KR102007161B1 - Monoclonal antibodies of specifically binding to spike S1 protein of MERS-CoV - Google Patents

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof having high affinity, neutralizing capacity, and a very high binding specificity to a spike protein of MERS-CoV; a nucleic acid encoding the same; a vector comprising the nucleic acid; a cell transformed with the vector; a method for producing the antibody or antigen-binding fragment thereof; and a composition for treating or preventing Middle East respiratory syndrome comprising the same.

Description

Monoclonal antibodies of specifically binding to spike S1 protein of MERS-CoV}

The present invention provides an antibody or antigen-binding fragment thereof having a very high binding specificity to a spike protein of MERS corona virus, and having a high affinity and neutralizing ability, a nucleic acid encoding the same, a vector comprising the nucleic acid, The present invention relates to a cell transformed with the vector, a method for producing the antibody or antigen-binding fragment thereof, a composition for treating or preventing MERS coronavirus disease, and a diagnostic kit.

After the discovery of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the Middle East in 2012, the new corona virus was introduced into Korea on May 20, 2015, infecting 186 people for about two months. 38 of them died and 16,693 people who were classified as contactors of confirmed patients were isolated, causing social turmoil.

In addition, there are still sporadic cases of MERS infections in the Middle East and other regions, and since they have already occurred in more than 22 countries, the possibility of reintroduction in Korea cannot be excluded. The MERS corona virus has been reported to cause severe respiratory symptoms with high fever, with a mortality rate of 20-50% higher than SARS virus, which shows a 10% mortality rate.

MERS corona virus has been developed various diagnostic techniques (molecular diagnosis, antigen and antibody detection ELISA, etc.), and the treatment is also actively studied. Specifically, researches on antiviral agents are actively underway at home and abroad, and non-specific antiviral agents that have been shown to be clinically effective have type I interferon and ribavirin, but have been reported to have a virus inhibitory effect only at the beginning of infection. Coronavirus Therapeutics and Therapeutics for SARS Corona Virus are drawing attention as recent development needs increase (A. Zumla et.al, Nature Reviews Drug Discovery, 15: 327-347, 2016). However, there are currently no domestic or foreign licensed therapeutics or vaccines, and the evaluation system for the developed materials has not been established.

Structural proteins of MERS coronavirus include S (spike), E (envelope), M (matrix) and NP (nucleocapsid) proteins. In particular, the spike protein of MERS corona virus consists of S1 protein which binds to the receptor protein on the cell surface to be infected and S2 protein which is a fusion part with the cell.

In human infection mechanisms, it is known that the receptor binding domain (RBD) of the S1 protein of MERS coronavirus binds to human DPP4 (Dipeptidyl peptidase 4) receptors for intracellular penetration (Wang N et al. Cell Research. 2013 : 23: 986).

The present inventors have attempted to develop antibodies specific for the domestic MERS coronavirus reported to have mutated the S protein portion. As a result, continuous research was conducted to develop antibodies with low affinity, excellent affinity and neutralizing ability from peripheral blood mononuclear cells (PBMC) of humans cured after MERS corona virus infection in Korea. The present invention has been completed.

The present invention seeks to provide novel antibodies or antigen binding fragments thereof against MERS corona virus.

Specifically, the antibody or antigen-binding fragment thereof of the present invention has a high affinity that specifically binds to the S1 protein of MERS corona virus.

Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.

Another object of the present invention is to provide a vector comprising the nucleic acid, a cell transformed with the vector, and a method of preparing the same.

It is another object of the present invention to provide a composition for treating or preventing an infectious disease of MERS corona virus comprising the antibody or antigen-binding fragment thereof.

Still another object of the present invention is to provide a diagnostic kit for mers corona virus comprising the antibody or antigen-binding fragment thereof.

The present invention provides a CDRH1 having a sequence of SEQ ID NO: 1; CDRH2 having the sequence of SEQ ID NO: 2; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 3; And CDRL1 having the sequence of SEQ ID NO: 22; CDRL2 having the sequence of SEQ ID NO: 23; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 24, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

The present invention provides a CDRH1 having a sequence of SEQ ID NO: 4; CDRH2 having the sequence of SEQ ID NO: 5; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 6; And CDRL1 having the sequence of SEQ ID NO: 25; CDRL2 having the sequence of SEQ ID NO: 26; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 27, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

The present invention provides a CDRH1 having a sequence of SEQ ID NO: 7; CDRH2 having the sequence of SEQ ID NO: 8; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 9; And CDRL1 having the sequence of SEQ ID NO: 28; CDRL2 having the sequence of SEQ ID NO: 29; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 30, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

The present invention provides a CDRH1 having a sequence of SEQ ID NO: 10; CDRH2 having the sequence of SEQ ID NO: 11; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 12; And CDRL1 having the sequence of SEQ ID NO: 31; CDRL2 having the sequence of SEQ ID NO: 32; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 33, which provides an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

The present invention provides a CDRH1 having a sequence of SEQ ID NO: 13; CDRH2 having the sequence of SEQ ID NO: 14; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 15; And CDRL1 having the sequence of SEQ ID NO: 34; CDRL2 having the sequence of SEQ ID NO: 35; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 36, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

The present invention provides a CDRH1 having a sequence of SEQ ID NO: 16; CDRH2 having the sequence of SEQ ID NO: 17; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 18; And CDRL1 having the sequence of SEQ ID NO: 37; CDRL2 having the sequence of SEQ ID NO: 38; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 39, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

The present invention provides a CDRH1 having a sequence of SEQ ID NO: 19; CDRH2 having the sequence of SEQ ID NO: 20; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 21; And CDRL1 having the sequence of SEQ ID NO: 40; CDRL2 having the sequence of SEQ ID NO: 41; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 42, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

The present invention specifically relates to the Spike S1 protein of MERS corona virus comprising a heavy chain variable region having an amino acid sequence of one of SEQ ID NOs: 43 to 49, and a light chain variable region having an amino acid sequence of one of SEQ ID NOs: 50 to 56 Provided are binding antibodies or antigen binding fragments thereof.

The present invention provides nucleic acids encoding such antibodies or antigen binding fragments thereof.

The present invention provides a vector comprising the nucleic acid.

The present invention provides a cell transformed with the vector.

The present invention provides a method of producing an antibody or antigen-binding fragment thereof comprising culturing the cell and recovering the antibody or antigen-binding fragment thereof from the cultured cell.

The present invention provides a composition for treating or preventing an infectious disease of MERS corona virus comprising the antibody or antigen-binding fragment thereof.

The present invention provides a kit for diagnosing MERS coronavirus comprising the antibody or antigen-binding fragment thereof.

The present invention has been developed from the patient B cells of the cure of MERS-Coronavirus infectious disease in Korea, and provides an antibody that has little inappropriate immune response in the human body and specifically recognizes the spike S1 protein. The antibody of the present invention is an antibody having a novel complementarity determining site (CDR) having high specificity due to no cross-reaction with human coronavirus and having high neutralizing ability against MERS coronavirus.

Antibodies of the present invention can be used for the treatment or prevention of viral infection diseases using high specificity and good neutralizing ability against Spike S1 protein of MERS corona virus. In addition, since the antibody of the present invention maintains a constant monomer form and maintains high stability at high temperature conditions, the antibody has an advantage of being utilized as a diagnostic kit.

