Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

Obinna C Ubah; Eric W Lake; Gihan S Gunaratne; Joseph P Gallant; Marie Fernie; Austin J Robertson; Jonathan S Marchant; Tyler D Bold; Ryan A Langlois; William E Matchett; Joshua M Thiede; Ke Shi; Lulu Yin; Nicholas H Moeller; Surajit Banerjee; Laura Ferguson; Marina Kovaleva; Andrew J Porter; Hideki Aihara; Aaron M LeBeau; Caroline J Barelle

doi:10.1038/s41467-021-27611-y

Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

Obinna C Ubah, Eric W Lake, Gihan S Gunaratne, Joseph P Gallant, Marie Fernie, Austin J Robertson, Jonathan S Marchant, Tyler D Bold, Ryan A Langlois, William E Matchett, Joshua M Thiede, Ke Shi, Lulu Yin, Nicholas H Moeller, Surajit Banerjee, Laura Ferguson, Marina Kovaleva, Andrew J Porter, Hideki Aihara^* (Corresponding Author), Aaron M LeBeau^* (Corresponding Author)Caroline J Barelle^* (Corresponding Author)

^*Corresponding author for this work

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Abstract

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

Original language	English
Article number	7325
Number of pages	12
Journal	Nature Communications
Volume	12
Issue number	1
Early online date	16 Dec 2021
DOIs	https://doi.org/10.1038/s41467-021-27611-y
Publication status	Published - 16 Dec 2021

Bibliographical note

Acknowledgements
This work was supported by the Chief Scientist Office, Scottish Government, Grant COV/ABN/20/01 (Elasmogen, Ltd.), a 2018 Prostate Cancer Foundation Challenge Award (AML), a 2013 Prostate Cancer Foundation Young Investigator Award (AML), NCI R01s CA237272, CA233562, and CA245922 (AML). WEM was supported by the NIH T32 HL007741 and JMT by the NIH T32 AI055433. JSM was funded by NIGMS R01 GM088790. HA was funded by NIGMS R35 GM118047 and NCI P01 CA234228. X-ray diffraction data were collected at the Northeastern Collaborative Access Team beamlines, which are funded by the US National Institutes of Health (NIGMS P30 GM124165). The Pilatus 6M detector on 24-ID-C beamline is funded by a NIH-ORIP HEI grant (S10 RR029205). We thank the Marco Pravetoni lab for providing training and access to the OctetRED96e for BLI experiments.

Keywords

Antibody fragment therapy
Protein design

UN SDGs

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

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Cite this

Ubah, O. C., Lake, E. W., Gunaratne, G. S., Gallant, J. P., Fernie, M., Robertson, A. J., Marchant, J. S., Bold, T. D., Langlois, R. A., Matchett, W. E., Thiede, J. M., Shi, K., Yin, L., Moeller, N. H., Banerjee, S., Ferguson, L., Kovaleva, M., Porter, A. J., Aihara, H., ... Barelle, C. J. (2021). Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography. Nature Communications, 12(1), Article 7325. https://doi.org/10.1038/s41467-021-27611-y

Ubah, OC, Lake, EW, Gunaratne, GS, Gallant, JP, Fernie, M, Robertson, AJ, Marchant, JS, Bold, TD, Langlois, RA, Matchett, WE, Thiede, JM, Shi, K, Yin, L, Moeller, NH, Banerjee, S, Ferguson, L , Kovaleva, M , Porter, AJ, Aihara, H, LeBeau, AM & Barelle, CJ 2021, 'Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography', Nature Communications, vol. 12, no. 1, 7325. https://doi.org/10.1038/s41467-021-27611-y

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abstract = "Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.",

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author = "Ubah, {Obinna C} and Lake, {Eric W} and Gunaratne, {Gihan S} and Gallant, {Joseph P} and Marie Fernie and Robertson, {Austin J} and Marchant, {Jonathan S} and Bold, {Tyler D} and Langlois, {Ryan A} and Matchett, {William E} and Thiede, {Joshua M} and Ke Shi and Lulu Yin and Moeller, {Nicholas H} and Surajit Banerjee and Laura Ferguson and Marina Kovaleva and Porter, {Andrew J} and Hideki Aihara and LeBeau, {Aaron M} and Barelle, {Caroline J}",

note = "Acknowledgements This work was supported by the Chief Scientist Office, Scottish Government, Grant COV/ABN/20/01 (Elasmogen, Ltd.), a 2018 Prostate Cancer Foundation Challenge Award (AML), a 2013 Prostate Cancer Foundation Young Investigator Award (AML), NCI R01s CA237272, CA233562, and CA245922 (AML). WEM was supported by the NIH T32 HL007741 and JMT by the NIH T32 AI055433. JSM was funded by NIGMS R01 GM088790. HA was funded by NIGMS R35 GM118047 and NCI P01 CA234228. X-ray diffraction data were collected at the Northeastern Collaborative Access Team beamlines, which are funded by the US National Institutes of Health (NIGMS P30 GM124165). The Pilatus 6M detector on 24-ID-C beamline is funded by a NIH-ORIP HEI grant (S10 RR029205). We thank the Marco Pravetoni lab for providing training and access to the OctetRED96e for BLI experiments.",

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AU - Ubah, Obinna C

AU - Lake, Eric W

AU - Gunaratne, Gihan S

AU - Gallant, Joseph P

AU - Fernie, Marie

AU - Robertson, Austin J

AU - Marchant, Jonathan S

AU - Bold, Tyler D

AU - Langlois, Ryan A

AU - Matchett, William E

AU - Thiede, Joshua M

AU - Shi, Ke

AU - Yin, Lulu

AU - Moeller, Nicholas H

AU - Banerjee, Surajit

AU - Ferguson, Laura

AU - Kovaleva, Marina

AU - Porter, Andrew J

AU - Aihara, Hideki

AU - LeBeau, Aaron M

AU - Barelle, Caroline J

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N2 - Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

AB - Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

KW - Antibody fragment therapy

KW - Protein design

U2 - 10.1038/s41467-021-27611-y

DO - 10.1038/s41467-021-27611-y

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SN - 2041-1723

VL - 12

JO - Nature Communications

JF - Nature Communications

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ER -

Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

Abstract

Bibliographical note

Keywords

UN SDGs

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