Structure based design of dengue subunit vaccines for inducing protective but not disease enhancing antibodies

摘要 由于存在4种血清型，登革病毒（DENV）疫苗的开发一直具有挑战性 （DENV 1 -4）和疫苗增强严重疾病的潜力。领先的四价登革减毒活疫苗 疫苗一直受到疫苗组分的不平衡复制的困扰， 疫苗引发了一些儿童的严重登革热疾病。该提案的目标是开发新的 重组DENV包膜（E）蛋白和病毒样颗粒（VLP）疫苗克服了 四价减毒活疫苗。我们已经发现，DENV E蛋白作为一种分泌的 蛋白质是一种差的疫苗，因为它是一种单体，不显示被靶向的主要四级表位。 人中和和保护性抗体（Abs）。这个建议是基于我们小组的新发现 关于如何基于结构，计算方法可以用来产生高度稳定和正确折叠， 从哺乳动物细胞有效分泌的DENV E二聚体。我们将使用登革病毒的小鼠模型 免疫接种和感染，以测试人工稳定的DENV E二聚体是否刺激类似于 暴露于初级和次级野生型的人群中的血型特异性和血型交叉保护性Ab应答 型DENV感染。稳定的E二聚体将被进一步修饰，以测试E二聚体的大规模表面置换是否 二聚体可用于将免疫应答集中在由有效中和Ab识别的表位上，而 消除对脱靶、疾病增强表位的反应。最后，我们将设计膜锚固 促进登革病毒样颗粒（VLP）疫苗候选物形成的稳定E二聚体变体 比用现有技术制备的VLP更类似于成熟的感染性病毒体。我们的研究， 探索如何设计和递送重组E抗原，以将宿主抗体反应集中在重要的 四级结构中和表位，将激发黄病毒亚基领域新的研究方向 疫苗。

ABSTRACT Dengue virus (DENV) vaccine development has been challenging because of the presence of 4 serotypes (DENV1-4) and the potential for vaccine enhanced severe disease. The leading live attenuated tetravalent DENV vaccines have been plagued by poorly balanced replication of vaccine components leading to variable efficacy and vaccine primed severe dengue disease in some children. The goal of this proposal is to develop novel recombinant DENV envelope (E) protein and virus like particle (VLP) vaccines that overcome barriers faced by live attenuated tetravalent vaccines. We have discovered that the DENV E protein produced as a secreted protein is a poor vaccine because it is a monomer that does not display major quaternary epitopes targeted by human neutralizing and protective antibodies (Abs). This proposal is based on new discoveries from our group about how structure based, computational approaches can be used to produce highly stable and properly folded DENV E dimers that are efficiently secreted from mammalian cells. We will use mouse models of DENV vaccination and infection to test if artificially stabilized DENV E dimers stimulate Ab responses that are similar to serotype-specific and serotype-cross-protective Ab responses in people exposed to primary and secondary wild type DENV infections. The stabilized E dimers will be further modified to test if large-scale resurfacing of the E dimer can be used to focus the immune response on epitopes recognized by potent neutralizing Abs, while eliminating responses to off target, disease enhancing epitopes. Finally, we will design membrane anchored variants of stabilized E dimers to promote the formation of dengue virus-like particle (VLP) vaccine candidates that better resemble mature infectious virions than VLPs made with current techniques. Our studies, which explore how to design and deliver recombinant E antigens to focus the host antibody response on important quaternary structure neutralizing epitopes, will stimulate new research directions in the field of flavivirus subunit vaccines.