Figure 1 shows the results of measuring the binding affinity of the antibodies KNIH-58, KNIH-68, KNIH-72, KNIH-78, KNIH-88, KNIH-90, KNIH-95 and S1 protein of the present invention.
Figure 2 shows the results of measuring the binding affinity between the antibody KNIH-58, KNIH-68, KNIH-72, KNIH-78, KNIH-88, KNIH-90, KNIH-95 and the receptor binding site (RDB) of the present invention Indicates.
Figure 3 shows the results of measuring the binding affinity of the antibodies KNIH-58, KNIH-68, KNIH-72, KNIH-78, KNIH-88, KNIH-90, KNIH-95 and S2 protein of the present invention.
Figure 4 shows the results of measuring the binding affinity between the KNIH-58 antibody and S1 protein by surface plasmon resonance (SPR).
Figure 5 shows the results of measuring the binding affinity of the KNIH-68 antibody and S1 protein by surface plasmon resonance (SPR).
Figure 6 shows the results of measuring the binding affinity between the KNIH-72 antibody and S1 protein by surface plasmon resonance (SPR)
7 shows the results of measuring the binding affinity between the KNIH-78 antibody and the S1 protein by surface plasmon resonance (SPR).
8 shows the results of measuring the binding affinity between the KNIH-88 antibody and the S1 protein by surface plasmon resonance (SPR).
9 shows the results of measuring the binding affinity between the KNIH-90 antibody and the S1 protein by surface plasmon resonance (SPR).
10 shows the results of measuring the binding affinity between the KNIH-95 antibody and the S1 protein by surface plasmon resonance (SPR).
Figure 11 shows the results of the Plaque Reduction Neutralization Assay (PRNT) of the MERS virus EMC strain.
12 shows the results of cross-reaction evaluation with human coronaviruses (OC43, 229E, NL63) for the evaluation of the specificity of the antibody.
Figure 13 shows the result of the neutralizing ability of the antibody.

The MERS coronavirus has a single positive strand gene of about 30 kb, which is a 5'-UTR (Untlanslated Region) protected with a Cap at the 5 'end and a 3'-UTR with a polyA-tail at the 3' end. Have MERS coronavirus has the longest gene among RNA viruses and uses a total of 8 mRNAs (1 full-length genome and 7 subgenome) to participate in replication (ORF1a / 1b), structural proteins and accessory proteins Create them.

The S1 protein of MERS coronavirus that binds to the receptor protein on the cell surface to be infected has the amino acid sequence of SEQ ID NO: 57.

Peripheral blood mononuclear cells (PBMCs) refer to cells with globular nuclei present in the blood, which include B cells, T cells, macrophage, dendritic cells, and natural killer. Immune cells such as natural killer cells (NK cells) are included.

A common method of separating PBMCs from blood is to dissolve Ficoll ^® in water to form a density gradient, and to use density gradient centrifugation. Because PBMCs are lighter than red blood cells, granulocytes, and heavier than plasma, they can be separated from blood using this specific gravity difference.

As used herein, the term “antibody” refers to a polypeptide that specifically binds to and recognizes an antigen such as a spike protein of MERS corona virus. The present invention includes not only complete antibody forms that bind to spike proteins, but also antigen binding fragments of such antibody molecules.

As used herein, an "antibody" may include both monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies). The antibody consists of two heavy chains and two light chains, and includes a variable region in which the amino acid sequence varies according to the type of antigen of interest, and a constant region in which the sequence does not change. Have

The variable regions of the light and heavy chains comprise three highly variable regions called "complementarity-determining regions (CDRs)". The CDRs bind to the epitope of the antigen, and the CDRs of each chain typically have three regions (CDR1, CDR2, and CDR3).

"Antigen binding fragment" of an antibody means a fragment that retains antigenic function and includes Fab, F (ab '), F (ab') 2, Fv, and the like. Fab in the antibody fragment has a structure having the variable region of the light and heavy chains, the constant region of the light chain and the first constant region (CH1) of the heavy chain, and has one antigen binding site. Fab 'differs from Fab in that it has a hinge region comprising at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. F (ab ') 2 antibodies are produced by disulfide bonds of cysteine residues in the hinge region of Fab'. Fv is at least an antibody fragment having only heavy and light chain variable regions.

As used herein, "epitope" refers to an antigenic determinant that reacts with the CDRs of an antibody. Since a single antigen may have more than one epitope, several different antibodies may bind to one antigen. In this case, different biological effects may be exhibited depending on the binding site. The epitope may refer to the location of the antigen to which the B and / or T cells respond, but in the present invention, the epitope may refer to the location of the S1 protein of MERS coronavirus.

The present invention provides seven antibodies KNIH-58, KNIH-68, KNIH-72, KNIH-78, KNIH-88, KNIH-90, and KNIH-95.

KNIH-58 antibody of the present invention is CDRH1 having a sequence of SEQ ID NO: 1; CDRH2 having the sequence of SEQ ID NO: 2; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 3; And CDRL1 having the sequence of SEQ ID NO: 22; CDRL2 having the sequence of SEQ ID NO: 23; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 24, an antibody or antigen binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

KNIH-68 antibody of the present invention is a CDRH1 having a sequence of SEQ ID NO: 4; CDRH2 having the sequence of SEQ ID NO: 5; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 6; And CDRL1 having the sequence of SEQ ID NO: 25; CDRL2 having the sequence of SEQ ID NO: 26; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 27, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

KNIH-72 antibody of the present invention is CDRH1 having a sequence of SEQ ID NO: 7; CDRH2 having the sequence of SEQ ID NO: 8; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 9; And CDRL1 having the sequence of SEQ ID NO: 28; CDRL2 having the sequence of SEQ ID NO: 29; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 30, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

KNIH-78 antibody of the present invention is a CDRH1 having a sequence of SEQ ID NO: 10; CDRH2 having the sequence of SEQ ID NO: 11; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 12; And CDRL1 having the sequence of SEQ ID NO: 31; CDRL2 having the sequence of SEQ ID NO: 32; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 33, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

KNIH-88 antibodies of the invention include CDRH1 having a sequence of SEQ ID NO: 13; CDRH2 having the sequence of SEQ ID NO: 14; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 15; And CDRL1 having the sequence of SEQ ID NO: 34; CDRL2 having the sequence of SEQ ID NO: 35; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 36, or an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

KNIH-90 antibodies of the invention include CDRH1 having a sequence of SEQ ID NO: 16; CDRH2 having the sequence of SEQ ID NO: 17; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 18; And CDRL1 having the sequence of SEQ ID NO: 37; CDRL2 having the sequence of SEQ ID NO: 38; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 39, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

KNIH-95 antibodies of the invention include CDRH1 having a sequence of SEQ ID NO: 19; CDRH2 having the sequence of SEQ ID NO: 20; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 21; And CDRL1 having the sequence of SEQ ID NO: 40; CDRL2 having the sequence of SEQ ID NO: 41; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 42, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.

The heavy chain variable region of the invention has the amino acid sequence of any one of SEQ ID NOs: 43-49.

The light chain variable region of the invention also has an amino acid sequence of any one of SEQ ID NOs: 50-56.

In the present invention, the term "cloning" is a genetic engineering technology that mass-produces the same gene, which is introduced into a plasmid or virus acting as a vector to mass-produce genes or DNA fragments, and genetically through cell division. Refers to the technique of cloning the same clone.

In the present invention, "clone" refers to the same copy, and refers to the same copy of the same cell population or a specific gene segment, In the present invention, a clone includes the same copy of the full-length or some genes of MERS coronavirus. It may be.

In the present invention, "cDNA" refers to DNA complementary to the mRNA, preferably DNA having a nucleotide sequence complementary to the mRNA of Mers Corona virus, but is not limited thereto.

In the present invention, "vector" is a means for introducing a foreign gene into a suitable host cell so that the foreign gene can be expressed. A plasmid vector, a cosmid vector, a bacteriophage vector capable of replicating itself And adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and the like.

