Novel immunotherapeutics against multi-drug resistant Neisseria gonorrhoeae

描述(申请人提供)：淋病奈瑟氏菌(Ng)是性传播感染(STI)淋病的病原体。在世界范围内，每年发生1亿例新病例。NG已经对几乎所有使用过的抗生素都产生了抗药性，现在已经达到了“超级细菌”的状态。特别是，头孢曲松耐药菌株的出现和传播预示着一个无法治愈的淋病时代的到来，并代表着一场全球公共卫生危机。迫切需要针对这种病原体的新的治疗方法。补体(C2)是Ng先天免疫防御系统的重要组成部分。NG使用几种机制来逃避C2，包括清除人类C2抑制因子H(Fh)。Fh由20个结构域组成--结构域18-20与孔蛋白B相互作用，结合通过脂寡糖唾液酸化增强，而结构域6-7与新序列表面蛋白A结合。通过将FH区6-7或18-20融合到免疫球蛋白Fc区而产生的嵌合分子可以对Ng产生C2依赖的杀伤作用。在全长天然FH的背景下，FH结构域18-20与宿主细胞结合并用于限制对宿主细胞的病理性损伤，这限制了FH/Fc中“未经修饰”的结构域18-20的治疗用途。我们通过在fH18-20/Fc的19区(D1119G)引入点突变来克服这一限制，该突变消除了C2介导的宿主细胞的裂解，而不改变抗Ng的效果，从而为我们提供了一种先导治疗分子。在目标1中，我们将通过在Fc区引入3个氨基酸点突变和消除Fc糖链的核心岩藻糖来最大限度地发挥fHD1119G/Fc的人IgG1Fc区介导吞噬细胞和C2依赖的杀伤多药耐药Ng分离株的能力。同样，在目标2a中，我们将优化与人FH结构域6和7(fH6，7/Fc)融合的Fc的效应功能，并评估其介导C2依赖的杀伤和对耐药Ng的吞噬作用的能力。目标2b将评估识别不同表位的fHD111G/Fc和fH67/Fc协同和刺激对Ng的杀菌活性的能力。在AIMS 1和2(R21阶段)取得的里程碑之后，将进行体内研究(R33阶段)，以确定FH/FC的安全性、有效性和作用机制。目标3将评估肾脏、眼睛和血液毒性(C2介导的损伤的靶器官)。引入D1119G突变可以创建新的表位并诱导自体抗体；这种可能性将使用我们的人fh转基因(TG)小鼠来解决。在目标4中，我们将使用我们的新型人Fh/C4BP(C4BP；经典途径C2抑制剂)“双重”TG小鼠(Ng仅结合人，而不结合小鼠C2抑制剂)，在Ng感染的小鼠模型中确定Fh/Fc融合蛋白对一组耐药Ng的疗效。利用缺乏补体C3、C5、Fc受体、中性粒细胞或巨噬细胞的小鼠，我们将定义Fh/Fc如何在体内清除Ng。许多医学上重要的微生物通过结构域6-7和/或18-20与人FH结合。虽然我们将使用Ng作为模式病原体，但这些研究的成功完成将为评估这些Fh/Fc分子对抗其他新出现的耐药病原体提供一个强大的平台。

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae (Ng) is the causative agent of the sexually transmitted infection (STI), gonorrhea. Worldwide, >100 million new cases occur annually. Ng has become resistant to almost every antibiotic that has been used and has now achieved "superbug" status. The emergence and spread of ceftriaxone-resistant strains, in particular, has heralded an era of untreatable gonorrhea and represents a global public health crisis. Novel therapeutics against this pathogen are urgently needed. Complement (C2) is a critical arm of innate immune defenses against Ng. Ng evade C2 using several mechanisms, including scavenging the human C2 inhibitor, factor H (fH). fH comprises 20 domains - domains 18-20 interact with porin B and binding is augmented by lipooligosaccharide sialylation, while domains 6-7 bind to neisserial surface protein A. All Ng we have tested bind to one or both of these regions in fH. Chimeric molecules created by fusing fH domains 6-7 or 18-20 to the Fc region of IgG can effect C2-dependent killing of Ng. fH domains 18-20 in the context of full-length native fH binds to and serves to limit pathological injury to host cells, which limits the therapeutic use of 'unmodified' domains 18-20 in fH/Fc. We have overcome this limitation by introducing a point mutation in domain 19 (D1119G) of fH18-20/Fc that eliminates C2-mediated lysis of host cells without altering anti-Ng efficacy, thus providing us with a lead therapeutic molecule. In Aim 1, we will maximize the ability of the human IgG1 Fc region of fHD1119G/Fc to mediate opsonophagocytosis and C2-dependent killing of multi-drug resistant Ng isolates by introducing 3 amino acid point mutations in the Fc and eliminating the core fucose of Fc glycans. Similarly, in Aim 2a, we will optimize the effector functions of the Fc that is fused to human fH domains 6 and 7 (fH6,7/Fc) and evaluate its ability to mediate C2-dependent killing and opsonophagocytosis of drug-resistant Ng. The ability of fHD111G/Fc and fH67/Fc, which recognize distinct epitopes, to synergize and stimulate bactericidal activity against Ng will be evaluated in Aim 2b. The milestones achieved in Aims 1 and 2 (the R21 phase) will be followed by studies in vivo (R33 phase) to determine the safety, efficacy and mechanism of action of fH/Fc. Renal, ocular and hematologic toxicity (target organs for C2-mediated injury) will be assessed in Aim 3. Introducing the D1119G mutation could create a neo-epitope and elicit auto-Abs; this possibility will be addressed using our human fH transgenic (Tg) mice. In Aim 4 we will determine efficacy of the fH/Fc fusion proteins against a panel of drug-resistant Ng in the mouse model of Ng infection using our novel human fH/C4BP (C4BP; classical pathway C2 inhibitor) "double" Tg mice (Ng bind only human, but not mouse C2 inhibitors). Using mice that lack complement C3, C5, Fc receptors, PMNs or macrophages we will define how fH/Fc clear Ng in vivo. Many medically important microbes bind human fH through domains 6-7 and/or 18-20. While we will use Ng as a model pathogen, successful completion of these studies will provide a strong platform to assess these fH/Fc molecules against other emerging drug-resistant pathogens.