EP3227318A1 - Dpp4 immunoadhesin compositions and methods - Google Patents

Abstract

Described herein fusion proteins comprising modified DPP4 binding sequence and the Fc of a human immunoglobulin, related compositions, and related methods for inhibiting MERS- CoV infection. In addition to the improved potency, the modified 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 and the modified DPPR-Fc decoy of the invention 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.

Description

DPP4 IMMUNOADHESIN COMPOSITIONS AND METHODS

CROSS-REFERENCE

The present PCT patent application claims priority benefit of the U.S. provisional application for patent serial number 62/124,01 1 , filed on 05-Dec-2014 under 35 U.S.C. 1 19(e). The contents of this related provisional application is incorporated herein by reference for all purposes to the extent that such subject matter is not inconsistent herewith or limiting hereof. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS ATEXT FILE

A sequence listing is provided herewith as a text file, "DPP4-lmmunoad_SeqList.txt" created on August 4^th, 2015, and having a size of 81 KB. The contents of the text file are incorporated by reference herein in their entirety. FIELD OF THE INVENTION

Middle East respiratory syndrome coronavirus (MERS-CoV), also termed hCoV-EMC, was first identified in humans in 2012 in the Middle East. To date over one thousand people have contracted MERS in 25 countries, with mortality approaching 40 percent. 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. IgG and IgA), which could be used as a "receptor decoy" to block the interaction of MERS-CoV with DPP4 on human cells and thus stop infection. BACKGROUND

A novel coronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV), was first identified in humans in 2012 in the Middle East, and later in Europe (Bermingham et al. 2012; de Groot et al. 2013). The virus is also known as human coronavirus-Erasmus Medical Center (hCoV-EMC) (Zaki et al. 2012). Preliminary epidemiology studies suggest human-to- human transmission of this deadly virus, leading to global concern about a MERS pandemic. Genetic and phylogenetic characterization shows that MERS-CoV belongs to lineage C of the betacoronavirus genus and is closely related to Tylonycteris bat coronavirus HKU4 and Pipistrellus bat coronavirus HKU5. The direct source and reservoirs of MERS-CoV remain enigmatic. As for SARS-CoV and hCoV-NL63, a bat origin, possibly combined with the existence of an intermediate animal reservoir in camels, seems feasible (Cui et al. 2013; Lau et al. 2013; Reusken et al. 2013; Ithete et al. 2013; Perera et al. 2013). A developing understanding of MERS-CoV biology has proceeded rapidly leading to numerous possibilities for therapeutic and vaccine development. Like other coronaviruses, the MERS-CoV virion uses a large surface spike (S) glycoprotein for interaction with and entry into target cells. The S glycoprotein consists of a globular S1 domain at its N-terminus, followed by a membrane-proximal S2 domain, a transmembrane domain and an intracellular domain at its C- terminus. Determinants for cellular tropism and interaction with the target cell are within the S1 domain, while mediators of membrane fusion are within the S2 domain (Qian et al. 2008). Through co-purification with the MERS-CoV S1 domain, Raj and colleagues determined that dipeptidyl peptidase 4 (DPP4, also called CD26) functions as a cellular receptor for MERS-CoV (Raj et al. 2013).

DPP4 is a serine protease belonging to the prolyl oligopeptidase family (Hopsu-Havu and Glenner 1966), but its enzymatic function does not appear to be essential for viral entry. It cleaves peptide bonds to release proline-containing dipeptides from the N-terminus of physiologically important polypeptides. Many peptides have been identified as DPP4 substrates in vitro and in vivo, and DPP4 has therefore been proposed as an important regulator of different physiological and pathophysiological conditions (Mentlein 1999; Miyazaki et al. 2012; Shigeta et al. 2012; Moran et al. 2012; Bengsch et al. 2012). There is considerable

pharmaceutical interest in DPP4 because it inactivates the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide in vivo. This makes DPP4 an important regulator of glucose homeostasis, as glucagon-like peptide 1 and glucose-dependent insulinotropic peptide have glucose-dependent insulinotropic as well as neogenetic effects on pancreatic β-cells (Ahren 2012).

DPP4 has a transmembrane domain and a seven amino acid intracellular domain. The extracellular domain is comprised of amino acids S39 to P766 (Figure 1 ). A soluble DPP4, comprised of the same amino acids, is found in serum (Lambeir et al. 1997; Durinx et al. 2000). The extracellular domain consists of an N-terminal eight-bladed β-propeller domain (S39 to D496) and a C-terminal α/β hydrolase domain (N497 to P766). The β-propeller domain's eight blades are each made of four antiparallel β-strands (Thoma et al. 2003).

The DPP4 β-propeller domain amino acid sequence is the primary determinant of MERS- CoV species-specificity. MERS-CoV will infect cell lines of human, bat, non-human primate or pig origin, but not cell lines from mice, hamsters, dogs or cats (Chan et al. 2013; Raj et al. 2013). The virus can infect humans and rhesus macaques (de Wit, Rasmussen, et al. 2013; Yao et al. 2014), as well as camels, goats, cows and sheep (van Doremalen et al. 2014), but not mice, hamsters or ferrets (de Wit, Prescott, et al. 2013; Enserink 2013; Scobey et al. 2013). Non-susceptible cells transformed to express cell-surface human or bat DPP4 became susceptible to infection (Raj et al. 2013). Expression of camel, goat, cow or sheep DPP4 on the surface of hamster cells rendered them susceptible. Hamster cells in which five DPP4 amino acids were replaced with the corresponding human amino acids were susceptible, while cells expressing human DPP4 with the five hamster amino acids were not (van Doremalen et al. 2014). Human, camel and horse DPP4 were potent and nearly equally effective MERS-CoV receptors, while goat and bat receptors were considerably less effective (Barlan et al. 2014).

DPP4 is expressed on the surface of several cell types, including those found in human airways. In support of its role as a receptor for MERS-CoV, a polyclonal antiserum directed against DPP4 inhibited MERS-CoV infection of primary human bronchial epithelial cells and human hepatoma-7 (Huh-7) cells, and soluble DPP4 inhibited Vera cell infection by MERS-CoV (Raj et al. 2013). At least one mouse monoclonal antibody against DPP4 almost completely inhibited viral entry, and a humanized anti-CD26 mAb, YS1 10, partially inhibited viral entry (Ohnuma et al. 2013). DPP4 has ectopeptidase activity, although this enzymatic function does not appear to be essential for viral entry.

The structure of the S glycoprotein bound to DPP4 was solved by two different groups that identified the same regions of contact, though their reports differ about exactly which amino acid residues are involved (Wang et al. 2013; Lu et al. 2013). The MERS-CoV Receptor Binding Domain (RBD) contacts blades 4 and 5 of the DPP4 β-propeller domain (italic lettering in Figure 1 ; consensus contact amino acids underlined) and has no contact with the hydrolase domain (bold lettering in Figure 1 ). Potential DPP4 glycosylation sites that are actually glycosylated are N85, N92, N150, N219, N229, N281 and N321 (in the β-propeller domain) and N520 (in the α/β hydrolase domain) (Thoma et al. 2003).

SUMMARY OF THE INVENTION

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human health threat with a more than 40% case fatality rate. MERS-CoV uses the cell surface protein dipeptidyl peptidase 4 (DPP4) 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. Using a fusion of a modified DPP4 binding sequence and the Fc of human immunoglobulin the present invention provides a superior inhibitor of MERS-CoV infection and a potency greater than the expected increased potency of DPP4-Fc due to the stoichiometry of DPP4 in the Fc fusion (two DPP4 binding domains per molecule). In addition to the improved potency, the modified 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 and the modified DPP4-Fc decoy of the invention 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 on cells, resulting in an attenuated virus.

Accordingly in one aspect described herein is a DPP4 peptide comprising human DPP4 consensus contact sequence for the MERS CoV S1 spike glycoprotein comprising at least one consensus contact residue substitution, wherein the peptide has higher affinity for the ME S CoV S1 spike glycoprotein than human DPP4 consensus contact sequence without the at least one substitution. In some embodiments the DPP4 peptide the at least one residue substitution is with a residue selected from contact residues unique to camel DPP4. In some embodiments the at least one contact residue substitution is at a position selected from 288, 295, 317, 336, and 346. In one embodiment the residue at position 288 is V. In another embodiment the residue at position 288 is N. In one embodiment the residue at position 295 is F. In one embodiment the residue position at 336 is Y. In one embodiment the residue at position 346 is E.

In some embodiments the at least one consensus contact residue is selected from residues 285 to 293. In one embodiment the consensus contact residue at position 285 is substituted with R. In another embodiment the consensus contact residue at position 289 is substituted with P. In another embodiment the consensus contact residue at position 293 is substituted with V. In one embodiment the consensus contact residue at position 285 is substituted with V, the residue at position 288 is substituted with V, the residue at position 289 is substituted with P, and the residue at position 293 is substituted with V. In one embodiment the amino residues at positions 285 to 293 correspond to the amino acid sequence of SEQ ID NO: 17 (RQIVPPASV). In some embodiments the amino acid sequence of the DPP4 peptide comprises one or more amino acid substitutions selected from the group consisting of 188R, 269H, 291V, 294F, 295F, 336Y, 3411, 344R, 346F, and 392E.

In some embodiments the DPP4 peptide comprises an amino acid substitution that reduces hydrolase activity of the DPP4 peptide. In some embodiments the amino acid substitution that reduces hydrolase activity is with an amino acid residue other than Y at position 547. In one amino embodiment the amino acid residue at position 547 is F. In some embodiments, where the amino acid residue at position 547 is F, the DPP4 peptide further comprises one or more amino acid substitutions selected from the group consisting of 188R, 269H, 291V, 294F, 295F, 336Y, 3411, 344R, 346F, and 392E. In other embodiments the amino acid substitution that reduces hydrolase activity is with an amino acid residue other than S at position 630. In one embodiment the amino acid residue at position 630 is A. In other embodiments, where the amino acid residue at position 630 is A, the DPP4 peptide further comprises one or more amino acid substitutions selected from the group consisting of 188R, 269H, 291 V, 294F, 295F, 336Y, 3411 , 344R, 346R, and 392E. In some embodiments, where the DPP4 peptide comprises an amino acid substitution that reduces hydrolase activity, e.g., where position 547 is F, the amino acid sequence of the DPP4 peptide further comprises one or more amino acid substitutions selected from the group consisting of 188R, 269H, 291V, 294F, 295F, 336Y, 3411, 344R, 346F, and 392E.

In a related aspect described herein is a nucleic acid encoding any of the above-described DPP4 peptides. In some embodiments an expression vector comprises the nucleic acid encoding a DPP4 peptide. In another related aspect described herein is a method for producing any of the above-mentioned DPP4 peptides, comprising introducing the just-mentioned expression vector into a cellular host, and expressing the DPP4 peptide. In some embodiments the cellular host for the just-mentioned production method is a plant.

In a related aspect described herein are any of the above-described DPP4 peptides further comprising an Fc linked to the DPP4 peptide. In some embodiments the Fc is selected from the group consisting of lgG1 , lgG2, lgA1 , lgA2, and IgM. In some embodiments the Fc further comprises a KDEL sequence at its carboxy terminus. In some embodiments, where the Fc comprises a KDEL sequence at its carboxy terminus, the Fc is a truncated IgA comprising a deletion of the 18 amino acid C-terminal IgA piece relative to full length IgA. In some embodiments the Fc is from an IgA. In some embodiments, the Fc is from an IgAl In other embodiments the Fc is from an lgA2.

In some embodiments, where the Fc is an IgA, the DPP4 peptide further comprises a J-chain linked to the DPP4-Fc. In some embodiments, the J-chain is linked to at least two linked DPP4- Fcs.

In some embodiments the Fc is from an IgM. In some embodiments, where the Fc is from an IgM, the DPP4-Fc further comprises a J chain linked to the DPP4-Fc. In some embodiments, the J-chain is linked to at least two linked DPP4-Fcs. In some embodiments, where the J-chain is linked to at least two linked DPP4-Fcs, the J-chains and DPP4-Fcs form multimers.

In a related aspect described herein is a method for reducing binding of ME S CoV to a host cell, the method comprising: contacting the MERS-CoV with any of the above-mentioned DPP4 peptides, whereby the DPP4 peptide binds to the MERS-CoV Receptor Binding Domain (RBD) and reduces the binding of MERS-CoV RBD to the host cell. In a further related aspect is the use of any of the foregoing DPP4 peptides as a medicament. In another aspect described herein is the use of any of the foregoing DPP4 peptides for preventing or treating a MERS CoV infection.

In another aspect described herein is a chimeric MERs-CoV receptor protein comprising: (i) an immunoglobulin complex, wherein the immunoglobulin complex comprises at least a portion of an immunoglobulin heavy chain; and (ii) a mutated dipeptidyl peptidase 4 (DPP4) peptide comprising human DPP4 consensus contact residues, wherein at least one of the consensus contact residues of the human DPP4 sequence comprises at least one amino acid substitution that increases the affinity of the mutated DPP4 peptide for the S1 spike protein of MERS-CoV relative to the affinity of an unmutated DPP4 peptide, and wherein the mutated human DPP4 is covalently associated with the immunoglobulin heavy chain. In some embodiments the chimeric MERs-CoV receptor protein is a dimer of the just-described chimeric MERS-CoV receptor protein. In some embodiments the immunoglobulin heavy chain and DPP4 peptide are human. In some embodiments the immunoglobulin complex further comprises at least a portion of an immunoglobulin light chain. I n some embodiments the immunoglobulin light chain is a kappa chain or a lambda chain. In some embodiments the covalent linkage between the mutated human DPP4 peptide and the immunoglobulin heavy chain is an immunoglobulin hinge.

