Structure-based design of broad flavivirus immunogens

总结 登革热病毒是一种蚊子传播的黄病毒，估计每年有3.9亿人感染 年有四种血清型登革热（DENV 1 -4）在高流行地区共同传播。初级 由单一DENV血清型感染导致发热性疾病和随后对该疾病的持久免疫， 血清型由异型血清型引起的继发感染可导致严重休克综合征和死亡。 严重的登革热部分是由原发性感染期间产生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 抗体促进进入和感染Fcγ受体阳性细胞，从而引起“抗体依赖性” 感染增强（ADE）。虽然最近有一种减毒活四组分嵌合疫苗， 在19个国家和欧洲部署，这种疫苗不能保护幼稚个体免受症状或 严重感染，在某些情况下甚至可能加剧疾病。此外，全球出现的 寨卡病毒（ZIKV）以及DENV和ZIKV之间的潜在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， 保护性的，在某些情况下，广泛中和的抗体表明，在E 糖蛋白可能具有引起广泛保护性应答的能力。在这里，我们利用创新的蛋白质 工程方法开发“免疫聚焦”抗原作为潜在的候选疫苗，其中 诱导非中和抗体的表位被工程化突变或糖基化掩蔽。我们 假设这些不利表位的掩蔽将使免疫反应偏向更强 中和、保护和广泛的反应。目的1和2聚焦于DENV和ZIKV E中的关键表位 Aim 3探索了ZIKV E融合前二聚体的聚糖掩蔽，以免疫聚焦于ZIKV E融合前二聚体。 E-二聚体表位（EDE）。EDIII对于亚单位疫苗设计是有吸引力的，因为它是有效的免疫调节的靶点。 针对DENV和ZIKV两者的中和性和保护性抗体。然而，用野生型EDIII免疫 蛋白导致中和和非中和抗体的诱导， 表位我们已经使用噬菌体展示通过突变来掩蔽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， 评价了纳米颗粒及其在小鼠中诱导中和和保护性抗体应答的能力。 为了使融合前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， 允许快速评价E二聚体构建体与EDE mAb的结合。我们将利用这一系统， 筛选具有多个工程化糖基化位点的变体，这些位点掩盖了EDE外部的表面。的 将测试最有希望的候选物在小鼠中诱导EDE样mAb的能力。这项工作将 为针对DENV、ZIKV和可能的其他亚单位疫苗候选物提供概念验证。 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.