Novel immunotherapeutics against multi-drug resistant Neisseria gonorrhoeae

描述（由申请人提供）：淋病奈瑟菌（Ng）是性传播感染（STI），淋病的病原体。在世界范围内，每年有1亿例新发病例。吴恩达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”的地位。尤其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。迫切需要新的治疗方法来对抗这种病原体。补体（C2）是先天免疫防御Ng的关键臂。Ng通过几种机制逃避C2，包括清除人类C2抑制剂因子H （fH）。fH包含20个结构域，结构域18-20与孔蛋白B相互作用，并通过脂寡糖唾液化增强结合，而结构域6-7与neisserial表面蛋白a结合。我们测试的所有Ng在fH中都与这些区域中的一个或两个结合。通过将fH结构域6-7或18-20与IgG的Fc区融合而产生的嵌合分子可以影响c2依赖性的Ng杀伤。在全长天然fH的情况下，fH结构域18-20结合并限制宿主细胞的病理损伤，这限制了fH/Fc中“未修饰”结构域18-20的治疗使用。我们通过在fH18-20/Fc结构域19 （D1119G）引入点突变克服了这一限制，该突变消除了c2介导的宿主细胞裂解而不改变抗ng的功效，从而为我们提供了一种先导治疗分子。在Aim 1中，我们将通过在Fc中引入3个氨基酸点突变并消除Fc聚糖的核心焦点，最大限度地提高fHD1119G/Fc的人IgG1 Fc区域介导抗噬细胞作用和c2依赖性杀伤多重耐药Ng分离株的能力。同样，在Aim 2a中，我们将优化融合到人fH结构域6和7 （fH6,7/Fc）的Fc的效应功能，并评估其介导耐药Ng的c2依赖性杀伤和调理吞噬作用的能力。识别不同表位的fHD111G/Fc和fH67/Fc协同和刺激对Ng的杀菌活性的能力将在Aim 2b中进行评估。在目标1和2 （R21期）达到里程碑之后，将进行体内研究（R33期），以确定fH/Fc的安全性、有效性和作用机制。肾、眼和血液学毒性（c2介导损伤的靶器官）将在Aim 3中进行评估。引入D1119G突变可以产生新的表位并引发自身抗体；这种可能性将通过我们的人类fH转基因（Tg）小鼠来解决。在Aim 4中，我们将使用我们的新型人类fH/C4BP （C4BP；经典途径C2抑制剂）“双”Tg小鼠（Ng仅结合人类，而不结合小鼠C2抑制剂）来确定fH/Fc融合蛋白对Ng感染小鼠模型中一组耐药Ng的功效。使用缺乏补体C3、C5、Fc受体、PMNs或巨噬细胞的小鼠，我们将确定fH/Fc如何在体内清除Ng。许多具有重要医学意义的微生物通过结构域6-7和/或18-20结合人类fH。虽然我们将使用Ng作为模型病原体，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.