I n some embodiments of the chimeric MERS-CoV receptor protein, the portion of an immunoglobulin heavy chain is selected from the group consisting of IgGs, IgAs, IgD. IgE, and IgM. In some embodiments the immunoglobulin heavy chain is an IgG and comprises heavy chain constant regions 2 and 3 thereof.

In a related aspect described herein is pharmaceutical composition comprising any of the above-described chimeric MERS-CoV receptor proteins and a pharmaceutically acceptable carrier. In another related aspect described herein any of the above-mentioned MERS-CoV receptor proteins is for use as a medicament. In a further related aspect any of the above- mentioned MERS-CoV receptor proteins is for use in preventing or treating a MERS-CoV infection. In yet another aspect described herein is an expression vector encoding any of the above-mentioned MERS-CoV receptor proteins. Also described is a method for producing any of the above-mentioned chimeric MERS-CoV receptor protein, comprising introducing the expression vector into a cellular host, and expressing a chimeric MERS-CoV receptor protein. In some embodiments the cellular host to be used in the production method is a plant.

In a related aspect described herein is a composition comprising any of the above- mentioned MERS-CoV receptor proteins and a plant material. In some embodiments the plant material in such a composition is selected from the group consisting of: plant cell walls, plant organelles, plant cytoplasm, intact plant cells, plant seeds, and viable plants.

In a related aspect described herein is a method for reducing binding of MERS CoV to a host cell, comprising: contacting the MERS-CoV with any of the above-mentioned chimeric MERS-CoV receptor proteins, whereby the chimeric MERS-CoV receptor protein binds to the MERS-CoV Receptor Binding Domain (RBD) and reduces the binding of MERS-CoV RBD to the host cell.

The anti-MERS-CoV inhibitory potency of the modified DPP4 fused to the Fc of three different immunoglobulin isotypes - lgG1 , lgA1 and lgA2 - is increased compared to the same Fc fusions of unmodified DPP4. Fusions of Fc and the full-length DPP4 extracellular domain (amino acids 39-766) as well as the DPP4 β-propeller domain (amino acids 39-504) are described, and genetic constructs capable of expression by eukaryotic host cells, tissues organs or organisms are provided. Purified modified DPPR-Fc fusions and formulations thereof are also shown. The ability of the DPP4-Fc variants to bind the S1 domain of the MERS-CoV spike protein in a functional ELISA as well as in cell culture is disclosed. In further preferred embodiments of the modified DPP4-Fc fusion, amino acid changes at specific positions in the human DPP4 are disclosed that further increase binding to the MERS-CoV spike protein.

In a preferred, but not limiting embodiment, the modified DPP4-Fc fusion is expressed using a rapid transient plant expression system. Nucleotide sequences encoding the DPP4-Fc fusions are cloned into a plant expression vector and the constructs transformed into Agrobacterium tumefaciens (A.t). The Agrobacterium strains transiently transform Nicotiana benthamiana plants, which express the recombinant proteins. In a preferred embodiment vacuum infiltration is used to transport the A.t. into the tissues of plants. After a suitable period of time the plant-produced fusion proteins are purified from extracts of plant tissue using standard procedures, including Protein A affinity chromatography in the case of DPP4-lgG Fc fusions. The plant-produced recombinant modified DPP4-Fc fusion proteins are assayed for binding to the recombinant S glycoprotein of MERS-CoV and evaluated in vitro and in vivo for MERS CoV neutralizing activity.

Genetic fusions of human DPP4 with human immunoglobulin sequences, and preferably immunoglobulin Fc sequences, which include the hinge, CH2 and CH3 of lgG1 , lgA1 and lgA2 have been produced. While numerous DPP4-Fc gene fusions can be designed, and include for example three incorporating the full-length DPP4 extracellular domain (amino acids 39-766 in Figure"! ) and three incorporating just the DPP4 β-propeller domain (amino acids 39-496 in Figure 1 ). Additional variants including modified DPP4 such as the DPP4 β-propeller domain (amino acids 39-504) may be fused with human immunoglobulin sequences. The activities of these DPP4-immunoglobulin variants may be characterized in vitro by binding assays, such as ELISAs, or cell-based assays such as inhibition of cytopathological effect caused by MERS-CoV infection of cells in the presence of soluble DPP4, DPP4-Fc, modified soluble DPP4 and modified DPP4-immunoadhesins such as modified DPP4-Fc.

The structural integrity of the DPP4-Fc proteins according to the invention is determined by reducing and non-reducing SDS-PAGE and immunoblotting with Fc-specific and DPP4- specific antibodies. Protein size is determined by analytical size exclusion chromatography. The ability of the DPP4-Fc variants to bind the S1 domain of the MERS-CoV spike protein is determined in a functional ELISA. The effect of making single or multiple amino acid changes at specific positions in the human DPP4 sequence of our fusion proteins, and their binding to the spike protein is also determined by these techniques.

All DPP4-Fc variants that specifically bind to S protein of MERS-CoV are tested for the ability to block infection of mammalian cells by a MERS-CoV pseudovirus that was developed in the laboratory of Shibo Jiang (Zhao et al. 2013). This pseudovirus bears the full-length S protein of MERS-CoV in an Env-defective, luciferase-expressing HIV-1 backbone. The recombinant DPP4 and modified DPP4 immunoglobulin Fc fusion proteins with inhibitory activity against the pseudotyped MERS-CoV are further tested for their antiviral activity against live MERS-CoV infection both in vitro and in vivo in a new animal model of the disease.

An anti-DPP4 antibody has been shown to block infection in vitro (Ohnuma et al. 2013), but there are potential problems with this approach. Blocking a widespread human cell-surface antigen with an antibody may have pleiotropic effects on the host or patient. Such an antibody may stimulate a receptor response upon binding or may interfere with or prevent binding of a normal ligand to the receptor. In addition, DPP4 circulates at about 6 μg/ml in serum (Javidroozi, Zucker, and Chen 2012) and could be bound by an anti-DPP4 antibody, requiring a greater parenteral dose to achieve a therapeutic effect. Furthermore, an unknown quantity of DPP4 is found on cell surfaces, so, a significant amount of antibody may be needed to block enough cell surface MERS-CoV-DPP4 binding sites to prevent infection.

An alternative approach is to provide an antibody against the S1 domain on the viral spike and it has been shown that S-protein-specific neutralizing antibodies are generated in MERS- CoV recovering patients (Gierer et al. 2013). However, the development of escape mutants of MERS-CoV, i.e. MERS-CoVs that mutate to carry a S1 domain-proteins that bind to the antibody yet are still able to bind to the DPP4 receptor, can occur, as has been seen previously with anti-SARS mAbs to the SARs Coronavirus (Rockx et al. 2010).

Du et al. created a recombinant protein containing a fragment of the viral receptor binding domain (RBD) (residues 377-588) fused with human IgG Fc. The S377-588-Fc protein efficiently bound to DPP4 and inhibited MERS-CoV infection (IC50 ^« 3.2 g/ml) in vitro (Du et al. 2013). This fusion protein would be expected to have the same potential drawbacks as an anti-DPP4 mAb. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection (Raj et al. 2014). Several small molecule inhibitors are being investigated and some show promise as therapeutics (Dyall et al. 2014; Hart et al. 2014; Lu et al. 2014).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the amino acid sequence of human DPP4 amino acid residues 39 to 776, with consensus contact sequences and catalytic domain indicated.

Fig. 2 shows the native human DPP4 amino acid sequence and modified human DPP4

Variant 1 sequence spanning residues 277 to 301 with residues to be changed underlined; (A) Original human DPP4 sequence spanning residues 277 to 301 ; (B) Modified human DPP4 Variant 1 sequence spanning residues 277 to 301 .

Fig. 3 shows the native human DPP4 amino acid sequence and modified human DPP4 Variant 2 sequence spanning residues 277 to 301 with residues to be changed underlined.

(A) Original human DPP4 sequence spanning residues 277 to 301 ; (B). Modified human DPP4 Variant 2 sequence spanning residues 277 to 301 .

Fig. 4 shows the plasmid maps of plant expression vectors pTRAk-DPP4-Fc and pTRA-P19. Fig. 5 shows images of Coomassie stained SDS-PAGE gels (reduced and non-reduced) of DPP4-lgG Fc fusions of different DP4 length [5(a)] DPP4(39-776)-lgA1 Fc and DPP4(39-776)- lgA2 Fc [5(b)] and Size Exclusion Chromatography curve of dimerized DPP4-lgG Fc 5(c). Fig. 6 shows enhanced binding of DPP(39-766)-Fc over soluble DPP4 and truncated DPP4(39- 496)-Fc.

Fig. 7 shows dose-dependent binding of DPP4-FC variants to the S1 protein of MERS-CoV in a ligand binding ELISA.

Fig. 8 shows DPP4-Fc V1 and DPP4-Fc V2 neutralization of cellular infection with MERS CoV- pseudovirus.

Fig. 9 shows inhibition of MERS-CoV (ECM isolate) infection of human cells by DPP4-Fcs of different Isotypes (lgG1 , lgA1 , and lgA2) and Variants.

Fig. 10 shows survival of Vero E6 cells after exposure to MERS-CoV Jordan Strain with or without DPP4-Fc variants at increasing concentration.

Fig. 11 shows the sequence of human DPP4(39-766) fused to human lgG1 Fc.

Fig. 12 shows sequence of human DPP4-Fc(39-766)V1 fused to human lgG1 Fc.

Fig. 13 shows the sequence of human DPP4-Fc(39-766)V2 fused to human lgG1 Fc.

Fig. 14 shows the sequence of human DPP4-Fc(39-766)V1 fused to human lgA1 Fc.

Fig. 15 shows the sequence of human DPP4-Fc(39-766)V2 fused to human lgA1 Fc.

Fig. 16 shows the sequence of human DPP4-Fc(39-766) fused to human lgA1 Fc.

Fig. 17 shows the sequence of human DPP4-Fc(39-766)V1 fused to human lgA2 Fc.

Fig. 18 shows the sequence of human DPP4-Fc(39-766)V2 fused to human lgA2 Fc.

Fig. 19 shows the sequence of human DPP4-Fc(39-766) fused to human lgA2 Fc. DETAILED DESCRIPTION OF THE INVENTION

The compositions of matter according to the invention utilize the ability of DPP4, the cell surface receptor for the S1 domain on the viral spike glycoprotein, to bind to the MERS-CoV RBD, to create a potent therapeutic to disrupt the initial steps of MERS-CoV infection. A preferred composition according to the invention is a recombinant protein comprised of the extracellular domain of DPP4 fused to of a portion of a human immunoglobulin which confers a biological or effector function (e.g. the hinge and Fc of lgG1 ), The composition can function as a "receptor decoy" to prevent the interaction of MERS-CoV with DPP4 on human cells and thus stop infection. This receptor decoy may bind to the MER-CoV spike protein thereby blocking its availability to bind to DPP4 on the cell surface. Recombinant soluble DPP4 inhibits MERS-CoV infection of Vera cell in vitro, but the concentration required to achieve 50% inhibition is fairly high: -10 μg/ml (Raj et al. 2013). Therefore, a DPP4 peptide sequence having increased affinity for the MERS-CoV RBD would be desirable.

The DPP4-Fc receptor decoy according to the invention has increased potency compared to soluble DPP4 because of the increase in binding interaction (avidity vs affinity) due to the stoichiometry of DPP4 in the Fc fusion (two DPP4 binding domains per homodimeric molecule). Furthermore a DPP4-Fc decoy will not subject the virus to selection for neutralization escape mutants, as any mutation of the viral spike protein that decreases binding to the decoy will likewise decrease virus binding to the native receptor, resulting in an attenuated virus.

In US Patent 7951378, herein incorporated by reference, it has been demonstrated, with human rhinovirus (HRV), and its cellular receptor, intercellular adhesion molecule 1 (ICAM1 ), that an ICAM-Fc fusion is a significantly more potent inhibitor of HRV infection than soluble ICAM. Like DPP4, ICAM1 is found on cells lining the upper respiratory tract. Recombinant soluble ICAM1 (sICAMI ) inhibits HRV infection of susceptible cells, with an in vitro EC50 (50% inhibition of the virus' cytopathic effect) of ~3 μg/ml against a standard HRV serotype. However, fusions of ICAM1 to human Fc are more potent and have significantly lower EC50. Recombinant ICAM1 -lgA2Fc, produced in our plant expression system, had an EC50 of 0.5 μg/ml, while ICAM1 -lgG1 Fc had an EC50 of 0.3 g/ml. An ICAM1 -lgA1 Fc had an EC50 of 0.08 μ9 πτιΙ (Martin et al. 1993).

These differences in in vitro virus neutralization may be related to structural differences in the immunoglobulin isotypes. For instance, studies of lgA1 and lgA2 in solution indicate that they have more of a T-shape than the Y shape typical of IgG. The arms of the T in lgA1 are more extended, due to its longer hinge, than the arms of lgA2 (Boehm et al. 1999; Furtado et al. 2004). Structural modeling indicates that the Fab-to-Fab center-to-center distance is 8.2 nm in lgA2, 16.9 nm in lgA1 and 7 to 9 nm in IgG, depending on the subtype (Boehm et al. 1999; Eryilmaz et al. 2013). Thus in a preferred embodiment significant increases in potency can be engineered into a DPP4-Fc fusion against MERS-CoV by using different Fc fusions from other immunoglobulin isotypes, such as lgG1 , lgG2 , lgA1 , lgA2, IgE, and IgM. Furthermore, DPP4-Fc may not just block virus binding to the cell, but multiple DPP4 ligands bound to the virus may trigger disruption of the viral particle and non-productive release of viral nucleic acid, as has been seen with ICAM-Fc disruption of HRV (Martin et al. 1993; Casasnovas and Springer 1994).

In one embodiment of the invention recombinant fusion proteins will have DPP4 at the amino terminal end and a portion of an immunoglobulin, for example an Fc, at the carboxyl terminal end of the fusion protein. It is not known from the literature whether the α/β hydrolase domain is required for effective binding of the MERS-CoV S glycoprotein.

