key: cord-1048844-n7ehxulc authors: Park, Byoung Kwon; Maharjan, Sony; Lee, Su In; Kim, Jinsoo; Bae, Joon-Yong; Park, Man-Seong; Kwon, Hyung-Joo title: Generation and characterization of a monoclonal antibody against MERS-CoV targeting the spike protein using a synthetic peptide epitope-CpG-DNA-liposome complex date: 2019-06-30 journal: BMB Rep DOI: 10.5483/bmbrep.2019.52.6.185 sha: 077a70059a89059bace1150d779cc911a2229c60 doc_id: 1048844 cord_uid: n7ehxulc Middle East respiratory syndrome coronavirus (MERS-CoV) uses the spike (S) glycoprotein to recognize and enter target cells. In this study, we selected two epitope peptide sequences within the receptor binding domain (RBD) of the MERS-CoV S protein. We used a complex consisting of the epitope peptide of the MERS-CoV S protein and CpG-DNA encapsulated in liposome complex to immunize mice, and produced the monoclonal antibodies 506-2G10G5 and 492-1G10E4E2. The western blotting data showed that both monoclonal antibodies detected the S protein and immunoprecipitated the native form of the S protein. Indirect immunofluorescence and confocal analysis suggested strong reactivity of the antibodies towards the S protein of MERS-CoV virus infected Vero cells. Furthermore, the 506-2G10G5 monoclonal antibody significantly reduced plaque formation in MERS-CoV infected Vero cells compared to normal mouse IgG and 492-1G10E4E2. Thus, we successfully produced a monoclonal antibody directed against the RBD domain of the S protein which could be used in the development of diagnostics and therapeutic applications in the future. Middle East respiratory syndrome coronavirus (MERS-CoV) is a fatal zoonotic pathogen that causes acute respiratory disease, MERS, in humans (1) (2) (3) . MERS-CoV, a lineage C betacoronavirus, first appeared in Saudi Arabia in June 2012 (3) (4) (5) . Since then, the cases of MERS-CoV human infection have been reported to the World Health Organization (WHO) from 27 countries, mostly from Middle East countries (6) . Globally, as of May 2018, MERS-CoV has caused a total of 2,220 cases of infections with at least 790 individual deaths (mortality rate: 35.6%), with majority being reported from Saudi Arabia (1844 cases with 716 deaths) (6) . Another major outbreak outside of the Middle East took place in South Korea in 2015 resulting in 186 cases with 36 fatalities (7, 8) . An elevation in the pathogenicity of MERS-CoV and absence of effective vaccines or therapeutics against the virus could be a risky combination inviting a pandemic in the future. Therefore, ideal and effective therapeutic measures for the prevention and treatment of MERS are urgently required. Previous reports have shown neutralizing monoclonal antibodies as potential candidates for the treatment of numerous diseases including virus infections, immune dysfunction and cancer (2, 9, 10) . The MERS-CoV genome consists of structural proteins such as the spike (S), membrane (M), envelope (E) and nucleocapsid (N) (2, 11) . The S glycoprotein of MERS-CoV portrays a crucial role in viral attachment and subsequent entrance into host cell (12, 13) . The S glycoprotein is comprised of two subunits: receptor recognizing S1 and membrane fusion S2 (12, 13) . The S1 subunit of MERS-CoV contains the receptor binding domain (RBD) which is responsible for interacting with the cellular receptor dipeptidyl peptidase 4 (DPP4; also called CD26) in the host cell membrane (13, 14) . The heptad repeat (HR) 1 and 2 regions of the S2 subunit mediate fusion between the viral and host cell membranes (12) . Due to the vital antigenic property, the S protein has been focus of the development of therapeutic strategies against MERS (2, 11) . Importantly, antibody production against the RBD has been a functional determinant factor for promising diagnostic and therapeutic targeting (2) . We previously generated antibodies against hepatitis C virus (15), influenza virus (16), respiratory syncytial virus (17) and MERS-CoV M protein (18) by immunization with a complex of a B cell epitope peptide coencapsulated with CpG-DNA in a dioleoyl phosphatidylethanolamine (DOPE): cholesterol hemihttp://bmbreports.org ssuccinate (CHEMS) complex (named as Lipoplex (O)). In this study, we selected novel B cell epitope peptide sequences, Spike-492 and Spike-492 (L506F), from the MERS-CoV S protein-RBD of representative the South Korean and Saudi Arabian strains, respectively. The B cell epitope peptide sequence, Spike-492 (L506F), has a single amino acid substitution of phenylalanine for leucine at residue 506. Herein, we generated monoclonal antibodies, 506-2G10G5 and 492-1G10E4E2, specific against the S protein of MERS-CoV by immunizing mice with a complex