MONOCLONAL ANTIBODY AGAINST S PROTEIN OF MERS-CORONAVIRUS, AND USE OF SAME

Abstract

The present invention relates to a monoclonal antibody specifically recognizing a spike protein of MERS coronavirus (MERS-CoV) or a part of the protein, or a functional fragment thereof, wherein the monoclonal antibody, or functional fragment of the monoclonal antibody characterized in that it comprises polypeptide sequence selected from the group consisting of the following polypeptide sequences: a heavy chain comprising a complementarity determining region 1(CDR 1) amino acid sequence consisting of the sequence of SEQ ID NO: 1, a CDR2 consisting of the sequence of SEQ ID NO: 2 and a CDR 3 consisting of the sequence of SEQ ID NO: 3; and a light chain comprising CDR1 amino acid sequence consisting of the sequence of SEQ ID NO: 4, a CDR2 consisting of the sequence of SEQ ID NO: 5 and a CDR 3 consisting of the sequence of SEQ ID NO: 6.and uses thereof.

Claims

1. In a monoclonal antibody that specifically recognizes a MERS coronavirus (MERS-CoV) protein or a part of the protein, or a functional fragment of the monoclonal antibody, the monoclonal antibody, or functional fragment of the monoclonal antibody characterized in that it comprises polypeptide sequence selected from the group consisting of the following polypeptide sequences: a heavy chain comprising a complementarity determining region 1(CDR 1) amino acid sequence consisting of the sequence of SEQ ID NO: 1, a CDR2 consisting of the sequence of SEQ ID NO: 2 and a CDR 3 consisting of the sequence of SEQ ID NO: 3; and a light chain comprising CDR1 amino acid sequence consisting of the sequence of SEQ ID NO: 4, a CDR2 consisting of the sequence of SEQ ID NO: 5 and a CDR 3 consisting of the sequence of SEQ ID NO: 6.

2. The monoclonal antibody or the functional fragment of the monoclonal antibody of claim 1, wherein said functional fragment is a single chain variable fragment (scFv).

3. The monoclonal antibody or the functional fragment of the monoclonal antibody of claim 1, wherein said functional fragment is a Fab.

4. The monoclonal antibody or the functional fragment of the monoclonal antibody of claim 1, wherein said functional fragment is a light chain or a heavy chain comprising the CDR region of claim 1.

5. The monoclonal antibody or the functional fragment of the monoclonal antibody of claim 1, wherein said functional fragment is a variable domain comprising the CDR region of claim 1.

6. The monoclonal antibody or the functional fragment of the monoclonal antibody of claim 1, wherein said monoclonal antibody comprises a heavy chain comprising an amino acid sequence consisting of the sequence of SEQ ID NO: 7; and a light chain comprising an amino acid sequence consisting of the sequence of SEQ ID NO: 8.

7. The monoclonal antibody or the functional fragment thereof of claim 1, wherein the protein of the MERS coronavirus (MERS-CoV) is a spike protein.

8. The monoclonal antibody or the functional fragment thereof of claim 7, wherein a part of the spike protein is a peptide sequence set forth in SEQ ID NO: 10.

9. A monoclonal antibody that recognizes the peptide sequence set forth in SEQ ID NO: 10 as an epitope.

10.-15. (canceled)

16. A MERS coronavirus diagnosis kit comprises the monoclonal antibody of claim 1 and a container.

17-18. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0061] FIG. 1. Production of B cell epitope-specific antibody. (A) Schematic representation of MERS-CoV S protein. S protein consists of S1 and S2 subunits. RBD, receptor binding domain; FP, fusion peptide; HR1 and HR2, heptad repeat region 1 and 2; TM, transmembrane; CP, cytoplasmic tail. (B) Selection of B cell epitope peptide sequence using the IEDB on the basis of epitope prediction, surface accessibility and antigenicity index. (C) ELISA analysis showing generation of MERS-CoV Spike-492- or Spike-492 (L506F)-specific antibodies using sera. Sera were isolated from BALB/c mice immunized with the complex of B cell epitope peptide (Spike-492 or Spike-492 (L506F)) and Lipoplex (O).