Accordingly, compositions according to the invention include constructs containing either the entire extracellular domain (84 kDa) or just the β-propeller domain (53 kDa) of DPP4, which is approximately the size of a Fab. Thus, in one embodiment, wherein a portion of the immunoglobulin heavy chain is an Fc or hinge and Fc, the composition will be approximately the size of a typical IgG, IgA, or dimeric IgM. When the fusion protein forms homo-dimers, as a result of dimerization of the Fc region, the two DPP4-MERS-CoV binding sites will be separated by about the same distance as the combining sites on normal dimeric antibodies. Because the spikes on a typical coronavirus virion are situated about 15 nM apart (Neuman et al. 2006), in a preferred embodiment the lgA1 fusion may be able to bind two spikes simultaneously. In another preferred embodiment lgA2 and IgG fusions containing the entire 84 kDa extracellular domain may also achieve improved neutralization.

In addition to the potential for superior virus neutralization, a fusion of DPP4 to the Fc of lgG1 has two additional advantages as a therapeutic: an increased circulating half-life due to the ability of Fc to bind to the neonatal Fc receptor (FcRn) for recycling (Rath et al. 2013) and a simplified purification using affinity chromatography, for example protein A affinity

chromatography. Furthermore, a fusion of DPP4 to the Fc of lgA1 has the additional advantage in purification using affinity ligands designed for human IgA purification.

Although application of the receptor-Fc fusion approach to MERS-CoV is novel, the approach has previously been applied to develop therapeutics for other pathogens, including HIV, Hepatitis A virus, Pneumocystis carinii and coxsackievirus (Rapaka et al. 2007; Silberstein et al. 2003; Ward et al. 1991 ; Lim et al. 2006).

The MERS-CoV Receptor Binding Domain (RBD) contacts blades 4 and 5 of the DPP4 β- propeller domain (italic lettering in Figure 1 ; consensus contact amino acids underlined) and has no contact with the hydrolase domain (bold lettering in Figure 1 ). Differences in the identity of the DPP4-RBD contact amino acids among species have been identified and present an opportunity to modify the affinity of binding of the various DPP4-Fcs. Although the MERS-CoV S1 glycoprotein binds to human DPP4, it is likely better adapted to bind to the DPP4 of its animal host. A fusion protein based on the binding surface of the animal DPP4 might be more potent at neutralizing MERS-CoV infection. For that reason single amino acid changes are made at specific positions in the human DPP4 of the fusion proteins, based on the best understanding of the MERS-CoV animal host, which has been identified as camel. A single amino acid difference allows the known MERS spike protein to bind more tightly to camel DPP4 than to human DPP4. Thus the invention includes functional human DPP4 sequences altered from the native human sequence that have a higher binding affinity for the MERS CoV spike protein and hence the MERS-CoV itself. Thus in a preferred embodiment ,the invention includes altered soluble human DPP4 having a higher binding affinity to MERS-CoV than native soluble human DPP4. Such high binding affinity-altered soluble human DPP4s of the invention may alone bind to and neutralize MERS-CoV. Furthermore, high binding affinity-altered soluble human DPP4s and the nucleic acid sequences that encode them herein disclosed are valuable as intermediates in the recombinant production of DPP4-Fc fusions.

The first of two sequence variants of DPP4 was made by substituting a single amino acid in human DPP4 with the corresponding camel residue in the contact region for spike protein There is only one difference between the camel and human sequences at the consensus amino acids (241 to 320) for contact to MERS-CoV S1 , at amino acid 288. In variant V1 aa 288 of human DPP4 is changed from threonine (T) to valine (V). Figure 2.

In variant V2 of DPP4 non-contact amino acids around aa 288 were modified, in addition to the T288V substitution, to more resemble camel DPP4. The stretch from 285-293 differs at four amino acids in human vs camel, while adjacent sequences are similar; therefore the human DPP4 sequence from 285-293 (IQITAPASM) was changed to RQIVPPASV.

It was not obvious that these amino acid changes would make DPP4 or the corresponding

DPP4-Fc fusiona better decoy for MERS-CoV, and thus better at blocking the virus from infecting cells. In fact, a recent publication claims that the MERS-CoV spike protein binds equally well to cell surface human and camel DPP4 (Barlan et al. 2014). Also, the crystal structure predicts that human DPP4 T288 forms a polar contact with spike protein K502 (Lu et al. 2013). Thus, a change at DPP4 residue 288 from Threonine, a polar amino acid, to Valine, a non-polar amino acid, was not predicted to improve binding and neutralization as it did in the Examples herein below.

Eleven additional single amino acid substitutions have been identified that may improve binding of DPP4-V1 -FcG1 to MERS-CoV spike protein: K392E, I25F, L294F, I346F, V341 I, Q344R, R336Y, V288N, F269H, A291V and T188R. The amino acid changes can be made to the corresponding nucleic acid codons via overlap extension PCR mutagenesis, by using a site- directed mutagenesis kit (Q5® Kit, New England Biolabs), or by commercially available de novo synthesis of the corresponding nucleic acid sequence by means well known in the industry. Single amino acid modifications that show improved binding may be combined with other such modifications in a DPP4 sequence.

The peptidase activity of DPP4 is retained by the DPP4 (39-766)-Fc dimer, which is able to cleave the chromogenic substrate Gly-Pro-pNA. DPP4 (39-504)-Fc, which lacks the hydrolase domain, showed no capacity for Gly-Pro-pNA cleavage but binds poorly to the MERS- COV RBD. To eliminate peptidase activity from DPP4 (39-766) and DPP4 (39-766) V1 or DPP4(39-766), single amino acid changes of from Y547 to a different amino acid or S630 to a different amino acid (residues Ser-630, His-740, Asp-708 make up the active site) will eliminate this hydrolase activity as shown by the peptidase assay of Gly-Pro-pNA, yet should have no effect on folding of the β-propeller domain and thus the S1 binding site. In a preferred embodiment, this conversion may be of Y547F or S630A in DPP4 (39-776). In another preferred embodiment this conversion may be of Y547F or S630A in DPP4 (39-776) V1 or DPP4 (39-776) V2. Furthermore the altered amino acid residues 547 or 630 or DPP4 (39-776) V1 or V2 may be fused to Fc of lgG1 , lgG2, lgA1 , lgA2, IgM or IgD as described above. Furthermore the combined sequence thus produced described above may also include any of the 1 1 additional single amino acid substitutions indicated above. The modification of the nucleic acid sequence encompassing the Y547 to an alternative amino acid change including but not limited to Y547 to F or S630 to an alternative amino acid change, including but not limited to S630 to A, can be accomplished by any of the methods previously mentioned above in connection with modification of specific DPP4 sequences.

Modification of the FC sequence with and without KDEL

The DPP4-Fc variants of the invention may include the ER retention signal KDEL, appended to the Fc C-terminus. The use of the ER retention signal KDEL results in the high- mannose form for the protein's N-glycans (Petruccelli et al. 2006). Alternatively the DPP4V-Fc variants of the invention may be produced without ER retention signal KDEL. The N-glycans of the DPP4-Fc variants lacking the ER retention signal KDEL will be of the complex type on both DPP4 and Fc regions of the protein. Antibodies with high-mannose glycans are cleared from circulation more rapidly than those with complex type glycans in mice (Kanda et al. 2007) and humans (Goetze et al. 201 1 ); DPP4-Fc variants with complex N-glycans should therefore possess improved pharmacodynamics characteristics.

In one embodiment of the invention, the DPP4-Fc-fusions of the invention may be expressed in eukaryotic cells, tissues, organs or organisms, including fungal, insect, plant cell or mammalian cell culture according to known cell culture conditions. In a preferred embodiment the DPP4-Fc-fusions according to the invention are made in intact plant cells. Such plants may be transformed so that the nucleic acid sequences encoding the DPP4-Fc-fusion are stably incorporated into the plant genome and expressed in the cells and tissues of the intact plant and are transmitted from one generation to the next through the development of seed incorporating the nucleic acid sequences encoding the DPP4-Fc-fusion.

In another preferred embodiment of the invention the DPP4-Fc-fusions according to the invention are made in intact plants that have been transfected with Agrobacterium tumefaciens wherein the Ti plasmid has been engineered to contain the nucleic acid sequences encoding the DPP4-Fc-fusion protein which are transiently expressed by the cells and tissues of the intact plant. According to this method of production in plants, the open reading frames encoding a DPP4-Fc fusion described above is cloned into the plant expression vector pTRAk with suitable promoters and expression control sequences and the resulting vectors are transformed into Agrobacterium tumefaciens. The Agrobacterium strains will be used for transient transformation of Nicotiana benthamiana plants, with the recombinant protein expressed in plant cells. The fusion protein will be purified from extracts of plant tissue using standard chromatographic procedures, including, if the DPP4-Fc fusion comprises an IgG heavy chain, Protein A affinity chromatography or if the DPP4-Fc fusion comprises an IgA heavy chain, other affinity reagents including for example Protein G, CaptureSelect IgA Affinity Matrix (Life Technologies) and the like.

Proper N-glycosylation of the Fc may be important for in vivo viral neutralization.

Accordingly it is preferred to produce the DPP4-Fc fusion proteins with N-glycans as similar to typical mammalian N-glycans as possible using an N. benthamiana line in which the endogenous β1 ,2-xylosyltransferase (XylT) and a1 ,3-fucosyltransferase (FucT) genes have been down-regulated by RNA interference. Such strains are produced as described in (Strasser et al. 2008). Glycoproteins produced in this line contain almost homogeneous N-glycan species without detectable plant-specific 31 ,2-xylose and a1 ,3-fucose residues. The expression of the XylT gene and FucT gene may be down regulated or eliminated by methods other than RNA interference, including by modification using the CRISPR/Cas system to alter the sequence of the genes encoding one or both proteins. Additionally, to ensure uniform addition of terminal β1 ,4-Gal residues to N-glycans (Strasser et al. 2009), it is additionally preferred to co-infiltrate this N. benthamiana with a binary vector that encodes a modified human 31 ,4-galactosyl- transferase (ST-GalT) to "humanize" plant-made N-glycans.

There is another reason why appropriate DPP4-Fc N-glycosylation may be important. According to one publication (Lu et al. 2013), the N-glycan of DPP4 N229 interacts with RBD amino acids W535 and E536 when DPP4 binds S1 , though the exact structure of the native N- glycan is unclear. DPP4 with complex N-glycans similar to typical human N-glycans may have increased affinity for S1 .

In another embodiment of the invention, the DPP4-Fc or modified DPP4-Fc may be delivered to the body by various routes including parenteral, preferably intravenous, intraarterial and intraperitoneal, or by mucosal administration. FcRn mediates the endocytic salvage pathway responsible for the long circulating half-life of IgGs (Goebl et al. 2008) and also mediates bi-directional IgG transcytosis across mucosal epithelial cells in a variety of adult human tissues. FcRn is expressed in the mucosal epithelial cells lining the conducting airways (the trachea and bronchioles) (Spiekermann et al. 2002) and is responsible for the high IgG concentration in airway surface liquid (up to 17% of total protein) (Goldblum and Garofolo 2004; Hand and Cantey 1974). Bidirectional IgG transport between the blood and the lumen of the airways is facilitated because the epithelium lies on top of the basement membrane, which lies directly above the highly vascularized lamina propria. For this reason parenterally administered DPP4-Fc will be delivered to airway mucosal surfaces, which is the site of ME S-CoV infection (Tao et al. 2013).

As used herein, the following abbreviations and terms include, but are not necessarily limited to, the following definitions.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods In Enzymology (Academic Press, Inc.); M. J. MacPherson, et al., eds. Per 2: A Practical Approach (1995); Harlow and Lane, eds, Antibodies: A Laboratory

Manual (1988), and H. Jones, Methods In Molecular Biology vol. 49, "Plant Gene Transfer And Expression Protocols" (1995).

Immunoadhesin: A complex containing a chimeric receptor protein molecule fused to a portion of an immunoglobulin constant region, and optionally containing secretory component and J chain.

Chimeric receptor protein: A receptor-based protein having at least a portion of its amino acid sequence derived from an extracellular receptor and at least a portion derived from an immunoglobulin complex.

Receptor: As used herein, the term refers to any polypeptide that binds to specific antigens as defined herein, or any proteins, lipoproteins, glycoproteins, polysaccharides or lipopolysaccharides that exert or lead to exertion of a biological or pathogenic effect with an affinity and avidity sufficient to allow a chimeric receptor protein to act as a receptor decoy. For example, a receptor may be a viral attachment receptor such as ICAM-1 , which is a receptor for human rhinovirus, or DPP4 which is a receptor for MERS-CoV spike glycoprotein 1 , or a receptor for a bacterial toxin, such as CMG2 which is one of the receptors for anthrax protective antigen, or tumor necrosis factor receptor superfamily (TNFRSF) is a group of cytokine receptors characterized by the ability to bind tumor necrosis factors (TNFs) via an extracellular cysteine-rich domain. The receptors as used herein shall at a minimum contain the functional elements for binding of a component or components of the molecule to which they bind but may optionally also include one or more additional polypeptides.

Immunoglobulin molecule or Antibody: A polypeptide or multimeric protein containing the immunologically active portions of an immunoglobulin heavy chain and immunoglobulin light chain covalently coupled together and capable of specifically combining with antigen. The immunoglobulins or antibody molecules are a large family of molecules that include several types of molecules such as IgM, IgD, IgG, IgA, secretory IgA (SlgA), and IgE.

Immunoglobulin complex: A polypeptide complex that can include a portion of an immunoglobulin heavy chain or both a portion of an immunoglobulin heavy chain and an immunoglobulin light chain. The two components can be associated with each other via a variety of different means, including covalent linkages such as disulfide bonds. Examples of an immunoglobulin complex include FaB' and FaB'2.

