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High-Throughput, Single-Copy Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral Immunity during Acute COVID-19

Sung Hee Ko, Elham Bayat Mokhtari, Prakriti Mudvari, Sydney Stein, Christopher D. Stringham, Danielle Wagner, Sabrina Ramelli, Marcos J. Ramos-Benitez, Jeffrey R. Strich, Richard T. Davey Jr., Tongqing Zhou, John Misasi, Peter D. Kwong, Daniel S. Chertow, Nancy J. Sullivan, View ORCID ProfileEli A. Boritz
doi: https://doi.org/10.1101/2021.02.21.432184
Sung Hee Ko
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Elham Bayat Mokhtari
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Prakriti Mudvari
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Sydney Stein
2Emerging Pathogens Section, Critical Care Medicine Department, National Institutes of Health Clinical Center, Bethesda, MD 20892, USA
3Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Christopher D. Stringham
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Danielle Wagner
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Sabrina Ramelli
2Emerging Pathogens Section, Critical Care Medicine Department, National Institutes of Health Clinical Center, Bethesda, MD 20892, USA
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Marcos J. Ramos-Benitez
2Emerging Pathogens Section, Critical Care Medicine Department, National Institutes of Health Clinical Center, Bethesda, MD 20892, USA
3Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Jeffrey R. Strich
2Emerging Pathogens Section, Critical Care Medicine Department, National Institutes of Health Clinical Center, Bethesda, MD 20892, USA
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Richard T. Davey Jr.
3Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Tongqing Zhou
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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John Misasi
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Peter D. Kwong
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Daniel S. Chertow
2Emerging Pathogens Section, Critical Care Medicine Department, National Institutes of Health Clinical Center, Bethesda, MD 20892, USA
3Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Nancy J. Sullivan
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Eli A. Boritz
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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  • ORCID record for Eli A. Boritz
  • For correspondence: boritze@mail.nih.gov
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Abstract

Tracking evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within infected individuals will help elucidate coronavirus disease 2019 (COVID-19) pathogenesis and inform use of antiviral interventions. In this study, we developed an approach for sequencing the region encoding the SARS-CoV-2 virion surface proteins from large numbers of individual virus RNA genomes per sample. We applied this approach to the WA-1 reference clinical isolate of SARS-CoV-2 passaged in vitro and to upper respiratory samples from 7 study participants with COVID-19. SARS-CoV-2 genomes from cell culture were diverse, including 18 haplotypes with non-synonymous mutations clustered in the spike NH2-terminal domain (NTD) and furin cleavage site regions. By contrast, cross-sectional analysis of samples from participants with COVID-19 showed fewer virus variants, without structural clustering of mutations. However, longitudinal analysis in one individual revealed 4 virus haplotypes bearing 3 independent mutations in a spike NTD epitope targeted by autologous antibodies. These mutations arose coincident with a 6.2-fold rise in serum binding to spike and a transient increase in virus burden. We conclude that SARS-CoV-2 exhibits a capacity for rapid genetic adaptation that becomes detectable in vivo with the onset of humoral immunity, with the potential to contribute to delayed virologic clearance in the acute setting.

Author Summary Mutant sequences of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) arising during any individual case of coronavirus disease 2019 (COVID-19) could theoretically enable the virus to evade immune responses or antiviral therapies that target the predominant infecting virus sequence. However, commonly used sequencing technologies are not optimally designed to detect variant virus sequences within each sample. To address this issue, we developed novel technology for sequencing large numbers of individual SARS-CoV-2 genomic RNA molecules across the region encoding the virus surface proteins. This technology revealed extensive genetic diversity in cultured viruses from a clinical isolate of SARS-CoV-2, but lower diversity in samples from 7 individuals with COVID-19. Importantly, concurrent analysis of paired serum samples in selected individuals revealed relatively low levels of antibody binding to the SARS-CoV-2 spike protein at the time of initial sequencing. With increased serum binding to spike protein, we detected multiple SARS-CoV-2 variants bearing independent mutations in a single epitope, as well as a transient increase in virus burden. These findings suggest that SARS-CoV-2 replication creates sufficient virus genetic diversity to allow immune-mediated selection of variants within the time frame of acute COVID-19. Large-scale studies of SARS-CoV-2 variation and specific immune responses will help define the contributions of intra-individual SARS-CoV-2 evolution to COVID-19 clinical outcomes and antiviral drug susceptibility.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://www.ncbi.nlm.nih.gov/sra

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
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Posted February 22, 2021.
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High-Throughput, Single-Copy Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral Immunity during Acute COVID-19
Sung Hee Ko, Elham Bayat Mokhtari, Prakriti Mudvari, Sydney Stein, Christopher D. Stringham, Danielle Wagner, Sabrina Ramelli, Marcos J. Ramos-Benitez, Jeffrey R. Strich, Richard T. Davey Jr., Tongqing Zhou, John Misasi, Peter D. Kwong, Daniel S. Chertow, Nancy J. Sullivan, Eli A. Boritz
bioRxiv 2021.02.21.432184; doi: https://doi.org/10.1101/2021.02.21.432184
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High-Throughput, Single-Copy Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral Immunity during Acute COVID-19
Sung Hee Ko, Elham Bayat Mokhtari, Prakriti Mudvari, Sydney Stein, Christopher D. Stringham, Danielle Wagner, Sabrina Ramelli, Marcos J. Ramos-Benitez, Jeffrey R. Strich, Richard T. Davey Jr., Tongqing Zhou, John Misasi, Peter D. Kwong, Daniel S. Chertow, Nancy J. Sullivan, Eli A. Boritz
bioRxiv 2021.02.21.432184; doi: https://doi.org/10.1101/2021.02.21.432184

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