[0062] FIG. 2. Screening of hybridoma clone producing anti-Spike-492(L506F)-specific monoclonal antibody (506-2G10G5). (A, B) Three BALB/c mice were immunized i.p. with Spike-492(L506F) peptide and MB-ODN 4531(O) coencapsulated in a DOPE:CHEMS complex on four occasions at 10 day intervals. ELISA results from the initial screening (HAT medium) of a cell-fusion experiment using splenocytes of Spike-492(L506F) peptide immunized mice. (C, D) Hybridoma clones were selected for the production of monoclonal antibody following subcloning by limiting dilution method in HT medium.

[0063] FIG. 3. Screening of hybridoma clone producing anti-Spike-492-specific monoclonal antibody (492-1G10E4E2). (A, B) Three BALB/c mice were immunized i.p. with Spike-492 peptide and MB-ODN 4531(O) coencapsulated in a DOPE:CHEMS complex on four occasions at 10 day intervals. ELISA results from the initial screening (HAT medium) of a cell-fusion experiment using splenocytes of Spike-492 peptide immunized mice. (C-F) Hybridoma clones were selected for the production of monoclonal antibody following subcloning by limiting dilution method in HT medium.

[0064] FIG. 4. Purification and characterization of anti-MERS-CoV Spike-492 (L506F) epitope-specific monoclonal antibody. (A) The pristine-primed mice was injected with 506-2G10G5 clone and ascites was obtained. Subsequently, 506-2G10G5 monoclonal antibody was purified using Protein A agarose column chromatography and performed SDS-PAGE analysis. R, reducing condition; NR, non-reducing conditions. (B) Isotype of purified 506-2G10G5 monoclonal antibody was determined by ELISA. (C) The binding affinity of 506-2G10G5 monoclonal antibody to MERS-CoV Spike-492 (L506F) peptide was measured by ELISA and EC.sub.50 value was evaluated with Sigma Plot program. (D-F) MERS-CoV-infected and non-infected Vero cells lysates were treated with PBS (−) or PNGase F (+). The lysates were immunoblotted with 506-2G10G5 monoclonal antibody (D). Lysates were subjected to western blotting with an anti-β actin antibody as control (E). Western blot analysis using 506-2G10G5 monoclonal antibody was performed following the immunoprecipitation of lysates with 506-2G10G5 monoclonal antibody (F). Lysates were subjected to western blotting with an anti-β actin antibody as control (G). (H, I) Cross-reactivity of the monoclonal antibodies. 96-well immunoplates coated with MERS-CoV Spike-492 (G) or Spike-492 (L506F) (H) peptides and incubated with either the 492-1G10E4E2 or 506-2G10G5 monoclonal antibody. The reactivity of 492-1G10E4E2 monoclonal antibody to Spike-492 (L506F) peptide and that of 506-2G10G5 monoclonal antibody to Spike-492 peptide was determined by ELISA assay.

[0065] FIG. 5. Purification and characterization of anti-MERS-CoV Spike-492 epitope-specific monoclonal antibody. (A) The 492-1G10E4E2 monoclonal antibody from ascites was purified using Protein A agarose column chromatography and analyzed by SDS-PAGE. R, reducing condition; NR, non-reducing conditions. (B) Determination of 492-1G10E4E2 monoclonal antibody isotype by ELISA. (C) The binding of 492-1G10E4E2 monoclonal antibody to MERS-CoV Spike-492 peptide was measured by ELISA and EC.sub.50 value was analyzed with Sigma Plot program. (D-F) MERS-CoV-infected and non-infected Vero cells lysates were treated with PBS (−) or PNGase F (+). The lysates were immunoblotted with 492-1G10E4E2 monoclonal antibody (D). Western blot analysis using 492-1G10E4E2 monoclonal antibody was performed following the immunoprecipitation of lysates with 492-1G10E4E2 monoclonal antibody (E). Lysates were subjected to western blotting with an anti-β actin antibody as control (F).

