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.
Middle East respiratory syndrome coronavirus (MERS-CoV), also termed hCoV-EMC, was first identified in humans in 2012 in the Middle East. To date 163 known people have contracted MERS in ten countries, resulting in 71 deaths. Preliminary epidemiology studies suggest human-to-human transmission of this deadly virus, leading to global concern about a MERS pandemic. We propose a novel therapeutic, a recombinant protein comprised of the extracellular domain of DPP4 (the MERS-CoV cellular receptor) fused to Fc of a human immunoglobulin (e.g. IgG1), which could be used as a receptor decoy to block the interaction of MERS-CoV with DPP4 on human cells and thus stop infection.
