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. 2020 Mar:278:197863.
doi: 10.1016/j.virusres.2020.197863. Epub 2020 Jan 13.

Characterization of novel monoclonal antibodies against MERS-coronavirus spike protein

Junghyun Goo  1 , Yuji Jeong  1 , Young-Shin Park  1 , Eunji Yang  1 , Dae-Im Jung  1 , Semi Rho  1 , Uni Park  2 , Hyeyeong Sung  3 , Pil-Gu Park  4 , Jung-Ah Choi  1 , Sang Hwan Seo  1 , Nam Hyuck Cho  5 , Hyeja Lee  3 , Jae Myun Lee  4 , Jae-Ouk Kim  6 , Manki Song  7
Affiliations

Characterization of novel monoclonal antibodies against MERS-coronavirus spike protein

Junghyun Goo et al. Virus Res. 2020 Mar.

Abstract

Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes severe pulmonary infection, with ∼35 % mortality. Spike glycoprotein (S) of MERS-CoV is a key target for vaccines and therapeutics because S mediates viral entry and membrane-fusion to host cells. Here, four different S subunit proteins, receptor-binding domain (RBD; 358-606 aa), S1 (1-751 aa), S2 (752-1296 aa), and SΔTM (1-1296 aa), were generated using the baculoviral system and immunized in mice to develop neutralizing antibodies. We developed 77 hybridomas and selected five neutralizing mAbs by immunization with SΔTM against MERS-CoV EMC/2012 strain S-pseudotyped lentivirus. However, all five monoclonal antibodies (mAb) did not neutralize the pseudotyped V534A mutation. Additionally, one mAb RBD-14F8 did not show neutralizing activity against pseudoviruses with amino acid substitution of L506 F or D509 G (England1 strain, EMC/2012 L506 F, and EMC/2012 D509 G), and RBD-43E4 mAb could not neutralize the pseudotyped I529 T mutation, while three other neutralizing mAbs showed broad neutralizing activity. This implies that the mutation in residue 506-509, 529, and 534 of S is critical to generate neutralization escape variants of MERS-CoV. Interestingly, all five neutralizing mAbs have binding affinity to RBD, although most mAbs generated by RBD did not have neutralizing activity. Additionally, chimeric antibodies of RBD-14F8 and RBD-43E4 with human Fc and light chain showed neutralizing effect against wild type MERS-CoV KOR/KNIH/002, similar to the original mouse mAbs. Thus, our mAbs can be utilized for the identification of specific mutations of MERS-CoV.

Keywords: Epitope; MERS-CoV; Monoclonal antibody; Neutralization; Neutralizing antibody; Pseudovirus.

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Conflict of interest statement