[0066] FIG. 6. The cDNA sequences for variable domains of heavy and light chains isolated from the hybridoma cell clone 492-1G10E4E2. (A) Sequence of the heavy chain variable domain. (B) Sequence of the light chain variable domain Predicted amino acid sequences are indicated under the cDNA sequences.

[0067] FIG. 7. The cDNA sequences for variable domains of heavy and light chains isolated from the hybridoma cell clone 506-2G10G5. (A) Sequence of the heavy chain variable domain. (B) Sequence of the light chain variable domain Predicted amino acid sequences are indicated under the cDNA sequences.

[0068] FIG. 8. Immunofluorescence assay and confocal microscopy for the detection of MERS-CoV-infected cells with Spike-492 (L506F) monoclonal antibody. (A) Indirect immunofluorescence assay. Mixture of MERS-CoV-infected and non-infected Vero cells were stained with 506-2G10G5 monoclonal antibody or normal mouse IgG, followed by incubation with Alexa488-conjugated goat anti-mouse IgG antibody (green). Cells were counterstained with Hoechst 33258 for nuclear staining (blue) Images were taken using fluorescence microscope. (B) Confocal microscopy. Vero cells were infected with MERS-CoV for 2 days. The cells were stained with 506-2G10G5 monoclonal antibody or normal mouse IgG and then incubated with Alexa488-conjugated goat anti-mouse IgG antibody (green). Nuclei were stained using Hoechst 33258 (blue). Scale bar, 10 μm.

[0069] FIG. 9. Immunofluorescence assay and confocal microscopy for the detection of MERS-CoV-infected cells with 492-1G10E4E2 monoclonal antibody. (A) Indirect immunofluorescence assay. Mixture of MERS-CoV-infected and non-infected Vero cells were stained with 492-1G10E4E2 monoclonal antibody or normal mouse IgG, followed by incubation with Alexa488-conjugated goat anti-mouse IgG antibody (green). Cells were counterstained with Hoechst 33258 for nuclear staining (blue) Images were taken using fluorescence microscope. (B) Confocal microscopy. Vero cells were infected with MERS-CoV for 2 days. The cells were stained with 492-1G10E4E2 monoclonal antibody or normal mouse IgG and then incubated with Alexa488-conjugated goat anti-mouse IgG antibody (green). Nuclei were stained using Hoechst 33258 (blue). Scale bar, 10 μm.

[0070] FIG. 10. Inhibition of MERS-CoV infection by 506-2G10G5 and 492-1G10E4E2 monoclonal antibody. MERS-CoVirus were pre-incubated with two fold serial diluted normal mouse IgG, 492-1G10E4E2 monoclonal antibody or 506-2G10G5 for 30 min at 37° C. The virus-antibody mixture was added to Vero cells and incubated for 1 h. After incubation medium was replaced with DMEM/F12 containing 0.6% oxoid agar. The plaques were stained with crystal violet following 4 days after infection. (A) A representative picture showing plaque reduction assay. (B) Quantification of plaque reduction assay against MERS-CoV after treatment with 100 μg/well to 0 μg/well of normal mouse IgG or 492-1G10E4E2 or 506-2G10G5 monoclonal antibody.

MODE FOR INVENTION

[0071] Hereinafter, the embodiments of the present invention will be described in detail with reference to the attached exemplary drawings, as such an example, a person skilled in the art to which the present invention pertains may be implemented in various different forms, it is not limited to the embodiment described here.

[0072] Vero cells, the African green monkey kidney cells, were obtained from the American Type Culture Collection (ATCC, Manassas, Va., USA). Dulbecco's modified Eagle's medium (DMEM) purchased from Life Technologies (Thermo Fisher Scientific, Waltham, Mass., USA) with supplementation of 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 25 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin was used in culture of Vero cells. The cells were incubated in an atmosphere of 5% CO.sub.2 and 95% air at 37° C. MERS-CoV/KOR/KNIH/002_05_2015 was obtained from the Korea Centers for Disease Control and Prevention (Permission No. 1-001-MER-IS-2015001).

