Engineered Dengue EDIIIs as Broad Immunogens

摘要 登革热病毒是一种由蚊子传播的黄病毒，导致全球数亿人感染- 每年都很宽。有四种登革热血清型(DENV1-4)在高度流行地区共同传播。 单一血清型的原发感染会导致发热性疾病和随后的终身免疫 血清型。然而，异型血清型的继发感染可能导致严重的休克综合征和 死亡。严重的登革热被认为是由初次感染时产生的抗体引起的。 它可以结合多种血清型，但不能中和它们，并促进Fcγ受体的进入和感染 因此，阳性细胞会引起感染的“抗体依赖增强”(ADE)。而一个活着的衰减器 四组分嵌合疫苗最近在三个国家实施，这种疫苗没有保护作用 天真的个体抵抗有症状的感染。因此，有很大的理由去探索替代方案 亚单位疫苗(免疫原)等平台作为下一代初级疫苗或加强免疫 改进现有减毒活病毒疫苗的试剂。此外，还发现和发展了 能诱导广谱中和抗体(BNAb)反应的DENV免疫原是非常理想的。DENV E 糖蛋白结构域III(EDIII)是一个小的(~100个残基)β-夹心结构域，是一个有吸引力的候选结构域 免疫基因设计，因为许多bNAb针对这一区域。然而，该结构域的免疫优势区域 存在于关键的中和表位之外，这阻碍了其作为亚单位疫苗的发展。我们 我为EDIII免疫原工程开发了一个噬菌体展示平台，并将其用于生产 “重新浮出水面的”EDIII(RsDIII)，其中非生产性表位被突变所掩盖。我们以结构为基础 设计策略包括生产和筛选基于EDIII的噬菌体文库，其中的残基是 不参与与模型bNAbs的相互作用随限制性氨基酸多样性而变化。这种方法 已经产生了一组rsDIII，它们对原型bNAb表现出反应性，从而保持了 广泛易感表位的构象完整性，但不指向特定类型或非中和 抗体。我们建议对该免疫原板进行完整的体外生物化学表征。 特性，如折叠稳定性和结合亲和力。单价、双价和多价 此外，还将探讨免疫生成演示文稿格式。最有希望的rsDIII免疫原候选者将 在小鼠身上测试免疫原性和在血清转移挑战中授予保护性免疫的能力 做实验。这项工作的首要目标是使用创新的蛋白质工程方法来 使用基于EDIII的免疫原克服传统障碍。此工作将在以下方面提供概念验证 针对DENV的新型亚单位疫苗候选小鼠，可能还有其他全球关注的黄病毒。

SUMMARY Dengue virus is a mosquito-transmitted flavivirus that causes hundreds of millions of human infections world- wide each year. There are four serotypes of Dengue (DENV1-4) that co-circulate in hyperendemic regions. Primary infection by a single serotype results in febrile illness and subsequent lifelong immunity to that serotype. Secondary infections by heterotypic serotypes, however, can lead to severe shock syndrome and death. Severe Dengue disease is thought to be caused by antibodies that are elicited during primary infection that can bind multiple serotypes but not neutralize them, and 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 implemented in three countries, this vaccine did not protect naïve individuals against symptomatic infection. There is therefore significant rationale to explore alternative platforms such as subunit vaccines (immunogens) as either next-generation primary vaccines or as boosting agents to improve existing live attenuated virus vaccines. Furthermore, identification and development of DENV immunogens that elicit broadly neutralizing antibody (bNAb) responses is highly desirable. DENV E glycoprotein domain III (EDIII) is a small (~100 residue) β-sandwich domain that is an attractive candidate for immunogen design, since many bNAbs target this region. However, immunodominant regions of this domain lie outside of critical neutralization epitopes, which has hampered its advancement as a subunit vaccine. We have developed a phage display platform for EDIII immunogen engineering and used it to produce "resurfaced" EDIIIs (rsDIIIs) in which non-productive epitopes are masked by mutation. Our structure-based design strategy involves production and screening of EDIII-based phage libraries in which residues that are not involved in interactions with model bNAbs are varied with restricted amino acid diversity. This approach has yielded a panel of rsDIIIs that exhibit reactivity toward a prototypic bNAb, thus maintaining the conformational integrity of the broadly susceptible epitope, but not toward type-specific or non-neutralizing antibodies. We propose to perform a full in vitro characterization of this immunogen panel for biochemical properties such as folding stability and binding affinity. Monovalent as well as bivalent and polyvalent immunogen presentation formats also will be explored. The most promising rsDIII immunogen candidates will be tested in mice for immunogenicity and ability to confer protective immunity in a serum transfer challenge experiment. The overarching goal of this work is to use innovative protein engineering approaches to overcome the traditional barriers with EDIII-based immunogens. This work will provide proof-of-concept in mice for novel subunit vaccine candidates against DENV, and possibly other flaviviruses of global concern.