Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations

Julia A. Tree; Jeremy E. Turnbull; Karen R. Buttigieg; Michael J. Elmore; Naomi Coombes; John Hogwood; Courtney J. Mycroft-West; Marcelo A. Lima; Mark A. Skidmore; Richard Karlsson; Yen Hsi Chen; Zhang Yang; Cosma Mirella Spalluto; Karl J. Staples; Edwin A. Yates; Elaine Gray; Dave Singh; Tom Wilkinson; Clive P. Page; Miles Carroll

doi:10.1111/bph.15304

Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations

Julia A. Tree^*, Jeremy E. Turnbull, Karen R. Buttigieg, Michael J. Elmore, Naomi Coombes, John Hogwood, Courtney J. Mycroft-West, Marcelo A. Lima, Mark A. Skidmore, Richard Karlsson, Yen Hsi Chen, Zhang Yang, Cosma Mirella Spalluto, Karl J. Staples, Edwin A. Yates, Elaine Gray, Dave Singh, Tom Wilkinson, Clive P. Page, Miles Carroll

^*Corresponding author for this work

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Abstract

Background and Purpose: Currently, there are no licensed vaccines and limited antivirals for the treatment of COVID-19. Heparin (delivered systemically) is currently used to treat anticoagulant anomalies in COVID-19 patients. Additionally, in the United Kingdom, Brazil and Australia, nebulised unfractionated heparin (UFH) is being trialled in COVID-19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild type SARS-CoV-2, in vitro, is needed.

Experimental Approach: Seven different heparin preparations including UFH and low MW heparins (LMWH) of porcine or bovine origin were screened for antiviral activity against live SARS-CoV-2 (Australia/VIC01/2020) using a plaque inhibition assay with Vero E6 cells. Interaction of heparin with spike protein RBD was studied using differential scanning fluorimetry and the inhibition of RBD binding to human ACE2 protein using elisa assays was examined.

Key Results: All the UFH preparations had potent antiviral effects, with IC₅₀ values ranging between 25 and 41 μg·ml⁻¹, whereas LMWHs were less inhibitory by ~150-fold (IC₅₀ range 3.4–7.8 mg·ml⁻¹). Mechanistically, we observed that heparin binds and destabilizes the RBD protein and furthermore, we show heparin directly inhibits the binding of RBD to the human ACE2 protein receptor.

Conclusion and Implications: This comparison of clinically relevant heparins shows that UFH has significantly stronger SARS-CoV-2 antiviral activity compared to LMWHs. UFH acts to directly inhibit binding of spike protein to the human ACE2 protein receptor. Overall, the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID-19.

Original language	English
Pages (from-to)	626-635
Number of pages	10
Journal	British Journal of Pharmacology
Volume	178
Issue number	3
Early online date	14 Dec 2020
DOIs	https://doi.org/10.1111/bph.15304
Publication status	Published - Feb 2021

Bibliographical note

Funding Information: We thank Professor Jan Shute (University of Portsmouth) for helpful comments on nebulised heparin. We add thanks to Dr Julian Druce, Doherty Institute, Melbourne, Australia, for donating the virus used in this study. We thank Dr Kevin Bewley, Public Health England, for designing and optimising the PRNT assay used in this study. We thank Mrs Elizabeth Penn for preparing the cell cultures, Mrs Harriet Garlant and Mrs Joanna McGlashan for technical assistance. We are grateful for receiving MERS convalescent sera from Dr Mark Page and Dr Giada Mattiuzzo, NIBSC, UK. D.S. is supported by the National Institute for Health Research (NIHR) Manchester Biomedical Research Centre (BRC). J.E.T. acknowledges the support of University of Liverpool, and M.A.S. the support of Keele University. Z.Y. acknowledges the Danish National Research Foundation (DNRF107) and the Lundbeck Foundation, Y.-H.C. the Innovation Fund Denmark and R.K. the European Commission (GlycoImaging H2020-MSCA-ITN-721297). This work was funded by Public Health England, the University of Liverpool and the University of Keele.

Open Access: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Publisher Copyright: © 2020 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society

Citation: Tree, JA, Turnbull, JE, Buttigieg, KR, et al. Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations. Br J Pharmacol. 2021; 178: 626– 635.