Example 1: B Cell Epitope Peptides Preparation

[0073] The selection, analysis and synthesis of a B cell epitopes peptide of MERS-CoV S protein was performed as described previously (Park B K, Lee S I, Bae J-Y et al (2018) Int J Pept Res Ther https://doi.org/10.1007/s10989-018-9731-8). The B cell epitope peptide sequences for the S protein of MERS-CoV are selected as Spike-492 (.sup.492TKPLKYSYINKCSRLLSDDRTEVPQ.sup.516: SEQ ID NO:9) from MERS-CV strain (MERS-CoV/KOR/KNIH/002_05_2015 (GI: 829021049)) and Spike-492 (L506F) (.sup.492TKPLKYSYINKCSRFLSDDRTEVPQ.sup.516: SEQ ID NO:10) from MERS-CV strain (Spike glycoprotein universal sequence (GI: 510785803)), and synthesized with an automated peptide synthesizer (Peptron III-R24, Peptron, Daejeon, Korea). The complex of B cell epitope peptide and CpG-DNA (MB-ODN 4531 (0)) co-encapsulated in the DOPE: CHEMS (named as Lipoplex (0)) were prepared.

Example 2: Mice Immunization

[0074] 4-week-old BALB/c (H-2.sup.b) female mice were purchased from Nara-Biotec (Seoul, Korea). The mice were maintained at animal facility Hallym University under specific pathogen-free conditions. All the experiments involving animals were carried out with approval from the Institutional Animal Care and Use Committee of Hallym University (Hallym2016-51). The mice were intraperitoneally immunized thrice at 10-day intervals with 200 μl of Spike-492 peptide (50 μg) or Spike-492 (L506F) peptide (50 μg) and Lipoplex (0) complex.

Example 3: Production of Mouse Anti-MERS-CoV S Protein Monoclonal Antibody

[0075] In accordance with standard hybridoma technique, hybridoma cells were selected to produce anti-Spike-492-specific monoclonal antibody (492-1G10E4E2) and anti-Spike-492(L506F)-specific monoclonal antibody (506-2G10G5) [Wu G, Kim D, Kim J N et al (2018) Theranostics 8, 78-91; W. M. Yokoyama, M. Christensen, G. D. Santos, et al., Curr. Protoc. Immunol., Chapter 2(2006), Unit 2.51. On three occasions at 10 day intervals, BALB/c mice were injected i.p. with MERS-Spike-492 peptide (50 μg) (or Spike-492(L506F) peptide) and MB-ODN 4531(O) (50 μg) encapsulated in DOPE:CHEMS complex. The spleens from the immunized mice were used for fusion in accordance with standard hybridoma technique. Hybridoma clone in HAT medium and HT medium were selected by a standard limiting dilution protocol to obtain clonal cell population. To obtain the ascites, the selected hybridoma clones (492-1G10E4E2 clone and 506-2G10G5) were injected in peritoneal cavity of BALB/c mouse. Anti-Spike-492-specific monoclonal antibody (492-1G10E4E2) and anti-Spike-492(L506F)-specific monoclonal antibody (506-2G10G5) were purified from the ascites fluid by protein-A column chromatography. To determine the isotype of the monoclonal antibodies, an isotyping kit (Southern Biotechnology Associates Inc, Birmingham, USA) was used.