In the present invention, "transformation" refers to a change in the genetic properties of the host cell by introducing a foreign gene into the host cell, the term "transfection" can be used when the host is eukaryotic. have. Transformation used in the present invention may include any method of introducing a foreign gene into an organism, cell, tissue or organ, preferably may mean to introduce the foreign gene into E. coli. As a transformation method, commonly used methods such as electroporation, liposomes, and DNA transport using Ca ⁺⁺ can be used.

In the present invention, "neutralizing capacity" refers to the ability of an antibody to bind to a specific antigen of a virus to inhibit the infectivity of the virus.

The prophylactic or therapeutic composition of the present invention may further comprise a carrier, diluent, excipient, and the like conventionally used in biological preparations. Specifically, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol or one or more thereof may be included. At this time, if necessary, other conventional additives such as antioxidants, buffers, bacteriostatic agents, and the like may be added.

The MERS virus diagnostic kit of the present invention may include not only the monoclonal antibody of the present invention but also tools, reagents, and the like generally used in the art for immunological analysis. Such tools and reagents include, but are not limited to, suitable carriers, labeling materials capable of producing detectable signals, solubilizers, detergents, buffers, stabilizers, and the like.

Suitable carriers are soluble carriers, for example physiologically acceptable buffers known in the art, for example PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, polyacrylonitrile, fluorine Resins, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other paper, glass, metals, agarose, and combinations thereof, but are not limited thereto.

Antigen-antibody complex formation may include tissue immunostaining, radioimmunoassay (RIA), enzyme-linked immunosorbent assay, western blotting, immunoprecipitation assay, and immunodiffusion assay (Immunodiffusion Assay). ), Complement Fixation Assay, FACS and protein chip, but are not limited thereto.

Labels that enable qualitative or quantitative determination of the formation of antigen-antibody complexes include, but are not limited to, enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes.

Enzymes available as detection labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxy Multidase, Hexokinase and GDPase, RNase, Glucose Oxidase and Luciferase, Phospho Fructokinase, Phosphoenolpyruvate Carboxylase, Aspartate Aminotransferase, Phosphopyruvate, Decarboxylase, β- Latamases and the like, but is not limited thereto.

Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like.

Ligands include biotin derivatives and the like, and luminescent materials include, but are not limited to, acridinium esters, luciferin, luciferase, and the like.

Microparticles include, but are not limited to, colloidal gold, colored latex, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited by these examples.

Example 1

Preparation of MERS Coronavirus Antibody Expression Recombinant Vector

The present inventors selected a domestic patient who had been cured for 1 year after infection with MERS coronavirus, and isolated peripheral blood mononuclear cells (PBMC) from the blood of the cure patients by Ficoll method. .

Thereafter, MERS-CoV spike S1 or RBD protein probe (PE fluorescent dye), anti-human IgG-FITC, and anti-human CD20 IgG-Cy55PerCP were used to isolate the Mers Corona virus antibody from PBMC isolated by flow cytometry (FACS). Exclusive B cells were separated into single cells. In addition, unwanted immune cells other than B cells were negatively selected by attachment of CD3, CD4, CD8 and CD14 markers and removed by FACS.

The variable regions of the light and heavy chains, which are complementarity-determining region (CDR) of the antibody obtained from the isolated memory B cells, were amplified by PCR. The nucleotide sequence of the amplified antibody gene was confirmed using IMGT (Immunogenetics, http://www.imgt.org) database to confirm the expression of the antibody, and finally seven genes were selected.

Seven nucleotide sequences of the selected antibody genes were cloned into a pFUSE vector (Invivogen) containing an antibody constant region sequence to prepare seven recombinant vectors.

Example 2

Monoclonal Antibody Preparation

The recombinant vector was transfected with animal cells (Expi293F). The host cells into which the expression vector was introduced were cultured in a large amount for 5 days to express a large amount of antibody, and a cell culture solution was obtained to purify the protein G agarose column to prepare a total of seven types of monoclonal antibodies. The antibodies thus obtained are named KNIH-58, KNIH-68, KNIH-72, KNIH-78, KNIH-88, KNIH-90, KNIH-95, and the heavy and light chain variable regions, the CDRs of the heavy chain and the CDRs of the light chain It is shown in Tables 1 to 3, respectively.

Antibodies Heavy chain variable region Light chain variable region Kappa Lambda 58 MYRMQLLSCIALSLALVT NS
QVQLVQSGAELKKPGSSVKVSCKAS GGTFKISA ISWVRQAPGQGLEWVGG IIPIFGTP HYAQKLQGRVTITADDSTATAYMELSSLRSEDTAVYYC AREGDLGGTTGFWQLAY WGQGTLVTVSS
(SEQ ID NO: 43) - MYRMQLLSCIALSLALVT NS SYELTQPLSVSVALGQTATLTCGGD NIGSKN VHWYQQKPGQAPVLVIY IND NRPSAIPERFSGSNSGNTATLTIRRAQAGDEADYYC QLWDGSTGV FGGGTELTVLL
(SEQ ID NO: 50) 68 MYRMQLLSCIALSLALVT NS EVQLVQSGAEVKKPGSSVKVSCKAS GGAFNTYT ISWVRQAPGQGLEWMGG IIPLFRSS SYAQRFQGRVTFTADESTNTVYMELSSLRSEDTAVYYC TRRGDGSGRLGYIHYDTDV WGQGTTVTVSS
(SEQ ID NO: 44) - MYRMQLLSCIALSLALVT NS QPVLTQLPSASGTPGQRVTISCSGS SSNIGGNY VYWYQQLAGAAPKLLIY KNN ERPSGVPDRFSGSKSGTSASLAISGLRSDDEGDYYC AAWDDSLSGVV FGGGTKVTVLL
(SEQ ID NO 51) 72 MYRMQLLSCIALSLALVT NS QVQLVQSGAEVKKPGASVKVSCKA SGYFSTYE INWVRQATGQGLEWLGW MNPKSGIT GYAPKFQGRVTMTGNTSINTTYMELSSLRSDDTAVYYC ARGFLGADYYGLDV WGQGTTVTRLLQL
(SEQ ID NO 45) MYRMQLLSCIALSLALVT NS EIVMTQSPSSLSASVGDRVTITCRASQ RVISTY LNWYQQSPGKAPKLLIY AAS TLQRGVPSRFSGSGSGTDFTLTISGLQPEDFATYYC QQTYNTPQT FGQGTKVEIKR
(SEQ ID NO 52) - 78 MYRMQLLSCIALSLALVT NS EVQLVQSGAEVKMPGSSVKVSCKAS GGTFSSSG IIWVRQAPGQGLEWMGG IIPFFGTT TYAQRFQGRFTITADKSTNTAYMELNNLRFEDTAIYFC ARDGGIPSARLNNHYGMDV WGQGTTVTVSS
(SEQ ID NO 46) MYRMQLLSCIALSLALVT NS EIVLTQSPGTLSLSPGERATLSCRAS QSVCTIC LAWYQQKPGQAPRLLIS GAS SRAAGIPDRFSGSGSGTDFTLIIDRLEPEDFAVYYC QQYGGSAWT FGQGTKVEIKR
(SEQ ID NO: 53) - 88 MYRMQLLSCIALSLALVT NS EVQLVESGGGLIQPGGSLRLSCAAS GFTVIDYY MTWVRQAPGKGLEWVSV IYAGGST YYADSVKGRFTVSRDYSRNTLYLQMNSLKAEDTAVYYC TRDYLAAGGL WGQGALVTVSS
(SEQ ID NO 47) MYRMQLLSCIALSLALVT NS EIVLTQSPSSLSASVGDRVTITCRAS QGIRNN LAWYQQKPGQVPELLIY GAS NLKPGVPSRFSGSASGTDFTLTISSMQPEDVATYYC QKYDSAPLT FGGGTKLEIKR
(SEQ ID NO 54) - 90 MYRMQLLSCIALSLALVT NS QVQLVQSGAEVKKPGTSVKVSCRAS GGTVSSYA ISWVRQAPGQGLEWMGG IIPLFGTV NYTQKFQGRVTITADASTSTAYMELSSLTSDDTAVYYC ARSTSGWYGGRNWLDP WGQGTLVTVSS
(SEQ ID NO 48) MYRMQLLSCIALSLALVT NS DIVMTQSPFSLSASVGDRVTITCRAS QAIYNS LAWYQQKPGKAPKLLLY GAS GLESGVPSRFSGSGSGSGTDYTLTISSLQPEDFATYYC QQYHSTPPWT FGQGTKLEIKR
(SEQ ID NO 55) - 95 MYRMQLLSCIALSLALVT NS QVQLVQSGAEVKKPGSSVKVSCKAS EDTFRRFA ISWVRQAPGQGLEWMGR IIAFFGSA NYAQKFQGRVTITADKSTSTVYMELGSLRFEDTAVYYC ARDGGFGVVTPPNIYSYGLDV WGQGTTVTVSS
(SEQ ID NO 49) MYRMQLLSCIALSLALVT NS EIVLTQSPGTLSLSPGERATLSCRAS QSVSSTY LAWYQQKPGQAPRLLIY GTS SRATGIPDRFSGSGSGTGTTLTLTITRLEPEDFAVYYC QQFGSSPQYT FGQGTKVEIKR
(SEQ ID NO 56) -

