Broad-Spectrum Therapeutic Human Antibodies for Dengue Virus Infections

描述（由申请人提供）：该项目的总体目标是开发抗体疗法来治疗危及生命的黄病毒感染。我们的工作假设是，识别特异性、高度保守的包膜蛋白表位的抗体可以预防和治疗几种不同黄病毒的感染，包括登革热病毒和西尼罗病毒（WNV）的所有4种血清型。被动免疫是一种公认的预防感染的医学做法。我们使用专有的重组西尼罗河病毒包膜蛋白（WNV- e）从人噬菌体展示抗体文库中选择了2个重组人单链可变区片段（scFv）， scFv 11和scFv 71。为了增加这些抗体片段的亲和力和血清半衰期，我们将每个scFv与人Fc结构域融合，产生了二价抗体scFv-Fc 11和scFv-Fc 71。这些抗体保护小鼠在感染前或感染后不受致命剂量的西尼罗河病毒感染。scFv-Fc 11和scFv-Fc 71抗体也能中和登革热病毒。因此，抗体scFv-Fc 11和scFv-Fc 71识别在不同黄病毒物种中保守的表位。我们认为，抗体11和抗体71作为抗多种黄病毒种类和亚种的先导广谱治疗药物，具有开发产品的潜力。为了寻找具有增强治疗特性的抗体，我们将首先确定登革热病毒血清2型包膜蛋白（DEN2-E）与scFv 11或scFv 71复合物的三维结构。这将揭示保守抗体表位的程度，以及中和登革热病毒和WN病毒的功能基础。同时，我们将寻找DEN2-E和WNV-E逃避抗体11和71中和的突变体。将逃逸突变映射到我们的结构上将使我们能够设计出不太容易产生耐药病毒株的抗体。在该项目的第二阶段，我们将利用第一阶段的结构数据来设计具有增强表位结合亲和力、更广泛的活性谱或增强的药代动力学特性的突变型scFv-Fc抗体。我们将寻找能够中和所有登革热和西WN病毒血清型的突变抗体，并在感染这些病毒后保护小鼠。该项目将为临床医生和公共卫生官员提供管理自然、意外和故意暴发黄病毒的新手段。

DESCRIPTION (provided by applicant): The overall goal of this project is to develop antibody therapeutics to treat life-threatening flavivirus infections. Our working hypothesis is that antibodies recognizing specific, highly conserved envelope protein epitopes can prevent and treat infection by several different flaviviruses, including all 4 serotypes of dengue virus and West Nile virus (WNV). Passive immunization is an accepted medical practice to protect against infection. We have used a proprietary recombinant WNV envelope protein (WNV-E) to select 2 recombinant human single-chain variable region fragments (scFv), scFv 11 and scFv 71, from a human phage display antibody library. To increase the avidity and serum half-life of these antibody fragments, we fused each scFv to a human Fc domain to produce bivalent antibodies scFv-Fc 11 and scFv-Fc 71. These antibodies protect mice against a lethal dose of WNV administered either pre-or post-infection. The scFv-Fc 11 and scFv-Fc 71 antibodies also neutralize dengue virus. Antibodies scFv-Fc 11and scFv-Fc 71 therefore, recognize epitopes that are conserved across different flavivirus species. We propose that antibodies 11 and 71 are promising for product development as lead broad-spectrum therapeutics against various flavivirus species and subspecies. In pursuit of antibodies with enhanced therapeutic properties, we will first determine the three-dimensional (3D) structure of dengue virus serotype 2 envelope protein (DEN2-E) in complex with either scFv 11 or scFv 71. This will reveal the extent of the conserved antibody epitopes, and the functional basis for neutralization of both dengue and WN viruses. In parallel, we will seek mutants of DEN2-E and WNV-E that escape neutralization by antibodies 11 and 71. Mapping the escape mutations onto our structure will allow us to design antibodies that are less prone to generating resistant viral strains. In Phase II of this project, we will use our structural data from Phase I to design mutant scFv-Fc antibodies with enhanced epitope binding affinities, a broader spectrum of activity, or enhanced pharmacokinetic properties. We will seek mutated antibodies that neutralize all dengue and WN virus serotypes, and protect mice after infection with these viruses. This project will provide clinicians and public health officials with new means to manage natural, accidental and intentional flavivirus outbreaks. Dengue is a mosquito-borne infection that recently emerged as a major international public health concern. The goal of this research is to develop an antibody therapeutic to treat life- threatening dengue virus infections. This project will provide clinicians and public health officials with new means to manage natural, accidental and intentional flavivirus outbreaks.