Example 4: ELISA Assay

[0076] To measure the epitope peptide-specific antibody titer, 96-well immunoplates (Thermo Fisher Scientific) were coated with 5 μg/well of MERS-CoV Spike-492 or Spike-492 (L506F) peptides and incubated at 4° C. overnight, washed with PBST (PBS supplemented with 0.05% Tween-20) and blocked with PBST containing 1% bovine serum albumin (BSA). The mice sera obtained by retro orbital bleeding were serially diluted at 1:3 ratio and added to wells of each plate followed by incubation for 2 h at room temperature. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch laboratories, West Grove, Pa., USA) was used as secondary antibody for 1 h. After washing with PBST, tetramethylbenidine (TMB) peroxidase substrate (KPL, SeraCare, Milford, Mass., USA) was treated and reactions were stopped with TMB-stop solution (KPL). The absorbance was read using Spectra Max 250 microplate reader (Molecular Devices, San Jose, Calif., USA) at 450 nm. For the identification of isotype of the monoclonal antibody, HRP-conjugated anti-mouse IgG (each isotype) antibody (Southern Biotech, Birmingham, Ala., USA) was used. For detection of cross-reactivity, 96-well immunoplates were coated with MERS-CoV Spike-492 or Spike-492 (L506F) peptides as described above. The coated wells were incubated with either the 492-1G10E4E2 monoclonal antibody or 506-2G10G5 monoclonal antibody for 2 h before the secondary antibody incubation.

Example 5: Affinity Constant Measurement by ELISA

[0077] To measure the binding affinity of the Anti-Spike-492-specific monoclonal antibody (492-1G10E4E2) and anti-Spike-492(L506F)-specific monoclonal antibody (506-2G10G5), 5 μg/well of Spike-492 and Spike-492 (L506F) epitope peptide was coated on 96-well immunoplates and then blocked with PBST containing 1% BSA. The monoclonal antibody was added to the each plate with serial 1:5 dilutions in PBST and then incubated for 2 h at room temperature. After washing with PBST, anti-IgG antibody conjugated with horseradish peroxidase was added to the each plate. The amounts of antibody in plates were determined by developing with tetramethylbenzidine (TMB) peroxidase substrate (KPL, SeraCare, Milford, Mass., USA). The absorbance was evaluated with the Spectra Max 250 microplate reader (Molecular Devices, San Jose, Calif., USA) at 405 nm and the calculated with the SigmaPlot program to determine the EC.sub.50 value.

Example 6: Western Blotting and Immunoprecipitation

[0078] MERS-CoV-infected Vero cell lysates were run on SDS-PAGE and subsequently transferred onto a nitrocellulose membrane. The membranes were blocked with 3% BSA in PBST. Then, the membrane were incubated with MERS-CoV Spike-492 monoclonal antibody or Spike-492 (L506F) monoclonal antibody, washed with PBST and treated with HRP-conjugated goat anti-mouse IgG antibody. The membrane was detected with chemiluminescence solution and picturized with ChemiDoc (Bio-Rad, Herculed, Calif., USA). For immunoprecipitation, virus infected-Vero cell lysates were incubated with MERS-CoV Spike-492 monoclonal antibody or Spike-492 (L506F) monoclonal antibody overnight at 4° C. and proteins (S protein of MERS-CoV) were precipitated with Protein-A beads (Repligen, Waltham, Mass., USA) for 1 h. The immunoprecipitated proteins were analyzed using MERS-CoV Spike-492 monoclonal antibody or Spike-492 (L506F) monoclonal antibody by western blotting.

Example 7: Deglycosylation Assay

[0079] MERS-CoV-infected and non-infected Vero cells were lysed with lysis buffer (0.5% SDS and 1% β-mercaptoethanol) and boiled at 100° C. for 10 min. The lysates were then subjected to peptide-N-glycosidase F (PNGase F) (Elpis Biotech, Daejeon, Korea) treatment at 37° C. for 2 h and boiled at 100° C. for 10 min. After the digestions, the samples were detected with MERS-CoV Spike-492 monoclonal antibody or Spike-492 (L506F) monoclonal antibody by western blot. For immunoprecipitation, the PNGase F digested samples were incubated with MERS-CoV Spike-492 monoclonal antibody or Spike-492 (L506F) monoclonal antibody overnight at 4° C. followed by western blot analysis with MERS-CoV Spike-492 monoclonal antibody or Spike-492 (L506F) monoclonal antibody.

