A MERS-CoV Receptor Decoy

描述(申请人提供)：中东呼吸综合征冠状病毒(MERS-CoV)是一种新出现的人类健康威胁，病死率超过40%。MERS冠状病毒利用细胞表面蛋白二肽基肽酶4(DPP4)进入细胞感染细胞。可溶性重组人DPP4结合MERS冠状病毒尖峰蛋白(S)糖蛋白并抑制MERS冠状病毒对Vero细胞的感染，但达到50%抑制所需的浓度较高。本项目的目的是设计、生产和测试一种使用DPP4和人免疫球蛋白Fc融合的高效MERS冠状病毒感染抑制剂。我们预计DPP4-Fc由于Fc融合中DPP4的化学计量比(每个分子有两个DPP4结合域)而具有更高的效力。DPP4-FC也有望具有优越的药代动力学，因为FC将赋予较长的循环半衰期和给药的能力 呼吸道粘膜表面，MERS冠状病毒感染部位。与MERS-CoV抗体不同，DPP4-Fc诱骗不会使病毒受到中和逃逸突变体的选择，因为任何减少与诱骗结合的突变都会减少与天然受体的结合，导致病毒减毒。我们将比较DPP4与三种不同免疫球蛋白同种类型的Fc融合的抗MERS冠状病毒的抑制效力：IgG1、IgA1和IgA2。我们将设计这些基因融合以整合全长DPP4胞外区(氨基酸39-766)或仅DPP4-螺旋桨结构域(氨基酸39-504)。我们将使用快速瞬时植物表达系统表达这些蛋白，并对其进行纯化。我们将通过还原和非还原的SDS-PAGE和Fc特异性抗体和DPP4特异性抗体的免疫印迹，以及大小排斥层析来监测DPP4-Fc蛋白的结构完整性和三级构象。我们将在功能性ELISA法中测量DPP4-Fc变体与hCoV-EMC刺突蛋白S1结构域的结合能力。我们还建议在我们的融合蛋白的人DPP4的特定位置进行单一氨基酸的改变，以最大限度地与Spike蛋白结合。我们与纽约血液中心的江世波博士建立了合作关系，他在MERS冠状病毒上发表了大量文章。江博士将测试所有与MERS冠状病毒S蛋白特异结合的DPP4-Fc变异体阻止MERS冠状病毒假病毒感染哺乳动物细胞的能力。

DESCRIPTION (provided by applicant): Middle East respiratory syndrome coronavirus (MERS-CoV) is newly emerging human health threat with a more than 40% case fatality rate. The cell surface protein dipeptidyl peptidase 4 (DPP4) is used by MERS-CoV to enter and infect cells. Soluble recombinant human DPP4 binds the MERS-CoV spike (S) glycoprotein and inhibits MERS-CoV infection of VERO cells, but the concentration required to achieve 50% inhibition is fairly high. The aim of this project is to design, produce and test a superior inhibior of MERS-CoV infection using a fusion of DPP4 and the Fc of human immunoglobulin. We expect DPP4-Fc to have increased potency due to the stoichiometry of DPP4 in the Fc fusion (two DPP4 binding domains per molecule). DPP4-Fc is also expected to have superior pharmacokinetics, as Fc will confer a long circulating half-life and the ability to be delivered to airway mucosal surfaces, the site of MERS-CoV infection. Unlike antibodies against MERS-CoV, a DPP4-Fc decoy will not subject the virus to selection for neutralization escape mutants, as any mutation that decreases binding to the decoy will decrease binding to the native receptor, resulting in an attenuated virus. We will compare the anti-MERS-CoV inhibitory potency of DPP4 fused to the Fc of three different immunoglobulin isotypes: IgG1, IgA1 and IgA2. We will design these genetic fusions to incorporate either the full-length DPP4 extracellular domain (amino acids 39-766) or just the DPP4 ¿-propeller domain (amino acids 39- 504). We will express the proteins using a rapid transient plant expression system and purify them. We will monitor the structural integrity and tertiary conformation of the DPP4-Fc proteins by reducing and non-reducing SDS-PAGE and immunoblotting with Fc-specific and DPP4-specific antibodies, along with size exclusion chromatography. We will measure the ability of the DPP4-Fc variants to bind the S1 domain of the hCoV-EMC spike protein in a functional ELISA. We also propose to make single amino acid changes at specific positions in the human DPP4 of our fusion proteins, to maximize binding to the spike protein. We have established a collaboration with Dr. Shibo Jiang of the New York Blood Center, who has published extensively on MERS-CoV. Dr. Jiang will test all of the DPP4-Fc variants that specifically bind to S protein of MERS-CoV for the ability to block infection of mammalian cells by a MERS-CoV pseudovirus.