Factor H Fc fusions as novel therapeutics for Burkholderia pseudomallei infections

假鼻疽伯克霍尔德氏菌(BP)是类鼻疽病的病原体，是引起疾病的主要原因之一。 全世界热带/亚热带地区的败血症死亡。它在糖尿病患者中尤其致命，也是 由于其潜在的生物武器用途，被认为是一级选择剂。这些细菌具有天然的抗药性。 对于许多抗生素，目前还没有疫苗可用。因此，人们对确定 治疗性/预防性治疗的目标。像许多人类病原体一样，英国石油公司擅长通过 补体，先天免疫的关键武器。由一名合作调查员就这笔赠款生成的初步数据 研究表明，强毒BP在结合宿主因子H(FH)方面非常有效，FH是一种抑制C3的调节蛋白 沉积，从而防止宿主细胞上的补体激活。FH由20个称为Short的域名组成 共识重复序列(SCR)，但只有四个N端的SCR(SCR 1-4)具有补体抑制活性。 通过SCR6-7或SCR18-20结合宿主FH的能力是几种病原体使用的共同机制 (现在包括英国石油公司)，以逃避补体介导的杀伤。 行星生物技术公司(PBI)正在开发含有特定FH的治疗性重组蛋白 SCR(与病原体结合的那些)与Ig G Fc融合。在初步实验中，我们已经证明了一个结构 与IgG3 Fc N端连接到ScR18-20(Fc3/Scr(18-20))，促进补体沉积并显示潜力 因调解杀害英国石油公司而被起诉。这项建议的目标是进一步修改FC3/SCR(18-20)，以使其更加有效 促进直接杀伤、吞噬细胞摄取和杀死BP，并潜在地激活宿主细胞胞浆 杀戮路径。 我们将首先生产FC3/SCR(19-20)的变种，既有短铰链，也有长铰链，旨在 分别优化直接补体介导的杀伤或吞噬细胞作用。我们还将制作一个 构建带有Fc修饰的结构，旨在提高与细胞内Fc受体TRIM21的结合，从而 增强抗体依赖的细胞内中和作用(Adin)。我们将提纯和刻画足够的 这些新的FH-FC变种的数量将在体外和体内进行测试，以对抗BP的毒力菌株。 我们将测试新的Fc3/Scr(19-20)变体的能力：1)与BP结合，2)促进C3沉积 和膜攻击复合体(MAC)对BP的形成，3)促进血清存在下的直接杀伤，4) 促进巨噬细胞和中性粒细胞的摄取和细胞内杀伤，以及5)促进小鼠对BP的杀伤 在感染前或感染后给予，以显著减少类鼻疽的发展。 为了评估Fc3/Scr(19-20)对“正常”鼻腔菌群的潜在活性，我们将获得 ATCC人类鼻部共生(非致病)菌种的代表性培养及评价 在Fc-SCR存在和不存在的情况下，对补体杀死的敏感性(19-20)。我们还将评估 Fc-SCR(19-20)促进正常人红细胞补体沉积的能力。

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a major cause of morbidity and septic death in tropical/subtropical regions worldwide. It is particularly virulent in diabetic patients and is also considered a Tier 1 select agent due to its potential use as a bioweapon. These bacteria are naturally resistant to many antibiotics and there are currently no vaccines available. Thus, there is a great interest in identifying targets for curative/preventative therapies. Like many human pathogens, Bp is adept at evading killing by complement, a critical arm of innate immunity. Preliminary data generated by a co-investigator on this grant showed that virulent Bp are very efficient at binding host Factor H (FH), a regulatory protein that inhibits C3 deposition, thus preventing complement activation on host cells. FH comprises 20 domains called short consensus repeats (SCR), but only the four N-terminal SCR (SCR 1-4) possess complement inhibiting activity. The ability to bind host FH, either via SCR6-7 or SCR18-20, is a common mechanism used by several pathogens (now including Bp) to escape complement-mediated killing. Planet Biotechnology Inc (PBI) is developing therapeutic recombinant proteins containing specific FH SCRs (those that bind to pathogens) fused to IgG Fc. In preliminary experiments we have shown that a construct with IgG3 Fc N-terminal to SCR18-20 (Fc3/SCR(18-20)), promotes complement deposition and show potential for mediating killing of Bp. The goal of this proposal is to further modify Fc3/SCR(18-20) to become more effective at promoting direct killing, phagocyte uptake and killing of Bp, and potentially activating host cell cytoplasmic killing pathways. We will start by producing variants of Fc3/SCR(19-20) with both short and long hinges, intended to optimize either direct complement-mediated killing or opsonophagocytosis, respectively. We will also produce a construct with Fc modifications intended to improve binding to the intracellular Fc receptor, TRIM21, and thus enhance antibody-dependent intracellular neutralization (ADIN). We will purify and characterize sufficient amounts of these new FH-Fc variants to be tested in vitro and in vivo against virulent strains of Bp. We will test the ability of the new Fc3/SCR(19-20) variants to: 1) bind to Bp, 2) promote C3 deposition and membrane attack complexes (MAC) formation on Bp, 3) promote direct killing in the presence of serum, 4) promote uptake and intracellular killing by macrophages and neutrophils, and 5) promote killing of Bp in mice if administered either before or after infection, so as to significantly decrease the development of melioidosis. To evaluate potential activity of Fc3/SCR(19-20) against the “normal” intranasal flora, we will obtain cultures representative of human nasal commensal (non-pathogenic) species from ATCC and evaluate their sensitivity to killing by complement in the presence and absence of Fc-SCR(19-20). We will also evaluate the ability of Fc-SCR(19-20) to enhance complement deposition on normal human erythrocytes.