Testing Borrelia recurrentis isolates by using a newly developed louse-borne relapsing fever mouse model

项目摘要 虱传回归热（LBRF）是由复发性疏螺旋体引起的人类疾病。LBRF仍然是一个 在几个非洲国家的沉重负担。LBRF可能占住院人数的27%。B。复发性 是通过体虱在人与人之间传播的，体虱是一种严格意义上的人类虱子， 寄生虫如果不治疗，死亡率可高达40%，而用抗微生物剂治疗时，死亡率低至1-5%。 治疗往往是有问题的，因为高达76%的患者表现出全身性过敏反应， J-H反应，与死亡率升高相关。LBRF研究受到阻碍 缺乏免疫活性的动物模型。最近，我们开发了一种免疫活性小鼠模型。 小鼠在感染后第1-3天持续发展螺旋体血症水平，并且抗-Bre IgM 反应然而，从感染后第4天到第10天未检测到Bre。与Bre相比，蜱传 回归热疏螺旋体（例如，B。hermsii）在7-14天内有多个螺旋体血症高峰， 由Vmp蛋白（vlsp和vsp基因的产物）的抗原变异驱动， 抗体的继发性螺旋体血症的缺乏可归因于Bre抗原的遗传组成 系统最近，A17（分离年份，1985年）和其他Bre菌株、PBeK（2004年）和PAbN的基因组， 对PAbJ、PMaC和PUfA（2015）进行了全基因组测序（Marosevic et al 2017），并与 参考菌株A1。结果，在10个基因组之间鉴定出低遗传变异性（仅29-38个SNP）。 所有这些菌株的染色体/质粒。然而，由于序列相似性和可变数目的 在LP 124中的VLP/VSL基因中，VLP/VSL基因的覆盖率与周围区域相比存在差异（高10- 20倍）， 这表明VLP/VSP拷贝数是高度可变的。后者和重新组装 该研究采用的方法排除了VLP/VSP（假）基因的确切数量和位置的鉴定 对于6种测序的Bre菌株（Marosevic et al 2017）。因此，有可能的是， 在EE小鼠（和大多数人类患者）中观察到的螺旋体血症是由于vlp/vsl系统的有限库 在某些Bree菌株中。我们假设，具有较高数量的完整vlp/vsp的Bre菌株， 会导致螺旋体血症复发为了测试它，我们将追求以下具体目标： SA 1：确定7种Bre菌株在新型LBRF中将发展的螺旋体血症复发次数 免疫活性小鼠模型。SA 2：确定完整vlp和vsp的确切拷贝数 利用PacBio测序技术对7株Bre的假基因进行了序列分析。这是一项重要而有影响力的提案 因为将提供对Bre发病机制的新见解。这项研究也可能改善我们的Bre小鼠 通过使Bre菌株发展一个以上的螺旋体血症循环，这将使我们能够研究抗原性， Bre的变化更详细。我们的方法是新颖的，因为除了我们的新的（唯一可用的） 在免疫活性小鼠模型中，我们还将使用PacBio对Bre vlp/vsp系统进行准确测序。

PROJECT SUMMARY Louse-borne relapsing fever (LBRF) is a human disease caused by Borrelia recurrentis. LBRF remains a significant burden in several African nations. LBRF could account for 27% of hospital admissions. B. recurrentis is transmitted between humans by the body louse, Pediculus humanus humanus, which is a strictly human parasite. The mortality can be as high as 40%, if left untreated, and low as 1-5% when treated with antimicrobials. Treatment is often problematic because up to 76% of patients exhibit a systemic antibiotic-induced reaction, the Jarisch-Herxeimer reaction, which is associated with an elevated mortality. LBRF research has been hampered by the lack of immunocompetent animal model. Recently, we developed an immunocompetent mouse model. The mice consistently developed an increasing level of spirochetemia during day 1-3 pi and anti-Bre IgM response. However, Bre was not detected from day 4 through day 10 post infection. In contrast to Bre, tick-borne relapsing fever Borrelia (e.g., B. hermsii) have multiple peaks of spirochetemia over 7-14-day period, which is driven by antigenic variation of Vmp proteins (products of vlsp and vsp genes) allowing spirochetes to evade antibodies. The lack of secondary spirochetemia could be accounted for by genetic make-up of Bre antigenic system. Recently, genomes of A17 (year of isolation, 1985) and other Bre strains, PBeK (2004), and PAbN, PAbJ, PMaC, and PUfA (2015) were whole-genome sequenced (Marosevic et al 2017) and compared to the reference strain A1. As a result, low genetic variability (only 29-38 SNPs) was identified between the chromosome/plasmids for all of these strains. However, due to sequence similarity and variable numbers of vlp/vsl genes in lp124, there was discrepant coverage of vlp/vsl genes than surrounding regions (10-20x higher), which indicated that the numbers of vlp/vsp copies were highly variable. The latter and de novo assembly approach taken by that study precluded identification of exact numbers and location of vlp/vsp (pseudo)genes for the 6 sequenced Bre strains (Marosevic et al 2017). Thus, it is possible that the single relapse of spirochetemia observed in the EE mice (and most human patients) is due to a limited repertoire of vlp/vsl system in some Bre strains. We hypothesize that the Bre strains that possess a higher number of intact vlp/vsp copies will develop more relapses of spirochetemia. To test it, we will pursue the following Specific Aims: SA1: Determine the number of spirochetemic relapses that 7 Bre strains will develop in the novel LBRF immunocompetent mouse model. SA2: Determine the exact copy numbers of intact vlp and vsp (pseudo)genes in 7 Bre strains by using the PacBio sequencing. This is a significant and impactful proposal because novel insights into Bre pathogenesis will be provided. This study may also improve our Bre mouse model by having Bre strain(s) develop more than one spirochetemic cycle, which will allow us study the antigenic variation of Bre in greater details. Our approach is novel since, in addition to our new (the only available) immunocompetent mouse model, we will also use PacBio to accurately sequence the Bre vlp/vsp system.