SARS-CoV-2 cell-to-cell spread occurs rapidly and is insensitive to antibody neutralization

  1. Laurelle Jackson
  2. Hylton Rodel
  3. Shi-Hsia Hwa
  4. Sandile Cele
  5. Yashica Ganga
  6. Houriiyah Tegally
  7. Mallory Bernstein
  8. Jennifer Giandhari
  9. COMMIT-KZN Team
  10. Bernadett I. Gosnell
  11. Khadija Khan
  12. Willem Hanekom
  13. Farina Karim
  14. Tulio de Oliveira
  15. Mahomed-Yunus S. Moosa
  16. Alex Sigal

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Abstract

Viruses increase the efficiency of close-range transmission between cells by manipulating cellular physiology and behavior, and SARS-CoV-2 uses cell fusion as one mechanism for cell-to-cell spread. Here we visualized infection using time-lapse microscopy of a human lung cell line and used live virus neutralization to determine the sensitivity of SARS-CoV-2 cell-to-cell spread to neutralizing antibodies. SARS-CoV-2 infection rapidly led to cell fusion, forming multinucleated cells with clustered nuclei which started to be detected at 6h post-infection. To compare sensitivity of cell-to-cell spread to neutralization, we infected either with cell-free virus or with single infected cells expressing on their surface the SARS-CoV-2 spike protein. We tested two variants of SARS-CoV-2: B.1.117 containing only the D614G substitution, and the escape variant B.1.351. We used the much smaller area of single infected cells relative to infection foci to exclude any input infected cells which did not lead to transmission. The monoclonal antibody and convalescent plasma we tested neutralized cell-free SARS-CoV-2, with the exception of B.1.351 virus, which was poorly neutralized with plasma from non-B.1.351 infections. In contrast, cell-to-cell spread of SARS-CoV-2 showed no sensitivity to monoclonal antibody or convalescent plasma neutralization. These observations suggest that, once cells are infected, SARS-CoV-2 may be more difficult to neutralize in cell types and anatomical compartments permissive for cell-to-cell spread.