Portion of an Immunoglobulin heavy chain: As used herein, the term refers to that region of a heavy chain which is necessary for conferring at least one of the following properties on the chimeric receptor proteins as described herein: ability to multimerize, effector functions such as binding to Fc receptors, neonatal Fc receptors or compliment fixation, proteins, ability to be purified by Protein G or A, or improved pharmacokinetics. Typically, this includes at least a portion of the heavy chain constant region.

Fc region The C-terminal portion of an immunoglobulin heavy chain that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

The presence of a Fc region in a chimeric immune complex should confer immunoglobulin effector functions to the complex, such as the ability to mediate the specific lysis of cells in the presence of complement. The heavy chain constant region domains of the immunoglobulins confer various properties known as antibody effector functions on a particular molecule containing that domain. Example effector functions include complement fixation, placental transfer, binding to staphyloccal protein, binding to streptococcal protein G, binding to mononuclear cells, neutrophils or mast cells and basophils. The association of particular domains and particular immunoglobulin isotypes with these effector functions is well known and for example, described in Immunology, Roitt et al., Mosby St. Louis, Mo. (1993 3rd Ed.) In addition, binding of the Fc to the Fc n should allow the immunoadhesins to persist in the circulation much longer (Ober, R. J., Martinez, C, Vaccaro, C, Zhou, J. & Ward, E. S.

Visualizing the Site and Dynamics of IgG Salvage by the MHC Class l-Related Receptor, FcRn. J Immunol 172, 2021 -2029 (2004)). This may allow the antitoxin to be used as a prophylactic.

Portion of an Immunoglobulin light chain: As used herein, the term refers to that region of a light chain which is necessary for increasing stability of the described chimeric receptor protein and thus increasing production yield. Typically, this includes at least a portion of the immunoglobulin light chain constant region.

Heavy chain constant region: A polypeptide that contains at least a portion of the heavy chain immunoglobulin constant region. Typically, in its native form, IgG, IgD and IgA

immunoglobulin heavy chain contain three constant regions joined to one variable region. IgM and IgE contain four constant regions joined to one variable region. As described herein, the constant regions are numbered sequentially from the region proximal to the variable domain. For example, in IgG, IgD, and IgA heavy chains, the regions are named as follows: variable region, constant region 1 , constant region 2, constant region 3. For IgM and IgE, the regions are named as follows: variable region, constant region 1 , constant region 2, constant region 3 and constant region 4.

Chimeric immunoglobulin heavy chain: An immunoglobulin derived heavy chain wherein at least a first portion of its amino acid sequence is a first antibody isotype or subtype and second peptide, polypeptide or protein or glycoprotein. The second polypeptide, protein or glycoprotein, may itself be derived from an immunoglobulin heavy chain of a different isotype or subtype antibody. Typically, a chimeric immunoglobulin heavy chain has its amino acid residue sequences derived from at least two different isotypes or subtypes of immunoglobulin heavy chain.

J chain: A polypeptide that is involved in the polymerization of immunoglobulins and transport of polymerized immunoglobulins through epithelial cells. See, The Immunoglobulin Helper: The J Chain in Immunoglobulin Genes, at pg. 345, Academic Press (1989). J chain is found in pentameric IgM and dimeric IgA and typically attached via disulfide bonds. J chain has been studied in both mouse and human.

Secretory component (SC): A component of secretory immunoglobulins that helps to protect the immunoglobulin against inactivating agents thereby increasing the biological effectiveness of secretory immunoglobulin. The secretory component may be from any mammal or rodent including mouse or human.

Linker: As used herein, the term refers to any polypeptide sequence used to facilitate the folding and stability of a recombinantly produced polypeptide. Preferably, this linker is a flexible linker, for example, one composed of a polypeptide sequence such as (Gly3Ser)3 or

(Gly4Ser)3.

Transgenic plant: Genetically engineered plant or progeny of genetically engineered plants. The transgenic plant usually contains material from at least one unrelated organism, such as a virus, bacterium, fungus, another plant or animal.

Plant Material: materials derived from plants including, plant cell walls, plant organelles, plant cytoplasm, intact plant cells, plant tissues, plant leaves, plant stems, plant roots, plant seeds, and viable plants.

Monocots: Flowering plants whose embryos have one cotyledon or seed leaf. Examples of monocots are: lilies; grasses; corn; grains, including oats, wheat and barley; orchids; irises; onions and palms.

Dicots: Flowering plants whose embryos have two seed halves or cotyledons. Examples of dicots are: tobacco; tomato; the legumes including alfalfa; oaks; maples; roses; mints;

squashes; daisies; walnuts; cacti; violets and buttercups.

Glycosylation: The modification of a protein by oligosaccharides. See, Marshall, Ann. Rev.

Biochem., 41 :673 (1972) and Marshall, Biochem. Soc. Symp., 40: 17 (1974) for a general review of the polypeptide sequences that function as glycosylation signals. These signals are recognized in both mammalian and in plant cells. Plant-specific glycosylation: The glycosylation pattern found on plant-expressed proteins, which is different from that found in proteins made in mammalian or insect cells. Proteins expressed in plants or plant cells have a different pattern of glycosylation than do proteins expressed in other types of cells, including mammalian cells and insect cells. Detailed studies characterizing plant-specific glycosylation and comparing it with glycosylation in other cell types have been performed by Cabanes-Macheteau et al., Glycobiology 9(4):365-372 (1999), Lerouge et al., Plant Molecular Biology 38:31-48 (1998) and Altmann, Glycoconjugate J.

14:643-646 (1997). Plant-specific glycosylation generates glycans that have xylose linked β(1 ,2) to mannose. Neither mammalian nor insect glycosylation generate xylose linked β(1 ,2) to mannose. Plants do not have a sialic acid linked to the terminus of the glycan, whereas mammalian cells do. In addition, plant-specific glycosylation results in a fucose linked a(1 ,3) to the proximal GlcNAc, while glycosylation in mammalian cells results in typically a fucose linked a(1 ,6) to the proximal GlcNAc.

Immunoglobulin Heavy Chain: The chimeric DPP4 and modified or altered DPP4 receptor proteins contain at least a portion of an immunoglobulin heavy chain constant region sufficient to confer either the ability to multimerize the attached anthrax receptor protein, confer antibody effector functions, stabilize the chimeric protein in the plant, confer the ability to be purified by Protein A or G, or to improve pharmacokinetics. These properties are conferred by the constant regions of the immunoglobulin heavy chains. If the chimeric toxin receptor protein contains only an immunoglobulin heavy chain, the portion of the heavy chain in the immunoglobulin complex preferably contains at least domains CH2 and CH3 and more preferably, only CH2 and CH3. If the chimeric toxin receptor protein contains both a heavy chain and a light chain, the portion of the heavy chain in the immunoglobulin complex preferably also contains a CH1 domain.

One of skill in the art will readily be able to identify immunoglobulin heavy chain constant region sequences. For example, a number of immunoglobulin DNA and protein sequences are available through GenBank. Table 1 shows the GenBank Accession numbers of

immunoglobulin heavy chain genes and the proteins encoded by the genes.

TABLE 1

GENBANK ACCESSION NO. HUMAN IMMUNOGLOBULIN SEQUENCE NAME

J00220 lg_a1 Heavy Chain Constant Region Coding Sequence

J00220 lg_a1 Heavy Chain Constant Region Amino Acid Sequence

J00221 lgA_∑ Heavy Chain Constant Region Coding Sequence

J00221 lgA∑ Chain Constant Region Amino Acid Sequence J00228 lg_Y1 Heavy Chain Constant Region Coding Sequence

J00228 Igy1 Heavy Chain Constant Region Amino Acid Sequence

J00230 lgG₂ Heavy Chain Constant Region Coding Sequence

J00230 lgG2 Heavy Chain Constant Region Amino Acid Sequence

V00554

X03604 lgG₃ Heavy Chain Constant Region Coding Sequence

M12958

X03604 lgG₃ Heavy Chain Constant Region Amino Acid Sequence

M12958

K01316 lgG₄ Heavy Chain Constant Region Coding Sequence

K01316 lgG₄ Heavy Chain Constant Region Amino Acid Sequence

K02876 IgD Heavy Chain Constant Region Coding Sequence

K02876 IgD Heavy Chain Constant Region Amino Acid Sequence

K02877 IgD Heavy Chain Constant Region Coding Sequence

K02877 IgD Heavy Chain Constant Region Amino Acid Sequence

K02878 Germline IgD Heavy Chain Coding Sequence

K02878 Germline IgD Heavy Chain Amino Acid Sequence

K02879 Germline IgD Heavy Chain C-S-3 Domain Coding Sequence

K02879 Germline IgD Heavy Chain C-S-3 Amino Acid Sequence

K0131 1 Germline IgD Heavy Chain J-S Region: C-S CH 1 Coding

K0131 1 Germline IgD Heavy Chain J-S Region:

C-S CH1 Amino Acid Sequence

K02880 Germline IgD Heavy Chain Gene, C-Region, Secreted Terminus

Coding Sequence

K02880 Germline IgD Heavy Chain Gene, C-Region, Secreted Terminus Amino Acid Sequence

K02881 Germline IgD-Heavy Chain Gene, C-Region, First Domain of

Membrane Terminus Coding Sequence

K02881 Germline IgD-Heavy Chain Gene, C-Region, First Domain

nf IVIpmhranp Tprmini i Aminn Arirl i^pm ipnrp

K02882 Germline IgD Heavy Chain Coding Sequence

K02882 Germline IgD Heavy Chain Amino Acid Sequence

K02875 Germline IgD Heavy Chain Gene, C-Region, C-S-1 Domain

Sequence

K02875 Germline IgD Heavy Chain Gene, C-Region, C-S-1 Domain

L00022 IgE Heavy Chain Constant Region Coding Sequence

J00227

V00555

L00022 IgE Heavy Chain Constant Region Amino Acid Sequence J00227

X171 15 IgM Heavy Chain Complete Coding Sequence

X171 15

IgM Heavy Chain Complete Amino Acid Sequence

Chimeric MERS-CoV spike glycoprotein 1 receptor protein: A protein having at least a portion of its amino acid sequence derived from the cell surface protein dipeptidyl peptidase 4 (DPP4) and at least a portion derived from an immunoglobulin complex. The immunoglobulin complex may contain only a portion of an immunoglobulin heavy chain or it may contain both a portion of a heavy chain and a portion of a light chain.

MERS-CoV Receptor Binding Domain (RBD) : residues 358 to 588 of the MERS CoV S1 spike protein and contains within this sequence the regions that contact amino acid residues located in blades 4 and 5 of the DPP4 β-propeller domain of DPP4 peptide. (Mou, H., Raj, V.S., van Kuppeveld, F.J., Rottier, P.J., Haagmans, B.L., and Bosch, B.J. 2013. The receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231 -residue region in the spike protein that efficiently elicits neutralizing antibodies. J Virol 87:9379-9383).

Consensus contact sequence of DPP4: those amino acid residues located in blades 4 and 5 of the DPP4 β-propeller domain that contact the MERS-CoV RBD, according to the deduced crystal structure of the MERS-CoV RBD/DPP4 complex. The crystal structure of human DPP4 indicates that blades 4 and 5 run from aa 1194 - E362. The amino acid residues of the consensus contact sequence of DPP4 include 288, 290, 293, 296, 297, 317, 335, 336, and 341 . (Lu, G., Hu, Y. , Wang, Q., Qi, J., Gao, F., Li, Y., Zhang, Y., Zhang, W., Yuan, Y. , Bao, J., et al. 2013. Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26. Nature 500:227-231 . Wang, N., Shi, X., Jiang, L, Zhang, S., Wang, D., Tong, P., Guo, D., Fu, L, Cui, Y. , Liu, X., et al. 2013. Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4. Cell Res 23:986-993.)

.Effective amount: An effective amount of an immunoadhesin of the present invention is sufficient to detectably inhibit viral attachment, viral cellular cytopathology or cellular cytotoxicity, or infection of an animal or to reduce the severity or duration of infection or symptoms of infection.

Construct or Vector: An artificially assembled DNA segment to be transferred into a target tissue or cell of a plant or animal, especially a mammal. Typically, the construct will include the gene or genes of a particular interest, a marker gene and appropriate control sequences.

Plasmid "An autonomous, self-replicating extrachromosomal DNA molecule. Plasmid constructs containing suitable regulatory elements are also referred to as "expression cassettes." In a preferred embodiment, a plasmid construct also contains a screening or selectable marker, for example an antibiotic resistance gene.

Selectable marker: A gene that encodes a product that allows the growth of transgenic tissue or cells on a selective medium. Non-limiting examples of selectable markers include genes encoding for antibiotic resistance, e.g., ampicillin, kanamycin, or the like. Other selectable markers will be known to those of skill in the art. EXAMPLES

The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

Example 1 Transient expression of DPP4-lqG1 fusion proteins in N. benthamiana

Briefly, sequences encoding the full-length DPP4 extracellular domain (amino acids 39- 766) (Sequence ID No.2) or the DPP4 β-propeller domain (amino acids 39-496) Sequence ID No. 3 were PCR-amplified from the human DPP4 sequence (Sequence ID No. 1 ) and then cloned into the pTRAkc plant binary vector (Maclean et al. 2007) in frame with an lgG1 Fc sequence optimized for expression in planta. Recombinant A. tumefaciens strains

(GV3101 ::pMP90RK) carrying these expression vectors were used to transiently express DPP4- Fc in whole N. benthamiana plants following vacuum-assisted agroinfiltration using known methods (Kapila et al. 1997; Vaquero et al. 1999). Co-infiltration of an additional A. tumefaciens strain (GV3101 ::pMP90RK) carrying the p19 silencing suppressor from tomato bushy stunt virus, was used to prevent post-transcriptional gene silencing and hence enhance expression levels (Voinnet et al. 2003). The transfected plants were harvested, and plant juice was extracted by grinding in a Waring blender, the juice was separated by filtration and the protein was purified by Protein A chromatography. Reduced and non-reduced samples were separated by SDS-PAGE and stained with Coomassie dye (a) or probed with anti-DPP4 antibodies (b). Monomer (reduced) and dimer bands were detected at the expected positions.

