Complement Activation on Neisseria meningitidis

描述(由申请人提供)：补体(C)是对抗侵袭性脑膜炎双球菌感染的天然免疫防御的关键环节。脑膜炎奈瑟菌(Nm)已经进化出几种复杂的机制来逃避宿主C。宿主对细菌激活C的努力和微生物的C逃避策略之间的平衡决定了有机体是从粘膜中清除出来，还是在鼻咽中保持无症状的定植，还是继续导致侵袭性疾病。在之前的资助期间，我们描述了Nm和C4b之间的相互作用，C4b是经典途径(CP)的一个组成部分。几乎所有从血液或脑脊液中回收的Nm分离株都有包膜，包膜对C菌的抗性是重要的。包膜介导的C药耐药性的分子基础仍不明确。在目标1中，我们将明确胶囊多糖(CPS)在调节C中的作用。首先，在目标1a中，我们将阐明抗CPS抗体和抗外膜抗体介导的CP激活水平不同的原因。在摩尔基础上，针对CPS的单抗对膜结构的C4b的固定作用比单抗少，这表明有效的C4b结合可能需要激活作为C4b受体的脑膜炎球菌分子附近的C4，如LOS和不透明蛋白。含有人IgG1Fc的针对CPS或Nm候选疫苗的嵌合单抗(FHBP)将被用来对称地比较这些单抗的C激活功能。替代途径(AP)的正反馈环对疫苗Abs的杀菌作用也很重要。我们的初步研究表明，A、C、W-135和Y组(而不是B组)CPSS可以阻断纯化的人因子B和D对人C3的激活。在目标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抑制AP的分子基础。这些研究可能确定多阴离子阻断AP激活的共同作用机制。CPS对AP的调节是人类特有的；Aim 1c将研究兔AP(不受CPS调节)的组装，以更好地了解CPS调节AP的人特异性。表面蛋白也有助于C的抗性；fHBP与AP抑制因子H(Fh)结合并抑制C的激活。在目标2中，我们将利用我们对Fh-fHBP相互作用的知识，使用嵌合分子来阻止Fh与Nm的结合，并操纵C级联来精确定义CP和AP在杀死Nm中的作用。我们还将尝试‘增强’抗fHBP单抗(JAR 4)的杀菌功能。这些知识可能会导致疫苗策略的优化。最后，在目标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，我们将在这个水平上表征C抗性的分子基础。这些研究将有助于确定Nm逃避C的新机制；这些信息将促进对脑膜炎双球菌发病机制的了解，并改进正在进行的开发有效的基于蛋白质的疫苗的努力。

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.