Strategies for next-generation flavivirus vaccine development

登革热病毒（DENV; DV）是世界上最常见的蚊媒病毒，有40亿人生活在世界各地。 这些区域使它们处于被其四种血清型（DV 1 -4）中的任何一种感染的风险中。DV感染及其相关因素 寨卡病毒（ZIKV; ZV）可在临床上表现为轻度流感样症状、皮疹和关节痛。严重 DV病例可进展为出血热和休克综合征，而ZV感染可导致严重并发症 包括成人格林-巴利综合征和新生儿小头畸形。严重的登革热与 感染的抗体依赖性增强（ADE），这是一种被视为潜在致命的现象。响应于 在原发性DV感染后，免疫系统产生抗体，可以结合并中和该血清型， 解决感染。然而，在随后被异型DV血清型或ZV感染期间，一些抗体可能会在随后的感染中产生。 从原发性感染中重新引发，但可能很难中和异型感染;因此， 消除感染，这些抗体促进活性病毒进入表达Fcγ受体的免疫细胞的摄取 并增加病毒血症和促炎细胞因子的水平。目前，需要疫苗和抗- DV和ZV的病毒疗法，不具有诱导ADE的风险。为了避免ADE，DV的一个重要目标 ZV疫苗的开发是为了引起广泛的保护性抗体，而不是广泛的反应性，非免疫性抗体。 中和之一。我们确定了DV和ZV表达的两种糖蛋白， 免疫原设计针对DV和ZV的中和保护性抗体已显示靶向结构域III（DIII） E糖蛋白，这是病毒进入和细胞附着的关键。然而，非中和性、ADE诱导性 还鉴定出抗体与WT DIII上的其它基序结合。靶向野生型NS 1糖蛋白， 在病毒复制和内皮功能障碍中起关键作用，可能在无ADE的情况下降低疾病严重程度，但可能 引发非中和性甚至自身反应性抗体为了应对这些挑战，基于结构的方法 可用于免疫原诱变和这些免疫原的多价展示。这项建议会 研究增加在侧脊（LR）上发现的异源表位的免疫优势的方法 DIII和NS 1的β-ladder，同时掩蔽这些免疫原上的某些表位，以诱导广泛的体液免疫。 保护和减轻疾病的严重程度。我们以前证明了强大的免疫保护潜力 同型ZV免疫策略，其涉及LR聚焦的DIII突变体的多价展示 到纳米颗粒上。我们将研究各种异型纳米颗粒作为交叉免疫的有效性 针对ZV和DV的策略，并表征对小鼠免疫的潜在体液应答（目的 1）。然后，我们将探索迷你β的设计，一种新的NS 1突变体，可用于遏制疾病 进展而不引发ADE或自身反应性抗体（目的2）。这些研究将使我们进一步了解 对黄病毒感染的适应性免疫反应，并可能提供一个框架， 疫苗和基于抗体的疗法。

Dengue virus (DENV; DV) is the most common mosquito-borne virus in the world, with 4 billion people living in regions that put them at risk for infection by any of its four serotypes (DV1-4). Infection by DV and its related flavivirus Zika virus (ZIKV; ZV) may clinically manifest as mild flu-like symptoms, rash, and arthralgia. Severe DV cases can progress into hemorrhagic fever and shock syndrome, while severe complications of ZV infection include Guillain-Barre syndrome in adults and neonatal microcephaly. Severe dengue is associated with antibody-dependent enhancement (ADE) of infection, a phenomenon seen as potentially fatal. In response to a primary DV infection, the immune system produces antibodies that can bind and neutralize that serotype to resolve infection. However, during a subsequent infection by a heterotypic DV serotype or ZV, some antibodies from the primary infection are re-elicited but may poorly neutralize the heterotypic infection; thus, instead of eliminating infection, these antibodies promote uptake of active virus into Fcγ receptor-expressing immune cells and increase viremia and levels of pro-inflammatory cytokines. Currently, there is a need for vaccines and anti- viral therapies for DV and ZV that do not carry the risk of inducing ADE. To avoid ADE, a significant goal for DV and ZV vaccine development is to elicit broadly protective antibodies, as opposed to broadly reactive, non- neutralizing ones. We determined two glycoproteins expressed by DV and ZV as favorable candidates for immunogen design. Neutralizing, protective antibodies against DV and ZV have shown to target domain III (DIII) of E glycoprotein, which is critical for viral entry and cell attachment. However, non-neutralizing, ADE-inducing antibodies were also identified to bind to other motifs on WT DIII. Targeting WT NS1 glycoprotein, which plays a key role in viral replication and endothelial dysfunction, may reduce disease severity without ADE, but it may elicit non-neutralizing, even autoreactive antibodies. To address these challenges, structure-based approaches may be used for immunogen mutagenesis and multivalent display of these immunogens. This proposal will investigate methods to increase the immunodominance of heterologous epitopes found on the lateral ridge (LR) of DIII and the β-ladder of NS1, while masking certain epitopes on these immunogens, to induce broad humoral protection and mitigate severity of disease. We previously demonstrated the strong immunoprotecting potential of a homotypic ZV immunization strategy, which involved the multivalent display of a LR-focused DIII mutant onto nanoparticles. We will study the effectiveness of various heterotypic nanoparticles as cross-immunization strategies against ZV and DV and characterize underlying humoral responses to the immunizations in mice (Aim 1). We will then explore the design of miniβ, a novel NS1 mutant which may be employed to curb disease progression without eliciting ADE or autoreactive antibodies (Aim 2). These studies will further our understanding of the adaptive immune response to flavivirus infections and may provide a framework for developing novel vaccines and antibody-based therapeutics.