A MERS-CoV Receptor Decoy

MERS-CoV 受体诱饵

• 批准号: 8780733
• 负责人: KEITH WYCOFF
• 金额: $ 22.5万
• 依托单位: PLANET BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8780733
• 关键词: Affinity Chromatography; Agrobacterium; Amino Acids; Animal Model; Antibodies; Antiviral Agents; Attenuated; Base Sequence; Binding; Blood; Case Fatality Rates; Cell Surface Proteins; Cells; Cessation of life; Chimeric Proteins; Collaborations; Contracts; Coronavirus; Coronavirus Infections; Coronavirus spike protein; Country; Dipeptidyl Peptidases; Drug Kinetics; Enzyme-Linked Immunosorbent Assay; Escape Mutant; Extracellular Domain; Gene Fusion; Genetic; Glycoproteins; HIV-1; Half-Life; Health; Human; IgA1; IgA2; IgG1; Immunoblotting; Immunoglobulin Isotypes; Immunoglobulins; In Vitro; Infection; Laboratories; Length; Life; Luciferases; Mammalian Cell; Measures; Medical center; Middle East; Molecular Conformation; Molecular Sieve Chromatography; Monitor; Mutation; Netherlands; New York; Nicotiana; Plant Extracts; Plants; Positioning Attribute; Procedures; Protein Binding; Protein Conformation; Protein S; Proteins; Publishing; Recombinant Proteins; Recombinants; Research Personnel; Rhizobium radiobacter; Site; Surface; Syndrome; System; Testing; Ursidae Family; Variant; Vertebral column; Virus; coronavirus receptor; design; epidemiology study; expression vector; in vivo; inhibitor/antagonist; novel therapeutics; pandemic disease; public health relevance; receptor; respiratory; stoichiometry; transmission process

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.

描述（由申请人提供）：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-CoV利用细胞表面蛋白二肽基肽酶4（DPP 4）进入并感染细胞。可溶性重组人DPP 4结合MERS-CoV刺突（S）糖蛋白并抑制VERO细胞的MERS-CoV感染，但达到50%抑制所需的浓度相当高。本研究的目的是利用DPP 4与人免疫球蛋白Fc融合蛋白设计、制备和测试一种MERS-CoV感染的上级抑制剂。我们预期DPP 4-Fc由于Fc融合物中DPP 4的化学计量（每个分子两个DPP 4结合结构域）而具有增加的效力。还预期DPP 4-Fc具有上级药代动力学，因为Fc将赋予长的循环半衰期和递送至靶细胞的能力。 呼吸道粘膜表面，MERS-CoV感染的部位。与针对MERS-CoV的抗体不同，DPP 4-Fc诱饵不会使病毒经受中和逃逸突变体的选择，因为降低与诱饵结合的任何突变都会降低与天然受体的结合，从而产生减毒病毒。我们将比较与三种不同免疫球蛋白同种型（IgG 1、IgA 1和IgA 2）的Fc融合的DPP 4的抗MERS-CoV抑制效力。我们将设计这些基因融合体，以整合全长DPP 4胞外结构域（氨基酸39-766）或仅DPP 4-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蛋白质并纯化它们。我们将通过还原性和非还原性SDS-PAGE以及使用Fc特异性和DPP 4特异性抗体的免疫印迹法，沿着分子排阻色谱法，监测DPP 4-Fc蛋白的结构完整性和三级构象。我们将在功能性ELISA中测量DPP 4-Fc变体结合hCoV-EMC刺突蛋白S1结构域的能力。我们还建议在我们的融合蛋白的人DPP 4中的特定位置处进行单个氨基酸改变，以最大化与刺突蛋白的结合。我们与纽约血液中心的Shibo Jiang博士建立了合作关系，他在MERS CoV方面发表了大量文章。Jiang博士将测试所有特异性结合MERS-CoV S蛋白的DPP 4-Fc变体阻断MERS-CoV假病毒感染哺乳动物细胞的能力。