High-throughput, single-copy sequencing reveals SARS-CoV-2 spike variants coincident with mounting humoral immunity during acute COVID-19

  1. Sung Hee Ko
  2. Elham Bayat Mokhtari
  3. Prakriti Mudvari
  4. Sydney Stein
  5. Christopher D. Stringham
  6. Danielle Wagner
  7. Sabrina Ramelli
  8. Marcos J. Ramos-Benitez
  9. Jeffrey R. Strich
  10. Richard T. Davey
  11. Tongqing Zhou
  12. John Misasi
  13. Peter D. Kwong
  14. Daniel S. Chertow
  15. Nancy J. Sullivan
  16. Eli A. Boritz

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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 NH 2 -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.

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  1. Version published to 10.1371/journal.ppat.1009431
  2. ScreenIT

    SciScore for 10.1101/2021.02.21.432184: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Analysis of Serum Antibody Binding to SARS-CoV-2 Spike Protein: Domain-specific antibody competition assays using a His-tagged SARS-CoV-2 Spike protein ectodomain containing 2 proline stabilization mutations (S-2P) (43) were performed using a fortéBio Octet HTX instrument and His1K (anti-penta His) biosensors at 30°C with agitation set to 1,000 rpm.
    anti-penta
    suggested: (Bio-Rad Cat# MCA5995P, RRID:AB_2888689)
    Monoclonal antibodies included were composed of human IgG RBD-specific antibodies LY-CoV-555 (44), S309 (45), CR3022 (46), and CB6 (47), NTD-specific antibodies S652-118 (48), 4-8 (49) and 4A8 (24) and S2-specific antibody S652-112 (48).
    CR3022
    suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    CB6
    suggested: None
    4-8
    suggested: None
    S2-specific
    suggested: None
    Percent competition (% C) of serum antibody binding to S-2P by competitor mAb groups was calculated using the following formula: % C = [1 – (shift nm value at 4001.2 s in presence of competitor mAb)/(shift nm value at 4001.2 s in presence of negative control antibody)]*100.
    antibody)]*100
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Extracted RNA from the 4th Vero cell passage of the SARS-CoV-2 WA-1 clinical isolate was obtained from the BEI Resource (catalog #NR-52285).
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    Plates were read on the QX200 Droplet Reader (Bio-Rad) and analyzed using the freely available QuantaSoft Analysis Pro Software (Bio-Rad) to quantify copies of N1, N2, and RP genes per well, which was then normalized to mL of sample input.
    QuantaSoft Analysis Pro
    suggested: None
    The resulting FASTA files were reoriented into 5’-3’ direction using the usearch-orient command in USEARCH (v8.1.1861) (38).
    USEARCH
    suggested: (mubiomics, RRID:SCR_006785)
    Consensus sequences were then analyzed by searching the BLAST nt database, and non-coronavirus sequences thus identified were discarded.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    Raw PacBio CCS sequence data associated with this study have been deposited in the NCBI SRA database with the BioProject accession number PRJNA680710.
    NCBI SRA
    suggested: None
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Nevertheless, our relatively homogeneous cross-sectional sequencing findings in people with COVID-19 were not due entirely to intrinsic limitations on SARS-CoV-2 diversity. Instead, longitudinal analysis during the second and early third weeks of illness in one person revealed a transient increase in virus burden and multiple new virus variants in which 3 different mutations in an epitope of the spike NTD had arisen independently. The mutated epitope was previously shown to be a neutralizing antibody target (24), and was identified herein as a major target for antibodies in the autologous serum. Our results therefore suggest selection of SARS-CoV-2 spike variants by mounting antibody responses in the acute setting. Mutational evasion of adaptive immune responses by SARS-CoV-2 during acute COVID-19 has not been clearly documented previously. This relationship may have been overlooked in part due to the emphasis on tracking new mutations on a global scale, with a predominance of cross-sectional rather than longitudinal analyses of infected individuals. The early peak of SARS-CoV-2 RNA in respiratory secretions may also favor high-quality data acquisition in very early infection, leading to overrepresentation of individuals in whom virus populations have not yet been subjected to adaptive immune pressure. Another important consideration is the sequencing method used. Our method was specifically developed for high-throughput analysis of single virus RNA molecules, and incorporate...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 40 and 36. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

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  3. Version published to 10.1101/2021.02.21.432184v1 on bioRxiv