of the B cell epitope peptide and Lipoplex (O). Our data show the potential of the 506-2G10G5 monoclonal antibody for diagnostic and therapeutic use against emerging MERS-CoV infection. B cell epitope identification and selection are one of the most important factors in epitope-based antibody production. Hence, candidate B cell epitopes from the amino acid sequence of the MERS-CoV S protein were predicted using the Immune Epitope Database and Analysis Resources (IEDB) tool based on the epitope prediction, surface accessibility and antigenicity scale (http://tools.iedb.org/bcell). Because the RBD domain within the S protein is responsible for binding to the host cell (2, 18) , the Spike-492 and Spike-492 (L506F) peptide sequences corresponding to the 492th-516th amino acid residues within the RBD domain of MERS-CoV S protein were selected and synthesized ( Fig. 1A and B) . To determine the efficiency of the peptides as a B cell epitope, each peptide and the Lipoplex (O) complex were formulated and then immunized into the BALB/c mice. To screen the antibody titers, ELISA was performed using the sera of the immunized mice. Both of the peptides induced a robust production of peptide-specific IgGs (Fig. 1C) . Thus, the immunogenic epitope peptides were successfully designed and produced. http://bmbreports.org BMB Reports To further characterize whether the monoclonal antibody 506-2G10G5 or 492-1G10E4E2 recognizes the S protein of MERS-CoV, western blotting and immunoprecipitation were performed with MERS-CoV-infected and non-infected Vero cells. The western blotting results (Fig. 2D, Fig. S1D and Fig. S2A ) show that both monoclonal antibodies detected a protein band corresponding to the S protein in the molecular weight range in the MERS-CoV-infected Vero cells, however, no band was observed in the non-infected Vero cells. Treatment with peptide-N-glycosidase (PNGase F) resulted in the reduction of the apparent molecular weight of the S protein band compared with the untreated sample ( Fig. 2D and Fig. S1D ) suggesting that both monoclonal antibodies could recognize the 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. Both monoclonal antibodies immunoprecipitated the native form of the S protein from MERS-CoV-infected Vero cells lysates (Fig. S1E and Fig. S2B ). In addition, both monoclonal antibodies recognized the de-glycosylated protein band in the PNGase F treated immunoprecipitated lysate ( Fig. 2E and Fig. S1E ). To further determine the cross-reactivity of the 506-2G10G5 and 492-1G10E4E2 monoclonal antibodies with each corresponding epitope, ELISA was performed. The 506-2G10G5 monoclonal antibody showed remarkable cross-reactivity to Spike-492 peptides which was higher than the result shown by the 492-1G10E4E2 monoclonal antibody to Spike-492 (L506F) (Fig. 2G and H) . Therefore, both of the antibodies exhibited specific binding to the MERS-CoV S protein. To further confirm the reactivity of the 506-2G10G5 or 492-1G10E4E2 monoclonal antibody, we performed indirect immunofluorescence assay (IFA) in non-infected and MERS-CoV-infected Vero cells. Fluorescence microscopy showed the strong fluorescence signal in the virus infected cells incubated with either of the monoclonal antibodies, whereas no fluorescence was observed with the normal mouse IgG or secondary antibody control ( Fig. 3A and Fig. S3A ) normal mouse IgG or secondary antibody control ( Fig. 3B and Fig. S3B ). These results demonstrate that both monoclonal antibodies efficiently recognize the MERS-CoV S protein in MERS-CoV-infected cells. Given the specificity and binding affinity of the 506-2G10G5 or 492-1G10E4E2 monoclonal antibody towards the S protein of MERS-CoV, we investigated the inhibitory activities of both monoclonal antibodies against MERS-CoV infecting using a plaque reduction assay. In this assay, both monoclonal antibodies inhibited plaque formation when compared to the nomal mouse IgG in a concentration-dependent manner. However, better inhibition of plaque formation was observed when treated with the 506-2G10G5 monoclonal antibody compared with the 492-1G10E4E2 monoclonal antibody ( Fig. 4A and B) . Thus, the results demonstrate the efficacy of the 506-2G10G5 monoclonal antibody, signifying its potential therapeutic applications against MERS-CoV infection. It has been demonstrated that MERS-CoV infection can be severe health hazard to mankind worldwide (1, 3). As of today, effective therapeutics against MERS-CoV infection are unavailable which contributes to its high mortality rate (2) . Clearly, an effective approach for neutralizing the viral infection is needed. In recent years, there has been an increase in the development of monoclonal