity. However, we have demonstrated that Pv collected in Cambodia with multiple copies of the PvDBP gene were able to overcome in vitro neutralization by these humabs. These observations provided the first evidence for an evolutionary advantage for pvdbp amplification, widespread in Pv populations, and created a new paradigm in which to consider pathogen immune evasion mechanisms. These results raise the concern that implementation of a PvDBP vaccine may select for multi-pvdbp copy parasites. The overall goal of this proposal is precisely to determine if pvdbp amplification will likely compromise a PvDBP vaccine strategy. The first Specific Aim (SA) is to determine to what extent multi-pvdbp copy parasites genetically distant from Cambodian isolates respond to anti-PvDBP humabs and to evaluate if pvdbp amplification is associated to Duffy polymorphisms in human populations. By evaluating the in vitro neutralization by anti-PvDBP humabs of single and multi-pvdbp copy parasites from Ethiopia, we will be able to evaluate the extent of the immune evasion phenotype conferred by pvdbp amplification described with Cambodian Pv. By (i) associating in vitro invasion rates with the full-length Duffy sequences of invaded erythrocytes, and (ii) prospectively testing for association between pvdbp copy number and human Duffy sequences in participants enrolled in longitudinal cohorts in Cambodia and in Ethiopia, we will be able to determine the relation between pvdbp amplification and Duffy human polymorphism. Our second SA will be to evaluate the within-hosts and within-population dynamics of pvdbp amplification over time. Through the analysis of the serological dynamics of our longitudinal cohorts’ participants, the measure of Pv infections and the pvdbp copy number of infecting parasites, we will be able to test if the gene amplification is selected in vivo by the immune status of human hosts and how it correlates with changes in Pv prevalence in the population. In vitro experimental evolution of Pk lines will provide complementary evidence for selection of pvdbp amplification by anti-PvDBP humabs. Our third SA will be to decipher the molecular mechanisms enabling multi-copy parasites to evade anti-PvDBP humabs’ neutralization. We will specifically test if immune evasion results from increased protein quantity produced by multi-copy parasites and/or from epitope variations through multiple, different alleles and variants present simultaneously in a given parasite. Through a combination of phenotyping and genomic approaches, our results will provide invaluable data to inform on strategies to overcome this immune evasion in the context of vaccine development. .