Underline: signal peptide

Bold: CDR

Heavy chain CDRs Antibodies CDRH1 CDRH2 CDRH3 KNIH-58 GGTFKISA
(SEQ ID NO 1) IIPIFGTP
(SEQ ID NO: 2) AREGDLGGTTGFWQLAY
(SEQ ID NO: 3) KNIH-68 GGAFNTYT
(SEQ ID NO: 4) IIPLFRSS
(SEQ ID NO: 5) TRRGDGSGRLGYIHYDTDV
(SEQ ID NO: 6) KNIH-72 SGYFSTYE
(SEQ ID NO: 7) MNPKSGIT
(SEQ ID NO: 8) ARGFLGADYYGLDV
(SEQ ID NO: 9) KNIH-78 GGTFSSSG
(SEQ ID NO: 10) IIPFFGTT
(SEQ ID NO: 11) ARDGGIPSARLNNHYGMDV
(SEQ ID NO: 12) KNIH-88 GFTVIDYY
(SEQ ID NO: 13) IYAGGST
(SEQ ID NO: 14) TRDYLAAGGL
(SEQ ID NO: 15) KNIH-90 GGTVSSYA
(SEQ ID NO: 16) IIPLFGTV
(SEQ ID NO: 17) ARSTSGWYGGRNWLDP
(SEQ ID NO: 18) KNIH-95 EDTFRRFA
(SEQ ID NO: 19) IIAFFGSA
(SEQ ID NO: 20) ARDGGFGVVTPPNIYSYGLDV
(SEQ ID NO: 21)

CDR of the light chain Antibodies CDRL1 CDRL2 CDRL3 KNIH-58 NIGSKN
(SEQ ID NO: 22) IND
(SEQ ID NO: 23) QLWDGSTGV
(SEQ ID NO: 24) KNIH-68 SSNIGGNY
(SEQ ID NO: 25) KNN
(SEQ ID NO 26) AAWDDSLSGVV
(SEQ ID NO 27) KNIH-72 RVISTY
(SEQ ID NO 28) AAS
(SEQ ID NO 29) QQTYNTPQT
(SEQ ID NO: 30) KNIH-78 QSVCTIC
(SEQ ID NO 31) GAS
(SEQ ID NO: 32) QQYGGSAWT
(SEQ ID NO: 33) KNIH-88 QGIRNN
(SEQ ID NO 34) GAS
(SEQ ID NO 35) QKYDSAPLT
(SEQ ID NO: 36) KNIH-90 QAIYNS
(SEQ ID NO: 37) GAS
(SEQ ID NO: 38) QQYHSTPPWT
(SEQ ID NO 39) KNIH-95 QSVSSTY
(SEQ ID NO 40) GTS
(SEQ ID NO 41) QQFGSSPQYT
(SEQ ID NO 42)

As shown in Table 2, the heavy chain CDRs of KNIH-58, KNIH-58, KNIH-68, KNIH-72, KNIH-78, KNIH-88, KNIH-90, and KNIH-95 are amino acids of SEQ ID NOs: 1-21. Has a sequence.

In addition, as shown in Table 3, the light chain CDRs of KNIH-58, KNIH-58, KNIH-68, KNIH-72, KNIH-78, KNIH-88, KNIH-90, and KNIH-95 are SEQ ID NOs: 22-42 Has an amino acid sequence of

Experimental Example 1

Binding Affinity Review of Spark Proteins of MERS Corona Virus

The inventors performed the experiment using ELISA and SPR assay to measure the binding affinity of the monoclonal antibody prepared in Example 2 as follows.

1. Binding affinity measurement by ELISA method

In order to confirm whether or not the seven monoclonal antibodies prepared in Example 2 exhibit high binding affinity with MERS coronavirus S1 protein, the inventors of the S1 and S2 proteins of MERS coronavirus, and receptor binding domain, RBD) and affinity was measured by ELISA method.

Specifically, S1 antigen of MERS corona virus was put in a MaxiSorp 96-well plate (Nunc) to 0.2 μg / 100 μl PBS / well and reacted at 4 ° C. for 1 day. After the reaction was completed, the reaction solution was removed and blocked with a blocking solution containing 5% skim milk and 2% BSA in PBST for 1 hour at room temperature.

Next, the antibody prepared in Example 2 was diluted in a blocking solution to a concentration of 0.004, 0.016, 0.063, 0.25, 1.0, 4.0, or 16.0 nM, and placed in the S1 antigen-coated plate, which was reacted for 1 hour at room temperature. I was. Then, washed 6 times with PBST, rabbit anti-human IgG H & L-HRP (horse radish peroxidase) secondary antibody was added and reacted for 1 hour. When the reaction was completed, 100 μl of stop solution (Enzygost) was added, and the absorbance was measured at 450 nm to confirm the adhesion of the antibody to the antigen.

The results are shown in FIG. 1, and when the S1 antigen is 0.25 nM or more, the OD450 nm level of the KNIH-58 and KNIH-68 antibodies among the seven antibodies is higher than that of the other antibodies, resulting in high binding affinity to the MERS virus S1 antigen. It can be seen that it has a degree.

In addition, the results of measuring the binding affinity with the receptor binding domain (RBD) under the same conditions as shown in Figure 2, the KNIH-58 and KNIH-68 antibody showed a high affinity for RBD The remaining antibodies have very low affinity.

In addition, the results of measuring the binding affinity to S2 of the mers corona virus under the same conditions as shown in Figure 3, it was confirmed that all seven antibodies do not have affinity to the S2 antigen.

As a result, it can be seen that the antibodies of the present invention exhibit a high binding affinity specifically for the S1 protein of MERS corona virus.

2. Measurement of binding affinity by surface plasmon resonance

The present inventors confirmed the surface plasmon resonance (SPR) to determine whether the monoclonal antibody exhibits high binding affinity with the S1 protein of MERS coronavirus.