antibodies for the effective treatment of various infectious viruses such as rabies, HIV, SARS-CoV, influenza, and Ebola (9, 19) . Here, we produced and characterized the specificity and functionality of a monoclonal antibody against the S protein of MERS-CoV. In previous studies, we successfully screened B cell epitope peptides and produced antibodies based on our own strategy using epitopes and CpG-DNA encapsulated with a DOPE: CHEMS complex as an adjuvant (20) . In this study, we report generation and characterization of monoclonal antibodies against two selected B cell epitopes in the MERS-CoV S protein, Spike-492 and Spike-492 (L506F) peptides. Our data demonstrate that the hybridoma clones 492-1G10E4E2 and 506-2G10G5, obtained from mice immunized with the Spike-492 or Spike-492 (L506F) peptides and the Lipoplex (O) complex, produce antibodies specific to the MERS-CoV S protein and reciprocally cross-reactive to the epitopes representing the South Korean strains and the Saudi Arabian strains. The efficacy of the antibodies were evaluated based on the binding affinity and neutralizing activity. Of the two monoclonal antibodies, 506-2G10G5 had a higher binding affinity to the Spike-492 and Spike-492 (L506F) peptides than that of the 492-1G10E4E2. In addition, 506-2G10G5 exhibited better neutralization activity than that of 492-1G10E4E2 demonstrated by the plaque reduction assay. Considering that the 506-2G10G5 monoclonal antibody is raised against the peptide sequence within the RBD domain, the antibody could have a potential therapeutic effect in addition to its diagnostic value. However, further experimental evidence is required to determine the therapeutic function of the antibody. Vero cells, African green monkey kidney cells, were obtained from the American Type Culture Collection (ATCC Inc.). Dulbecco's modified Eagle's medium (DMEM) purchased from Life Technologies (Thermo Fisher Scientific Co.) with supplementation of 10% fetal bovine serum (FBS, Thermo Fisher Scientific Co.), 25 mM HEPES, 100 U/ml penicillin and 100 g/ml streptomycin was used in the culture of Vero cells. The cells were incubated in an atmosphere of 5% CO2 and 95% air at The selection, analysis and synthesis of the B cell epitope peptides of the MERS-CoV S protein were performed as described previously (21) . The B cell epitope peptide sequences for the MERS-CoV S protein were selected as Spike-492 ( )) were prepared as previously described (17) . Four-week-old BALB/c (H-2 b ) female mice were purchased from Nara-Biotec Co. Ltd. The mice were maintained at an animal facility under specific pathogen-free conditions. All the experiments involving the animals were carried out with the approval from the Institutional Animal Care and Use Committee of Hallym University (Hallym2016-51) . The mice were intraperitoneally immunized three times at 10-day intervals with 200 l of the Spike-492 peptide (50 g) or Spike-492 (L506F) peptide (50 g) and the Lipoplex (O) complex (17, 22, 23) . The monoclonal antibodies were produced in accordance with standard method as previously described (24, 25) . Splenocytes isolated from the mice were fused with mouse SP2/0 cells (ATCC Inc.) in presence of polyethylene glycol solution (PEG, Sigma Chemical Co.). To acquire positive clones producing the MERS-CoV Spike-492-or Spike-492 (L506F)-specific monoclonal antibody, HAT medium (Sigma Chemical Co.) and HT medium (Sigma Chemical Co.) were used for the cultures and for the selection of hybridoma cells. For a large-scale production of the monoclonal antibodies, ascites fluid was collected from mice injected intraperitoneally with the selected hybridomas and purified by protein A column chromatography as previously described (17) . To measure the epitope peptide-specific antibody titer, 96-well immunoplates (Thermo Fisher Scientific Co.) were coated with 5 g/well of the MERS-CoV Spike-492 or Spike-492 (L506F) peptides. The mice sera were obtained by retro orbital bleeding and analyzed as previously described (22) . To identify the isotype of the monoclonal antibody, the HRPconjugated anti-mouse IgG (each isotype) antibody (Southern Biotechnology Associates Inc.) was used. For detection of cross-reactivity, 96-well immunoplates were coated with the MERS-CoV Spike-492 or Spike-492 (L506F) peptides and incubated with either 492-1G10E4E2 or 506-2G10G5 monoclonal antibody for 2 h before incubation with the secondary antibody. The absorbance was evaluated with the Spectra Max 250 microplate reader (Molecular Devices Co.) at 450 nm and then calculated with the SigmaPlot program to determine the affinity constant (EC50 value) as described previously (17) . MERS-CoV-infected Vero cell