In greater detail, expression vectors were produced as follows. Sequences encoding the DPP4 extracellular domain (aa 39-766), or variant V1 (Figure 2) or variant,V2 (Figure 3), or truncated variants encoding only the DPP4 β-propeller domain (either aa 39-496 or 39-504) were PCR-amplified using the published human DPP4 sequence. The DPP4 sequences or altered DPP4 sequences were cloned into the pTRAk plant binary vector alone (Maclean et al. 2007), or upstream of and in-frame with human Fc sequences (hinge, CH2 and CH3) from human lgG1 , lgA1 or lgA2. The complete amino sequence of the DPP4 and DPP4 Variants 1 and 2 in-frame Fc fusion lgG1 , lgA1 and lgA2 is shown in Figures 12 though 19. The corresponding DNA sequence is inserted in pTRAk as shown in Figure 4 in the region denoted by DPP4 and Fc in the open reading frame (ORF). The IgA constructs were truncated to remove the 18-amino acid C-terminal IgA tail-piece, a sequence that enables dimeric IgA formation but significantly reduces IgA expression in plants (Hadlington et al. 2003) and is not required for binding Fc alpha receptors (Brunke et al. 2013). All constructs included a C-terminal KDEL peptide for endoplasmic reticulum (ER) retention, resulting in high mannose N-glycans. Alternatively, without KDEL, the fusion protein is targeted to the plant cell secretory pathway via a signal peptide from a mouse antibody heavy chain. See Figure 4, Plasmid maps for pTRAk- DPP4 Fc and pTRA-P19.

The resulting plasmids are transformed into A. tumefaciens GV3101 : :pMP90RK (Maclean et al. 2007) and the resulting A. tumefaciens strains are vacuum infiltrated into N. benthamiana for transient expression of the DPP4-Fc fusions. For high levels of expression, an

Agrobacterium strain carrying a vector encoding the p19 protein of the tomato bushy stunt virus (Voinnet et al. 2003) to suppress post-transcriptional gene silencing is co-infiltrated. The Agrobacterium cell suspensions are combined and diluted to appropriate concentrations in infiltration buffer. Whole N. benthamiana plants (3-6 plants per pot), inverted and submerged into the bacterial suspension, are subjected to two sequences of vacuum (to 20 in. Hg for 10 sec) followed by slow vacuum release (~2 kPa/second) to draw the bacterial suspension into the spongy leaf interstitial space. Following infiltration, plants are grown for up to 8 days in a greenhouse.

Example 2 Extraction and Purification

Briefly, N. benthamiana extracts are obtained by homogenizing the leaves with an aqueous buffer in a blender, which results in a mixture of DPP4-Fc and plant material. The mixture is clarified by centrifugation or other appropriate means such as filtration, which may be followed by micro filtration or ultrafiltration and or sterile filtratration, followed by DPP4-Fc captured on columns of the appropriate affinity chromatography medium. lgG1 Fc fusions are purified using Protein A-Sepharose and IgA Fc fusions are purified using for example

CaptureSelect™ Human IgA Affinity Matrix (Life Technologies) (Reinhart, Weik, and Kunert 2012). Other affinity chromatography resins, such as CaptureSelect IgA Affinity Matrix (Life Technologies) may be used for DPP4 IgA-Fc. fusions The DPP4-Fc fusions are eluted at low pH, neutralized, and dialyzed into PBS. Purity of 90-95% at >50% overall yield may be achieved. These affinity matrices work well with Fc-fusions and both have low affinity for plant proteins. If needed, an additional purification step, such as cation exchange chromatography, can be used.

In greater detail, upstream processing consists of grinding and pressing biomass, with an appropriate buffers (such as Tris, soytone, ethylenediamine, PBS, pH 7.2-9.5) that maintain the stability and recovery of the DPP4-Fc in order to segregate solids from the product-containing Raw Juice. The Raw Juice may be treated with acid to pH 4.0-5.0 followed by base treatment to pH 7.2-8.5 or polyethyleneimine (PEI) at 0.025-0.1 % (w/v) to agglomerate additional solids followed by centrifugation at 10K RPM for at least 15 min to remove solids and produce a clarified, product-containing liquid (centrate). The centrate is loaded onto Protein A, or other appropriate, affinity chromatography matrix.

The column is washed with 10-30 column volumes (CV) wash buffer containing PBS. Elution is carried out with 0.1 M glycine (acetic acid or citrate may also be used), 0.075-0.3 M NaCI, pH 2.0-3.0 and neutralized with 1 M HEPES, pH 8.0 or 1 M Tris, pH 8.5 (eluate). The eluate may be further purified via ion exchange chromatography and eluted via a salt or pH gradient. The polished eluate is buffer exchanged into the final formulation buffer and treated to remove endotoxin through a ToxinEraser (GenScript) column. Other excipients may be added to the final formulation to enhance stability and/or potency. The buffer exchanged eluate may be concentrated to the desired protein concentration and filtered through a 0.1 -0.2 micron PES membrane prior to storage at or below -65°C.

Alternatively, the Protein A column is washed with 5-10 CV wash buffer containing 1 % detergent (4 parts TX: 1 14 to 1 part TX: 100) in PBS. A second wash consist of 5-10 CV of 0.2 mg/ml Polymixin B in PBS. Lastly, 20 CV of PBS is used to wash away residual Polymixin B and/or detergent from the column prior to elution. Elution is carried out with 0.05-0.1 M glycine, 0.075-0.15 M NaCI, pH 2.0-3.0 and neutralized with 1 M HEPES, pH 8.0 or 1 M Tris, pH 8.5. The column may also be eluted using 0.75 M arginine (instead of glycine), 3.6 M MgCI2 in 0.2 M acetate, pH 6.6, or combination thereof. The eluate is buffer exchanged into PBS via dialysis or diafiltration using 3.5-100 kDa cut-off regenerated cellulose, cellulose ester, or polyethersulfone (PES) membranes. Other excipients may be added to the final formulation to enhance stability and/or potency. The buffer exchanged eluate may be concentrated to the desired protein concentration and filtered through a 0.1 -0.2 micron PES membrane prior to storage at or below -65°C.

Example 3 Characterization of Fc-fusions In Vitro

The structural integrity of the DPP4-Fc proteins is determined by reducing and non- reducing SDS-PAGE (Bio- ad) and immunoblotting with Fc-specific antibodies (Southern Biotechnology) and DPP4-specific antibodies (R & D Systems). Protein size, purity and assembly are determined by image analysis (Bio-Rad) of Coomassie stained (reduced and non- reduced) SDS-PAGE gels. The DPP4-Fc fusions, derived from lgG1 (see figure 5 a), lgA1 , and lgA2 (see figure 5 b) heavy chains, form homodimers under non-reducing conditions via disulfide bonds between hinge cysteines and have dimeric molecular weights predicted to be 160-225 kDa, depending on whether the complete extracellular domain or just the β-propeller domain is used. The proteins ran at the positions predicted by their theoretical molecular weight and presence of numerous N-linked glycans found on the β-propeller domain and in the hydrolase domain. See Figure 5 (a) and (b).

Additional protein conformation characterization included analytical size exclusion

chromatography (SEC) using a Shodex™ 8 x 300 mm column on a SpectraSYSTEM™ gradient HPLC (Thermo-Fisher). This column separates proteins between 500 and 1 ,000,000 Da. DPP4- Fc components were detected spectrophotometrically at 280 nm and quantified by measuring the area of individual peaks. Calibration of the column using protein molecular size standards allows accurately estimated sizes of DPP-Fc monomers, dimers, aggregates and fragments. See Figure 5(c). The major peak comprises approximately 93% of the sample in fully dimeric form.

Example 4 Binding ELISA for DPP4 Variants

The ability of soluble DPP4 (Sino Biological, Cat # 10688-HNCH) and the DPP4-Fc variants to bind to the S1 domain of the MERS-CoV S protein was determined in a functional ELISA. Briefly, Spike protein S1 domain (Sino Biological, Cat # 40069-V08B1 ) was coated on standard ELISA plates, 2.5 μg/mL, overnight at 4°C. The wells were blocked for an hour at room temperature (RT). Dilutions of DPP4-Fc were added to the plates and incubated for an hour at 37 °C. The wells were washed, and bound DPP4 or DPP4-Fc was detected using polyclonal goat anti-DPP4 IgG (R&D Systems, Cat # AF1 180) and reported with donkey anti-goat IgG labeled with HRP. OPD (o-Phenylenediamine dihydrochloride) substrate was added and absorbance at 490 nm was read on a Synergy™ HT Multi-Detection Microplate Reader (BioTek Instruments). The data was plotted and fitted to a 4-parameter logistic model

(GraphPad, San Diego, CA). An EC50 (the DPP4-Fc concentration for 50% maximal binding) was calculated for each variant. For DPP4 (39-766)-Fc, containing the full DPP4 extracellular domain, an EC50 of 0.04 ng/ml, showed significantly enhanced binding over soluble DPP4

(EC50 of 1 .2 ng/ml). DPP4 (39-496)-Fc, containing a truncated DPP4 domain, did not bind well to the MERS spike protein (EC50 of 3.2 ng/ml). See Figure 6

EC50 values from the binding ELISA indicate that the DPP4-Fc V1 (DPP4 (39-766) V1 - FcG1 is 8.7-fold better than the DPP4-Fc wild type (DPP4 (39-766)-FcG1 ) in the same assay. Also, DPP4 (39-766) V1 -FcG1 and DPP4 (39-766) V2-FcG1 (not shown) have comparable binding curves in the ELISA (Figure 7). Furthermore DPP4 (39-766) V1 -FcA1 and DPP4 (39- 766) V1 -FcA2 have binding comparable to wild type DPP4 (39-766)-FcG1 . Lastly all of the DPP4 (39-766)-Fcs, regardless of Fc heavy chain isotype, have binding to S1 protein superior to that of the truncated DPP4 (39-504)-FcG1 and soluble DPP4. See Figure 7.

Example 5 -DPP4-Fc neutralization of cellular infection with MERS CoV-pseudovirus.

Generation of MERS-CoV pseudovirus was done as previously described with some modifications (Du et al. 2010). Briefly, 293T cells (ATCC, Manassas, VA) were co-transfected with 20 μg of plasmid encoding Env-defective, luciferase-expressing HIV-1 (pNL4-3.luc.RE) and 20 μg of rMERS-CoV-S plasmid (pcDNA3.1-MERS-CoV-S), respectively, into a T175 tissue culture flask using the calcium phosphate method. Cells were changed into fresh DMEM 8 h later. Supernatants were harvested 72 h post-transfection and used for single- cycle infection. To detect the inhibitory activity of DPP4-Fc, DPP4-Fc(V1 ) and DPP4-Fc(V2) on infection by ME S pseudovirus, DPP4-expressing Huh-7 cells (104/well in 96-well plates) were infected with MERS-CoV pseudovirus in the presence or absence of DPP4-Fc variants at the indicated concentrations. The culture was re-fed with fresh medium 12 h post-infection and incubated for an additional 72 h. Cells were washed with PBS and lysed using lysis reagent included in a luciferase kit (Promega). Aliquots of cell lysates were transferred to 96-well flat-bottom luminometer plates (Costar), followed by addition of luciferase substrate (Promega). Relative light units were determined immediately using an Ultra 384 luminometer (Tecan USA). Viral replication was quantified by the amount of light measured.

DPP4-Fc V1 and V2 (described earlier) differ by one and five amino acids, respectively, from wild-type DPP4-Fc. All three DPP4-Fc variants neutralized pseudovirus infection, but with different potencies. The results are graphed in Figure 8.

The 50% inhibitory concentration (IC50) for the three variants was calculated using the dose-response software GraphPad Prism (GraphPad Software); DPP4-Fc was 0.46 μg/ml, while the IC50 for DPP4-Fc(V1 ) was 0.05 μg/ml and DPP4-Fc(V2) was 0.02 μ9/ηιΙ. The 90% inhibitory concentration (IC90) for DPP4-Fc was 4.2 g/ml, while the IC90 for DPP4-Fc (V1 ) and DPP4-Fc (V2) were 0.45 and 0.21 μg/ml, respectively. This compares to an IC90 of 0.039 μg/ml for the most potent monoclonal antibody against the MERS CoV S1 protein (Ying et al. 2014). Example 6 Inhibition of MERS-CoV Infection by DPP4-Fc and Modified DPP4 Fes

DPP4 variants were tested in an assay that measures inhibition of MERS-CoV infection. Virus stocks of MERS-CoV (EMC isolate) were prepared and diluted to 10,000 TCID50/ml and incubated with serial dilutions of our DPP4-Fc variants for 1 hour. The MERS-CoV/DPP4-Fc mixtures were added to Huh-7 cells in 96-well plates and incubated for 1 hour. The inoculation mixture was removed, replaced with fresh medium and 8 hours later the cells were fixed with 4% formaldehyde in PBS for 10 min and 70% ethanol for 30 min. Cells were stained for newly synthesized viral antigen as a measure for infection, using rabbit anti-MERS-CoV antiserum, followed by FITC-conjugated swine anti-rabbit antibody as a second step (Raj et al. 2013). The number of MERS-CoV infected cells per well were counted using an inverted fluorescent microscope, then the inhibitory effect was calculated based on the control group. This assay is a measure of the ability of the different DPP4-Fc variants to block MERS-CoV infection of human cells. In this assay (Figure 7) the IC50 for DPP4(39-766)-FcG1 , DPP4(39-766)V1 -FcG1 and DPP4(39-766)V2-FcG1 were 0.66, 0.05 and 0.03 μg/ml, respectively. The IC50 for DPP4(39- 766)-FcA1 was 0.30 μg/ml while DPP4(39-766)-FcA2 did not inhibit infection at any

concentration. See Figure 9.

