Disruption of the CCL5/RANTES-CCR5 Pathway Restores Immune Homeostasis and Reduces Plasma Viral Load in Critical COVID-19

  1. Bruce K. Patterson
  2. Harish Seethamraju
  3. Kush Dhody
  4. Michael J. Corley
  5. Kazem Kazempour
  6. Jay Lalezari
  7. Alina P.S. Pang
  8. Christopher Sugai
  9. Edgar B. Francisco
  10. Amruta Pise
  11. Hallison Rodrigues
  12. Mathew Ryou
  13. Helen L. Wu
  14. Gabriela M. Webb
  15. Byung S. Park
  16. Scott Kelly
  17. Nader Pourhassan
  18. Alena Lelic
  19. Lama Kdouh
  20. Monica Herrera
  21. Eric Hall
  22. Enver Aklin
  23. Lishomwa C. Ndhlovu
  24. Jonah B. Sacha

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is now pandemic with nearly three million cases reported to date 1 . Although the majority of COVID-19 patients experience only mild or moderate symptoms, a subset will progress to severe disease with pneumonia and acute respiratory distress syndrome (ARDS) requiring mechanical ventilation 2 . Emerging results indicate a dysregulated immune response characterized by runaway inflammation, including cytokine release syndrome (CRS), as the major driver of pathology in severe COVID-19 3,4 . With no treatments currently approved for COVID-19, therapeutics to prevent or treat the excessive inflammation in severe disease caused by SARS-CoV-2 infection are urgently needed. Here, in 10 terminally-ill, critical COVID-19 patients we report profound elevation of plasma IL-6 and CCL5 (RANTES), decreased CD8+ T cell levels, and SARS-CoV-2 plasma viremia. Following compassionate care treatment with the CCR5 blocking antibody leronlimab, we observed complete CCR5 receptor occupancy on macrophage and T cells, rapid reduction of plasma IL-6, restoration of the CD4/CD8 ratio, and a significant decrease in SARS-CoV-2 plasma viremia. Consistent with reduction of plasma IL-6, single-cell RNA-sequencing revealed declines in transcriptomic myeloid cell clusters expressing IL-6 and interferon-related genes. These results demonstrate a novel approach to resolving unchecked inflammation, restoring immunologic deficiencies, and reducing SARS-CoV-2 plasma viral load via disruption of the CCL5-CCR5 axis, and support randomized clinical trials to assess clinical efficacy of leronlimab-mediated inhibition of CCR5 for COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.05.02.20084673: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Informed consent was obtained from patient or their legally authorized representative per 21 CFR Part 50.
    IRB: The Albert Einstein College of Medicine Institution Review Board (IRB) reviewed and approved this study.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Monocyte Blocker (BioLegend, San Diego, CA), and the following surface marker antibodies: anti-CD19 (PE-Dazzle594), anti-CD3 (APC), anti-CD16 (Alexa700), HLA-DR (APC/Fire750), and anti-CTLA-4 (PE-Cy7).
    anti-CD19
    suggested: (BD Biosciences Cat# 340546, RRID:AB_400053)
    anti-CD3 ( APC
    suggested: None
    anti-CD16
    suggested: None
    HLA-DR
    suggested: None
    anti-CTLA-4
    suggested: None
    PE-Cy7
    suggested: None
    The following antibodies were then added to each tube individually: anti-CD8 (BUV496), anti-CD4 (BUV661), anti-CD45 (BUV805), anti-CD103 (BV421)
    anti-CD8
    suggested: (BD Biosciences Cat# 741627, RRID:AB_2871032)
    BUV496
    suggested: (BD Biosciences Cat# 741083, RRID:AB_2870687)
    anti-CD4
    suggested: (Thermo Fisher Scientific Cat# MA1-18657, RRID:AB_1072314)
    anti-CD45
    suggested: None
    BUV805
    suggested: (BD Biosciences Cat# 741948, RRID:AB_2871259)
    anti-CD103
    suggested: None
    BV421
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    The detector reads the droplets to determine which contain target (positive) and which do not (negative) for each of the targets identified with the Bio-Rad SARS-CoV-2 ddPCR Test: N1, N2 and RP.
    N2
    suggested: None
    Software and Algorithms
    SentencesResources
    Analysis was performed with Kaluza version 2.1 software.
    Kaluza
    suggested: (Kaluza, RRID:SCR_016182)
    The fluorescence data is then analyzed by the QuantaSoft 1.7 and QuantaSoft Analysis Pro 1.0 Software to determine the presence of SARS-CoV-2 N1 and N2 in the specimen.
    QuantaSoft Analysis Pro
    suggested: None

    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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04343651Active, not recruitingStudy to Evaluate the Efficacy and Safety of Leronlimab for …
    NCT04347239RecruitingStudy to Evaluate the Efficacy and Safety of Leronlimab for …

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

    Results from JetFighter: We did not find any issues relating to colormaps.