lysates were run on SDS-PAGE and subsequently transferred onto a nitrocellulose membrane as described previously (26, 27) , and detailed information is provided in the Supplementary Material. MERS-CoV-infected and non-infected Vero cells were lysed with lysis buffer (0.5% SDS and 1% -mercaptoethanol) and For analysis of the indirect immunofluorescence assay, a mixture of MERS-CoV-infected and non-infected Vero cells at a ratio of 3:1 was seeded onto slide glasses. The cells were then fixed with acetone and incubated with the normal mouse IgG, 492-1G10E4E2 or 506-2G10G5 monoclonal antibody at 37 o C for 2 h. The samples were further incubated with Alexa Flour 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific Co.). Finally, the samples were mounted and analyzed using a fluorescence microscope (1X70, Olympus Co.) (28, 29) . To visualize the confocal microcopy, Vero cells (5 × 10 4 ) were seeded onto cover glasses in 12 well plates 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 the presence of the 492-1G10E4E2 or 506-2G10G5 monoclonal antibody for 2 h, washed and then incubated with the Alexa Flour 488conjugated goat anti-mouse IgG antibody for 1 h. Hoechst 33258 (Thermo Fisher Scientific) was used to stain the nuclei. The slides were examined by Carl Zeiss LSM710 (Carl Zeiss Co. Ltd.). 6 × 10 5 Vero cells/ well were plated on six-well plates (Thermo Fisher Scientific Co.) and cultured for 12 h. Prior to infection, the MERS-CoVirus was pre-incubated with two fold serial diluted normal mouse IgG, 492-1G10E4E2 or 506-2G10G5 monoclonal antibodies for 30 min at 37 o C. The virus-antibody mixture was added to the Vero cells with 500 l of PBS. After 1 h incubation, the supernatant was removed and 3 ml of DMEM/F12 medium (Thermo Fisher Scientific Co.) containing 0.6% oxoid agar were added. The plaques formed in each wells were stained with crystal violet (20) 4 days after infection. The plaques were counted and the percentage was calculated. Coronavirusesdrug discovery and therapeutic options MERS-CoV spike protein: a key target for antivirals MERS coronavirus: diagnostics, epidemiology and transmission Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia Middle East respiratory syndrome coronavirus (MERS-CoV): animal to human interaction World Health Organization, Middle East respiratory syndrome coronavirus (MERS-CoV) MERS outbreak in Korea: hospital-tohospital transmission Middle East respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control Converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play Monoclonal antibodies in cancer therapy MERS-CoV spike protein: Targets for vaccines and therapeutics Structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the S protein of Middle East respiratory syndrome coronavirus Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4 Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC Activation of Toll-like receptor 9 and production of epitope specific antibody by liposome-encapsulated CpG-DNA Immunization with a hemagglutinin-derived synthetic peptide formulated with a CpG-DNA-liposome complex induced protection against lethal influenza virus infection in mice Monoclonal antibodies against the human respiratory syncytial virus obtained by immunization with epitope peptides and CpG-DNA-liposome complex Production of a Monoclonal Antibody targeting the M protein of MERS-CoV for detection of MERS-CoV using a synthetic peptide epitope formulated with a CpG-DNA-liposome complex Antibody therapies for the prevention and treatment of viral infections Production of an anti-TM4SF5 monoclonal antibody and its application in the detection of TM4SF5 as a possible marker of a poor prognosis in colorectal cancer A Novel monoclonal antibody targets mucin1 and attenuates growth in pancreatic cancer model Production of anti-c-Myc monoclonal antibody inhibiting DNA binding of c-Myc and max dimer by epitope peptide-CpG-DNA-liposome complex without carriers A Mucin1 C-terminal subunit-directed monoclonal antibody targets overexpressed Mucin1 in breast cancer Production of antibodies with peptide-CpG-DNA-liposome complex without carriers Production of monoclonal antibodies Gomisin G inhibits the growth of triple-negative breast cancer cells by suppressing AKT phosphorylation and decreasing cyclin D1 Gomisin G suppresses the growth of colon cancer cells by attenuation of AKT phosphorylation and arrest of cell cycle progression Effects of PEP-1-FK506BP on cyst formation in polycystic kidney disease Phosphorylation of p53 at threonine 155 is required for Jab1-mediated nuclear export of p53 This work was supported by grants from the National Research The authors have no conflicting interests.