Factor H Fc fusions as novel therapeutics for Burkholderia pseudomallei infections

类鼻疽伯克霍尔德氏菌（Bp）是类鼻疽病的病原体，类鼻疽病是发病的主要原因， 全世界热带/亚热带地区的脓毒性死亡。它在糖尿病患者中特别致命， 被认为是一级选择剂，因为它可能用作生物武器。这些细菌天生就有抵抗力 许多抗生素，目前没有可用的疫苗。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，Bp擅长通过以下方式逃避死亡： 补体，先天免疫的关键武器。本研究资助的合作研究者生成的初步数据 表明毒性Bp在结合宿主H因子（FH）方面非常有效，H因子是一种抑制C3 沉积，从而防止宿主细胞上的补体活化。FH由20个称为短的结构域组成 共有重复序列（SCR），但只有四个N-末端SCR（SCR 1-4）具有补体抑制活性。 通过SCR 6 -7或SCR 18 -20结合宿主FH的能力是几种病原体使用的常见机制 (now包括Bp）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 与IgG Fc融合的SCR（与病原体结合的那些）。在初步的实验中，我们已经表明， 与IgG 3 Fc N-末端连接至SCR 18 -20（Fc 3/SCR（18-20）），促进补体沉积，并显示出潜在的 介导了Bp的死亡。该提案的目标是进一步修饰Fc 3/SCR（18-20）以变得更有效 促进直接杀伤、吞噬细胞摄取和Bp杀伤，并潜在激活宿主细胞质 杀戮之路 我们将首先生产具有短铰链和长铰链的Fc 3/SCR（19-20）的变体， 分别优化直接补体介导的杀伤或调理吞噬作用。我们还将制作一个 具有Fc修饰的构建体，旨在改善与细胞内Fc受体TRIM 21的结合，从而 增强抗体依赖性细胞内中和（ADIN）。我们将充分净化和表征 这些新的FH-Fc变体的量将在体外和体内针对Bp的强毒株进行测试。 我们将测试新的Fc 3/SCR（19-20）变体的能力：1）结合Bp，2）促进C3沉积 和膜攻击复合物（MAC）对Bp的形成，3）在血清存在下促进直接杀伤，4） 促进巨噬细胞和嗜中性粒细胞的摄取和细胞内杀伤，和5）促进小鼠中Bp的杀伤，如果 在感染之前或之后施用，从而显著减少类鼻疽病的发展。 为了评价Fc 3/SCR（19-20）对“正常”鼻内植物群的潜在活性，我们将获得 来自ATCC的代表人鼻粘膜（非致病性）物种的培养物，并评估它们的 在存在和不存在Fc-SCR的情况下对补体杀伤的敏感性（19-20）。我们还将评估 Fc-SCR（19-20）增强正常人红细胞上补体沉积的能力。

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.