Th1 Dominant Nucleocapsid and Spike Antigen-Specific CD4+ and CD8+ Memory T Cell Recall Induced by hAd5 S-Fusion + N-ETSD Infection of Autologous Dendritic Cells from Patients Previously Infected with SARS-CoV-2

  1. Peter Sieling
  2. Lise Zakin
  3. Annie Shin
  4. Brett Morimoto
  5. Helty Adisetiyo
  6. Hermes Garban
  7. Philip Liu
  8. Adrian Rice
  9. Justin Taft
  10. Roosheel Patel
  11. Sofija Buta
  12. Marta Martin-Fernandez
  13. Dusan Bogunovic
  14. Elizabeth Gabitzsch
  15. Jeffrey T. Safrit
  16. Lennie Sender
  17. Patricia Spilman
  18. Shahrooz Rabizadeh
  19. Kayvan Niazi
  20. Patrick Soon-Shiong

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

To address the need for a safe, efficacious vaccine against SARS-CoV-2 infection with the critical properties of enabling both blocking viral entry into cells and clearing virus from cells already infected, we have developed a bivalent, human adenovirus serotype 5 (hAd5) SARS-CoV- 2 S-Fusion + N-ETSD vaccine that is currently in clinical testing. This vaccine uses the next- generation hAd5 [E1-, E2b-, E3-] platform previously used successfully in cancer patients with pre-existing adenovirus immunity, engineered to express both SARS-CoV-2 spike (S) protein modified to improve the generation of neutralizing antibodies to block entry of the virus, and nucleocapsid (N) protein with an Enhanced T cell Stimulation Domain (ETSD) to activate CD4+ and CD8+ T cells to clear the virus and block replication by killing infected cells. The targeting of N to endosomes and lysosomes to enhance CD4+ and CD8+ T-cell responses distinguishes our vaccine. In our previously reported pre-clinical studies we showed that in mice, the hAd5 S-Fusion + N-ETSD vaccine elicits both humoral and T-cell responses that are robust and T helper cell 1 (Th1) dominant. Here we report that the hAd5 S-Fusion + N-ETSD vaccine is recognized by anti-sera and T cells from previously SARS-CoV-2 infected patients, and that the presence of N is vital for T-cell recall. The findings presented herein: (i) demonstrate specific recognition of hAd5 S-Fusion + N-ETSD infected cells by plasma antibodies from previously SARS-CoV-2 infected patients, but not antibodies from virus-naïve subjects; (ii) show enhanced binding of plasma SARS-CoV-2 antibodies from previously infected patients to monocyte-derived dendritic cells (MoDCs) expressing the hAd5 S-Fusion + N-ETSD vaccine as compared to hAd5 S-Fusion alone; (iii) reveal N-ETSD localizes to vesicles associated with MHC class II antigen presentation, including endosomes, lysosomes and autophagosomes in MoDCs; (iv) demonstrate endosome/lysosome-targeted N-ETSD elicits higher interferon-γ T-cell responses than cytoplasm-localized N; and (v) N-ETSD alone or in the hAd5 S-Fusion + N-ETSD construct induces both CD4+ and CD8+ T cell memory recall. This recognition of hAd5 S-Fusion + N-ETSD vaccine antigens by T cells from previously SARS-CoV-2 infected patients, together with the ability of this vaccine candidate to elicit de novo immune responses in naïve mice suggests that it re-capitulates the natural immune response to SARS-CoV-2 to activate both B and T cells towards viral neutralization and recognition of infected cells, critical for prevention of COVID-19 disease. Intriguingly, our hAd5 S-Fusion + N-ETSD T-cell biased vaccine has the potential to not only provide protection for uninfected individuals, but also to be utilized as a therapeutic for already infected patients to induce rapid clearance of the virus by activating T cells to kill the virus-infected cells, thereby reducing viral replication and lateral transmission.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.11.04.20225417: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: The constructs created included: Collection of plasma and peripheral blood mononuclear cells from patients with confirmed previous SARS-CoV-2 infection and from virus-naïve volunteers: Blood was collected with informed consent via venipuncture from volunteers who had either not been exposed (UNEX) to SARS-CoV-2 as confirmed by ELISA and multiple negative SARS-CoV-2 tests or who had recovered from COVID-19 as indicated by recent medical history and a positive SARS-CoV-2 antibody test (Patients, Pt).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Infection of MoDCs with hAd5 N-WT or N-ETSD and labeling with anti-N, anti-CD71, anti-LAMP-1, and Anti-LC3a/b antibodies: Freshly thawed MoDCs were plated on 4-well Lab-Tek II CC2 Chamber Slides, using 3 ×104 cells per well and transduction performed at MOI 5000 one hour after plating using hAd5 N-ETSD or hAd5 N.
    anti-N
    suggested: None
    anti-CD71
    suggested: (Miltenyi Biotec Cat# 130-104-152, RRID:AB_2660547)
    anti-LAMP-1
    suggested: None
    Anti-LC3a/b
    suggested: None
    To label N, cells were then incubated with an anti-flag monoclonal (Anti-Flag M2 produced in mouse) antibody at 1:1000 in phosphate buffered saline (PBS) with 3% BSA, 0.5% Triton X100 and 0.01% saponin overnight at 4°C, followed by three washes in PBS and a 1 hour incubation with a goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 (Life Technologies) at 1:500.
    anti-flag
    suggested: (Advanced Targeting Systems Cat# AB-450-1000, RRID:AB_10584603)
    anti-Mouse IgG
    suggested: None
    For co-localization studies, cells were also incubated overnight at 4°C with a rabbit anti-CD71 (transferrin receptor, recycling/sorting endosomal marker) antibody (ThermoFisher) at 1:200; sheep anti-Lamp1 Alexa Fluor 488-conjugated (lysosomal marker) antibody (R&D systems) at 1:10; or a rabbit monoclonal anti human LC3a/b (Light Chain 3, autophagy marker) antibody (Cell Signaling Tech #12741S) used at 1:100.
    anti-Lamp1
    suggested: None
    After removal of the primary antibody, two washes in PBS and three washes in PBS with 3% BSA, cells were incubated with fluor-conjugated secondary antibodies when applicable at 1:500 (Goat anti-Rabbit IgG (H+L) secondary antibody, Alexa Fluor 488; 1:500 dilution) for 1 hour at room temperature.
    anti-Rabbit IgG
    suggested: None
    One day after infection, the MoDCs were detached using EDTA (0.5 mM), gently pipetted and transferred for incubation with previously infected patient plasma at 1:100 dilution from a single patient (Pt4) at (4°C) for 30 minutes, and plasma antibodies detected on the MoDC surface by goat anti-human IgG (phycoerythrin conjugated).
    anti-human IgG
    suggested: None
    IL-4 was measured by ELISpot using a kit (MabTech) with wells precoated with anti-IL-4 antibody and following the manufacturer’s instructions.
    anti-IL-4
    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: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 11 and 44. 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.
    • Thank you for including a protocol registration statement.

    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. Version published to 10.1101/2020.11.04.20225417v1 on medRxiv