Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Protein co-immunization in rhesus macaques

  1. Margherita Rosati
  2. Mahesh Agarwal
  3. Xintao Hu
  4. Santhi Devasundaram
  5. Dimitris Stellas
  6. Bhabadeb Chowdhury
  7. Jenifer Bear
  8. Robert Burns
  9. Duncan Donohue
  10. Laurent Pessaint
  11. Hanne Andersen
  12. Mark G. Lewis
  13. Evangelos Terpos
  14. Meletios Athanasios Dimopoulos
  15. Alexander Wlodawer
  16. James I. Mullins
  17. David J. Venzon
  18. George N. Pavlakis
  19. Barbara K. Felber

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Abstract

The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized Wuhan-Hu-1 SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The antibodies recognized and potently neutralized a panel of different Spike variants including Alpha, Delta, Epsilon, Eta and A.23.1, but to a lesser extent Beta and Gamma. The DNA-only vaccine regimens were compared to a regimen that included co-immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques.

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  1. Version published to 10.1371/journal.ppat.1009701
  2. ScreenIT

    SciScore for 10.1101/2021.06.11.448032: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Ethics Statement: The mouse studies were approved by the National Cancer Institute-Frederick Animal Care and Use Committee and were conducted in accordance with the ACUC guidelines and the NIH Guide for the Care and Use of Laboratory Animals (Approval ASP#20-024).
    Field Sample Permit: The non- human primate studies were conducted in compliance with all relevant local, state, and federal regulations and were approved by the BIOQUAL Institutional Animal Care and Use Committee (IACUC) (Approval #20-042).
    Consent: Donors gave written informed consent.
    Sex as a biological variableVaccination of mice: Female BALB/c 6 weeks-old were obtained from Charles River Laboratory (Frederick, MD).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Protein production was measured by Western immune-blotting by loading same amount of cell lysate or supernatant on Novex Bolt 8% Bis-tris (Thermo Fischer Scientific) and blotted onto nitrocellulose membranes which were probed with the SARS-CoV2/2019-nCoV Spike rabbit polyclonal Ab (PAb, cat# 40591-T62, Sino Biologicals; dilution 1:1,000) followed by anti-Rabbit IgG-HRP conjugated antibody (1:15,000 dilution, GE Healthcare, Piscataway, NJ).
    anti-Rabbit IgG-HRP
    suggested: None
    Anti-Spike antibodies ELISA: An in-house ELISA assay described elsewhere [40, 41] was used to detect the Spike receptor binding domain (Spike-RBD) (AA 319-525) and the complete trimeric Spike (amino acid (AA) 15-1208_2P) using mammalian Expi293-cells produced proteins [64].
    Anti-Spike
    suggested: None
    The following antibodies were used in the cocktail for surface staining: CD3-allophycocyanin (APC)-Cy7 (clone SP34-2, Cat#557757, BD Pharmingen, San Jose, CA), CD4-PE-CF594 (clone L200, Cat#562402, BD Horizon, San Jose, CA), CD8-Brilliant Violet 650 (BV650) (clone RPA-T8; Cat#301042, BioLegend, San Diego, CA), CD28-peridinin chlorophyll protein (PerCP) Cy5.5 (clone CD28.2; Cat#302922, BioLegend, San Diego, CA), CD95-fluorescein isothiocyanate (FITC) (clone DX2; Cat#556640, BD Pharmingen, San Jose, CA) and CD107a-phycoerythrin (PE) monoclonal antibody (clone eBioH4A3, Cat#12-1079-42, eBioscience, San Diego, CA).
    CD3-allophycocyanin
    suggested: None
    APC)-Cy7
    suggested: None
    CD4-PE-CF594
    suggested: None
    BV650
    suggested: (BioLegend Cat# 301042, RRID:AB_2563505)
    CD28-peridinin chlorophyll protein (PerCP) Cy5.5
    suggested: None
    CD95-fluorescein
    suggested: None
    CD107a-phycoerythrin (PE
    suggested: None
    After cell permeabilization, intracellular staining was performed using IFN-γ-phycoerythrin (PE) Cy7 (clone B27, Cat#557643, BD Pharmingen, San Jose, CA), Granzyme B- allophycocyanin (APC) (clone GB12, Cat#MHGB05, Invitrogen), and Ki67-Alexa Fluor-700 (clone B56, Cat#561277, BD Pharmingen, San Jose, CA) antibodies.
    IFN-γ-phycoerythrin
    suggested: None
    Cy7
    suggested: None
    Ki67-Alexa
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    In vitro analysis of the Spike DNA plasmids: Expression of Spike DNA plasmids was analyzed upon transient transfection of HEK293T clone17 cells using the Calcium Phosphate DNA co-precipitation and DNA (1 microG DNA/60 mm plate) as described [32, 63].
    HEK293T
    suggested: None
    Briefly, pseudotyped viruses carrying different Spike proteins were generated in HEK293T/clone17 cells by co-transfection with pHIVNLΔEnv- Nanoluc.
    HEK293T/clone17
    suggested: None
    The undiluted and the 7 dilutions (100 microL; 1x105 Luminescence (RLU)) were used in triplicate to transduce HEK293T/ACE2wt cells.
    HEK293T/ACE2wt
    suggested: None
    Plaque reduction neutralization test (PRNT): The PRNT assay were conducted in Vero E6 cells (ATCC, cat# CRL-1586) plated in 24-well plates at 175,000 cells/well in DMEM + 10% FBS + Gentamicin reaching 80-100% confluency the following day.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Vaccination of mice: Female BALB/c 6 weeks-old were obtained from Charles River Laboratory (Frederick, MD).
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Recombinant DNA
    SentencesResources
    SARS-COV2 DNA vaccine: All plasmids contain RNA/codon-optimized genes inserted between the human CMV promoter and the BGH polyadenylation signal of the expression vector pCMV.kan [61].
    pCMV.kan
    suggested: None
    Plasmid S-RBD_ΔF (plasmid C36) produces a protein comprising the Spike CoV-2 signal (AA1- 14), Spike AA 295-1171 and the T4 foldon.
    S-RBD_ΔF
    suggested: None
    Software and Algorithms
    SentencesResources
    The images of the blot were acquired on the ChemiDoc XRS using Bio-Rad ImageLab software (Bio-Rad laboratories, Inc).
    Bio-Rad ImageLab
    suggested: None
    Vaccination of mice: Female BALB/c 6 weeks-old were obtained from Charles River Laboratory (Frederick, MD).
    Charles River Laboratory
    suggested: None
    Five mice per group were vaccinated by the in vivo electroporation method (AgilePulseTM System; BTX, Harvard Bioscience, Inc) at weeks 0 and 3.
    Harvard Bioscience
    suggested: None
    The 50% Inhibitory dose (ID50) was calculated using GraphPad Prism version 9.0.2 for MacOS X (GraphPad Software, Inc, La Jolla, CA) with nonlinear regression curve fit using inhibitor vs responses variable slope (four parameters).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Samples were acquired on a Fortessa or Symphony flow cytometer (BD Biosciences, San Jose, CA), and the data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).
    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: 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
    NCT04408209RecruitingConvalescent Plasma for the Treatment of Patients With Sever…

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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

    About SciScore

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  3. Version published to 10.1101/2021.06.11.448032v1 on bioRxiv