Specifically, after attaching the S1 antigen to the surface of the CM5 chip, the antibody of the present invention was injected. Thereafter, the association time was 3 minutes, the dissociation time was set forth in the conditions described in Table 4, the running buffer was HBS-EP, and the regeneration solution was set forth in Table 4 using the SPR assay Was performed, and the results are shown in FIGS. 4 to 10 and Table 5.

Antibodies Nautical hour
(min) Regeneration solution KNIH-58 60 10 mM glycine pH 2.0 KNIH-68 120 15 mM NaOH KNIH-72 60 10 mM glycine pH 2.0 + 1 M NaOH KNIH-78 60 10 mM glycine pH 1.5 + 1M NaOH
15 mM NaOH KNIH-88 60 10 mM glycine pH 2.0 + 1 M NaOH KNIH-90 60 10 mM glycine pH 2.0 + 1 M NaOH KNIH-95 60 10 mM glycine pH 2.0 + 1 M NaOH

In Table 5, Ka (1 / Ms) represents the association (association) rate, Kd (1 / s) represents the dissociation rate. K _D (M) is a value calculated by Ka / Kd, and the lower the value, the higher the antigen-antibody affinity (affinity), which means that the poor.

Thus, all seven antibodies showed very strong binding strength of 19 pM (picomolar) to 264 pM to the S1 protein. The highest binding antibody was antibody 95 (≦ 19.64 pM).

As such, it can be seen that the antibodies of the present invention have a high binding affinity for the S1 protein of MERS coronavirus.

Experimental Example 2

MERS virus neutralization evaluation experiment

The present inventors performed a plaque reduction neutralization test (PRNT) on the mers corona virus EMC strain to measure the neutralizing ability of the monoclonal antibody prepared in Example 2 against the mers corona virus.

Specifically, Vero cells were first divided into 24 well plates at a concentration of 10 ⁵ cells / well, and then cultured. Remove the culture medium of the cultured cells, MERS- diluted with 50 PFU / well and 7 antibodies of the present invention prepared at concentrations of 0.001, 0.004, 0.016, 0.063, 0.25, 1.0 or 4.0 ng / ul diluted with cell culture. CoV (EMC strain) was mixed to prepare a mixed solution.

100 μl / well of the mixed solution was dispensed to the cultured cells and reacted at 37 ° C. for 1 hour. Then, DMEM culture medium containing 1.5% CMC (carboxymethyl cellulose) was added at a concentration of 1 ml / well, and cultured for 3 days. When the incubation was completed, the plaques were identified through crystal violet staining, and the number of plaques of each well was measured, and the neutralizing ability was calculated by the same method as in Equation 1 below. Based on the graphPad Prism program was calculated the inhibitory concentration (inhibition concentration 50, IC50) 50% inhibition of MERS virus infection.

As a result, as shown in Figure 5, all seven antibodies showed a neutralizing ability against the MERS virus EMC strain, among which KNIH-58, KNIH-68, KNIH-72 and KNIH-88 antibodies were MERS virus EMC It showed high neutralizing ability against the strain.

When the antibody concentration was 0.156 ng / μl, KNIH-58 antibody showed the highest neutralizing capacity to block 100% MERS virus infection. In addition, KNIH-68 antibody showed a neutralizing ability of 85.7 ± 4.6%, KNIH-72 antibody 81.3 ± 4.7%, KNIH-88 antibody 79.2 ± 7.9%. However, when the antibody concentration was low at 0.0025 ng / μl, KNIH-88 antibody showed about 50% neutralization ability.

Therefore, when a small amount of KNIH-88 antibody in combination with other antibodies the antibody epitope, it can be seen that the effect of suppressing MERS virus infection is more excellent.

Experimental Example 3 Analysis of Physical Properties of Monoclonal Antibody

Physical properties of all seven antibodies of the present invention were analyzed by SEC-HPLC (size exclusion chromatography-HPLC) and PTS (protein thermal shift assay).

1. Morphology analysis of antibody by SEC-HPLC

Antibody morphology was analyzed using a Water SEC-HPLC system. The HPLC analysis column was Agilent Bio SEC3 and the detector was an ultraviolet absorbance meter (280 nm). At this time, 10 µl of antibody at a concentration of 1 mg / ml was injected as a sample, and the flow rate was 0.3 ml / min. In addition, isocratic elution was performed using 1X PBS as the mobile phase.

As a result, as shown in Table 5 below, it was confirmed that all seven antibodies maintained a monomer and almost no aggregated form.

2. Confirmation of the Stability of Antibodies through PTS Assay

The stability of the antibody was confirmed in PBS (phosphate buffered saline) containing pH 7.4, which is a storage solution of the antibodies and does not contain calcium and magnesium. Specifically, 0.75 μg of antibody was used, and PTS assay was performed according to the manufacturer's method using Protein Thermal Shift ™ Dye Kit (Catalog No. 4466038, Life technologies).

As a result, as shown in Table 6, the melting point (melting point, T _m ) all showed a result of more than 62.46 ℃. That is, it was confirmed that stability was maintained even at a relatively high temperature.

Experimental Example 4

Specificity test of monoclonal antibody

In order to evaluate the specificity of the monoclonal antibody prepared in Example 2, the present inventors performed cross-reaction evaluation (Immunofluorescence assay) with human corona virus OC43, 229E, NL63 strains other than MERS corona virus as follows.

Each cell line was seeded on a Lab-Tek II Chamber slide with 2x10 ⁴ cells / well, and after 24hr, each cell was infected with 200 μl of coronavirus and MERS virus at a 1/10000 ratio. CPE formation was confirmed after 4 hours adsorption and washed with DPBS. Each infected cell was fixed with 4% paraformaldehyde buffer. 50 μl of each antibody of the present invention was injected at a concentration of 2.5 ng / ml to react with the fixed cells at 37 ° C. for 30 minutes. The cells were then washed with PBS buffer and subjected to a secondary antibody reaction with goat anti-human-IgG-FITC. After washing with PBS buffer again, the results of observation using a fluorescence microscope are shown in FIG. 6.

As shown in FIG. 6, the antibodies of the present invention did not adhere to each cell infected with the human coronavirus OC43, 229E, and NL63 strains similar to the MERS virus. On the other hand, it was confirmed that all the antibodies of the present invention attached to cells infected with the MERS virus EMC strain showed fluorescence. Thus, it can be seen that the monoclonal antibody of the present invention has excellent specificity for MERS coronavirus.

Experimental Example 5

Neutralization comparison test

A neutralization test was performed with antibodies 90-F1 and 90-B2 obtained from blood isolated from early patients of MERS coronavirus infection. Antibodies 90-F1 and 90-B2 have the amino acid sequences of Table 8 below.

Antibodies Heavy chain CDR Light chain CDR 90-F1 QVQLVQSGAEVKRPGSSVKVSCKTSGGTFNNNAINWVRQAPGQGLEWMGGIIPFFGIAKYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDLPRESSYGSGSYYTHYYAMDVWGQGTTVTVSSAS EIVLTQSPATLSLSPGERATLSCGASQSVSSSYLAWYQQKPGLAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRT 90-B2 QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGNTYYNPSLKSRVTISVDTSKNHFSLRLSSVTAADTAVYFCARSLPHYDSTGYLLYWGQGTLVTVSSAS EIVLTQSPATLSLSPGGRATLSCRASQSVSRYLAWYQQKPGRAPRLLIYDASNRAPGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPQTTFGPGTKVDIKRT

FIG. 7 shows the results of comparative evaluation of the neutralizing ability of the antibodies 90-F1 and 90-B2 and the antibodies KNIH-58 and KNIH-72 of the present invention by the method described in Example 2.