Example 8: Cloning of the Variable Heavy and Light Domains of Anti-MERS CoV S Protein Monoclonal Antibody

[0080] Hybridoma clones (492-1G10E4E2 clone and 506-2G10G5) producing Anti-Spike-492 peptide-specific monoclonal antibody (492-1G10E4E2) and anti-Spike-492(L506F) peptide-specific monoclonal antibody (506-2G10G5) were cultured and isotyped using a mouse monoclonal antibody isotyping kit (Dipstick format, Bibco BRL or Roche, Mannheim, Germany) Total RNAs were extracted from hybridoma cells (492-1G10E4E2 clone and 506-2G10G5) with an RNeasy Mini Kit (Qiagen), and the cDNAs were generated. To clone the sequences for the variable heavy and light domains (V.sub.H and V.sub.L) of anti-Spike-492 peptide-specific monoclonal antibody (492-1G10E4E2), the resultant cDNAs were amplified using Vent polymerase (NEB) with the following primer sets. For heavy chain primers, IGG2A (5′-GGA AGA TCT CTT GAC CAG GCA TCC TAG AGT CA-3″SEQ ID NO:11) and 5′MH2 (5′-CTT CCG GAA TTC SAR GTN MAG CTG SAG SAG TCW GG-3″SEQ ID NO:12) were used. For kappa chain primers, 3′Kc (5′-GGT GCA TGC GGA TAC AGT TGG TGC AGC ATC-3′ SEQ ID NO:13) and 5′Mk (5′-GG GAG CTC GAY ATT GTG MTS ACM CAR WCT MCA-3″SEQ ID NO:14) were used. To clone the sequences for the variable heavy and light domains (V.sub.H and VL) of anti-Spike-492(L506F) peptide-specific monoclonal antibody (506-2G10G5), the resultant cDNAs were amplified using Vent polymerase (NEB) with the following primer sets. For heavy chain primers, IGG1 (5′-GGA AGA TCT ATA GAC AGA TGG GGG TGT CGT TTT GGC-3″SEQ ID NO:11) and 5′MH2 (5′-CTT CCG GAA TTC SAR GTN MAG CTG SAG SAG TCW GG-3″SEQ ID NO:12) were used. For kappa chain primers, 3′Kc (5′-GGT GCA TGC GGA TAC AGT TGG TGC AGC ATC-3″SEQ ID NO:13) and 5′Mk (5′-GG GAG CTC GAY ATT GTG MTS ACM CAR WCT MCA-3″SEQ ID NO:14) were used. The standard PCR reaction was performed for 25 cycles. The PCR products were directly ligated into the pGEM-T easy vector (Promega). Cloned mouse Ig inserts were analyzed by DNA sequencing.

Example 9: Indirect Immunofluorescence Assay and Confocal Images

[0081] For analysis of indirect immunofluorescence assay, mixture of MERS-CoV-infected and non-infected Vero cells at the ratio of 3:1 were seeded onto slide glasses. The cells were then fixed with acetone and incubated with normal mouse IgG or MERS-CoV Spike-492 monoclonal antibody at 37° C. for 2 h. The samples were further incubated with Alexa Flour 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific, Waltham, Mass., USA). Finally, the samples were mounted and analyzed using fluorescence microscope (1×70, Olympus, Tokyo, Japan) (30, 31). To visualize confocal microcopy, Vero cells (5×10.sup.4) were seeded onto cover glass in 12 well plate and infected with MERS-CoV (0.1 MOI). After two days, the infected cells were fixed with 4% paraformaldehyde and subsequently blocked with 1% BSA and 0.1% triton X-100 in PBS. The slides were incubated in presence of MERS-CoV Spike-492 monoclonal antibody for 2 h, washed and then incubated with Alexa Flour 488-conjugated goat anti-mouse IgG antibody for 1 h. Hoechst 33258 (Thermo Fisher Scientific) was utilized to stain nuclei. The slides were examined by Carl Zeiss LSM710 (Carl Zeiss, Oberkochen, DE).