Declaration of Competing Interest The authors declare they have no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1
Production of SΔTM, S1, S2, and RBD subunit recombinant proteins by baculovirus system. A. Schematic diagram for the domain structure of MERS-CoV Spike (S) protein. B. SDS-PAGE and Coomassie blue staining of purified recombinant S subunit proteins from the insect cell culture supernatant: SΔTM (1–1296 aa), S1 (1–751 aa), S2 (752–1296 aa), and RBD (358–606 aa).
Fig. 2
Fig. 2
Pseudovirus neutralizing activity of hybridomas generated using splenocytes of mice that were immunized with SΔTM, S1, S2, and RBD recombinant protein. A. Distribution of neutralizing activity of 77 hybridomas against MERS-CoV EMC/2012 Spike (S)-pseudotyped lentivirus. Hybridoma culture supernatant was incubated with MERS-CoV EMC/2012 pseudovirions for 60 min at 37 °C and added to 786O cells, incubated for 6 h, replaced with fresh medium, and then analyzed for luciferase-reporter activity 3 days later. The relative luminescence unit (RLU) was converted to % neutralizing activity. B. Neutralizing activity of the selected hybridomas against EMC/2012 (left) and KOR/KNIH/002 (right) strain of pseudovirions. Among 77 hybridomas, selected 15 hybridomas (1 by S1, 11 by SΔTM, and 2 by S2) were tested for their neutralizing activity against indicated pseudovirions. Four ten-fold serial dilutions from 1/5 of hybridoma culture supernatant were used for EMC/2012 pseudovirions, and undiluted culture supernatant was used for KOR/KNIH/002 pseudovirions. C. Binding domain of selected mAbs within S protein. Seven mAbs were purified, and each binding region was assessed using ELISA. The value indicates the OD after coating the 96-well plate with each S subunit antigen.
Fig. 3
Fig. 3
Dose-dependent neutralizing activity of purified mAbs against 11 different MERS-CoV Spike (S)-pseudotyped virions. Each mAb was diluted 2-fold from 50 ng and tested. mAb was incubated with indicated MERS-CoV S-pseudotyped virions for 60 min at 37 °C and added to 786O cells, incubated for 6 h, replaced with fresh medium, and analyzed for luciferase-reporter activity 3 days later. The RLU value was converted to % neutralizing activity. The data is represented as the mean ± standard deviation from at least two independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s test.
Fig. 4
Fig. 4
Dose-response curve of neutralizing activity of mAbs against three pseudoviruses with MERS-CoV RBD identified in 2015 during the Korean MERS outbreak (D510 G, I529 T, and V530 L respectively). Each mAb was diluted 2-fold from 50 ng and tested. mAb was incubated with indicated MERS-CoV S-pseudotyped virions for 60 min at 37 °C and added to 786O cells, incubated for 6 h, replaced with fresh medium, and analyzed for luciferase-reporter activity 3 days later. The RLU value was converted to % neutralizing activity. The data is represented as the mean ± standard deviation from at least two independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s test.
Fig. 5
Fig. 5
Epitope-specific neutralizing activity of chimeric h14F8 and h43E4 against MERS-CoV Spike (S)-pseudotyped virions. A. Fc/light chain-humanized chimeric h14F8 (left) and h43E4 (right) mAb purified from 293 F cell culture supernatant after transfection with relevant DNA plasmids and run on the 4∼20 % SDS-PAGE followed by Coomassie-blue staining. Re, reducing condition; NR, non-reducing condition. B. h14F8 (left) and h43E4 (right) mAb was diluted 4-fold from 12.5 ng and were neutralized against EMC/2012, KOR/KNIH/002, L506 F, D509 G, D510 G, I529 T, and V530 L pseudotyped virions. The luciferase activity of pseudovirus was converted to % neutralizing activity. C. Neutralizing activity of both mouse and chimeric 14F8 and 43E4 against wild type MERS-CoV KOR/KNIH/002 strain was assessed by PRNT. mAbs were diluted 4-fold from 100 ng and were tested. The data is representative of at least two independent experiments.
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References

    1. Chen Y., Lu S., Jia H., Deng Y., Zhou J., Huang B., Yu Y., Lan J., Wang W., Lou Y., Qin K., Tan W. A novel neutralizing monoclonal antibody targeting the N-terminal domain of the MERS-CoV spike protein. Emerg. Microbes Infect. 2017;6(6):e60. - PMC - PubMed
    1. Cho S.Y., Kang J.M., Ha Y.E., Park G.E., Lee J.Y., Ko J.H., Kim J.M., Kang C.I., Jo I.J., Ryu J.G., Choi J.R., Kim S., Huh H.J., Ki C.S., Kang E.S., Peck K.R., Dhong H.J., Song J.H., Chung D.R., Kim Y.J. MERS-CoV outbreak following a single patient exposure in an emergency room in South Korea: an epidemiological outbreak study. Lancet. 2016;388(10048):994–1001. - PMC - PubMed
    1. Dang V.T., Mandakhalikar K.D., Ng O.W., Tan Y.J. A simple methodology for conversion of mouse monoclonal antibody to human-mouse chimeric form. Clin. Dev. Immunol. 2013;2013 - PMC - PubMed
    1. de Groot R.J., Baker S.C., Baric R.S., Brown C.S., Drosten C., Enjuanes L., Fouchier R.A., Galiano M., Gorbalenya A.E., Memish Z.A., Perlman S., Poon L.L., Snijder E.J., Stephens G.M., Woo P.C., Zaki A.M., Zambon M., Ziebuhr J. Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group. J. Virol. 2013;87(14):7790–7792. - PMC - PubMed
    1. de Wit E., Feldmann F., Okumura A., Horne E., Haddock E., Saturday G., Scott D., Erlandson K.J., Stahl N., Lipsich L., Kyratsous C.A., Feldmann H. Prophylactic and therapeutic efficacy of mAb treatment against MERS-CoV in common marmosets. Antiviral Res. 2018;156:64–71. - PMC - PubMed

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