DOI: https://doi.org/10.1111/bph.15304

Keywords

COVID-19
LMWH
SARS-CoV-2
UFH
heparin
nebulised

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Tree, J. A., Turnbull, J. E., Buttigieg, K. R., Elmore, M. J., Coombes, N., Hogwood, J., Mycroft-West, C. J., Lima, M. A., Skidmore, M. A., Karlsson, R., Chen, Y. H., Yang, Z., Spalluto, C. M., Staples, K. J., Yates, E. A., Gray, E., Singh, D., Wilkinson, T., Page, C. P., & Carroll, M. (2021). Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations. British Journal of Pharmacology, 178(3), 626-635. https://doi.org/10.1111/bph.15304

@article{8d6e026940734898a31035fb728b2ccc,

title = "Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations",

abstract = "Background and Purpose: Currently, there are no licensed vaccines and limited antivirals for the treatment of COVID-19. Heparin (delivered systemically) is currently used to treat anticoagulant anomalies in COVID-19 patients. Additionally, in the United Kingdom, Brazil and Australia, nebulised unfractionated heparin (UFH) is being trialled in COVID-19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild type SARS-CoV-2, in vitro, is needed. Experimental Approach: Seven different heparin preparations including UFH and low MW heparins (LMWH) of porcine or bovine origin were screened for antiviral activity against live SARS-CoV-2 (Australia/VIC01/2020) using a plaque inhibition assay with Vero E6 cells. Interaction of heparin with spike protein RBD was studied using differential scanning fluorimetry and the inhibition of RBD binding to human ACE2 protein using elisa assays was examined. Key Results: All the UFH preparations had potent antiviral effects, with IC50 values ranging between 25 and 41 μg·ml−1, whereas LMWHs were less inhibitory by ~150-fold (IC50 range 3.4–7.8 mg·ml−1). Mechanistically, we observed that heparin binds and destabilizes the RBD protein and furthermore, we show heparin directly inhibits the binding of RBD to the human ACE2 protein receptor. Conclusion and Implications: This comparison of clinically relevant heparins shows that UFH has significantly stronger SARS-CoV-2 antiviral activity compared to LMWHs. UFH acts to directly inhibit binding of spike protein to the human ACE2 protein receptor. Overall, the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID-19.",

keywords = "COVID-19, LMWH, SARS-CoV-2, UFH, heparin, nebulised",

author = "Tree, {Julia A.} and Turnbull, {Jeremy E.} and Buttigieg, {Karen R.} and Elmore, {Michael J.} and Naomi Coombes and John Hogwood and Mycroft-West, {Courtney J.} and Lima, {Marcelo A.} and Skidmore, {Mark A.} and Richard Karlsson and Chen, {Yen Hsi} and Zhang Yang and Spalluto, {Cosma Mirella} and Staples, {Karl J.} and Yates, {Edwin A.} and Elaine Gray and Dave Singh and Tom Wilkinson and Page, {Clive P.} and Miles Carroll",

note = "Funding Information: We thank Professor Jan Shute (University of Portsmouth) for helpful comments on nebulised heparin. We add thanks to Dr Julian Druce, Doherty Institute, Melbourne, Australia, for donating the virus used in this study. We thank Dr Kevin Bewley, Public Health England, for designing and optimising the PRNT assay used in this study. We thank Mrs Elizabeth Penn for preparing the cell cultures, Mrs Harriet Garlant and Mrs Joanna McGlashan for technical assistance. We are grateful for receiving MERS convalescent sera from Dr Mark Page and Dr Giada Mattiuzzo, NIBSC, UK. D.S. is supported by the National Institute for Health Research (NIHR) Manchester Biomedical Research Centre (BRC). J.E.T. acknowledges the support of University of Liverpool, and M.A.S. the support of Keele University. Z.Y. acknowledges the Danish National Research Foundation (DNRF107) and the Lundbeck Foundation, Y.-H.C. the Innovation Fund Denmark and R.K. the European Commission (GlycoImaging H2020-MSCA-ITN-721297). This work was funded by Public Health England, the University of Liverpool and the University of Keele. Open Access: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Publisher Copyright: {\textcopyright} 2020 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society Citation: Tree, JA, Turnbull, JE, Buttigieg, KR, et al. Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations. Br J Pharmacol. 2021; 178: 626– 635. DOI: https://doi.org/10.1111/bph.15304 ",

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month = feb,

doi = "10.1111/bph.15304",

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volume = "178",

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Tree, JA, Turnbull, JE, Buttigieg, KR , Elmore, MJ , Coombes, N, Hogwood, J, Mycroft-West, CJ, Lima, MA, Skidmore, MA, Karlsson, R, Chen, YH, Yang, Z, Spalluto, CM, Staples, KJ, Yates, EA, Gray, E, Singh, D, Wilkinson, T, Page, CP & Carroll, M 2021, 'Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations', British Journal of Pharmacology, vol. 178, no. 3, pp. 626-635. https://doi.org/10.1111/bph.15304

TY - JOUR

T1 - Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations

AU - Tree, Julia A.

AU - Turnbull, Jeremy E.

AU - Buttigieg, Karen R.

AU - Elmore, Michael J.

AU - Coombes, Naomi

AU - Hogwood, John

AU - Mycroft-West, Courtney J.

AU - Lima, Marcelo A.

AU - Skidmore, Mark A.

AU - Karlsson, Richard

AU - Chen, Yen Hsi

AU - Yang, Zhang

AU - Spalluto, Cosma Mirella

AU - Staples, Karl J.

AU - Yates, Edwin A.

