Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model

  1. Ami Patel
  2. Jewell Walters
  3. Emma L. Reuschel
  4. Katherine Schultheis
  5. Elizabeth Parzych
  6. Ebony N. Gary
  7. Igor Maricic
  8. Mansi Purwar
  9. Zeena Eblimit
  10. Susanne N. Walker
  11. Diana Guimet
  12. Pratik Bhojnagarwala
  13. Arthur Doan
  14. Ziyang Xu
  15. Dustin Elwood
  16. Sophia M. Reeder
  17. Laurent Pessaint
  18. Kevin Y. Kim
  19. Anthony Cook
  20. Neethu Chokkalingam
  21. Brad Finneyfrock
  22. Edgar Tello-Ruiz
  23. Alan Dodson
  24. Jihae Choi
  25. Alison Generotti
  26. John Harrison
  27. Nicholas J. Tursi
  28. Viviane M. Andrade
  29. Yaya Dia
  30. Faraz I. Zaidi
  31. Hanne Andersen
  32. Mark G. Lewis
  33. Kar Muthumani
  34. J Joseph Kim
  35. Daniel W. Kulp
  36. Laurent M. Humeau
  37. Stephanie Ramos
  38. Trevor R.F. Smith
  39. David B. Weiner
  40. Kate E. Broderick

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Abstract

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health, social, and economic infrastructures. Here, we assess immunogenicity and anamnestic protective efficacy in rhesus macaques of the intradermal (ID)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800. INO-4800 is an ID-delivered DNA vaccine currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and neutralizing antibody responses against both the D614 and G614 SARS-CoV-2 spike proteins. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T and B cell responses. These responses were associated with lower viral loads in the lung and with faster nasal clearance of virus. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system which are likely important for providing durable protection against COVID-19 disease.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.28.225649: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Animals: All rhesus macaque experiments were approved by the Institutional Animal Care and Use Committee at Bioqual (Rockville, Maryland), an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited facility.
    RandomizationImmunizations, sample collection and viral challenge: Ten Chinese rhesus macaques (ranging from 4.55kg-5.55kg) were randomly assigned to be immunized (3 males and 2 females) or naïve (2 males and 3 females).
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableImmunizations, sample collection and viral challenge: Ten Chinese rhesus macaques (ranging from 4.55kg-5.55kg) were randomly assigned to be immunized (3 males and 2 females) or naïve (2 males and 3 females).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ACE2 Competition ELISA-Non-humanprimates: 96-well half area plates (Corning) were coated at room temperature for 3 hours with 1 μg/mL PolyRab anti-His antibody (ThermoFisher, PA1-983B), followed by overnight blocking with blocking buffer containing 1x PBS, 5% SM, 1% FBS, and 0.2% Tween-20.
    anti-His
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Flow cytometry-based ACE2 receptor binding inhibition assay: HEK-293T cells stably expressing ACE2-GFP were generated using retroviral transduction.
    HEK-293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Data was acquired using a BD LSRII and analyzed by FlowJo (version 10).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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:
    Despite these limitations, this study demonstrated significant reduction in peak BAL sgmRNA and overall viral mRNA. Wolfel et al reported nasal titers in patients of an average 6.5 x 105 copies/swab days 1-5 following onset of symptoms (Wolfel et al., 2020). These titers are significantly lower than our NHP challenge dose and support the potential for the vaccine to exhibit great impact in the field against natural SARS-CoV-2 challenge. T cell immunity has been shown to be an important mediator of protection against betacoronaviruses, and recent studies have specifically identified a role in protection against COVID-19 disease (Channappanavar et al., 2014a; Channappanavar et al., 2014b; Sekine et al., 2020; Sun et al., 2020). Sekine et al reported SARS-CoV-2-reactive T cells in asymptomatic and mild COVID-19 convalescent patients who were antibody seronegative (Sekine et al., 2020). These results from convalescent patient studies collectively suggest that vaccine candidates which could generate balanced humoral and cellular immune responses could be important in providing protection against COVID-19 disease. Our study and other published reports show that DNA vaccination with candidates targeting the full-length SARS-CoV-2 spike protein likely increase the availability of T cell immunodominant epitopes leading to a broader and more potent immune response, compared to partial domains and truncated immunogens. In this study, we also observe T cell cross-reactivity to SARS-CoV-1...

    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: 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 10.1101/2020.07.28.225649: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementAnimals: All rhesus macaque experiments were approved by the Institutional Animal Care and Use Committee at Bioqual (Rockville, Maryland), an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited facility.RandomizationTen Chinese rhesus macaques (ranging from 4.55kg-5.55kg) were randomly assigned to be immunized (3 males and 2 females) or naïve (2 males and 3 females).Blindingnot detected.Power Analysisnot detected.Sex as a biological variableTen Chinese rhesus macaques (ranging from 4.55kg-5.55kg) were randomly assigned to be immunized (3 males and 2 females) or naïve (2 males and 3 females).Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    This vaccine candidate induced neutralizing and ACE2-blocking antibodies, as well as T cell responses in mice and guinea pigs.
    ACE2-blocking
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>guinea pigs.</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cross-reactive antibodies were also detected against SARS-CoV S1 protein and RBD, but not MERS-CoV (Supplementary Figure 1).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>SARS-CoV S1 protein</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 pseudovirus-neutralizing antibodies were detected in the serum of vaccinated animals at week 12 following immunization (Figure 1D).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>SARS-CoV-2 pseudovirus-neutralizing</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In this assay, anti-spike antibodies are bound to SARS-CoV-2 spike protein in an ELISA plate followed by addition of a soluble version of the ACE2 receptor.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-spike</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We observe that sera from 80% of immunized NHPs had reduced AUC indicating the presence of antibodies that can block the SARS-CoV-2 spike protein from interacting with ACE2 (Figure 1F).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>ACE2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">One day after viral challenge, 3/5 of INO-4800 vaccinated animals displayed an increase in antibody titers against the SARS-CoV-2 full-length ECD.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>ECD</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 Competition ELISA-Non-human primates 96-well half area plates (Corning) were coated at room temperature for 3 hours with 1 µg/mL PolyRab anti-His antibody (ThermoFisher, PA1-983B), followed by overnight blocking with blocking buffer containing 1x PBS, 5% SM, 1% FBS, and 0.2% Tween-20.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-His</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</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">ACE2-expressing 293T cells were co-incubated with Spike with or without the presence of sera.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T</b></div>
        <div>suggested: KCB Cat# KCB 200744YJ, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0063">CVCL_0063</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK-293T cells stably expressing ACE2-GFP were generated using retroviral transduction.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK-293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus Neutralization Assay SARS-CoV-2 pseudovirus was produced using HEK293T cells transfected with GeneJammer (Agilent) using IgE-SARS-CoV-2 spike plasmid (Genscript) and pNL4-3.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CHO cells stably expressing ACE2 were used as target cells (Creative Biolabs, Catalog No. VCeL-Wyb019).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>CHO</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">10,000 CHO-ACE2 cells/well were plated in 96- well plates in 100ul D10 media and rested overnight at 37˚C and 5% CO2 for 24 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>CHO-ACE2</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">Data was acquired using a BD LSRII and analyzed by FlowJo (version 10).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>FlowJo</b></div>
        <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization titers (ID50) were calculated using GraphPad Prism 8 and defined as the reciprocal serum dilution at which RLU were reduced by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad Prism</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr></table>

    Data from additional tools added to each annotation on a weekly basis.

    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 10.1101/2020.07.28.225649v1 on bioRxiv