Dissection of Epitope-Specific Mechanisms of Neutralization of Influenza Virus by Intact IgG and Fab Fragments.
Dissection of Epitope-Specific Mechanisms of Neutralization of Influenza Virus by Intact IgG and Fab Fragments.
复制标题
完整 IgG 和 Fab 片段中和流感病毒的表位特异性机制的剖析。
DOI：
10.1128/jvi.02006-17
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发表时间：
2018
影响因子：
5.4
通讯作者：
Lee,KellyK
中科院分区：
文献类型：
--
作者：
Williams,JamesA;Gui,Long;Hom,Nancy;Mileant,Alexander;Lee,KellyK
中和抗体（nAb）对流感病毒血凝素（HA）融合糖蛋白的反应对预防病毒感染很重要，但我们对这些抗体的作用机制缺乏全面的了解。在这里，我们研究了针对不同HA表位的nAb结合和IgG二价在抑制HA功能中的作用。HC19靶向HA末端的受体结合袋，而FI6v3主要结合HA2融合亚基向茎的基部。令人惊讶的是，HC19通过阻止HA在融合激活条件下的结构重排，抑制了HA诱导脂质混合的能力。这些结果表明，像HC19这样的nab不仅可以阻断受体结合，还可以抑制融合所需的关键晚期HA构象变化。完整的HC19 IgG也被证明可以交联分离的病毒颗粒，将大比例的HA埋在聚集体中，从而阻止它们与靶膜相互作用；fab没有产生这种聚集，并且表现出比IgG更弱的中和作用，强调了二价对病毒中和能力的影响。相比之下，茎靶向nAb FI6v3不聚集颗粒。Fab片段在防止膜破坏和融合方面的效果明显低于IgG。我们推断，FI6v3 IgG在特定颗粒内的刺间交联可能是其有效中和的关键，因为在fab中没有发生明显的中和。这些结果表明，IgG二价通过重要的功能模式增强了HA抑制作用，而在减少fab可溶性HA结构中并不明显。流感病毒血凝素（HA）融合糖蛋白介导进入靶细胞，是中和抗体（nab）的主要抗原靶点。我们目前对抗体（Ab）介导的中和机制的结构理解在很大程度上依赖于抗原结合（Fab）片段与可溶性分离抗原复合物的高分辨率表征，通过低温电子显微镜（EM）单粒子重建或x射线晶体学。全长IgG和整个病毒粒子之间的相互作用尚未得到很好的表征，我们对完整抗体如何中和病毒和预防感染的理解仍然存在空白。利用结构和生物物理方法，我们观察到抗体介导的HA功能抑制和病毒感染的中和是通过多种共存的机制发生的，在很大程度上依赖于靶向的特定表位，并且高度依赖于IgG分子的二价性质。
The neutralizing antibody (nAb) response against the influenza virus hemagglutinin (HA) fusion glycoprotein is important for preventing viral infection, but we lack a comprehensive understanding of the mechanisms by which these antibodies act. Here we investigated the effect of nAb binding and the role of IgG bivalency in the inhibition of HA function for nAbs targeting distinct HA epitopes. HC19 targets the receptor binding pocket at the distal end of HA, while FI6v3 binds primarily to the HA2 fusion subunit toward the base of the stalk. Surprisingly, HC19 inhibited the ability of HA to induce lipid mixing by preventing the structural rearrangement of HA under fusion-activating conditions. These results suggest that nAbs such as HC19 not only act by blocking receptor binding but also inhibit key late-stage HA conformational changes required for fusion. Intact HC19 IgG was also shown to cross-link separate virus particles, burying large proportions of HA within aggregates where they are blocked from interacting with target membranes; Fabs yielded no such aggregation and displayed weaker neutralization than IgG, emphasizing the impact of bivalency on the ability to neutralize virus. In contrast, the stem-targeting nAb FI6v3 did not aggregate particles. The Fab fragment was significantly less effective than IgG in preventing both membrane disruption and fusion. We infer that interspike cross-linking within a given particle by FI6v3 IgG may be critical to its potent neutralization, as no significant neutralization occurred with Fabs. These results demonstrate that IgG bivalency enhances HA inhibition through functionally important modes not evident in pared-down Fab-soluble HA structures.IMPORTANCEThe influenza virus hemagglutinin (HA) fusion glycoprotein mediates entry into target cells and is the primary antigenic target of neutralizing antibodies (nAbs). Our current structural understanding of mechanisms of antibody (Ab)-mediated neutralization largely relies on the high-resolution characterization of antigen binding (Fab) fragments in complex with soluble, isolated antigen constructs by cryo-electron microscopy (EM) single-particle reconstruction or X-ray crystallography. Interactions between full-length IgG and whole virions have not been well characterized, and a gap remains in our understanding of how intact Abs neutralize virus and prevent infection. Using structural and biophysical approaches, we observed that Ab-mediated inhibition of HA function and neutralization of virus infectivity occur by multiple coexisting mechanisms, are largely dependent on the specific epitope that is targeted, and are highly dependent on the bivalent nature of IgG molecules.