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（由适用提供）：中东呼吸综合征冠状病毒（MERS-COV）是新出现的人类健康威胁，病例死亡率超过40％。 MERS-COV使用细胞表面蛋白二肽基肽4（DPP4）进入和感染细胞。可溶性重组人DPP4结合MERS-COV峰值（S）糖蛋白并抑制Vero细胞的MERS-COV感染，但是实现50％抑制所需的浓度相当高。该项目的目的是使用DPP4和人类免疫球蛋白的FC设计，生产和测试MERS-COV感染的优质抑制剂。我们预计，由于FC融合中DPP4的化学计量，DPP4-FC的效力会提高（每个分子两个DPP4结合域）。 DPP4-FC也有望拥有出色的药代动力学，因为足球俱乐部将召集长期循环的半衰期，并能够交付给 气道粘膜表面，MERS-COV感染的部位。与针对MERS-COV的抗体不同，DPP4-FC诱饵不会使病毒选择进行谈判逃生突变体，因为任何降低与诱饵结合的突变都会降低与天然受体的结合，从而导致病毒衰减。我们将比较DPP4与三种不同免疫球蛋白同型的FC的抗MERS-COV抑制效力：IgG1，IgA1和IgA2。我们将设计这些遗传融合，以结合全长DPP4细胞外结构域（氨基酸39-766）或仅DPP4€-Propeller结构域（氨基酸39-504）。我们将使用快速的瞬态植物表达系统表达蛋白质并净化它们。我们将通过减少和非还原的SDS-PAGE以及使用FC特异性和DPP4特异性抗体以及尺寸排除色谱法监测DPP4-FC蛋白的结构完整性和第三构构象。我们将测量DPP4-FC变体在功能性ELISA中结合HCOV-EMC SPIKE蛋白的S1结构域的能力。我们还建议在融合蛋白的人类DPP4的特定位置进行单个氨基酸变化，以最大程度地结合峰值蛋白。我们已经与纽约血液中心的Shibo Jiang博士建立了合作，后者在MERS-COV上广泛出版。 Jiang博士将测试所有DPP4-FC变体，这些变体特异性结合了MERS-COV的S蛋白，能够通过MERS-COV PSEUDOVIRUS阻断哺乳动物细胞的感染。