AU - Gray, Elaine

AU - Singh, Dave

AU - Wilkinson, Tom

AU - Page, Clive P.

AU - Carroll, Miles

N1 - Funding Information: We thank Professor Jan Shute (University of Portsmouth) for helpful comments on nebulised heparin. We add thanks to Dr Julian Druce, Doherty Institute, Melbourne, Australia, for donating the virus used in this study. We thank Dr Kevin Bewley, Public Health England, for designing and optimising the PRNT assay used in this study. We thank Mrs Elizabeth Penn for preparing the cell cultures, Mrs Harriet Garlant and Mrs Joanna McGlashan for technical assistance. We are grateful for receiving MERS convalescent sera from Dr Mark Page and Dr Giada Mattiuzzo, NIBSC, UK. D.S. is supported by the National Institute for Health Research (NIHR) Manchester Biomedical Research Centre (BRC). J.E.T. acknowledges the support of University of Liverpool, and M.A.S. the support of Keele University. Z.Y. acknowledges the Danish National Research Foundation (DNRF107) and the Lundbeck Foundation, Y.-H.C. the Innovation Fund Denmark and R.K. the European Commission (GlycoImaging H2020-MSCA-ITN-721297). This work was funded by Public Health England, the University of Liverpool and the University of Keele. Open Access: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Publisher Copyright: © 2020 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society Citation: Tree, JA, Turnbull, JE, Buttigieg, KR, et al. Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations. Br J Pharmacol. 2021; 178: 626– 635. DOI: https://doi.org/10.1111/bph.15304

PY - 2021/2

Y1 - 2021/2

N2 - Background and Purpose: Currently, there are no licensed vaccines and limited antivirals for the treatment of COVID-19. Heparin (delivered systemically) is currently used to treat anticoagulant anomalies in COVID-19 patients. Additionally, in the United Kingdom, Brazil and Australia, nebulised unfractionated heparin (UFH) is being trialled in COVID-19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild type SARS-CoV-2, in vitro, is needed. Experimental Approach: Seven different heparin preparations including UFH and low MW heparins (LMWH) of porcine or bovine origin were screened for antiviral activity against live SARS-CoV-2 (Australia/VIC01/2020) using a plaque inhibition assay with Vero E6 cells. Interaction of heparin with spike protein RBD was studied using differential scanning fluorimetry and the inhibition of RBD binding to human ACE2 protein using elisa assays was examined. Key Results: All the UFH preparations had potent antiviral effects, with IC50 values ranging between 25 and 41 μg·ml−1, whereas LMWHs were less inhibitory by ~150-fold (IC50 range 3.4–7.8 mg·ml−1). Mechanistically, we observed that heparin binds and destabilizes the RBD protein and furthermore, we show heparin directly inhibits the binding of RBD to the human ACE2 protein receptor. Conclusion and Implications: This comparison of clinically relevant heparins shows that UFH has significantly stronger SARS-CoV-2 antiviral activity compared to LMWHs. UFH acts to directly inhibit binding of spike protein to the human ACE2 protein receptor. Overall, the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID-19.

AB - Background and Purpose: Currently, there are no licensed vaccines and limited antivirals for the treatment of COVID-19. Heparin (delivered systemically) is currently used to treat anticoagulant anomalies in COVID-19 patients. Additionally, in the United Kingdom, Brazil and Australia, nebulised unfractionated heparin (UFH) is being trialled in COVID-19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild type SARS-CoV-2, in vitro, is needed. Experimental Approach: Seven different heparin preparations including UFH and low MW heparins (LMWH) of porcine or bovine origin were screened for antiviral activity against live SARS-CoV-2 (Australia/VIC01/2020) using a plaque inhibition assay with Vero E6 cells. Interaction of heparin with spike protein RBD was studied using differential scanning fluorimetry and the inhibition of RBD binding to human ACE2 protein using elisa assays was examined. Key Results: All the UFH preparations had potent antiviral effects, with IC50 values ranging between 25 and 41 μg·ml−1, whereas LMWHs were less inhibitory by ~150-fold (IC50 range 3.4–7.8 mg·ml−1). Mechanistically, we observed that heparin binds and destabilizes the RBD protein and furthermore, we show heparin directly inhibits the binding of RBD to the human ACE2 protein receptor. Conclusion and Implications: This comparison of clinically relevant heparins shows that UFH has significantly stronger SARS-CoV-2 antiviral activity compared to LMWHs. UFH acts to directly inhibit binding of spike protein to the human ACE2 protein receptor. Overall, the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID-19.

KW - COVID-19

KW - LMWH

KW - SARS-CoV-2

KW - UFH

KW - heparin

KW - nebulised

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DO - 10.1111/bph.15304

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AN - SCOPUS:85096138013

SN - 0007-1188

VL - 178

SP - 626

EP - 635

JO - British Journal of Pharmacology

JF - British Journal of Pharmacology

IS - 3

ER -

Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations

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Bibliographical note

Keywords

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