Complement Activation on Neisseria meningitidis

描述（由申请人提供）：补体(C)是先天免疫防御侵袭性脑膜炎球菌感染的关键臂。脑膜炎奈瑟菌（Neisseria meningitidis, Nm）已经进化出几种复杂的机制来逃避宿主C。宿主在细菌上激活C的努力与微生物逃避C的策略之间的平衡决定了该生物是被从粘膜中清除，还是在鼻咽中保持无症状的定菌，还是继续引起侵袭性疾病。在之前的资助期内，我们描述了Nm与C4b（经典途径（CP）的一个组成部分）之间的相互作用。几乎所有从血液或脑脊液中分离出来的奈米菌都被包膜，胶囊对C的耐药性很重要。胶囊介导的C耐药性的分子基础尚不清楚。目标1中定义的角色荚膜多糖(CPS)在调节c。首先,在目标1,我们将阐明CP激活水平差异的原因由anti-CPS anti-outer膜Abs,基于摩尔马伯针对CPS修复C4b低于马伯对膜结构,表明高效C4b绑定可能需要激活的C4近似脑膜炎球菌分子作为C4b受体如洛杉矶和不透明性的蛋白质。含有人IgG1 Fc的嵌合单克隆抗体，针对CPS或Nm候选疫苗因子h结合蛋白（fHbp），将用于对称比较这些单克隆抗体的c激活功能。我们的初步研究表明，A组、C组、W-135组和Y组（但不包括B组）的CPS可阻断纯化的人因子B和d对人C3的激活。在Aim 1b中，我们将通过检测CPS与纯化的AP组分的相互作用来确定AP抑制的分子基础。这些研究可能会确定聚阴离子阻断AP活化的共同作用机制。CPS对AP的调节是人类特有的；兔AP的组装（不受CPS调节）将在Aim 1c中进行检查，以更好地了解CPS调节AP的人类特异性。表面蛋白也有助于抵抗C；fHbp结合AP抑制剂，因子H (fH)，抑制C活化。在目标2中，我们将利用我们对fH- fHbp相互作用的了解，利用嵌合分子阻断fH与Nm的结合，并操纵C级联，以精确定义CP和AP在杀死Nm中的作用。我们还将尝试“增强”一种非抗菌的抗fhbp单抗（JAR 4）的杀菌功能。这些知识可能导致疫苗策略的优化。最后，在Aim 3中，我们将在表达低水平fHbp的菌株中定义新的C逃避策略。初步证据表明，低fHbp表达/低fH结合Nm在C5b-9的形成或插入水平上调节C，我们将在此水平上表征C抗性的分子基础。这些研究将有助于定义Nm逃避C的新机制；这些信息将促进对脑膜炎球菌发病机制的了解，并改善正在进行的开发有效蛋白质疫苗的努力。

DESCRIPTION (provided by applicant): Complement (C) is a key arm of innate immune defenses against invasive meningococcal infections. Neisseria meningitidis (Nm) have evolved several sophisticated mechanisms to evade host C. A balance between the hosts' efforts to activate C on the bacterium and the microbe's C evasion strategies dictates whether the organism is cleared from the mucosa, remains an asymptomatic colonizer in the nasopharynx, or proceeds to cause invasive disease. In the previous funding period we characterized interactions between Nm and C4b, a component of the classical pathway (CP). Almost all Nm isolates recovered from the blood or cerebrospinal fluid are encapsulated and capsule is important for resistance to C. The molecular basis for capsule-mediated C resistance remains undefined. In Aim 1 will define the role of capsular polysaccharide (CPS) in regulating C. First, in Aim 1a, we will elucidate the reason for differences in the level of CP activation mediated by anti-CPS and anti-outer membrane Abs. On a molar basis, mAbs directed against CPS fix less C4b than mAbs against membrane structures, suggesting that efficient C4b binding may require activation of C4 proximate to the meningococcal molecules that serve as C4b acceptors such as LOS and opacity protein. Chimeric mAbs containing human IgG1 Fc and directed against either CPS or the Nm vaccine candidate, factor H-binding protein (fHbp) will be used to symmetrically compare the C-activating functions of these mAbs. The positive feedback loop of the alternative pathway (AP) is also important for bacterial killing by vaccine Abs. Our preliminary studies indicate that the group A, C, W-135 and Y (but not B) CPSs block activation of human C3 by purified human factors B and D. In Aim 1b we will define the molecular basis of AP inhibition by examining the interaction of CPS with purified AP components. These studies may identify a common mechanism of action for polyanions that block AP activation. AP regulation by CPS is human-specific; assembly of the rabbit AP (not regulated by CPS) will be examined in Aim 1c to provide a better understanding of the human-specificity of AP regulation by CPS. Surface proteins also contribute to C resistance; fHbp binds to the AP inhibitor, factor H (fH) and inhibits C activation. In Aim 2, we will we will exploit our knowledge of fH- fHbp interactions and use chimeric molecules to block fH binding to Nm and manipulate the C cascade to precisely define the roles of the CP and AP in killing Nm. We will also attempt to 'boost' the bactericidal function of an otherwise nonbactericidal anti-fHbp mAb (JAR 4). Such knowledge could lead to optimization of vaccine strategies. Finally, in Aim 3 we will define novel C evasion strategies in strains that express low levels of fHbp. Preliminary evidence indicates that low fHbp expressing/low fH binding Nm regulate C at the level of C5b-9 formation or insertion and we will characterize the molecular basis for C resistance at this level. These studies will help define novel mechanisms of C evasion by Nm; information that will advance understanding of meningococcal pathogenesis and improve ongoing efforts to develop effective protein-based vaccines. PUBLIC HEALTH RELEVANCE: Meningococcal meningitis and sepsis is a major health problem worldwide. It mainly affects children and young adults and is a significant cause of morbidity and mortality. Although a good vaccine exists against several meningococcal serogroups, there is no effective vaccine against serogroup B disease. The emergence of disease caused by strains that lack a capsule following vaccination campaigns using capsular polysaccharide-based vaccines is of concern. Complement forms an important arm of host defenses against the meningococcus. The studies proposed in this application will help us better understand how this pathogen escapes killing by complement and will ultimately aid in the development of better vaccines against meningococcal meningitis.