Example 7 Cell Based Viral Neutralization Assay with Live MERS-CoV Jordan Strain DPP4-Fc variants were assayed in a preliminary cell-based viral neutralization assay with live MERS-CoV Jordan strain, in a biosafety level 3 laboratory. This assay measures survival of Vero E6 (African Green Monkey) cells 48 hr after exposure to MERS-CoV with or without DPP4-Fc variants at increasing concentration. Vero E6 cells are seeded in 96-well plates and incubated overnight. MERS-CoV at an MOI of 0.1 is incubated with eight 2-fold serial dilutions of each variant (final concentrations between 10 ng/ml and 10 μg/ml) in duplicate for one hour, after which the virus/variant mixtures are added to cells. Cell survival is quantified at 48 hours post-infection using CellTiter-Glo® reagent (Promega). Controls include cells incubated with 1 ) virus alone, 2) virus plus an anti-DPP4 mAb (Sino Biologicals), or 3) media only. Supernatants are collected at 24 and 48 hours for titering of virus growth by TCID50 to confirm cell viability results. Data is fit to a 4-parameter logistic model to calculate the IC50 for each variant. As with the binding ELISA and pseudovirus infection experiment, DPP4(39- 766)V1-FcG1 and DPP4(39-766)V2-FcG1 performed better than DPP4(39-766)-FcG1 . In this assay the lgA1 fusion variant, DPP4(39-766)-FcA1 , had comparable potency to the lgG1 fusion, DPP4(39-766)-FcG1 , while the lgA2 fusion variant did not protect against cell death at any concentration (not shown). See Figure 10.

Example 8 - Production in Plants Modified for Altered Glvcosylation.

Although the N-glycans in DPP4 do not make contact with the S1 RBD, proper N- glycosylation of the Fc may be important for in vivo viral neutralization. Accordingly it is preferred to produce fusion proteins with N-glycans as similar to typical mammalian N-glycans as possible using an N. benthamiana line in which the endogenous R1 ,2-xylosyltransferase (XylT) and a1 ,3-fucosyltransferase (FucT) genes have been down-regulated by RNA interference. Such strains are produced as described in (Strasser et al. 2008). Glycoproteins produced in this line contain almost homogeneous N-glycan species without detectable plant- specific 31 ,2-xylose and a1 ,3-fucose residues. To ensure uniform addition of terminal 31 ,4-Gal residues to N-glycans, it is additionally preferred to co-infiltrate this N. benthamiana with a binary vector that encodes a modified human β1 ,4-galactosyl-transferase (ST-GalT) to

"humanize" plant-made N-glycans (Strasser et al. 2009).

Example 9 Construction of Additional DPP4 Fc Variants

Removal of peptidase activity from DPP4

To eliminate peptidase activity from DPP4 (39-766) and DPP4 (39-766) V1 Fc, a single amino acid change to Y547F or S630A will eliminate this hydrolase activity as shown by the standard peptidase assay using Gly-Pro-paranitroanaline as substrate in a colorimetric assay, yet has no effect on folding of the β-propeller domain and thus the S1 binding site. The amino acid changes can be made to the corresponding nucleic acid codons via overlap extension PCR mutagenesis, by using a site-directed mutagenesis kit (Q5® Kit, New England Biolabs), or by commercially available de novo synthesis of the corresponding nucleic acid sequence by means well know in the industry. The resulting peptidase altered proteins are hereafter referred to as DPP4m(39-766) and DPP4m(39-766)V1 -FcG1 and DPP4m(39-766) V2-FcG1 Additional amino acid changes in the DPP4 sequence

Eleven additional single amino acid substitutions have been identified that may improve binding of DPP4m(39-766) and DPP4m(39-766)V1 -FcG1 and DPP4m(39-766)V2-FcG1 to MERS-CoV spike protein: K392E, I295F, L294F, I346F, V341 I , Q344R, R336Y, V288N, F269H, A291V and T188R. Changes to the corresponding nucleic acid residues encoding any of the amino acid modifications may be made as described immediately above. The nucleic acid sequence including the codon modifications encoding one or more of these amino acid changes may be incorporated into the expression vectors previously described and used to stably or transiently transform a plant to express the desired protein with the corresponding amino acid modification.

Example 10 Selection of new DPP4-Fc variants

The functionality of all new DPP4-Fc variants is evaluated by binding to S1 protein of MERS-CoV by ELISA as described in Example 3. The binding to S1 of the DPP4 Fc variants is first evaluated to determine whether the mutation reduces binding. If the Y547F or S620A mutation does not reduce the binding of DPP4m(39-766)V1 -FcG1 it is further evaluated.

The DPP4m(39-766)V1-FcG1 is expressed transiently as described in Example 1 in the N. benthamiana strains described in Example 7 with the KDEL-containing pTrak vector that produces proteins with high mannose or with the pTrak vector lacking KDEL that produces proteins with complex N-glycans. The high mannose and complex N-glycan variants are recovered and purified as described in Example 1 , and are compared for binding of S1 in the ELISA described in Example 3 above. As long as a complex N-glycan variant expressed in the N. benthamiana strain that has reduced expression of the XylT gene or FucT gene or both, binds at least as well as the high mannose variant to MERS-CoV S1 protein in the ELISA, we select the complex N-glycan variant for further evaluation. The same procedure is followed to produce and evaluate to the corresponding DPP4m(39-766)V2- FcG1

We then prepare each of the 1 1 variants with single amino acid substitutions as described above in this Example 8 in the best of the glycosylation forms and test each of them for binding in the ELISA assay. Any additional mutations that reduce the EC50 of DPP4m(39- 766)V1-FcG1 or DPP4m(39-766)V2-FcG1 at least 25% are combined in a new construct and binding of the combined variant is tested. Our aim is to gain maximum binding with minimal changes to the DPP4(39-766)-V1-FcG1 or DPP4m(39-766)V1 -FcG1 or DPP4m(39-766)V2- FcG1 . Any sequence variant or combined variant that results in a reduction in EC50 of 50% or more is carried forward into in vitro efficacy testing along with DPP4-V1 -FcG1 using the following method.

Vero E6 cells are seeded in 96-well plates and incubated overnight. MERS-CoV at an MOI of 0.1 is incubated with eight 2-fold serial dilutions of each variant (final concentrations between 10 ng/ml and 10 μg/ml) in duplicate for one hour, after which the virus/variant mixtures are added to cells. Cell survival is quantified at 48 hours post-infection using CellTiter-Glo® reagent (Promega). Controls include cells incubated with 1 ) virus alone, 2) virus plus an anti- DPP4 mAb (Sino Biologicals), or 3) media only. Supernatants are collected at 24 and 48 hours for titering of virus growth by TCID50 to confirm cell viability results. Data is fit to a 4-parameter logistic model to calculate the IC50 for each variant.

Example 1 1 In Vivo Efficacy testing of DPP4 (39-766)-Fcs and Variants

A mouse model using an adenovirus (Ad) vector delivering human DPP4 (hDPP4) into the lungs of mice is used to test DPP4-Fc inhibition in vivo. This model has demonstrated that Ad/hDPP4 transduced mice infected with MERS-CoV at 105 pfu/mouse showed virus MERS- CoV replication in the lungs through 4 days post-infection (dpi), with lung titers of 5 x 106 at 4 dpi, MERS-CoV specific transcripts were present at high levels in the lungs These mice had no weight loss or clinical disease; however, at 4 days post-infection their lungs displayed significant inflammation consisting of eosinophils, neutrophils and macrophages. Inflammation was present throughout the lung parenchyma and alveoli. Alveolar spaces displayed infiltrating cells and thickening of the alveolar walls. This model is being utilized to identifying therapeutics that inhibit MERS-CoV replication and pathogenesis.

Adenovirus/hDPP4 transduced mice are treated with the chosen DPP4-Fc variants prior to infection with MERS-CoV, and/or at various times after virus challenge, to determine whether DPP4-Fc can inhibit infection and pathology. Four groups of Ad/hDPP4 transduced mice (n=5 mice per group) are treated once each with the chosen DPP4-Fc or with sham on the day before, day of or day after infection with MERS-CoV. Animals are dosed initially with 20 μg/mouse (1 mg/kg) DPP4-Fc.

To compare the effect of delivering DPP4-Fcs directly into the lungs of infected mice to the effect of parenteral delivery of DPP4-Fcs, duplicate groups of Ad/huDPP4 transduced mice are treated with 2 different delivery methods, one by intraperitoneal injection (IP) and the other by intranasal aspiration (IN) Mice are challenged by intranasal infection on day 0 with either PBS or MERS-CoV (Jordan strain) at 1x 105 pfu per mouse. Mice in all groups are weighed daily and scored for clinical disease.

The lungs of both treated and control mice are harvested at 4 days dpi to characterize the lung pathology in the Ad/hDPP4 mice and follow viral titers through the experiment. Lungs are analyzed for viral load by plaque assay on Vero cells and fixed in 4% paraformaldehyde for paraffin embedding and sectioning. Histological slides are stained with hematoxylin and eosin (H&E) and scored for pathologic damage. Additionally, lung sections are stained with anti- ME S-CoV Spike protein antibodies to identify infected cells during the course of the infection, and with antibodies to hDPP4 to analyze expression kinetics of the receptor during infection, to determine whether receptor expression changes in different cell types during infection and response. A reduction of 1 log in virus titer by day two post-infection is sufficient to protect mice from disease (Sui et al. 2014). As a corollary to that model, a 1 log reduction in virus titer with measurable reduction in lung pathology is the metric used to evaluate the activity of the DPP4- Fcs described herein. While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

References:

Ahren, B. 2012. 'DPP-4 inhibition and islet function', J Diabetes Investig, 3:

3-10.

Barlan, A., J. Zhao, M. K. Sarkar, K. Li, P. B. McCray, Jr., S. Perlman, and

T. Gallagher. 2014. 'Receptor variation and susceptibility to MERS coronavirus infection', J Virol, 88: 4953-61.

Bengsch, B., B. Seigel, T. Flecken, J. Wolanski, H. E. Blum, and R.

Thimme. 2012. 'Human Th17 cells express high levels of

enzymatically active dipeptidylpeptidase IV (CD26)', J Immunol, 188:

5438-47.

Bermingham, A., M. A. Chand, C. S. Brown, E. Aarons, C. Tong, C.

Langrish, K. Hoschler, K. Brown, M. Galiano, R. Myers, R. G.

Pebody, H. K. Green, N. L. Boddington, R. Gopal, N. Price, W.

Newsholme, C. Drosten, R. A. Fouchier, and M. Zambon. 2012.

'Severe respiratory illness caused by a novel coronavirus, in a patient transferred to the United Kingdom from the Middle East, September

2012', Euro Surveill, 17: 20290.

Boehm, M. K., J. M. Woof, M. A. Kerr, and S. J. Perkins. 1999. 'The Fab and Fc fragments of lgA1 exhibit a different arrangement from that in

IgG: a study by X-ray and neutron solution scattering and homology modelling', J Mol Biol, 286: 1421 -47.

Brunke, C, S. Lohse, S. Derer, M. Peipp, P. Boross, C. Kellner, T. Beyer,

M. Dechant, L. Royle, L. P. Liew, J. H. Leusen, and T. Valerius.

2013. 'Effect of a tail piece cysteine deletion on biochemical and functional properties of an epidermal growth factor receptor-directed lgA2 m(1 ) antibody', MAbs, 5: 936-45.

Casasnovas, J. M., and T. A. Springer. 1994. 'Pathway of rhinovirus

disruption by soluble intercellular adhesion molecule 1 (ICAM-1 ): an intermediate in which ICAM-1 is bound and RNA is released', J Virol, 68: 5882-9.

Chan, J. F., K. H. Chan, G. K. Choi, K. K. To, H. Tse, J. P. Cai, M. L.

Yeung, V. C. Cheng, H. Chen, X. Y. Che, S. K. Lau, P. C. Woo, and K. Y. Yuen. 2013. 'Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications for disease pathogenesis and clinical manifestation', J Infect Dis, 207: 1743-52. Cui, J., J. S. Eden, E. C. Holmes, and L. F. Wang. 2013. 'Adaptive

evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of Middle East respiratory syndrome

coronavirus', Virol J, 10: 304.

de Groot, R. J., S. C. Baker, R. S. Baric, C. S. Brown, C. Drosten, L.

Enjuanes, R. A. Fouchier, M. Galiano, A. E. Gorbalenya, Z. A.

Memish, S. Perlman, L. L. Poon, E. J. Snijder, G. M. Stephens, P. C. Woo, A. M. Zaki, M. Zambon, and J. Ziebuhr. 2013. 'Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group', J Virol, 87: 7790-2.

de Wit, E., J. Prescott, L. Baseler, T. Bushmaker, T. Thomas, M. G.

Lackemeyer, C. Martellaro, S. Milne-Price, E. Haddock, B. L.

Haagmans, H. Feldmann, and V. J. Munster. 2013. 'The Middle East respiratory syndrome coronavirus (MERS-CoV) does not replicate in

Syrian hamsters', PLoS ONE, 8: e69127.

de Wit, E., A. L. Rasmussen, D. Falzarano, T. Bushmaker, F. Feldmann, D.

L. Brining, E. R. Fischer, C. Martellaro, A. Okumura, J. Chang, D. Scott, A. G. Benecke, M. G. Katze, H. Feldmann, and V. J. Munster.