Example 10: Plaque Reduction Assay

[0082] 6×10.sup.5 Vero cells/well were plated on six-well plates (Thermo Fisher Scientific) and cultured for 12 h. Prior to infection, MERS-CoVirus were pre-incubated with two fold serial diluted normal mouse IgG, 492-1G10E4E2 monoclonal antibody or 506-2G10G5 for 30 min at 37° C. The virus-antibody mixture was added to Vero cells with 500 μl of PBS. After 1 h incubation, supernatant was removed and 3 ml of DMEM/F12 medium (Thermo Fisher Scientific) containing 0.6% oxoid agar were added. The plaques formed in each wells were stained with crystal violet following 4 days after infection. The plaques were counted and the percentage was calculated.

[0083] Analysis of B Cell Epitope and Production of Antibody Targeting S Protein Epitope of MERS-CoV

[0084] B cell epitope identification and selection is one of the important considerations in epitope-based antibody production. Hence, the explicit B cell epitope amino acid sequence of MERS-CoV S protein was predicted utilizing Immune Epitope Database and Analysis Resources (IEDB) tool on the basis of epitope prediction, surface accessibility and antigenicity scale. Since RBD domain within the S protein are responsible for binding to host, Spike-492 and Spike-492 (L506F) peptide sequences with 492-516 amino acids within the RBD domain of MERS-CoV S protein was selected and synthesized (FIGS. 1A and B). For the determination of the efficiency as a B cell epitope, each peptides and Lipoplex (0) complex was formulated and then immunized into the BALB/c mice. In order to screen the antibody titers, ELISA was done from the serum of immunized mice. Both of the peptides were able to induce production of the peptide-specific IgGs (FIG. 1C). Thus, immunogenic epitope peptide were successfully designed and produced.

[0085] Production of Monoclonal Antibody Specific to Spike-492 (L506F) or Spike-492 Epitope of MERS-CoV

[0086] To produce MERS-CoV S protein epitope-specific monoclonal antibody, splenocytes were collected from a complex of MERS-CoV Spike-492 epitope peptide (or Spike-492 (L506F) epitope peptide) and CpG-DNA co-encapsulated in liposome (DOPE;CHEMS)-immunized mice. The mouse splenocytes were fused with SP2/0, and Spike-492 (L506F) epitope peptide-specific antibody producing 506-2G10G5 clone was selected by HAT and HT supplements (FIG. 2). The mouse splenocytes were fused with SP2/0, and Spike-492 epitope peptide-specific antibody producing 492-1G10E4E2 clone was selected by HAT and HT supplements (FIG. 3).

[0087] Characterization of Monoclonal Antibody Specific to Spike-492 (L506F) or Spike-492 Epitope of MERS-CoV

[0088] To obtain monoclonal antibody in large scale, 506-2G10G5 clone or 492-1G10E4E2 clone was injected into mouse peritoneal cavity for production of ascites. Ascites fluid were collected from mice injected with 506-2G10G5 clone or 492-1G10E4E2 clone and purified (FIG. 4A and FIG. 5A). Next, isotype classification of 506-2G10G5 or 492-1G10E4E2 monoclonal antibody was performed using ELISA and found to be IgG1 and IgG2a, respectively (FIG. 4B and FIG. 5B). The binding affinity of the monoclonal antibody targeting the Spike-492 (L506F) peptide or Spike-492 was determined with ELISA and an EC50 value of the monoclonal antibody 506-2G10G5 and 492-1G10E4E2 was found to have 178 μm and 3.3 nM, respectively (FIG. 4C and FIG. 5C).

[0089] Detection of MERS-CoV S Protein by the Monoclonal Antibody Specific to Spike-492 (L506F) or Spike-492 Epitope of MERS-CoV

