Structure-based design of broad flavivirus immunogens

摘要 登革热病毒是一种由蚊子传播的黄病毒，估计每年导致 3.9 亿人感染 年。登革热有四种血清型 (DENV1-4) 在高流行地区共同流行。小学 单一 DENV 血清型感染会导致发热性疾病以及随后对该病毒的持久免疫力 血清型。异型血清型的继发感染可导致严重休克综合征和死亡。 严重登革热病部分是由初次感染期间引起的交叉反应抗体引起的， 可以结合异源 DENV 血清型，但不能中和它们。相反，这些非中和性 抗体有利于Fcγ受体阳性细胞的进入和感染，从而引起“抗体依赖性” 感染的增强”（ADE）。虽然最近推出了一种减毒活四组分嵌合疫苗 该疫苗已在 19 个国家和欧洲部署，但不能保护未接触过症状或疾病的个体 严重感染，在某些情况下甚至可能加剧病情。此外，全球出现 寨卡病毒 (ZIKV) 以及 DENV 和 ZIKV 之间潜在的 ADE 引起了对疫苗策略的担忧 含有E糖蛋白中的大部分或全部表位。尽管如此，分离和表征 保护性抗体，在某些情况下，广泛中和抗体表明，E 内的某些表位 糖蛋白可能具有引发广泛保护性反应的能力。在这里，我们利用创新蛋白质 开发“免疫聚焦”抗原作为潜在候选疫苗的工程方法，其中 诱导非中和抗体的表位被工程突变或糖基化所掩盖。我们的 假设是，掩盖这些不利的表位将使免疫反应偏向于更强的方向。 中和性、保护性和广泛的反应。目标 1 和 2 重点关注 DENV 和 ZIKV E 中的关键表位 结构域 III (EDIII) 和 Aim 3 探索 ZIKV E 融合前二聚体的聚糖掩蔽，以免疫聚焦 E-二聚体表位（EDE）。 EDIII 对于亚单位疫苗设计很有吸引力，因为它是有效的目标 DENV 和 ZIKV 的中和和保护性抗体。然而，用野生型 EDIII 进行免疫 蛋白质导致中和和非中和抗体的诱导，这些抗体参与多种 表位。我们使用噬菌体展示通过突变来掩盖 DENV 和 ZIKV EDIII 的非生产性表位， 同时保持中和表位。这些“表面重塑的 EDIII”（rsDIII）将与蛋白质结合 评估了纳米颗粒及其在小鼠中诱导中和和保护性抗体反应的能力。 为了将预融合 E 二聚体免疫聚焦在 EDE 上，我们开发了一种哺乳动物展示系统 允许快速评估 E 二聚体构建体与 EDE mAb 的结合。我们将利用这个系统 筛选具有多个工程糖基化位点的变体，这些位点掩盖了 EDE 外部的表面。的 最有前途的候选者将测试其在小鼠体内诱导 EDE 样单克隆抗体的能力。这项工作将 为针对 DENV、ZIKV 和其他可能的新型亚单位候选疫苗提供概念验证 全球关注的黄病毒。

SUMMARY Dengue virus is a mosquito-transmitted flavivirus that causes an estimated 390 million human infections each year. There are four serotypes of Dengue (DENV1-4) that co-circulate in hyperendemic regions. Primary infection by a single DENV serotype results in febrile illness and subsequent durable immunity to that serotype. Secondary infections by heterotypic serotypes can lead to severe shock syndrome and death. Severe Dengue disease is caused in part by cross-reactive antibodies elicited during primary infection that can bind heterologous DENV serotypes but cannot neutralize them. Instead, these non-neutralizing antibodies facilitate entry and infection in Fcγ receptor-positive cells, thus causing "antibody-dependent enhancement" (ADE) of infection. While a live-attenuated four-component chimeric vaccine was recently deployed in 19 countries and Europe, this vaccine does not protect naïve individuals against symptomatic or severe infection, and may even exacerbate disease in some cases. Furthermore, the global emergence of Zika virus (ZIKV), and the potential for ADE between DENV and ZIKV, raises concerns for vaccine strategies containing most or all epitopes in the E glycoprotein. Nonetheless, the isolation and characterization of protective and, in some cases, broadly-neutralizing antibodies indicates that certain epitopes within the E glycoprotein may have the capacity to elicit broadly protective responses. Here, we utilize innovative protein engineering approaches to develop “immune-focused” antigens as potential vaccine candidates, in which epitopes that induce non-neutralizing antibodies are masked by engineered mutations or glycosylation. Our hypothesis is that masking of these unfavorable epitopes will skew the immune response toward a stronger neutralizing, protective, and broad response. Aims 1 and 2 focus on critical epitopes in DENV and ZIKV E domain III (EDIII), and Aim 3 explores glycan masking of the ZIKV E prefusion dimer to immune focus on the E-dimer epitope (EDE). EDIII is attractive for subunit vaccine design because it is the target of potently neutralizing and protective antibodies for both DENV and ZIKV. However, immunization with wild-type EDIII protein results in induction of both neutralizing and non-neutralizing antibodies that engage a variety of epitopes. We have used phage display to mask unproductive epitopes of DENV and ZIKV EDIIIs by mutation, while maintaining neutralizing epitopes. These “resurfaced EDIIIs” (rsDIIIs) will be conjugated to protein nanoparticles and their capacity to induce neutralizing and protective antibody response in mice evaluated. To immune focus the prefusion E dimer on the EDE, we have developed a mammalian display system that allows for rapid evaluation of E dimer constructs for binding to EDE mAbs. We will utilize this system to screen variants with multiple engineered glycosylation sites that mask the surface outside of the EDE. The most promising candidates will be tested for their capacity to induce EDE-like mAbs in mice. This work will provide a proof-of-concept for novel subunit vaccine candidates against DENV, ZIKV, and possibly other flaviviruses of global concern.