Immune defenses against Neisseria gonorrhoeae

 描述（由申请人提供）：淋病奈瑟菌（Ng）是性传播感染淋病的病原体。淋病每年在全世界造成1亿多新病例，并对妇女的生殖健康产生不利影响。Ng对几乎所有使用过的抗生素都产生了耐药性，现在已经达到了“超级细菌”的地位。目前迫切需要针对Ng的疫苗。补体（C）是针对Ng的先天免疫防御的关键臂。淋病的特征是多形核白细胞（PMNs）涌入生殖道，这与Ab和C一起可能有助于清除感染。由于缺乏对保护相关性的了解，针对Ng的疫苗开发受到阻碍。我们的候选疫苗是Ng脂寡糖（LOS）表位的肽模拟物（模拟位），其在小鼠中激发杀菌和调理吞噬Ab。在具体目标1中，我们将阐明中性粒细胞，C和C受体在疫苗抗体效力中的作用，以确定体内保护的相关因素。将在缺乏PMN、C3（调理素）或C5 a（趋化因子）受体的小鼠中评价疫苗Ab。Ng以人类特异性方式结合C抑制剂、因子H（fH）和C4 b结合蛋白（C4 BP）。人C抑制剂的结合减弱了疫苗Ab的杀菌活性。我们已经开发了表达人fH和C4 BP的转基因小鼠，其将用于评估疫苗Ab在体内的功效并模拟Ab必须克服人C抑制剂的人体条件。Ng LOS在淋病的发病机制中起核心作用。Ng通过相位变化改变其LOS聚糖延伸的能力有助于其在免疫逃避中的多功能性。我们已经构建了八个Ng突变体，其中LOS聚糖表达已经“基因型固定”。在特定目标2a中，将检查这些突变体中的每一种感染小鼠的能力，这将阐明由固定基因型驱动的确定的LOS结构如何有助于体内细菌适应性。根据我们的数据，改变LOS聚糖可以调节杀菌活性， 我们将研究疫苗抗体对特定目标2b中每种“合适”LOS基因型的效力。目标2中的研究将使LOS基因/表型与适应性相关，并使我们能够鉴定可以补充我们目前候选疫苗的LOS结构。具有针对Ng保守和普遍存在的外膜蛋白（称为还原修饰蛋白（Rmp））的Ab的个体显示出对Ng的杀菌活性受损，并且具有较高的感染淋病的风险。在具体目标3中，我们将确定针对Rmp的Ab阻断Ng的C依赖性Ab杀伤并对疫苗Ab的效力产生负面影响的分子基础。有趣的是，抗Rmp Ab不阻断N.脑膜炎（Nm）。将在两个物种中进行rmp的等位基因置换，以确定Ng和Nm Rmp之间的细微序列差异是否有助于两种病原体之间阻断的差异。相应的Ng和Nm PorB分子也将在两个物种之间交换，以评估物种特异性PorB对阻断效应的共同依赖性。拟议研究的完成将代表着开发抗淋病新疫苗的重要一步。

 DESCRIPTION (provided by applicant): Neisseria gonorrhoeae (Ng) is the causative agent of the sexually transmitted infection, gonorrhea. Gonorrhea causes over 100 million new cases worldwide annually and adversely affects the reproductive health of women. Ng has become resistant to almost every antibiotic that has been used and has now achieved "superbug" status. Vaccines against Ng are urgently needed. Complement (C) is a critical arm of innate immune defenses against Ng. Gonorrhea is characterized by an influx of polymorphonuclear leukocytes (PMNs) into the genital tract, which in concert with Ab and C may facilitate clearance of infection. Vaccine development against Ng has been hindered by lack of knowledge of the correlates of protection. Our vaccine candidate is a peptide mimic (mimitope) of a Ng lipooligosaccharide (LOS) epitope that elicits bactericidal and opsonophagocytic Ab in mice. In Specific Aim 1, we will elucidate the roles of PMNs, C and C receptors in vaccine Ab efficacy to define the correlates of protection in vivo. Vaccine Ab will be evaluated in mice that lack either PMNs, C3 (opsonin) or C5a (chemotaxin) receptor. Ng binds the C inhibitors, factor H (fH) and C4b- binding protein (C4BP) in a human-specific manner. Binding of human C inhibitors blunts the bactericidal activity of vaccine Ab. We have developed transgenic mice that express both human fH and C4BP, which will be used to evaluate the efficacy of vaccine Ab in vivo and simulate conditions in humans where Ab must surmount human C inhibitors. Ng LOS plays a central role in the pathogenesis of gonorrhea. The ability of Ng to alter its LOS glycan extensions through phase variation contributes to its versatility in immune evasion. We have constructed eight Ng mutants where LOS glycan expression has been 'genotypically fixed'. In Specific Aim 2a the ability of each of these mutants to infect mice will be examined, which will elucidate how defined LOS structures driven by fixed genotypes contribute to bacterial fitness in vivo. In light of our data which show that altering LOS glycans modulates the bactericidal activity of vaccine anti-LOS Ab, we will study the efficacy of the vaccine Ab against each 'fit' LOS genotype in Specific Aim 2b. Studies in Aim 2 will correlate LOS geno/phenotypes with fitness and enable us to identify LOS structures that could supplement our current vaccine candidate. Individuals who possess Ab directed against an Ng conserved and ubiquitous outer membrane protein called reduction modifiable protein (Rmp) show impaired bactericidal activity against Ng and are at a higher risk of contracting gonorrhea. In Specific Aim 3 we will determine the molecular basis whereby Abs directed against Rmp block C-dependent Ab killing of Ng and negatively impact the efficacy of vaccine Ab. Interestingly, anti-Rmp Ab does not block killing of N. meningitidis (Nm). Allelic replacement of rmp across the two species will be performed to define whether subtle sequence differences between Ng and Nm Rmp contribute to differences in blocking across the two pathogens. Corresponding Ng and Nm PorB molecules will also be exchanged across the two species to assess co-dependence of species specific PorB on the blocking effect. Completion of the proposed studies will represent an important step forward in the development of novel vaccines against gonorrhea.