[0090] To further characterize the monoclonal antibody 506-2G10G5 or 492-1G10E4E2 recognizes S protein of MERS-CoV, western blotting and immunoprecipitation with MERS-CoV infected and non-infected Vero cells was performed. Western blotting results (FIG. 4D, FIG. 5D) showed that both monoclonal antibodies detected band specific to S protein in MERS-CoV-infected Vero cells but no band was observed in non-infected Vero cells. Treatment with peptide-N-glycosidase (PNGase F) resulted in the reduction of apparent molecular weight of S protein band in comparison to untreated sample (FIG. 4D and FIG. 5D) suggesting that both monoclonal antibodies could recognize S protein in its glycosylated and de-glycosylated form. Furthermore, immunoprecipitation was done to evaluate if the monoclonal antibodies could recognize the S protein in its native form. Here, we found that both monoclonal antibodies immunoprecipitated the native form of S protein from MERS-CoV-infected Vero cells lysates (FIG. 4F and FIG. 5E). To further determine the cross-reactivity of 506-2G10G5 and 492-1G10E4E2 monoclonal antibodies with each corresponding epitope, ELISA was performed. 506-2G10G5 monoclonal antibody showed remarkable cross-reactivity to Spike-492 peptides than shown by 492-1G10E4E2 monoclonal antibody to Spike-492 (L506F) (FIGS. 4H and I). Therefore, both the antibodies displayed specific binding to S protein of MERS-CoV.

[0091] Cloning of the Variable Domains of Anti-Spike-492 (L506F) Peptide-Specific and Anti-Spike-492 Peptide-Specific Monoclonal Antibody

[0092] The cDNA sequences encoding the variable domains of heavy and light chains (V.sub.H and V.sub.L) were cloned from hybridoma cells (506-2G10G5) producing anti-Spike-492 (L506F) peptide-specific monoclonal antibody using common heavy and light chain primers. The sequences confirmed by DNA sequencing are shown in FIG. 6. The cDNA sequences encoding the variable domains of heavy and light chains (V.sub.H and V.sub.L) were cloned from hybridoma cells (492-1G10E4E2) producing anti-Spike-492 peptide-specific monoclonal antibody using common heavy and light chain primers. The sequences confirmed by DNA sequencing are shown in FIG. 7. The sequences were analyzed for homology to known sequences by protein BLAST program. The cDNAs encoding variable domains of 506-2G10G5 and 492-1G10E4E2 heavy and light chains exhibited about 80˜95% and 93˜98% homology with the sequences of reported immunoglobulin variable heavy and light domains, respectively.

[0093] Reactivity of 506-2G10G5 or 492-1G10E4E2 Monoclonal Antibody to S Protein in the MERS-CoV-Infected Cells

[0094] We further analyzed breadth of reactivity of 506-2G10G5 or 492-1G10E4E2 monoclonal antibodies with indirect immunofluorescence assay (IFA). Fluorescence microscopy displayed the strong fluorescence signal in the virus infected cells incubated with both monoclonal antibodies, whereas no fluorescence was observed when with normal mouse IgG (FIG. 8A and FIG. 9A). To further substantiate the specificity of both monoclonal antibodies to S protein of MERS-CoV, confocal microscopy was performed. MERS-CoV-infected or non-infected Vero cells were stained with normal mouse IgG or 506-2G10G5 or 492-1G10E4E2 monoclonal antibody. Images of confocal microscopy clearly showed the fluorescence signal within the cytosolic region of the MERS-CoV infected cells stained with 506-2G10G5 or 492-1G10E4E2 monoclonal antibody. No staining was observed in cells incubated with normal mouse IgG (FIG. 8B and FIG. 9B). These results demonstrated the specificity of both monoclonal antibodies in efficient detection of S protein of MERS-CoV in MERS-CoV-infected cells.

[0095] 506-2G10G5 Monoclonal Antibody Inhibited MERS-CoV Infection in Vero Cells

[0096] We investigated the inhibitory activities of both the monoclonal antibodies against MERS-CoV in plaque reduction assay. In this assay, both monoclonal antibodies inhibited plaque formation when compared to normal mouse IgG in concentration dependent manner. However, better inhibition of plaque formation was observed when treated with 506-2G10G5 monoclonal antibody in comparison to 492-1G10E4E2 monoclonal antibody in concentration dependent manner (FIGS. 10A and B). Thus, the results demonstrated the efficacy of 506-2G10G5 monoclonal antibody, signifying its potential therapeutic applications against MERS-CoV infection.