2013. 'Middle East respiratory syndrome coronavirus (MERS-CoV) causes transient lower respiratory tract infection in rhesus

macaques', Proc Natl Acad Sci U S A, 1 10: 16598-603. Du, L, Z. Kou, C. Ma, X. Tao, L. Wang, G. Zhao, Y. Chen, F. Yu, C. T. Tseng, Y. Zhou, and S. Jiang. 2013. Ά Truncated Receptor-Binding Domain of MERS-CoV Spike Protein Potently Inhibits MERS-CoV Infection and Induces Strong Neutralizing Antibody Responses:

Implication for Developing Therapeutics and Vaccines', PLoS ONE,

8: e81587.

Du, L, G. Zhao, X. Zhang, Z. Liu, H. Yu, B. J. Zheng, Y. Zhou, and S.

Jiang. 2010. 'Development of a safe and convenient neutralization assay for rapid screening of influenza HA-specific neutralizing monoclonal antibodies', Biochem Biophys Res Commun, 397: 580-5.

Durinx, C, A. M. Lambeir, E. Bosmans, J. B. Falmagne, R. Berghmans, A.

Haemers, S. Scharpe, and I. De Meester. 2000. 'Molecular

characterization of dipeptidyl peptidase activity in serum: soluble

CD26/dipeptidyl peptidase IV is responsible for the release of X-Pro dipeptides', Eur J Biochem, 267: 5608-13.

Dyall, J., C. M. Coleman, B. J. Hart, T. Venkataraman, M. R. Holbrook, J.

Kindrachuk, R. F. Johnson, G. G. Olinger, Jr., P. B. Jahrling, M.

Laidlaw, L. M. Johansen, C. M. Lear-Rooney, P. J. Glass, L. E.

Hensley, and M. B. Frieman. 2014. 'Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection', Antimicrob Agents Chemother, 58: 4885-93. Enserink, M. 2013. 'Emerging diseases. New coronavirus reveals some of its secrets', Science, 340: 17-8.

Eryilmaz, E., A. Janda, J. Kim, R. J. Cordero, D. Cowburn, and A.

Casadevall. 2013. 'Global structures of IgG isotypes expressing identical variable regions', Mol Immunol, 56: 588-98.

Furtado, P. B., P. W. Whitty, A. Robertson, J. T. Eaton, A. Almogren, M. A.

Kerr, J. M. Woof, and S. J. Perkins. 2004. 'Solution structure determination of monomeric human lgA2 by X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human lgA1 ', J Mol Biol, 338: 921 -41.

Gierer, S., S. Bertram, F. Kaup, F. Wrensch, A. Heurich, A. Kramer-Kuhl, K. Welsch, M. Winkler, B. Meyer, C. Drosten, U. Dittmer, T. von Hahn, G. Simmons, H. Hofmann, and S. Pohlmann. 2013. The spike protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2, and is targeted by neutralizing antibodies', J Virol, 87: 5502-1 1.

Goebl, N. A., C. M. Babbey, A. Datta-Mannan, D. R. Witcher, V. J.

Wroblewski, and K. W. Dunn. 2008. 'Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells', Mol Biol Cell, 19: 5490-505.

Goetze, A. M., Y. D. Liu, Z. Zhang, B. Shah, E. Lee, P. V. Bondarenko, and G. C. Flynn. 201 1. 'High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans',

Glycobiology, 21 : 949-59.

Goldblum, R.M., and R.P. Garofolo. 2004. 'The mucosal defense system.' in R.E. Stiehm, H.D. Ochs and J.A. Winkelstein (eds.), Immunologic disorders in Infants & Children (Elsevier: Philadelphia, PA).

Hadlington, J. L., A. Santoro, J. Nuttall, J. Denecke, J. K. Ma, A. Vitale, and L. Frigerio. 2003. 'The C-terminal extension of a hybrid

immunoglobulin A/G heavy chain is responsible for its Golgi- mediated sorting to the vacuole', Mol Biol Cell, 14: 2592-602.

Hand, W. L., and J. R. Cantey. 1974. 'Antibacterial mechanisms of the

lower respiratory tract. I. Immunoglobulin synthesis and secretion', J

Clin Invest, 53: 354-62.

Hart, B. J., J. Dyall, E. Postnikova, H. Zhou, J. Kindrachuk, R. F. Johnson, G. G. dinger, Jr., M. B. Frieman, M. R. Holbrook, P. B. Jahrling, and L. Hensley. 2014. 'Interferon-beta and mycophenolic acid are potent inhibitors of Middle East respiratory syndrome coronavirus in cell- based assays', J Gen Virol, 95: 571 -7.

Hopsu-Havu, V. K., and G. G. Glenner. 1966. Ά new dipeptide

naphthylamidase hydrolyzing glycyl-prolyl-beta-naphthylamide', Histochemie, 7 : 197-201.

Ithete, N. L, S. Stoffberg, V. M. Corman, V. M. Cottontail, L. R. Richards, M. C. Schoeman, C. Drosten, J. F. Drexler, and W. Preiser. 2013. 'Close relative of human middle East respiratory syndrome

coronavirus in bat, South Africa', Emerg Infect Dis, 19: 1697-9.

Javidroozi, M., S. Zucker, and W. T. Chen. 2012. 'Plasma seprase and DPP4 levels as markers of disease and prognosis in cancer', Dis Markers, 32: 309-20.

Kanda, Y., T. Yamada, K. Mori, A. Okazaki, M. Inoue, K. Kitajima-Miyama, R. Kuni-Kamochi, R. Nakano, K. Yano, S. Kakita, K. Shitara, and M. Satoh. 2007. 'Comparison of biological activity among

nonfucosylated therapeutic lgG1 antibodies with three different relinked Fc oligosaccharides: the high-mannose, hybrid, and complex types', Glycobiology, 17: 104-18.

Kapila, J., R De Rycke, M.M. Van Monatagu, and G. Angenon. 1997. 'An Agrobacterium-mediated transient gene expression system for intact leaves', Plant Science, 122: 101 -08.

Lambeir, A. M., J. F. Diaz Pereira, P. Chacon, G. Vermeulen, K.

Heremans, B. Devreese, J. Van Beeumen, I. De Meester, and S. Scharpe. 1997. Ά prediction of DPP IV/CD26 domain structure from a physico-chemical investigation of dipeptidyl peptidase IV (CD26) from human seminal plasma', Biochim Biophys Acta, 1340: 215-26.

Lau, S. K., K. S. Li, A. K. Tsang, C. S. Lam, S. Ahmed, H. Chen, K. H.

Chan, P. C. Woo, and K. Y. Yuen. 2013. 'Genetic characterization of Betacoronavirus lineage C viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus HKU5 in Japanese pipistrelle: implications for the origin of the novel Middle East respiratory syndrome coronavirus', J Virol, 87: 8638-50.

Lim, B. K., J. H. Choi, J. H. Nam, C. O. Gil, J. O. Shin, S. H. Yun, D. K.

Kim, and E. S. Jeon. 2006. 'Virus receptor trap neutralizes

coxsackievirus in experimental murine viral myocarditis', Cardiovasc Res, 71 : 517-26.

Lu, G., Y. Hu, Q. Wang, J. Qi, F. Gao, Y. Li, Y. Zhang, W. Zhang, Y. Yuan, J. Bao, B. Zhang, Y. Shi, J. Yan, and G. F. Gao. 2013. 'Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26', Nature, 500: 227-31.

Lu, L, Q. Liu, Y. Zhu, K. H. Chan, L. Qin, Y. Li, Q. Wang, J. F. Chan, L.

Du, F. Yu, C. Ma, S. Ye, K. Y. Yuen, R. Zhang, and S. Jiang. 2014. 'Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor', Nat Commun, 5: 3067.

Maclean, J., M. Koekemoer, A. J. Olivier, D. Stewart, Hitzeroth, II, T.

Rademacher, R. Fischer, A. L. Williamson, and E. P. Rybicki. 2007. 'Optimization of human papillomavirus type 16 (HPV-16) L1

expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization', J Gen

Virol, 88: 1460-9.

Martin, S., J. M. Casasnovas, D. E. Staunton, and T. A. Springer. 1993.

'Efficient neutralization and disruption of rhinovirus by chimeric ICAM-

1/immunoglobulin molecules', J Virol, 67: 3561 -8.

Mentlein, R. 1999. 'Dipeptidyl-peptidase IV (CD26)-role in the inactivation of regulatory peptides', Regul Pept, 85: 9-24.

Miyazaki, M., M. Kato, K. Tanaka, M. Tanaka, M. Kohjima, K. Nakamura,

M. Enjoji, M. Nakamuta, K. Kotoh, and R. Takayanagi. 2012.

'Increased hepatic expression of dipeptidyl peptidase-4 in non- alcoholic fatty liver disease and its association with insulin resistance and glucose metabolism', Mol Med Rep, 5: 729-33.

Moran, G. W., C. O'Neill, P. Padfield, and J. T. McLaughlin. 2012.

'Dipeptidyl peptidase-4 expression is reduced in Crohn's disease', Regul Pept, 177: 40-5.

Neuman, B. W., B. D. Adair, C. Yoshioka, J. D. Quispe, G. Orca, P. Kuhn, R. A. Milligan, M. Yeager, and M. J. Buchmeier. 2006.

'Supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy', J Virol, 80: 7918- 28.

Ohnuma, K., B. L. Haagmans, R. Hatano, V. S. Raj, H. Mou, S. Iwata, N.

H. Dang, B. Jan Bosch, and C. Morimoto. 2013. 'Inhibition of Middle

East Respiratory Syndrome Coronavirus (MERS-CoV) Infection by

Anti-CD26 Monoclonal Antibody', J Virol, 87: 13892-9.

Perera, R. A., P. Wang, M. R. Gomaa, R. El-Shesheny, A. Kandeil, O.

Bagato, L. Y. Siu, M. M. Shehata, A. S. Kayed, Y. Moatasim, M. Li, L.

L. Poon, Y. Guan, R. J. Webby, M. A. Ali, J. S. Peiris, and G. Kayali.

2013. 'Seroepidemiology for MERS coronavirus using

microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt,

June 2013', Euro Surveill, 18: pii=20574.

Petruccelli, S., M. S. Otegui, F. Lareu, O. Tran Dinh, A. C. Fitchette, A.

Circosta, M. Rumbo, M. Bardor, R. Carcamo, V. Gomord, and R. N.

Beachy. 2006. Ά KDEL-tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds', Plant

Biotechnol J, 4: 511 -27. Qian, K., F. Xie, A. W. Gibson, J. C. Edberg, R. P. Kimberly, and J. Wu.

2008. 'Functional expression of IgA receptor FcalphaRI on human platelets', J Leukoc Biol, 84: 1492-500.

Raj, V. S., H. Mou, S. L. Smits, D. H. Dekkers, M. A. Muller, R. Dijkman, D.

Muth, J. A. Demmers, A. Zaki, R. A. Fouchier, V. Thiel, C. Drosten,

P. J. Rottier, A. D. Osterhaus, B. J. Bosch, and B. L. Haagmans.

2013. 'Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC, Nature, 495: 251-4.

Raj, V. S., S. L. Smits, L. B. Provacia, J. M. van den Brand, L. Wiersma, W.

J. Ouwendijk, T. M. Bestebroer, M. I. Spronken, G. van Amerongen,

P. J. Rottier, R. A. Fouchier, B. J. Bosch, A. D. Osterhaus, and B. L.

Haagmans. 2014. 'Adenosine deaminase acts as a natural

antagonist for dipeptidyl peptidase 4-mediated entry of the Middle

East respiratory syndrome coronavirus', J Virol, 88: 1834-8.

Rapaka, R. R., E. S. Goetzman, M. Zheng, J. Vockley, L. McKinley, J. K.

Kolls, and C. Steele. 2007. 'Enhanced defense against Pneumocystis carinii mediated by a novel dectin-1 receptor Fc fusion protein', J

Immunol, 178: 3702-12.

Rath, T., K. Baker, J. A. Dumont, R. T. Peters, H. Jiang, S. W. Qiao, W. I.

Lencer, G. F. Pierce, and R. S. Blumberg. 2013. 'Fc-fusion proteins and FcRn: structural insights for longer-lasting and more effective therapeutics', Crit Rev Biotechnol.

Reinhart, D., R. Weik, and R. Kunert. 2012. 'Recombinant IgA production: single step affinity purification using camelid ligands and product characterization', J Immunol Methods, 378: 95-101.

Reusken, C. B., B. L. Haagmans, M. A. Muller, C. Gutierrez, G. J. Godeke,

B. Meyer, D. Muth, V. S. Raj, L. S. Vries, V. M. Corman, J. F. Drexler,

S. L. Smits, Y. E. El Tahir, R. De Sousa, J. van Beek, N. Nowotny, K. van Maanen, E. Hidalgo-Hermoso, B. J. Bosch, P. Rottier, A. Osterhaus, C. Gortazar-Schmidt, C. Drosten, and M. P. Koopmans. 2013. 'Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study', Lancet Infect Dis, 13: 859-66.

Rockx, B., E. Donaldson, M. Frieman, T. Sheahan, D. Corti, A.

Lanzavecchia, and R. S. Baric. 2010. 'Escape from human

monoclonal antibody neutralization affects in vitro and in vivo fitness of severe acute respiratory syndrome coronavirus', J Infect Dis, 201 : 946-55.

Scobey, T., B. L. Yount, A. C. Sims, E. F. Donaldson, S. S. Agnihothram, V. D. Menachery, R. L. Graham, J. Swanstrom, P. F. Bove, J. D. Kim, S. Grego, S. H. Randell, and R. S. Baric. 2013. 'Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus', Proc Natl Acad Sci U S A, 1 10: 16157-62. Shigeta, T., M. Aoyama, Y. K. Bando, A. Monji, T. Mitsui, M. Takatsu, X.

