A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions.
A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions.
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DOI：
10.1371/journal.pntd.0004264
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发表时间：
2015-12
影响因子：
3.8
通讯作者：
Rayner JC
中科院分区：
文献类型：
--
作者：
Hostetler JB;Sharma S;Bartholdson SJ;Wright GJ;Fairhurst RM;Rayner JC
针对间日疟原虫的疫苗将是任何全面消除疟疾计划的重要组成部分，但我们对间日疟原虫生物学的理解存在重大差距，包括介导裂殖子入侵网织红细胞的蛋白质-蛋白质相互作用，阻碍了候选抗原的寻找。仅鉴定了一种配体-受体相互作用，即间日疟原虫达菲结合蛋白（PvDBP）与红细胞达菲趋化因子抗原受体（DARC）之间的相互作用，并且对PvDBP的菌株特异性免疫应答使其成为复杂的疫苗靶标。为了拓宽潜在的间日疟原虫裂殖子阶段疫苗靶标的库，我们利用了最近在哺乳动物细胞中以功能活性形式表达疟原虫蛋白的全长胞外域的突破，并启动了对间日疟原虫裂殖子蛋白的大规模研究，所述裂殖子蛋白可能参与网织红细胞结合和侵袭。我们选择了39个预测定位于裂殖子表面或侵入性分泌细胞器的间日疟原虫蛋白，其中一些显示与恶性疟原虫疫苗候选物同源。其中，我们能够在哺乳动物表达系统中表达37个全长蛋白质胞外域，该系统先前已用于表达恶性疟原虫侵袭配体如PfRH 5。为了确定表达的蛋白质是否正确折叠，我们评估了它们是否被来自柬埔寨急性间日疟患者的抗体识别。这些样本中的IgG在检测的34种抗原中的27种中显示出与未处理对照相比至少两倍的反应性变化，并且大多数显示出热不稳定IgG免疫反应性，表明存在构象敏感表位和天然三级蛋白结构。使用专门设计用于检测低亲和力的细胞外蛋白质-蛋白质相互作用的方法，我们证实了间日疟原虫6-半胱氨酸蛋白P12和P41之间的预测相互作用，进一步表明蛋白质是天然折叠和功能性的。该筛选还鉴定了P12和PVX_110945之间以及MSP 3.10和MSP 7.1之间的两种新的蛋白质-蛋白质相互作用，后者通过表面等离子体共振证实。我们产生了一个新的重组全长间日疟原虫胞外域文库，确定其中大多数含有三级结构，并使用它们来鉴定预测的和新的蛋白质-蛋白质相互作用。除了为进一步的生物学研究鉴定新的相互作用外，该文库将有助于鉴定具有疫苗潜力的间日疟原虫蛋白，以及研究间日疟原虫疟疾发病机制和免疫。ClinicalTrials.gov NCT00663546 间日疟原虫每年导致数百万人患上疟疾，主要发生在东南亚和中南美洲。间日疟原虫有一个休眠的肝脏阶段，即使没有蚊子传播，也会导致感染个体的疾病复发。因此，针对血液阶段间日疟原虫的疫苗的开发可能是全球根除疟疾努力的重要组成部分。研究间日疟原虫是非常困难的，因为这种寄生虫在实验室中生长很差，只能侵入患者体内少量的年轻红细胞。由于这些和其他挑战，只有少数间日疟原虫蛋白被测试为潜在的疫苗。为了产生更多的候选疫苗，我们表达了37种蛋白质的整个胞外域，这些蛋白质被预测参与间日疟原虫入侵红细胞。来自柬埔寨间日疟原虫疟疾患者的抗体识别了大多数这些蛋白质中的热敏表位，表明它们是天然折叠的。我们还使用这些蛋白质来筛选预测的和新的蛋白质-蛋白质相互作用，证实这些蛋白质是功能性的，并进一步支持它们作为候选疫苗的潜力。作为一个新的社区资源，该间日疟原虫重组蛋白库将有助于未来间日疟原虫致病性和免疫性的研究，并大大扩展了候选疫苗抗原的名单。
A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further suggesting that the proteins are natively folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance. We produced a new library of recombinant full-length P. vivax ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying P. vivax proteins with vaccine potential, and studying P. vivax malaria pathogenesis and immunity. ClinicalTrials.gov NCT00663546 Plasmodium vivax causes malaria in millions of people each year, primarily in Southeast Asia and Central and South America. P. vivax has a dormant liver stage, which can lead to disease recurrence in infected individuals even in the absence of mosquito transmission. The development of vaccines that target blood-stage P. vivax parasites is therefore likely to be an essential component of any worldwide effort to eradicate malaria. Studying P. vivax is very difficult as this parasite grows poorly in the laboratory and invades only small numbers of young red blood cells in patients. Due to these and other challenges, only a handful of P. vivax proteins have been tested as potential vaccines. To generate more vaccine candidates, we expressed the entire ectodomains of 37 proteins that are predicted to be involved in P. vivax invasion of red blood cells. Antibodies from Cambodian patients with P. vivax malaria recognized heat-sensitive epitopes in the majority of these proteins, suggesting that they are natively folded. We also used the proteins to screen for both predicted and novel protein-protein interactions, confirming that the proteins are functional and further supporting their potential as vaccine candidates. As a new community resource, this P. vivax recombinant protein library will facilitate future studies of P. vivax pathogenesis and immunity, and greatly expands the list of candidate vaccine antigens.