Genetic and functional studies of var2csa in placental malaria

描述（由申请人提供）：恶性疟原虫寄生在胎盘中，每年危及数百万孕妇和新生儿。隔离由插入红细胞表面的恶性疟原虫VAR 2CSA蛋白介导，其结合胎盘硫酸软骨素A（CSA），产生胎盘炎症，并导致低出生体重（LBW）婴儿。对VAR 2CSA的适应性免疫保护孕妇和新生儿免受疟疾发病。VAR 2CSA是一个大而多样的抗原性和免疫原性结构域的不完全描述，但一个2.4 kb的var 2csa区域，编码ID 1-DBL 2X-ID 2，它结合CSA在体外表达时具有高亲和力。这种重组蛋白能产生有效的结合抑制性抗体，因此作为候选疫苗具有很大的前景。然而，由于VAR 2CSA是高度多样化的，可能需要多价疫苗。在这里，我们建议询问var 2csa ID 1-DBL 2X-ID 2的多样性，并确定潜在的致病性变异在马拉维孕妇，然后测量CSA结合和免疫原性的重组表达。将从一项1600人的试验中选择200名妊娠中期和分娩时均患有寄生虫血症的孕妇，该试验比较了间歇性筛查和治疗（IST）与间歇性预防治疗（IPT）。它们将被分为4组：有和无低出生体重（LBW）后代的经产妇和有或无LBW后代的经产妇。我们将从每名女性中采集3份寄生虫样本：妊娠中期和分娩时的外周血以及胎盘血。对于第一个目标，将合并每组的每种样本类型（12个池），并在ID 1-DBL 2X-ID 2区域进行深度测序。我们将确定与妊娠失败和LBW相关的变异。在第二个目标中，我们将使用qPCR和逆转录酶qPCR来比较每个妇女在怀孕过程中50种最常见变异的群体大小。我们推测，最致病的变异将更常见于第二个三个月比在交付。在第三个目标中，我们将合成和表达最适合这些标准的变体的重组体，并测量它们的体外CSA结合和免疫原性。该提案代表了拥有流行病学和深度测序专业知识的CNAS小组与var 2csa生物学和疫苗开发领导者哥本哈根大学小组之间的新合作。这一建议将有助于阐明妊娠期疟疾的发病机制，并直接为正在进行的妊娠相关疟疾疫苗的开发提供信息。

DESCRIPTION (provided by applicant): Falciparum malaria parasites sequester in placentae and imperil millions of pregnant women and newborns each year. Sequestration is mediated by P. falciparum VAR2CSA proteins inserted on the erythrocyte surface which bind to placental chondroitin sulfate A (CSA), produce placental inflammation, and result in low birth weight (LBW) babies. Adaptive immunity to VAR2CSA protects pregnant women and newborns from malaria morbidity. VAR2CSA is large and diverse with incompletely described antigenic and immunogenic domains, but one 2.4 kb region of var2csa, encodes ID1-DBL2X-ID2, which binds CSA with high affinity when expressed in vitro. This recombinant protein elicits potent binding inhibitory antibodies and thus has great promise as a vaccine candidate. However, because VAR2CSA is highly diverse, a polyvalent vaccine might be necessary. Here we propose to interrogate var2csa ID1-DBL2X-ID2 diversity and identify potentially pathogenic variants in Malawian pregnant women, and then measure the CSA binding and immunogenicity of expressed recombinants. 200 pregnant women with both 2nd trimester and delivery parasitemias will be selected from a 1600-person trial comparing Intermittent Screening and Treatment (IST) with Intermittent Preventive Therapy (IPT). They will be divided into 4 groups: primigravidae with and without low-birth weight (LBW) offspring and multigravidae with or without LBW offspring. From each woman, we will obtain 3 parasite samples: peripheral blood from the 2nd trimester and from delivery, and from placental blood. For the first aim, each sample type from each group will be pooled (12 pools) and deep-sequenced across the ID1-DBL2X-ID2 region. We will identify variants associated with primigravidity and LBW. In the second aim, we will use qPCR and reverse-transcriptase qPCR to compare population sizes of the 50 most common variants within each woman over the course of pregnancy. We hypothesize that the most pathogenic variants will be more common in the 2nd trimester than at delivery. In the third aim, we will synthesize and express recombinants of the variants that best fit these criteria and measure both their in vitro CSA-binding and immunogenicity. This proposal represents a new collaboration between the UNC group, with expertise in epidemiology and deep sequencing, and the University of Copenhagen group, a leader in var2csa biology and vaccine development. This proposal will help elucidate the pathogenesis of malaria in pregnancy and directly inform the on-going development of a vaccine against pregnancy-associated malaria.