W. Cheng, T. Okumura, A. Hirashiki, K. Nagata, and T. Murohara. 2012. 'Dipeptidyl peptidase-4 modulates left ventricular dysfunction in chronic heart failure via angiogenesis-dependent and -independent actions', Circulation, 126: 1838-51.

Silberstein, E., L. Xing, W. van de Beek, J. Lu, H. Cheng, and G. G.

Kaplan. 2003. 'Alteration of hepatitis A virus (HAV) particles by a soluble form of HAV cellular receptor 1 containing the immunoglobin- and mucin-like regions', J Virol, 77: 8765-74.

Spiekermann, G. M., P. W. Finn, E. S. Ward, J. Dumont, B. L. Dickinson, R. S. Blumberg, and W. I. Lencer. 2002. 'Receptor-mediated

immunoglobulin G transport across mucosal barriers in adult life:

functional expression of FcRn in the mammalian lung', J Exp Med, 196: 303-10. Strasser, R., A. Castilho, J. Stadlmann, R. Kunert, H. Quendler, P.

Gattinger, T. Rademacher, F. Altmann, L. Mach, and H. Steinkellner. 2009. 'Improved virus neutralization by plant-produced anti-HIV antibodies with a homogeneous beta1 ,4-galactosylated N-glycan profile', J Biol Chem, 284: 20479-85.

Strasser, R., J. Stadlmann, M. Schahs, G. Stiegler, H. Quendler, L. Mach, J. Glossl, K. Weterings, M. Pabst, and H. Steinkellner. 2008.

'Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure', Plant Biotechnol J, 6: 392-402.

Sui, J., M. Deming, B. Rockx, R. C. Liddington, Q. K. Zhu, R. S. Baric, and W. A. Marasco. 2014. 'Effects of human anti-spike protein receptor binding domain antibodies on severe acute respiratory syndrome coronavirus neutralization escape and fitness', J Virol, 88: 13769-80. Tao, X., T. E. Hill, C. Morimoto, C. J. Peters, T. G. Ksiazek, and C. T.

Tseng. 2013. 'Bilateral entry and release of Middle East respiratory syndrome coronavirus induces profound apoptosis of human bronchial epithelial cells', J Virol, 87: 9953-8.

Thoma, R., B. Loffler, M. Stihie, W. Huber, A. Ruf, and M. Hennig. 2003.

'Structural basis of proline-specific exopeptidase activity as observed in human dipeptidyl peptidase-IV, Structure, 1 1 : 947-59.

van Doremalen, N., K. L. Miazgowicz, S. Milne-Price, T. Bushmaker, S.

Robertson, D. Scott, J. Kinne, J. S. McLellan, J. Zhu, and V. J.

Munster. 2014. 'Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through its Receptor Dipeptidyl Peptidase 4',

J Virol, 88: 9220-32.

Vaquero, C, M. Sack, J. Chandler, J. Drossard, F. Schuster, M. Monecke, S. Schillberg, and R. Fischer. 1999. 'Transient expression of a tumor- specific single-chain fragment and a chimeric antibody in tobacco leaves', Proceedings of the National Acadamy of Sciences USA, 96: 1 1 128-33.

Voinnet, O., S. Rivas, P. Mestre, and D. Baulcombe. 2003. 'An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus', The Plant

Journal, 33: 949-56.

Wang, N., X. Shi, L. Jiang, S. Zhang, D. Wang, P. Tong, D. Guo, L. Fu, Y.

Cui, X. Liu, K. C. Arledge, Y. H. Chen, L. Zhang, and X. Wang. 2013.

'Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4', Cell Res, 23: 986-93.

Ward, R. H., D. J. Capon, C. M. Jett, K. K. Murthy, J. Mordenti, C. Lucas,

S. W. Frie, A. M. Prince, J. D. Green, and J. W. Eichberg. 1991.

'Prevention of HIV-1 NIB infection in chimpanzees by CD4

immunoadhesin', Nature, 352: 434-6.

Yao, Y., L. Bao, W. Deng, L. Xu, F. Li, Q. Lv, P. Yu, T. Chen, Y. Xu, H.

Zhu, J. Yuan, S. Gu, Q. Wei, H. Chen, K. Y. Yuen, and C. Qin. 2014.

'An animal model of MERS produced by infection of rhesus

macaques with MERS coronavirus', J Infect Dis, 209: 236-42.

Ying, T., L. Du, T. W. Ju, P. Prabakaran, C. C. Lau, L. Lu, Q. Liu, L. Wang, Y. Feng, Y. Wang, B. J. Zheng, K. Y. Yuen, S. Jiang, and D. S.

Dimitrov. 2014. 'Exceptionally potent neutralization of MERS-CoV by human monoclonal antibodies', J Virol, 88: 7796-805.

Zaki, A. M., S. van Boheemen, T. M. Bestebroer, A. D. Osterhaus, and R.

A. Fouchier. 2012. 'Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia', N Engl J Med, 367: 1814-20.

Zhao, G., L. Du, C. Ma, Y. Li, L. Li, V. K. Poon, L. Wang, F. Yu, B. J.

Zheng, S. Jiang, and Y. Zhou. 2013. Ά safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV, Virol J, 10: 266.

Claims

What is claimed is:

I . A DPP4 peptide comprising human DPP4 consensus contact sequence for the MERS CoV S1 spike glycoprotein comprising at least one consensus contact residue substitution, wherein the peptide has higher affinity for the MERS CoV S1 spike glycoprotein than human DPP4 consensus contact sequence without the at least one substitution. 2. The DPP4 peptide of claim 1 , wherein the at least one contact residue substitution is with a residue selected from contact residues unique to camel DPP4.

3. The DPP4 peptide of claim 2, wherein the at least one contact residue substitution is at a position selected from 288, 295, 317, 336, and 346.

4. The DPP4 peptide of claim 3, wherein residue 288 is V.

5. The DPP4 peptide of claim 3, wherein residue 288 is N.

6. The DPP4 peptide of claim 3, wherein residue 295 is F.

7. The DPP4 peptide of claim 3, wherein residue 336 is Y. 8. The peptide of claim 3, wherein residue 346 is E.

9. The DPP4 peptide of claim 1 , wherein the at least one consensus contact residue substitution is selected from residues at positions 288, 295, 317, 336 and 346. 10. The DPP4 peptide of claim 9, wherein the at least one consensus contact residue substitution is at position 288.

I I . The DPP4 peptide of claim 10, wherein the consensus contact residue substitution at position 288 is a substitution with Valine.

12. The DPP4 peptide of claim 1 , wherein the at least one consensus contact residue is selected from residues 285 to 293.

13. The DPP4 peptide of claim 12, wherein the consensus contact residue at position 285 is substituted with R.

14. The DPP4 peptide of claim 12, wherein the consensus contact residue at position 289 is substituted with P.

15. The DPP4 peptide of claim 12, wherein the consensus contact residue at position 293 is substituted with V. 16. The DPP4 peptide of claim 12, wherein the consensus contact residue at position 285 is substituted with V, the residue at position 288 is substituted with V, the residue at position 289 is substituted with P, and the residue at position 293 is substituted with V.

17. The DPP4 peptide of claim 12, wherein amino acid residues 285 to 293 correspond to SEQ ID NO:17.

18. The DPP4 peptide of claim 1 or claim 2, comprising an amino acid substitution that reduces hydrolase activity of the DPP4 peptide. 19. The DPP4 peptide of claim 18, wherein the amino acid substitution is with an amino acid other than Y at position 547.

20. The DPP4 peptide of claim 19, wherein the amino acid residue at position 547 is F. 21 . The DPP4 peptide of claim 18, wherein the amino acid sequence of the DPP4

peptide further comprises one or more amino acid substitutions selected from the group consisting of 188R, 269H, 291V, 294F, 295F, 336Y, 341 1, 344R, 346F, and 392E. 22. The DPP4 peptide of claim 20, wherein the amino acid sequence of the DPP4

peptide further comprises one or more amino acid substitutions selected from the group consisting of 188R, 269H, 291V, 294F, 295F, 336Y, 341 1, 344R, 346F, and 392E. 23. The DPP4 peptide of claim 18, wherein the amino acid substitution is with an amino acid other than S at position 630.

24. The DPP4 peptide of claim 23, wherein the amino acid residue at position 630 is A.

25. The DPP4 peptide of claim 24, wherein the amino acid sequence of the DPP4 peptide further comprises one or more amino acid substitutions selected from the group consisting of 188R, 269H, 291V, 294F, 295F, 336Y, 3411, 344R, 346R, and 392E.

26. The DPP4 peptide of any one claims 1 to 25, further comprising an Fc linked to the DPP4 peptide. 27. The DPP4 peptide of claim 26, wherein the Fc is selected from the group consisting of lgG1 , lgG2, lgA1 , lgA2, and IgM.

28. The DPP4 peptide of claim 27, wherein the Fc further comprises a KDEL sequence at its carboxy terminus.

29. The DPP4 peptide of claim 28, wherein the Fc is a truncated IgA comprising a deletion of the 18 amino acid C-terminal IgA piece relative to a full length IgA.

30. The DPP4 peptide of claim 29 wherein said Fc is from an lgA1.

31. The DPP4 peptide of claim 29 wherein said Fc is from an lgA2.

32. The DPP4 peptide of claim 27 wherein said Fc is from an IgA. 33. The DPP4 peptide of claim 32, further comprising a J-chain linked to the DPP4-Fc.

34. The DPP4 peptide of claim 33, wherein the J-chain is linked to at least two linked DPP4-Fcs. 35. The DPP4 peptide of Claim 27, wherein said Fc is from an IgM.

36. The DPP4 peptide of claim 35, further comprising a J-chain linked to the DPP4-Fc.

37. The DPP4 peptide of claim 36 wherein the J-chain is linked to at least two linked DPP4-Fcs.

38. The DPP4 of claim 37, wherein said J-chains and DPP4-Fcs form multimers.

39. A nucleic acid encoding the DPP4 peptide of claim 1 to 25.

40. An expression vector comprising the nucleic acid sequence of claim 39.

41 . A chimeric MERs-CoV receptor protein comprising: (i) an immunoglobulin

complex, wherein the immunoglobulin complex comprises at least a portion of an immunoglobulin heavy chain; and (ii) a mutated dipeptidyl peptidase 4 (DPP4) peptide comprising human DPP4 consensus contact residues, wherein at least one of the consensus contact residues of the human DPP4 sequence comprises at least one amino acid substitution that increases the affinity of the mutated DPP4 peptide for the S1 spike protein of MERS-CoV relative to the affinity of an unmutated DPP4 peptide, and wherein the mutated human DPP4 is covalently associated with the immunoglobulin heavy chain.

42. A dimer of the chimeric MERs-CoV receptor protein of claim 41 .

43. The chimeric MERs-CoV receptor protein of claim 41 or claim 42, wherein the immunoglobulin complex further comprises at least a portion of an

immunoglobulin light chain. 44. The chimeric MERs-CoV receptor protein of claim 43, wherein the immunoglobulin light chain is a kappa chain or a lambda chain.

45. The chimeric MERs-CoV receptor protein of any one of claims 41 to 44,

wherein the covalent linkage between the mutated human DPP4 peptide and the immunoglobulin heavy chain is an immunoglobulin hinge.

46. The chimeric MERS-CoV receptor protein of any one of claims 41 to 44, wherein the portion of an immunoglobulin heavy chain is selected from the group consisting of IgGs, IgAs, IgD. IgE, and IgM.

47. The chimeric MERS-CoV receptor protein of any one of claims 41 to 44, wherein the immunoglobulin heavy chain is an IgG and comprises heavy chain constant regions 2 and 3 thereof. 48. The chimeric MERS-CoV receptor protein of any one of claims 41 to 44, wherein the immunoglobulin heavy chain and DPP4 peptide are human.

49. A composition comprising the chimeric MERS-CoV receptor protein of any one of claims 41 to 48 and a plant material.

50. The composition of claim 49, wherein the plant material is selected from the group consisting of: plant cell walls, plant organelles, plant cytoplasm, intact plant cells, plant seeds, and viable plants.

51 . A method for reducing binding of MERS CoV to a host cell, comprising: contacting the MERS-CoV with the chimeric MERS-CoV receptor protein of any one of claims 41 to 48, whereby the chimeric MERS-CoV receptor protein binds to the MERS-

CoV Receptor Binding Domain (RBD) and reduces the binding of MERS-CoV RBD to the host cell.

52. The chimeric MERS-CoV receptor protein of any one of claims 41 to 48 for use as a medicament.

53. The chimeric MERS-CoV receptor protein of any one of claims 41 to 48 for use in preventing or treating a MERS-CoV infection. 54. An expression vector encoding chimeric MERS-CoV receptor protein of any one of claims 41 to 48.

55. A method for producing a chimeric MERS-CoV receptor protein, comprising

introducing the expression vector of claim 54 into a cellular host, and expressing the chimeric MERS-CoV receptor protein.

56. The method of claim 55, wherein the cellular host is a plant.

57. A pharmaceutical composition comprising the chimeric MERS-CoV receptor protein of any one of claims 41 to 48 and a pharmaceutically acceptable carrier.

58. A method for producing the DPP4 peptide of any one of claims 1 to 25, comprising introducing the expression vector of claim 40 into a cellular host, and expressing the DPP4 peptide.

59. The method of claim 58, wherein the cellular host is a plant.

60. A method for reducing binding of MERS CoV to a host cell, the method comprising contacting the MERS-CoV with the DPP4 peptide of any of claims 26 to 38, whereby the DPP4 peptide binds to the MERS-CoV Receptor Binding Domain (RBD) and reduces the binding of MERS-CoV RBD to the host cell.

61. The DPP4 peptide of any one of claims 26 to 38 for use as a medicament.

62. The DPP4 peptide of any one of claims 26 to 38 for use in preventing or treating a MERS-CoV infection.