KNIH-58 antibody attaches to the RBD site that is highly related to the neutralizing ability of S1 virus of MERS virus, and has high neutralizing ability (IC50: 0.028 ng) similar to that of 90-F1 (IC50: 0.009 ng / μl) with high neutralizing ability. / Μl). On the other hand, when comparing KNIH-72 of the present invention to the S1 protein other than RBD and the existing 90-B2, the KNIH-72 antibody (IC50: 0.016 ng / uL) of the present invention is 90-B2 (IC50: 0.25 ng / uL) was found to show a higher neutralizing capacity.

As a result, when the results of FIGS. 5 and 7 are combined, it can be seen that the antibodies of the present invention have better neutralizing ability than the antibodies developed in the past.

<110> Korea Centers for Disease Control and Prevention <120> Monoclonal antibodies of specifically binding to spike S1 protein          of MERS-CoV <130> P180047 <160> 57 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-58 CDRH1 <400> 1 Gly Gly Thr Phe Lys Ile Ser Ala   1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-58 CDRH2 <400> 2 Ile Ile Pro Ile Phe Gly Thr Pro   1 5 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-58 CDRH3 <400> 3 Ala Arg Glu Gly Asp Leu Gly Gly Thr Thr Gly Phe Trp Gln Leu Ala   1 5 10 15 Tyr     <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-68 CDRH1 <400> 4 Gly Gly Ala Phe Asn Thr Tyr Thr   1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-68 CDRH2 <400> 5 Ile Ile Pro Leu Phe Arg Ser Ser   1 5 <210> 6 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-68 CDRH3 <400> 6 Thr Arg Arg Gly Asp Gly Ser Gly Arg Leu Gly Tyr Ile His Tyr Asp   1 5 10 15 Thr Asp Val             <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-72 CDRH1 <400> 7 Ser Gly Tyr Phe Ser Thr Tyr Glu   1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-72 CDRH2 <400> 8 Met Asn Pro Lys Ser Gly Ile Thr   1 5 <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-72 CDRH3 <400> 9 Ala Arg Gly Phe Leu Gly Ala Asp Tyr Tyr Gly Leu Asp Val   1 5 10 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-78 CDRH1 <400> 10 Gly Gly Thr Phe Ser Ser Ser Gly   1 5 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-78 CDRH2 <400> 11 Ile Ile Pro Phe Phe Gly Thr Thr   1 5 <210> 12 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-78 CDRH3 <400> 12 Ala Arg Asp Gly Gly Ile Pro Ser Ala Arg Leu Asn Asn His Tyr Gly   1 5 10 15 Met asp val             <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-88 CDRH1 <400> 13 Gly Phe Thr Val Ile Asp Tyr Tyr   1 5 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-88 CDRH2 <400> 14 Ile Tyr Ala Gly Gly Ser Thr   1 5 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-88 CDRH3 <400> 15 Thr Arg Asp Tyr Leu Ala Ala Gly Gly Leu   1 5 10 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-90 CDRH1 <400> 16 Gly Gly Thr Val Ser Ser Tyr Ala   1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-90 CDRH2 <400> 17 Ile Ile Pro Leu Phe Gly Thr Val   1 5 <210> 18 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-90 CDRH3 <400> 18 Ala Arg Ser Thr Ser Gly Trp Tyr Gly Gly Arg Asn Trp Leu Asp Pro   1 5 10 15 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-95 CDRH1 <400> 19 Glu Asp Thr Phe Arg Arg Phe Ala   1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-95 CDRH2 <400> 20 Ile Ile Ala Phe Phe Gly Ser Ala   1 5 <210> 21 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-95 CDRH3 <400> 21 Ala Arg Asp Gly Gly Phe Gly Val Val Thr Pro Pro Asn Ile Tyr Ser   1 5 10 15 Tyr Gly Leu Asp Val              20 <210> 22 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-58 CDRL1 <400> 22 Asn Ile Gly Ser Lys Asn   1 5 <210> 23 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-58 CDRL2 <400> 23 Ile asn asp   One <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-58 CDRL3 <400> 24 Gln Leu Trp Asp Gly Ser Thr Gly Val   1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-68 CDRL1 <400> 25 Ser Ser Asn Ile Gly Gly Asn Tyr   1 5 <210> 26 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-68 CDRL2 <400> 26 Lys Asn Asn   One <210> 27 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-68 CDRL3 <400> 27 Ala Ala Trp Asp Asp Ser Leu Ser Gly Val Val   1 5 10 <210> 28 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-72 CDRL1 <400> 28 Arg Val Ile Ser Thr Tyr   1 5 <210> 29 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-72 CDRL2 <400> 29 Ala Ala Ser   One <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-72 CDRL3 <400> 30 Gln Gln Thr Tyr Asn Thr Pro Gln Thr   1 5 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-78 CDRL1 <400> 31 Gln Ser Val Cys Thr Ile Cys   1 5 <210> 32 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-78 CDRL2 <400> 32 Gly ala ser   One <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-78 CDRL3 <400> 33 Gln Gln Tyr Gly Gly Ser Ala Trp Thr   1 5 <210> 34 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-88 CDRL1 <400> 34 Gln Gly Ile Arg Asn Asn   1 5 <210> 35 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-88 CDRL2 <400> 35 Gly ala ser   One <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-88 CDRL3 <400> 36 Gln Lys Tyr Asp Ser Ala Pro Leu Thr   1 5 <210> 37 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-90 CDRL1 <400> 37 Gln Ala Ile Tyr Asn Ser   1 5 <210> 38 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-90 CDRL2 <400> 38 Gly ala ser   One <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-90 CDRL3 <400> 39 Gln Gln Tyr His Ser Thr Pro Pro Trp Thr   1 5 10 <210> 40 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-95 CDRL1 <400> 40 Gln Ser Val Ser Ser Thr Tyr   1 5 <210> 41 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-95 CDRL2 <400> 41 Gly thro ser   One <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-95 CDRL3 <400> 42 Gln Gln Phe Gly Ser Ser Pro Gln Tyr Thr   1 5 10 <210> 43 <211> 144 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-58 <400> 43 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys              20 25 30 Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr          35 40 45 Phe Lys Ile Ser Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly      50 55 60 Leu Glu Trp Val Gly Gly Ile Ile Pro Ile Phe Gly Thr Pro His Tyr  65 70 75 80 Ala Gln Lys Leu Gln Gly Arg Val Thr Ile Thr Ala Asp Asp Ser Thr                  85 90 95 Ala Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala             100 105 110 Val Tyr Tyr Cys Ala Arg Glu Gly Asp Leu Gly Gly Thr Thr Gly Phe         115 120 125 Trp Gln Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser     130 135 140 <210> 44 <211> 146 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-68 <400> 44 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys              20 25 30 Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ala          35 40 45 Phe Asn Thr Tyr Thr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly      50 55 60 Leu Glu Trp Met Gly Gly Ile Ile Pro Leu Phe Arg Ser Ser Ser Tyr  65 70 75 80 Ala Gln Arg Phe Gln Gly Arg Val Thr Phe Thr Ala Asp Glu Ser Thr                  85 90 95 Asn Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala             100 105 110 Val Tyr Tyr Cys Thr Arg Arg Gly Asp Gly Ser Gly Arg Leu Gly Tyr         115 120 125 Ile His Tyr Asp Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val     130 135 140 Ser Ser 145 <210> 45 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-72 <400> 45 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys              20 25 30 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Phe          35 40 45 Ser Thr Tyr Glu Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu      50 55 60 Glu Trp Leu Gly Trp Met Asn Pro Lys Ser Gly Ile Thr Gly Tyr Ala  65 70 75 80 Pro Lys Phe Gln Gly Arg Val Thr Met Thr Gly Asn Thr Ser Ile Asn                  85 90 95 Thr Thr Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Gly Phe Leu Gly Ala Asp Tyr Tyr Gly Leu Asp         115 120 125 Val Trp Gly Gln Gly Thr Thr Val Thr Arg Leu Leu Gln Leu     130 135 140 <210> 46 <211> 146 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-78 <400> 46 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys              20 25 30 Met Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr          35 40 45 Phe Ser Ser Ser Gly Ile Ile Trp Val Arg Gln Ala Pro Gly Gln Gly      50 55 60 Leu Glu Trp Met Gly Gly Ile Ile Pro Phe Phe Gly Thr Thr Thr Tyr  65 70 75 80 Ala Gln Arg Phe Gln Gly Arg Phe Thr Ile Thr Ala Asp Lys Ser Thr                  85 90 95 Asn Thr Ala Tyr Met Glu Leu Asn Asn Leu Arg Phe Glu Asp Thr Ala             100 105 110 Ile Tyr Phe Cys Ala Arg Asp Gly Gly Ile Pro Ser Ala Arg Leu Asn         115 120 125 Asn His Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val     130 135 140 Ser Ser 145 <210> 47 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-88 <400> 47 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile              20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr          35 40 45 Val Ile Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly      50 55 60 Leu Glu Trp Val Ser Val Ile Tyr Ala Gly Gly Ser Thr Tyr Tyr Ala  65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Tyr Ser Arg Asn                  85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Thr Arg Asp Tyr Leu Ala Ala Gly Gly Leu Trp Gly Gln         115 120 125 Gly Ala Leu Val Thr Val Ser Ser     130 135 <210> 48 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-90 <400> 48 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys              20 25 30 Lys Pro Gly Thr Ser Val Lys Val Ser Cys Arg Ala Ser Gly Gly Thr          35 40 45 Val Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly      50 55 60 Leu Glu Trp Met Gly Gly Ile Ile Pro Leu Phe Gly Thr Val Asn Tyr  65 70 75 80 Thr Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Ala Ser Thr                  85 90 95 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala             