Dissecting Chloroquine Resistance in the Plasmodium vivax Cross

描述（由申请人提供）：尽管最近对疟疾的兴趣激增，但与恶性疟疾相比，间日疟原虫仍然相对被忽视。超过25亿人面临间日疟原虫感染的风险，每年造成8 000万至3亿病例。目前治疗间日疟的主要药物是氯喹（CQ）， 受到不断发展的抵抗的威胁。在过去的20年里，CQ耐药性出现在西太平洋和东南亚，现在正在全球范围内蔓延。在柬埔寨和印度尼西亚等一些国家，CQ疗法的疗效下降，促使人们改用青蒿素综合疗法作为间日疟的一线疗法，因为青蒿素综合疗法的费用要高得多。间日疟原虫CQ抗性的分子基础仍然未知。有证据表明，间日疟原虫CQ抗性背后的遗传事件与恶性疟原虫不同，因为在体内药物应答与间日疟原虫氯喹抗性转运蛋白（pvcrt）（恶性疟原虫氯喹抗性转运蛋白（pfcrt）的间日疟原虫直系同源物）的密码子变化之间没有发现关联。由于缺乏关键的体外培养、DNA转染和克隆方法，对间日疟原虫CQ抗性的进一步研究受到严重阻碍。因此，间日疟原虫CQ抗性的研究依赖于非人灵长类动物模型。最近，美国国立卫生研究院（NIH）投入了大量资源，在黑猩猩中产生了第一个CQ敏感株和CQ抗性株之间的间日疟原虫遗传杂交。使用这种交叉，我们的建议旨在通过利用亲本菌株的全基因组测序和药物压力前后的体内混合后代感染进行连锁群选择（LGS）分析来确定间日疟原虫中CQ抗性的遗传位点。在不需要传统的克隆分析和确定的IC 50的情况下，LGS鉴定了在用指示与CQ抗性相关的遗传基因座的CQ选择后向抗性亲本等位基因的转变，并且已用于在疟疾啮齿动物模型中多次成功鉴定抗性等位基因/突变。这一提议显然是重要的，原因有多个：1）CQ抗性间日疟原虫是一种主要的 全球公共卫生问题，造成高发病率和死亡率， 影响，2）这将是第一个全基因组评估CQ抗性遗传位点， 使用确定的遗传杂交的间日疟，3）它利用了来自黑猩猩中产生的独特且不可替代的遗传杂交的一组样品，该方法鉴定了恶性疟原虫对CQ和其他抗疟药的抗性的关键突变，4）CQ抗性机制的鉴定将有助于开发其他抗疟药，和5）鉴定与CQ抗性相关的基因座将提供抗性的分子标记，这是研究抗性传播的重要公共卫生工具。该提案也具有创新性，因为它利用新的工具进行全基因组测序，使用混合捕获来富集寄生虫DNA并去除污染的宿主DNA。了解间日疟原虫CQ抗性的分子基础将是了解间日疟原虫生物学的一个重大进展，并有可能对全球公共卫生产生深远影响。

DESCRIPTION (provided by applicant): Despite the recent surge of interest in malaria, Plasmodium vivax has remained relatively neglected compared to falciparum malaria. Over 2.5 billion persons are at risk of vivax infection, which causes between 80 and 300 million cases per year. The principal drug in use today for vivax malaria is chloroquine (CQ) but its efficacy in now threatened by evolving resistance. Over the past 20 years, CQ resistance emerged in Western Pacifica and Southeast (SE) Asia and is now spreading worldwide. In some countries, such as Cambodia and Indonesia, the diminished efficacy of CQ therapy has prompted a change to artemisinin combination therapies (ACTs), which are significantly more costly, as first-line therapy for vivax malaria. The molecular basis of P. vivax CQ resistance remains unknown. Evidence indicates that the genetic events underlying P. vivax CQ resistance differs from P. falciparum as no association was found between in vivo drug responses and codon changes in Plasmodium vivax chloroquine resistance transporter (pvcrt), the P. vivax ortholog of Plasmodium falciparum chloroquine resistance transporter (pfcrt). Further research on CQ-resistant P. vivax has been severely hampered by the lack of critical tools to study the parasites including in vitro culture, DNA transfection and cloning methods. Therefore research on P. vivax CQ resistance depends on non-human primate models. Recently, the National Institutes of Health (NIH) invested significant resources to generate the first P. vivax genetic cross between a CQ sensitive strain and a CQ resistant strain in a chimpanzee. Using this cross, our proposal is aimed at determining the genetic loci underlying CQ resistance in P. vivax by conducting a linkage group selection (LGS) analysis leveraging whole genome sequencing of parental strains and in vivo mixed progeny infections before and after drug pressure. Without the need for traditional clonal analysis and defined IC50s, LGS identifies shifts toward resistant parental alleles after selection with CQ indicative of genetic loci associated with CQ resistance and has been used to successfully identify resistance alleles/mutations multiple times in malaria rodent models. This proposal is clearly significant for multiple reasons: 1) CQ resistant vivax is a major global public health problem causing significant morbidity and mortality with significant financial implications, 2) this will be the first genome-wide assessment for genetic loci of CQ resistance in vivax malaria using a defined genetic cross, 3) it leverages a set of samples from a unique and irreplaceable genetic cross generated in a chimpanzee, the approach which identified the key P. falciparum mutations for resistance to CQ and other antimalarials, 4) identification of CQ resistance mechanisms will aid in the development of additional antimalarials, and 5) identification of loci associated with CQ resistance will provide molecular markers of resistance, an important public health tool for studying the spread of resistance. The proposal is also innovative as it leverages new tools for conducting whole genome sequencing using hybrid capture to enrich parasite DNA and remove contaminating host DNA. Understanding the molecular basis for CQ resistance in P. vivax would be a major advance in understanding of vivax biology and has the potential to have profound impacts on global public health.