Studying the role of anti-capsular antibodies in mediating pathogenesis of carbapenem-resistant Klebsiella pneumoniae

项目总结/摘要 碳青霉烯类耐药肺炎克雷伯菌（CR-Kp）引起严重的医院获得性感染， 死亡率超过50%，需要新的策略来治疗它。 由弗里斯实验室开发的与荚膜多糖（CPS）结合的最突出的 CR-Kp克隆在美国，ST 258。mAb已显示与ST 258菌株广泛反应， 多种血清型，通过体外吞噬作用和其他机制杀死CR-Kp，并保护小鼠免受CR- 肺炎克雷伯菌在肠内和肠外传播模型中的感染。然而， 这种mAb的保护作用如何在体内发生，如果17 H12用于治疗，这些问题至关重要 CR-Kp主要影响的免疫功能低下患者。 此外，我们的合作还发现了一个合成的寡糖表位，17 H12 识别并可引发对该ST 258亚群的CPS的免疫原性应答。该数据 这表明，17 H12的目标可能作为一个更便宜，更容易生产的替代全尺寸 CPS也可以覆盖更广泛的菌株，但这种可能性仍有待检验。 该提议寻求确定17 H12减轻体内CR-Kp感染的手段，并测试17 H12在体内的作用。 17 H12的合成表位可以作为潜在的疫苗。我将检验我的核心假设， 抗体通过专职吞噬细胞的作用保护小鼠免于CR-Kp肠外播散， 并且这些单克隆抗体识别的表位可用于防止传播。在目标1中，我将 检查吞噬细胞群和Fc受体是否是抗体介导的保护所必需的 相对于补体介导的机制。这一目标将利用各种调查工具，从流动 流式细胞术和转化体，以检查哪些常驻吞噬细胞群体在细胞中摄取GFP标记的CR-Kp。 抗体的存在、IgG亚类转换变体和Fc受体敲除在离体 吞噬作用测定和体内感染研究。我还将应用活体显微镜观察mAb- 介导的调理吞噬作用。在目标2中，我将评估是否接种小分子疫苗， 17 H12识别可以保护免受ST 258进化枝2 CR-Kp的感染。我会决定我们的合成人 结合17 H12的寡糖通过疫苗接种激发对全尺寸CPS的上级免疫应答， 挑战.此外，我将利用噬菌体展示来鉴定CPS表位的肽模拟物，并测试是否 17 H12以与其识别寡糖相同的方式识别该模拟表位。这项研究的结果 该提案不仅使我们能够检查抗荚膜免疫在CR-Kp清除中的作用，而且 使我们能够识别可能从mAb治疗中获益最大的患者，促进疫苗接种工作， 了解ST 258克隆体的胶囊。

PROJECT SUMMARY/ABSTRACT Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) causes serious hospital-acquired infections with mortality rates exceeding 50%, and new strategies are required to treat it. The monoclonal antibody (mAb) 17H12 developed by the Fries Laboratory binds to the capsular polysaccharide (CPS) to a subset of the most prominent CR-Kp clone in the United States, ST258. The mAb has been shown to broadly react with ST258 strains of multiple serotypes, kill CR-Kp through phagocytosis and other mechanisms in vitro, and protect mice from CR- Kp infection in both an intratracheal and extra-intestinal dissemination model. However, it remains unknown how protection by this mAb occurs in vivo, and such questions are crucial if 17H12 is to be used to treat immunocompromised patients that CR-Kp primarily affects. Furthermore, our collaborations have also uncovered a synthetic oligosaccharide epitope that 17H12 recognizes and can elicit an immunogenic response to the CPS of this ST258 subset as well. This data suggests that the target of 17H12 may serve as a cheaper, more easily producible alternative to full-sized CPS that can also cover a wider range of strains, but this possibility remains to be tested. This proposal seeks to determine the means by which 17H12 mitigates CR-Kp infection in vivo, and test synthetic epitopes of 17H12 that can act as potential vaccines. I will test my central hypothesis that anti-capsular antibodies protect mice from CR-Kp extra-intestinal dissemination through the action of professional phagocytes, and that epitopes that these mAbs recognize can be used to protect against dissemination. In Aim 1, I will examine whether phagocyte populations and the Fc receptor are required for antibody-mediated protection relative to complement-mediated mechanisms. This Aim will utilize a variety of investigative tools, from flow cytometry and transformants to examine which resident phagocyte populations take up GFP-labeled CR-Kp in the presence of antibody, to the use of IgG subclass switch variants and Fc receptor knockouts in ex-vivo phagocytosis assays and in vivo infection studies. I will also apply intra-vital microscopy to observe mAb- mediated opsonophagocytosis in real time. In Aim 2 I will assess whether vaccination with small molecules that 17H12 recognizes can protect against infection by ST258 clade 2 CR-Kp. I will determine whether our synthetic oligosaccharide that binds 17H12 elicits a superior immune response to full-sized CPS through vaccination and challenge. Additionally, I will utilize phage display to identify a peptide mimic of the CPS epitope and test whether 17H12 recognizes this mimotope in the same way as it recognizes the oligosaccharide. The findings of this proposal will not only allow us to examine the role of anti-capsular immunity in the clearance of CR-Kp, but also allow us to identify patients who may benefit best from mAb therapy, promote vaccination efforts, and better understand the capsule of the ST258 clone.