100 105 110 Val Tyr Tyr Cys Ala Arg Ser Thr Ser Gly Trp Tyr Gly Gly Arg Asn         115 120 125 Trp Leu Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser     130 135 140 <210> 49 <211> 148 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-95 <400> 49 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys              20 25 30 Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Glu Asp Thr          35 40 45 Phe Arg Arg Phe Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly      50 55 60 Leu Glu Trp Met Gly Arg Ile Ile Ala Phe Phe Gly Ser Ala Asn Tyr  65 70 75 80 Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr                  85 90 95 Ser Thr Val Tyr Met Glu Leu Gly Ser Leu Arg Phe Glu Asp Thr Ala             100 105 110 Val Tyr Tyr Cys Ala Arg Asp Gly Gly Phe Gly Val Val Thr Pro Pro         115 120 125 Asn Ile Tyr Ser Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val     130 135 140 Thr Val Ser Ser 145 <210> 50 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-58 <400> 50 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val              20 25 30 Ala Leu Gly Gln Thr Ala Thr Leu Thr Cys Gly Gly Asp Asn Ile Gly          35 40 45 Ser Lys Asn Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val      50 55 60 Leu Val Ile Tyr Ile Asn Asp Asn Arg Pro Ser Ala Ile Pro Glu Arg  65 70 75 80 Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Arg Arg                  85 90 95 Ala Gln Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Leu Trp Asp Gly             100 105 110 Ser Thr Gly Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Leu         115 120 125 <210> 51 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-68 <400> 51 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Gln Pro Val Leu Thr Gln Leu Pro Ser Ala Ser Gly              20 25 30 Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn          35 40 45 Ile Gly Gly Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Ala Gly Ala Ala      50 55 60 Pro Lys Leu Leu Ile Tyr Lys Asn Asn Glu Arg Pro Ser Gly Val Pro  65 70 75 80 Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile                  85 90 95 Ser Gly Leu Arg Ser Asp Asp Glu Gly Asp Tyr Tyr Cys Ala Ala Trp             100 105 110 Asp Asp Ser Leu Ser Gly Val Val Phe Gly Gly Gly Thr Lys Val Thr         115 120 125 Val Leu Leu     130 <210> 52 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 KNIH-72 <400> 52 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Glu Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser              20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Arg          35 40 45 Val Ile Ser Thr Tyr Leu Asn Trp Tyr Gln Gln Ser Pro Gly Lys Ala      50 55 60 Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Arg Gly Val Pro  65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile                  85 90 95 Ser Gly Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr             100 105 110 Tyr Asn Thr Pro Gln Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys         115 120 125 Arg     <210> 53 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Mers-cov spike S1 KNIH-78 <400> 53 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser              20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser          35 40 45 Val Cys Thr Ile Cys Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala      50 55 60 Pro Arg Leu Leu Ile Ser Gly Ala Ser Ser Arg Ala Ala Gly Ile Pro  65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ile Ile                  85 90 95 Asp Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr             100 105 110 Gly Gly Ser Ala Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys         115 120 125 Arg     <210> 54 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Mers-cov spike S1 KNIH-88 <400> 54 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser              20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly          35 40 45 Ile Arg Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro      50 55 60 Glu Leu Leu Ile Tyr Gly Ala Ser Asn Leu Lys Pro Gly Val Pro Ser  65 70 75 80 Arg Phe Ser Gly Ser Ala Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser                  85 90 95 Ser Met Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp             100 105 110 Ser Ala Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg         115 120 125 <210> 55 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Mers-cov spike S1 KNIH-90 <400> 55 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Asp Ile Val Met Thr Gln Ser Pro Phe Ser Leu Ser              20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala          35 40 45 Ile Tyr Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro      50 55 60 Lys Leu Leu Leu Tyr Gly Ala Ser Gly Leu Glu Ser Gly Val Pro Ser  65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser                  85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His             100 105 110 Ser Thr Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys         115 120 125 Arg     <210> 56 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> Mers-cov spike S1 KNIH-95 <400> 56 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu   1 5 10 15 Val Thr Asn Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser              20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser          35 40 45 Val Ser Ser Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala      50 55 60 Pro Arg Leu Leu Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro  65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile                  85 90 95 Thr Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe             100 105 110 Gly Ser Ser Pro Gln Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile         115 120 125 Lys arg     130 <210> 57 <211> 752 <212> PRT <213> Artificial Sequence <220> <223> MERS-CoV Spike S1 <400> 57 Met Ile His Ser Val Phe Leu Leu Met Phe Leu Leu Thr Pro Thr Glu   1 5 10 15 Ser Tyr Val Asp Val Gly Pro Asp Ser Val Lys Ser Ala Cys Ile Glu              20 25 30 Val Asp Ile Gln Gln Thr Phe Phe Asp Lys Thr Trp Pro Arg Pro Ile          35 40 45 Asp Val Ser Lys Ala Asp Gly Ile Ile Tyr Pro Gln Gly Arg Thr Tyr      50 55 60 Ser Asn Ile Thr Ile Thr Tyr Gln Gly Leu Phe Pro Tyr Gln Gly Asp  65 70 75 80 His Gly Asp Met Tyr Val Tyr Ser Ala Gly His Ala Thr Gly Thr Thr                  85 90 95 Pro Gln Lys Leu Phe Val Ala Asn Tyr Ser Gln Asp Val Lys Gln Phe             100 105 110 Ala Asn Gly Phe Val Val Arg Ile Gly Ala Ala Ala Asn Ser Thr Gly         115 120 125 Thr Val Ile Ile Ser Pro Ser Thr Arg Ala Thr Ile Arg Lys Ile Tyr     130 135 140 Pro Ala Phe Met Leu Gly Ser Ser Val Gly Asn Phe Ser Asp Gly Lys 145 150 155 160 Met Gly Arg Phe Phe Asn His Thr Leu Val Leu Leu Pro Asp Gly Cys                 165 170 175 Gly Thr Leu Leu Arg Ala Phe Tyr Cys Ile Leu Glu Pro Arg Ser Gly             180 185 190 Asn His Cys Pro Ala Gly Asn Ser Tyr Thr Ser Phe Ala Thr Tyr His         195 200 205 Thr Pro Ala Thr Asp Cys Ser Asp Gly Asn Tyr Asn Arg Asn Ala Ser     210 215 220 Leu Asn Ser Phe Lys Glu Tyr Phe Asn Leu Arg Asn Cys Thr Phe Met 225 230 235 240 Tyr Thr Tyr Asn Ile Thr Glu Asp Glu Ile Leu Glu Trp Phe Gly Ile                 245 250 255 Thr Gln Thr Ala Gln Gly Val His Leu Phe Ser Ser Arg Tyr Val Asp             260 265 270 Leu Tyr Gly Gly Asn Met Phe Gln Phe Ala Thr Leu Pro Val Tyr Asp         275 280 285 Thr Ile Lys Tyr Tyr Ser Ile Ile Pro His Ser Ile Arg Ser Ile Gln     290 295 300 Ser Asp Arg Lys Ala Trp Ala Ala Phe Tyr Val Tyr Lys Leu Gln Pro 305 310 315 320 Leu Thr Phe Leu Leu Asp Phe Ser Val Asp Gly Tyr Ile Arg Arg Ala                 325 330 335 Ile Asp Cys Gly Phe Asn Asp Leu Ser Gln Leu His Cys Ser Tyr Glu             340 345 350 Ser Phe Asp Val Glu Ser Gly Val Tyr Ser Val Ser Ser Phe Glu Ala         355 360 365 Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu Cys Asp     370 375 380 Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn Phe Lys 385 390 395 400 Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu Leu Ser                 405 410 415 Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro Ala Ala             420 425 430 Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe Ser Tyr         435 440 445 Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly Pro Ile     450 455 460 Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys Leu Ile 465 470 475 480 Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro Leu Lys                 485 490 495 Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp Arg Thr             500 505 510 Glu Val Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser Pro Cys Val Ser         515 520 525 Ile Leu Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg Lys Gln     530 535 540 Leu Ser Pro Leu Glu Gly Gly Gly Trp Leu Val Ala Ser Gly Ser Thr 545 550 555 560 Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr Val Gln                 565 570 575 Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Glu Phe Ala Asn             580 585 590 Asp Thr Lys Ile Ala Ser Gln Leu Gly Asn Cys Val Glu Tyr Ser Leu         595 600 605 Tyr Gly Val Ser Gly Arg Gly Val Phe Gln Asn Cys Thr Ala Val Gly     610 615 620 Val Arg Gln Gln Arg Phe Val Tyr Asp Ala Tyr Gln Asn Leu Val Gly 625 630 635 640 Tyr Tyr Ser Asp Asp Gly Asn Tyr Tyr Cys Leu Arg Ala Cys Val Ser                 645 650 655 Val Pro Val Ser Val Ile Tyr Asp Lys Glu Thr Lys Thr His Ala Thr             660 665 670 Leu Phe Gly Ser Val Ala Cys Glu His Ile Ser Ser Thr Met Ser Gln         675 680 685 Tyr Ser Arg Ser Thr Arg Ser Met Leu Lys Arg Arg Asp Ser Thr Tyr     690 695 700 Gly Pro Leu Gln Thr Pro Val Gly Cys Val Leu Gly Leu Val Asn Ser 705 710 715 720 Ser Leu Phe Val Glu Asp Cys Lys Leu Pro Leu Gly Gln Ser Leu Cys                 725 730 735 Ala Leu Pro Asp Thr Pro Ser Thr Leu Thr Pro Arg Ser Val Arg Ser             740 745 750

