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，估计每种病毒可导致3.9亿人感染。 年。有四种登革热血清型(DENV1-4)在高度流行地区共同传播。主要 感染单一DENV血清型会导致发热性疾病和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。 严重登革热的部分原因是在初次感染过程中产生的交叉反应抗体 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Fc-γ受体阳性细胞的进入和感染，从而导致“抗体依赖” 感染的“增强”(ADE)。而一种活减毒四组分嵌合疫苗最近被 这种疫苗在19个国家和欧洲部署，并不能保护幼稚的人免受症状或 严重感染，在某些情况下甚至可能加重疾病。此外，全球范围内出现了 寨卡病毒(ZIKV)以及DENV和ZIKV之间可能出现的ADE，引发了对疫苗策略的担忧 包含E糖蛋白中的大部分或全部表位的。尽管如此，病毒的分离和表征 保护性抗体，在某些情况下，广谱中和抗体表明E基因内的某些表位 糖蛋白可能有能力引起广泛的保护性反应。在这里，我们利用创新的蛋白质 开发“以免疫为中心”的抗原作为潜在的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 假说是，掩盖这些不利的表位将使免疫反应偏向更强的 中和、保护和广泛的反应。目标1和目标2关注DENV和ZIKV E的关键表位 结构域III(EDIII)，和Aim 3探索了ZIKV E预融合二聚体的糖链掩蔽，以免疫聚焦于 E-二聚体表位(EDE)EDIII对于亚单位疫苗的设计很有吸引力，因为它是有效的靶标 针对DENV和ZIKV的中和抗体和保护性抗体。然而，用野生型EDIII免疫 蛋白质导致中和和非中和抗体的诱导，这些抗体参与了各种 表位。我们已经使用噬菌体展示通过突变来掩盖DENV和ZIKV EDIII的无效表位， 同时维持中和表位。这些“重新出现的EDIII”(RsDIII)将与蛋白质结合 评价纳米颗粒及其诱导小鼠中和和保护性抗体反应的能力。 为了免疫聚焦在EDE上的预融合E二聚体，我们开发了一种哺乳动物展示系统 允许快速评估与EDE单抗结合的E-二聚体构建体。我们将利用这个系统来 筛选具有多个工程糖基化位点的变异体，这些变异体掩盖了EDE外部的表面。这个 最有希望的候选人将接受测试，看看他们是否有能力在小鼠身上诱导类似EDE的单抗。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.