Developing protective monoclonal antibodies against Gram- and Gram+ bacteria

项目总结/摘要： 抗生素耐药性对人类健康的威胁越来越大，导致难以治疗的感染，其中一些感染是由抗生素引起的。 多药耐药病原体对所有或大多数抗生素具有耐药性。为了应对这一日益严重的危机， U19正在采取综合方法来产生和开发针对关键毒力的疗法 细菌的机制。具体而言，该核心方案将与项目1和项目2采取综合办法，力求 开发针对不同细菌粘附素的单克隆抗体（mAb）疗法， 显示对于病原体结合到宿主组织和表面并抵抗从宿主组织和表面去除的能力是关键的。 肾盂肾炎大肠大肠杆菌（UPEC）利用CUP粘附性皮利与粘附素蛋白结合， 组织，包括未发炎的膀胱、发炎的膀胱、肾、导尿管和 胃肠道（GIT）。此外，克雷伯氏菌表达甘露糖结合FimH粘附素，其对于 克雷伯氏菌引起尿路感染的能力。此外，鲍曼不动杆菌中的CUP粘附素CupD和PrpD，如 以及革兰氏阳性肠球菌的分选酶组装菌毛尖端的EbpA粘附素， 纤维蛋白原，以促进生物膜形成和导管相关性UTI（UTI）。在 与项目1合作，该CORE将使用最先进的高通量克隆和选择方法 以产生针对所有这些粘附素的mAb并评估它们作为抗生素节约治疗剂的潜力。 这些单克隆抗体也将阐明未来抗粘附素疫苗设计的重要表位。此外，所有 CUP粘附皮利是通过同源系统组装的，包括周质伴侣和外膜蛋白。 膜引导器。Usher是一个用于CUP菌毛聚合的五域组装平台， 跨膜结构域用作用于挤出组装的纤维的孔。这个小孔是由一个塞子堵住的 只有当引导器组装菌毛纤维时，才离开孔的结构域。因此，引导蛋白将被靶向 并且选择mAb的能力是结合和抑制引导者介导的菌毛 组装和/或在没有生长的菌毛纤维的情况下“打开”引导孔的能力。具有开口 没有菌毛纤维的毛孔将使细菌对我们现有武器库中的抗生素敏感，而这些抗生素不是 通常能够穿过革兰氏阴性外膜，因此通常不能有效地对抗 革兰氏阴性病原体。粘附素及其组装的靶向具有以下优点： 靶向粘附素的结合口袋中或引导器中的关键组装位点中，以防止靶向粘附素的结合。 治疗剂可能破坏粘附素功能，从而使病原体无毒力。的 所产生的单克隆抗体有望成为治疗耐药性感染的可行替代方案。 治疗性mAb尚未完全用于治疗感染性疾病，这可能是由于 抗生素的历史性成功然而，随着抗生素耐药性的上升，是时候应用这些药物了。 潜在的预防传染病的工具。

PROJECT SUMMARY/ ABSTRACT SUMMARY: Antibiotic resistance is increasingly a threat to human health resulting in difficult to treat infections, with some multidrug resistant pathogens being resistant to all or most all antibiotics. To combat this growing crisis, this U19 is taking an integrated approach to the generation and development of therapeutics against key virulence mechanism of bacteria. Specifically, this CORE will in an integrated approach with Projects 1 and 2, seek to develop monoclonal antibody (mAb) therapies against different bacterial adhesins that extensive studies have shown to be critical for the ability of pathogens to bind to and resist removal from host tissues and surfaces. Uropathogenic E. coli (UPEC) use CUP adhesive pili tipped with adhesin proteins to bind to a variety of human tissues including the uninflammed bladder, the inflamed bladder, the kidney, urinary catheters and the gastrointestinal tract (GIT). In addition, Klebsiella expresses a mannose binding FimH adhesin that is critical for the ability of Klebsiella to cause UTIs. Further, CUP adhesins in Acinetobacter baumanii, CupD and PrpD, as well as the EbpA adhesin at the tip of the sortase assembled pilus of the Gram-positive Enterococci, bind to fibrinogen on implanted catheters to promote biofilm formation and catheter-associated UTIs (CAUTIs). In collaboration with Project 1 this CORE will use state of the art high throughput cloning and selection methods to generate mAbs against all of these adhesins and evaluate their potential as antibiotic sparing therapeutics. These mAbs will also elucidate important epitopes for future anti-adhesin vaccine design. Further, all of the CUP adhesive pili are assembled by homologous systems involving a periplasmic chaperone and and outer membrane usher. The usher is a five domain assembly platform for CUP pilus polymerization and its transmembrane domain functions as a pore for extrusion of the assembled fiber. This pore is gated by a plug domain that only exits the pore when the usher is assembly a pilus fiber. Thus, usher proteins will be targeted for mAb generation and mAbs will be selected for both their ability to bind and inhibit usher mediated pilus assembly and/or for the ability to “open” the usher pore in the absence of a growing pilus fiber. Having an open pore without a pilus fiber will render the bacteria sensitive to antibiotics from our existing arsenal that are not generally able to cross the Gram-negative outer membrane and thus are not generally efficacious against Gram-negative pathogens. Targeting of adhesins and their assembly has the advantage that mutations in the binding pocket of targeted adhesins or in critical assembly sites in the usher to prevent binding of the therapeutic to its target would likely destroy adhesin function and thus render the pathogen non-virulent. The mAbs generated have the promise to be a viable alternative for the treatment of antibiotic-resistant infecitons. Therapeutic mAbs have not yet been fully harnessed for treating infectious diseases, perhaps due to the historic success of antibiotics. However, with antibiotic resistance on the rise, it is time to apply these potentially antibiotic-sparing tools to infectious disease.