Claims (13)

CDRH1 having the sequence of SEQ ID NO: 1; CDRH2 having the sequence of SEQ ID NO: 2; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 3; And
CDRL1 having the sequence of SEQ ID NO: 22; CDRL2 having the sequence of SEQ ID NO: 23; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 24, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers Corona virus.
CDRH1 having the sequence of SEQ ID NO: 4; CDRH2 having the sequence of SEQ ID NO: 5; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 6; And
CDRL1 having the sequence of SEQ ID NO: 25; CDRL2 having the sequence of SEQ ID NO: 26; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 27, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.
CDRH1 having the sequence of SEQ ID NO: 7; CDRH2 having the sequence of SEQ ID NO: 8; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 9; And
CDRL1 having the sequence of SEQ ID NO: 28; CDRL2 having the sequence of SEQ ID NO: 29; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 30, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.
CDRH1 having the sequence of SEQ ID NO: 10; CDRH2 having the sequence of SEQ ID NO: 11; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 12; And
CDRL1 having the sequence of SEQ ID NO: 31; CDRL2 having the sequence of SEQ ID NO: 32; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 33, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.
CDRH1 having the sequence of SEQ ID NO: 13; CDRH2 having the sequence of SEQ ID NO: 14; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 15; And
CDRL1 having the sequence of SEQ ID NO: 34; CDRL2 having the sequence of SEQ ID NO: 35; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 36, or an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers Corona virus.
CDRH1 having the sequence of SEQ ID NO: 16; CDRH2 having the sequence of SEQ ID NO: 17; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 18; And
CDRL1 having the sequence of SEQ ID NO: 37; CDRL2 having the sequence of SEQ ID NO: 38; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 39, an antibody or antigen-binding fragment thereof that specifically binds to the Spike S1 protein of Mers corona virus.
CDRH1 having the sequence of SEQ ID NO: 19; CDRH2 having the sequence of SEQ ID NO: 20; And a heavy chain variable region comprising CDRH3 having the sequence of SEQ ID NO: 21; And
CDRL1 having the sequence of SEQ ID NO: 40; CDRL2 having the sequence of SEQ ID NO: 41; And a light chain variable region comprising CDRL3 having the sequence of SEQ ID NO: 42, an antibody or antigen-binding fragment thereof that specifically binds to Spike S1 protein of Mers corona virus.
An antibody comprising a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 43 and a light chain variable region having an amino acid sequence represented by SEQ ID NO: 50;
An antibody comprising a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 44 and a light chain variable region having an amino acid sequence represented by SEQ ID NO: 51;
An antibody comprising a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 45 and a light chain variable region having an amino acid sequence represented by SEQ ID NO: 52;
An antibody comprising a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 46 and a light chain variable region having an amino acid sequence represented by SEQ ID NO: 53;
An antibody comprising a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 47 and a light chain variable region having an amino acid sequence represented by SEQ ID NO: 54;
An antibody comprising a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 48 and a light chain variable region having an amino acid sequence represented by SEQ ID NO: 55; And
An antibody comprising a heavy chain variable region having the amino acid sequence represented by SEQ ID NO: 49 and a light chain variable region having the amino acid sequence represented by SEQ ID NO: 56
An antibody or antigen-binding fragment thereof selected from the group consisting of:
A nucleic acid encoding the antibody of claim 1 or an antigen binding fragment thereof.
An expression vector comprising the nucleic acid of claim 9.
An isolated cell transformed with the expression vector of claim 10.
A pharmaceutical composition for preventing or treating MERS coronavirus infection comprising the antibody of any one of claims 1 to 8.
A kit for diagnosing mers coronavirus infection comprising the antibody of any one of claims 1 to 8.

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