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. ScreenIT

    SciScore for 10.1101/2020.05.02.20084673: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementInformed consent was obtained from patient or their legally authorized representative per 21 CFR Part 50 .RandomizationThese results demonstrate a novel approach to resolving unchecked inflammation, restoring immunologic deficiencies, and reducing SARS-CoV-2 plasma viral load via disruption of the CCL5-CCR5 axis, and support randomized clinical trials to assess clinical efficacy of leronlimab-mediated inhibition of CCR5 for COVID-19. MAIN TEXT Since the initial cases of COVID-19 were reported from Wuhan, China in December 20192BlindingIndeed , randomized , double blind , placebo controlled clinical trials are underway to assess the efficacy of leronlimab treatments in patients with mild to moderate ( NCT04343651)24 and severe to critical ( NCT04347239)25 COVID-19 .Power Analysisnot detected.Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Following compassionate care treatment with the CCR5 blocking antibody leronlimab, we observed complete CCR5 receptor occupancy on macrophage and T cells, rapid reduction of plasma IL-6, restoration of the CD4/CD8 ratio, and a significant decrease in SARS-CoV-2 plasma viremia.
    CCR5
    suggested: None
    Leronlimab , formerly PRO 140 , is a CCR5-specific human IgG4 monoclonal antibody in development for HIV therapy as a once-weekly , at-home subcutaneous injection .
    human IgG4
    suggested: None
    Monocyte Blocker ( BioLegend , San Diego , CA) , and the following surface marker antibodies: anti-CD19 ( PE-Dazzle594) , anti-CD3 ( APC) , anti-CD16 ( Alexa700) , HLA-DR ( APC/Fire750) , and anti-CTLA-4 ( PE-Cy7) .
    anti-CD19
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>anti-CD3 ( APC</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-CD16</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>HLA-DR</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-CTLA-4</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>PE-Cy7</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The following antibodies were then added to each tube individually: anti-CD8 ( BUV496) , antiCD4 ( BUV661) , anti-CD45 ( BUV805) , anti-CD103 ( BV421)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-CD8</b></div>
        <div>suggested: (BD Biosciences Cat# 564804, <a href="https://scicrunch.org/resources/Any/search?q=AB_2744460">AB_2744460</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>BUV496</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>antiCD4</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>BUV661</b></div>
        <div>suggested: (BD Biosciences Cat# 565079, <a href="https://scicrunch.org/resources/Any/search?q=AB_2739057">AB_2739057</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-CD45</b></div>
        <div>suggested: (BD Biosciences Cat# 564914, <a href="https://scicrunch.org/resources/Any/search?q=AB_2744401">AB_2744401</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>BUV805</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-CD103</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>BV421</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Finally , to establish an unbiased gene repertoire for these COVID-19 patients , we performed 10X Genomics 5’ single cell RNA-sequencing of peripheral blood mononuclear cells to evaluate transcriptional changes between an uninfected healthy donor and two of the severe COVID-19 patients ( P2 and P4 ) for which sufficient baseline , pre-leronlimab treatment COVID-19 samples were available for this analysis .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Genomics</b></div>
        <div>suggested: (CLC Genomics Server, <a href="https://scicrunch.org/resources/Any/search?q=SCR_017396">SCR_017396</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed with Kaluza version 2.1 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Kaluza</b></div>
        <div>suggested: (Kaluza, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016182">SCR_016182</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The fluorescence data is then analyzed by the QuantaSoft 1.7 and QuantaSoft Analysis Pro 1.0 Software to determine the presence of SARS-CoV-2 N1 and N2 in the specimen .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>QuantaSoft Analysis Pro</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Ndhlovu has received compensation for serving on a scientific advisory board for Abbvie.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Abbvie</b></div>
        <div>suggested: (AbbVie, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010484">SCR_010484</a>)</div>
      </div>
    </td></tr></table>

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

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2020.05.02.20084673v1 on medRxiv