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  1. ScreenIT

    SciScore for 10.1101/2021.06.01.446516: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethical statement: Nasopharyngeal/oropharyngeal swab samples and plasma samples were obtained from 11 hospitalized adults with PCR-confirmed SARS-CoV-2 infection enrolled in a prospective cohort study approved by the Biomedical Research Ethics Committee (BREC) at the University of KwaZulu-Natal (reference BREC/00001275/2020).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The antibody-virus or antibody-donor cell mixtures were incubated for 1 hour at 37°C, 5% CO2.
    antibody-virus
    suggested: None
    For staining of foci, a rabbit anti-spike monoclonal antibody (mAb BS-R2B12, GenScript A02058) was used at 0.5μg/mL as the primary detection antibody.
    anti-spike
    suggested: None
    Cells were incubated with ACE2 antibody overnight at 4°C with rocking.
    ACE2
    suggested: None
    Cells were then washed three times with DPBS and incubated with secondary antibody, goat anti-rabbit IgG Alexa Fluor 555 (Abcam ab150078) at a concentration of 4 μg/mL in an antibody staining solution made up of 1% Bovine Serum Albumin (Sigman-Aldrich) in DPBST.
    anti-rabbit IgG
    suggested: (Abcam Cat# ab150078, RRID:AB_2722519)
    Experimental Models: Cell Lines
    SentencesResources
    Cells: Vero E6 cells (ATCC CRL-1586, obtained from Cellonex in South Africa) were propagated in complete DMEM with 10% fetal bovine serum (Hylone) containing 1% each of HEPES, sodium pyruvate, L-glutamine, and non-essential amino acids (Sigma-Aldrich).
    Vero E6
    suggested: None
    H1299 and H1299 subclones were propagated in complete RPMI with 10% fetal bovine serum containing 1% each of HEPES, sodium pyruvate, L-glutamine, and non-essential amino acids and and passaged every second day.
    H1299
    suggested: None
    HEK-293 (ATCC CRL-1573) cells were propagated in complete DMEM with 10% fetal bovine serum containing 1% each of HEPES, sodium pyruvate, L-glutamine, and non-essential amino acids and and passaged every second day.
    HEK-293
    suggested: ATCC Cat# CRL-1573, RRID:CVCL_0045)
    Single cell cloning to create H1299-ACE2 clones: The H1299-H2AZ clone with nuclear labelled YFP was constructed to overexpress ACE2 as follows: VSVG-pseudotyped lentivirus containing the human ACE2 was generated by co-transfecting 293T cells with the pHAGE2-EF1alnt-ACE2-WT plasmid along with the lentiviral helper plasmids HDM-VSVG, HDM-Hgpm2, HDM-tat1b and pRC-CMV-Rev1b using TransIT-LT1 (Mirus) transfection reagent.
    293T
    suggested: None
    ACE-2 transduced H1299-H2AZ cells were then sub-cloned at the single cell density in 384-well plates (Greiner Bio-One) in conditioned media derived from H1299 confluent cells.
    H1299-H2AZ
    suggested: None
    LVNA using focus forming assay: To quantify neutralization of virus either in cell-free or cell-to-cell infections, H1299-C7 cells were plated in an 96-well plate (Corning) at 30,000 target cells per well 1 day pre-infection.
    H1299-C7
    suggested: None
    Recombinant DNA
    SentencesResources
    Single cell cloning to create H1299-ACE2 clones: The H1299-H2AZ clone with nuclear labelled YFP was constructed to overexpress ACE2 as follows: VSVG-pseudotyped lentivirus containing the human ACE2 was generated by co-transfecting 293T cells with the pHAGE2-EF1alnt-ACE2-WT plasmid along with the lentiviral helper plasmids HDM-VSVG, HDM-Hgpm2, HDM-tat1b and pRC-CMV-Rev1b using TransIT-LT1 (Mirus) transfection reagent.
    pHAGE2-EF1alnt-ACE2-WT
    suggested: None
    pRC-CMV-Rev1b
    suggested: RRID:Addgene_164443)
    Software and Algorithms
    SentencesResources
    Image analysis of multinucleated H1299-ACE2 cells in time lapse microscopy images: Timelapse microscopy images were analysed using custom Matlab script.
    Matlab
    suggested: (MATLAB, RRID:SCR_001622)
    Data generated in this way was graphed in R using the ggplot2 package.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    A caveat to our results is that we used a human cancer cell line, and modified it to express ACE-2. However, syncytia are a common feature of SARS-CoV-2 lung pathology [11, 12, 13], showing that the cell fusion mechanism of cell-to-cell spread operates in this environment. We used infected cells after they already expressed the SARS-CoV-2 spike protein on the cell surface, providing a target for neutralization. Despite this, there was no appreciable neutralization. Both the earlier D614G variant and the B.1.351 variant showed similar insensitivity of cell-to-cell spread in D614G and B.1.351 elicited plasma, indicating that the insensitivity of cell-to-cell spread to neutralization is not specific to the infecting variant or the elicited neutralizing antibodies. We have also verified previous observations about the drop in B.1.351 neutralization by non-B.1.351 elicited plasma in the H1299-ACE2 human lung cell system, as well as the cross-neutralization of D614G by B.1.351-elicited plasma. Interestingly, in these experiments, neutralization of B.1.351 by its matched plasma was weaker than neutralization of D614G by its matched plasma. Despite the overall weaker neutralization capacity, the B.1.351-elicited plasma could still effectively cross-neutralize, showing that cross-neutralization is not necessarily linked to absolute neutralization capacity. Like with other viruses, cell-to-cell spread of SARS-CoV-2 may prove to play a role in pathology and possibly persistence. Future ...

    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 page 17. 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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.06.01.446516v1 on bioRxiv