Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp)

Wen Meng; Siying Guo; Simon Cao; Masahiro Shuda; Lindsey R. Robinson-McCarthy; Kevin R. McCarthy; Yoko Shuda; Alberto E. Paniz Mondolfi; Clare Bryce; Zachary Grimes; Emilia M. Sordillo; Carlos Cordon-Cardo; Pengfei Li; Hu Zhang; Stanley Perlman; Haitao Guo; Shou Jiang Gao; Yuan Chang; Patrick S. Moore

doi:10.1002/jmv.28246

Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp)

Wen Meng, Siying Guo, Simon Cao, Masahiro Shuda, Lindsey R. Robinson-McCarthy, Kevin R. McCarthy, Yoko Shuda, Alberto E. Paniz Mondolfi, Clare Bryce, Zachary Grimes, Emilia M. Sordillo, Carlos Cordon-Cardo, Pengfei Li, Hu Zhang, Stanley Perlman, Haitao Guo, Shou Jiang Gao, Yuan Chang, Patrick S. Moore

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

Abstract

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.

Original language	English
Article number	e28246
Journal	Journal of Medical Virology
Volume	95
Issue number	1
DOIs	https://doi.org/10.1002/jmv.28246
State	Published - Jan 2023

Keywords

COVID-19
NSP12
RNA-dependent RNA polymerase, RdRp
SARS-CoV-2
monoclonal antibody

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10.1002/jmv.28246

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Meng, W., Guo, S., Cao, S., Shuda, M., Robinson-McCarthy, L. R., McCarthy, K. R., Shuda, Y., Paniz Mondolfi, A. E., Bryce, C., Grimes, Z., Sordillo, E. M., Cordon-Cardo, C., Li, P., Zhang, H., Perlman, S., Guo, H., Gao, S. J., Chang, Y., & Moore, P. S. (2023). Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp). Journal of Medical Virology, 95(1), [e28246]. https://doi.org/10.1002/jmv.28246

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title = "Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp)",

abstract = "SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.",

keywords = "COVID-19, NSP12, RNA-dependent RNA polymerase, RdRp, SARS-CoV-2, monoclonal antibody",

author = "Wen Meng and Siying Guo and Simon Cao and Masahiro Shuda and Robinson-McCarthy, {Lindsey R.} and McCarthy, {Kevin R.} and Yoko Shuda and {Paniz Mondolfi}, {Alberto E.} and Clare Bryce and Zachary Grimes and Sordillo, {Emilia M.} and Carlos Cordon-Cardo and Pengfei Li and Hu Zhang and Stanley Perlman and Haitao Guo and Gao, {Shou Jiang} and Yuan Chang and Moore, {Patrick S.}",

note = "Funding Information: We thank the Pitt Biospecimen Core for technical support. This study was supported by the University of Pittsburgh Medical Center (UPMC) Hillman Cancer Center Startup Fund (S.J.G.); the Pittsburgh Foundation Endowed Chair in Drug Development for Immunotherapy (S.J.G.); the Pittsburgh Foundation Endowed Chair in Innovative Cancer Research (P.S.M.); UPMC Endowed Chair in Cancer Virology (Y.C.); and by the NIH P01 AI060699, R01 AI129269 (S.P.). This work used the UPMC Hillman Cancer Center and Tissue and Research Pathology/Pitt Biospecimen Core shared resource, which is supported in part by award P30CA047904. Publisher Copyright: {\textcopyright} 2022 Wiley Periodicals LLC.",

year = "2023",

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doi = "10.1002/jmv.28246",

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journal = "Journal of Medical Virology",

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Meng, W, Guo, S, Cao, S, Shuda, M, Robinson-McCarthy, LR, McCarthy, KR, Shuda, Y, Paniz Mondolfi, AE, Bryce, C, Grimes, Z, Sordillo, EM, Cordon-Cardo, C, Li, P, Zhang, H, Perlman, S, Guo, H, Gao, SJ, Chang, Y & Moore, PS 2023, 'Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp)', Journal of Medical Virology, vol. 95, no. 1, e28246. https://doi.org/10.1002/jmv.28246

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AU - Meng, Wen

AU - Guo, Siying

AU - Cao, Simon

AU - Shuda, Masahiro

AU - Robinson-McCarthy, Lindsey R.

AU - McCarthy, Kevin R.

AU - Shuda, Yoko

AU - Paniz Mondolfi, Alberto E.

AU - Bryce, Clare

AU - Grimes, Zachary

AU - Sordillo, Emilia M.

AU - Cordon-Cardo, Carlos

AU - Li, Pengfei

AU - Zhang, Hu

AU - Perlman, Stanley

AU - Guo, Haitao

AU - Gao, Shou Jiang

AU - Chang, Yuan

AU - Moore, Patrick S.

N1 - Funding Information: We thank the Pitt Biospecimen Core for technical support. This study was supported by the University of Pittsburgh Medical Center (UPMC) Hillman Cancer Center Startup Fund (S.J.G.); the Pittsburgh Foundation Endowed Chair in Drug Development for Immunotherapy (S.J.G.); the Pittsburgh Foundation Endowed Chair in Innovative Cancer Research (P.S.M.); UPMC Endowed Chair in Cancer Virology (Y.C.); and by the NIH P01 AI060699, R01 AI129269 (S.P.). This work used the UPMC Hillman Cancer Center and Tissue and Research Pathology/Pitt Biospecimen Core shared resource, which is supported in part by award P30CA047904. Publisher Copyright: © 2022 Wiley Periodicals LLC.

PY - 2023/1

Y1 - 2023/1

N2 - SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.

AB - SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.

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ER -

Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp)

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Keywords

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