Enhanced immunogenicity of a synthetic DNA vaccine expressing consensus SARS-CoV-2 Spike protein using needle-free immunization

  1. Sawsan S Alamri
  2. Khalid A Alluhaybi
  3. Rowa Y Alhabbab
  4. Abdullah Algaissi
  5. Sarah Almahboub
  6. Mohamed A Alfaleh
  7. Turki S Abujamel
  8. Wesam Abdulaal
  9. M-Zaki ElAssouli
  10. Rahaf Alharbi
  11. Mazen Hassanain
  12. Anwar M Hashem

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Abstract

The ongoing global pandemic of Coronavirus Disease 2019 (COVID-19) calls for an urgent development of effective and safe prophylactic and therapeutic measures. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is a major immunogenic and protective protein, and plays a crucial role in viral pathogenesis. In this study, we successfully constructed a synthetic codon-optimized DNA-based vaccine as a countermeasure against SARS-CoV-2; denoted as VIU-1005. The design was based on the synthesis of codon-optimized coding sequence for optimal mammalian expression of a consensus full-length S glycoprotein. The successful construction of the vaccine was confirmed by restriction digestion and sequencing, and the protein expression of the S protein was confirmed by western blot and immunofluorescence staining in mammalian cells. The immunogenicity of the vaccine was tested in two mouse models (BALB/c and C57BL/6J). Th1-skewed systemic S-specific IgG antibodies and neutralizing antibodies (nAbs) were significantly induced in both models four weeks post three injections with 100 μg of the VIU-1005 vaccine via intramuscular needle injection but not intradermal or subcutaneous routes. Importantly, such immunization induced long-lasting IgG response in mice that lasted for at least 6 months. Interestingly, using a needle-free system, we showed an enhanced immunogenicity of VIU-1005 in which lower doses such as 25-50 μg or less number of doses were able to elicit significantly high levels of Th1-biased systemic S-specific IgG antibodies and nAbs via intramuscular immunization compared to needle immunization. Compared to the intradermal needle injection which failed to induce any significant immune response, intradermal needle-free immunization elicited robust Th1-biased humoral response similar to that observed with intramuscular immunization. Furthermore, immunization with VIU-1005 induced potent S-specific cellular response as demonstrated by the significantly high levels of IFN-γ, TNF and IL-2 cytokines production in memory CD8 + and CD4 + T cells in BALB/c mice. Together, our results demonstrate that the synthetic VIU-1005 candidate DNA vaccine is highly immunogenic and capable of inducing long-lasting and Th1-skewed immune response in mice. Furthermore, we show that the use of needle-free system could enhance the immunogenicity and minimize doses needed to induce protective immunity in mice, supporting further preclinical and clinical testing of this candidate vaccine.

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  1. ScreenIT

    SciScore for 10.1101/2021.02.01.429219: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All animal experiments were conducted in accordance with the guidelines and approval of the Institutional Animal Care and Use Committee (IACUC) at KFMRC and the ethical approval from the bioethical committee at KAU (approval number 04-CEGMR-Bioeth-2020).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAnimal Studies: Six to 8-week-old female BALB/c or C57BL/6J mice were obtained from and housed in the animal facility in King Fahd Medical Research Center (KFMRC), King Abdulaziz University (KAU), Jeddah, Saudi Arabia.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The harvested cell lysates were subjected to western blot analysis to verify the expression of S protein using in house mouse anti-S (SARS-CoV-2) polyclonal antibodies at a 1:2000 dilution.
    anti-S
    suggested: None
    SARS-CoV-2
    suggested: None
    After three washes with PBS-T, cells were incubated with Alexa Fluor-488 labeled goat anti-mouse IgG H&L secondary antibody (Abcam, UK) at 1:500 dilution in blocking buffer in the dark at room temperature for 1 h.
    anti-mouse IgG
    suggested: (Abcam Cat# ab19639, RRID:AB_2040847)
    peroxidase-conjugated rabbit anti-mouse IgG secondary antibodies as well as anti-IgG1, IgG2a or IgG2b antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) were added at dilutions recommended by the manufacturer and incubated for 1 h at 37°C as 100 μl/well.
    peroxidase-conjugated rabbit anti-mouse IgG secondary antibodies
    suggested: None
    anti-IgG1 , IgG2a
    suggested: None
    IgG2b
    suggested: None
    After washing with PBS, PB-conjugated anti-mouse CD8, PB-conjugated anti-mouse CD4, APC-conjugated anti-mouse CD44 antibody and Pe-Cy7-conjugated anti-mouse CD62L antibodies (BioLegend, UK) were used for surface markers staining.
    anti-mouse CD8
    suggested: None
    anti-mouse CD4
    suggested: (Gen-Probe Cat# 873.045.050, RRID:AB_10408047)
    anti-mouse CD44
    suggested: None
    anti-mouse CD62L
    suggested: None
    PE-conjugated anti-mouse TNF-α (clone MP6-XT22) and Pe-Cy7–conjugated anti-mouse IL-2 (clone JES6-5H4) antibodies (BioLegend, UK) for 20 min at 4°C.
    anti-mouse TNF-α
    suggested: (Leinco Technologies Cat# T798, RRID:AB_2832121)
    anti-mouse IL-2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells: Baby Hamster kidney BHK-21/WI-2 cell line (Kerafast, EH1011), African Green monkey kidney-derived Vero E6 cell line (ATCC, CRL-1586) and Human embryonic kidney 293 cells (ATCC, CRL-1573) were cultured in Dulbecco’s modified essential medium (DMEM) containing penicillin (100 U/ml) and streptomycin (100 μg/ml) and supplemented with 5 or 10% fetal bovine serum (FBS) in a 5% CO2 environment at 37°C.
    BHK-21/WI-2
    suggested: RRID:CVCL_HB78)
    Immunofluorescence analysis: HEK-293 cells (70% confluent) on a 8-well cell culture slide [growth area/well (cm²): 0.98 and working volume/well (ml): 0.20 - 0.60] were transfected with 0.2 μg of VIU-1005 or control plasmid using JetPRIME® Transient Transfection Protocol and Reagents (Polyplus, New York, NY) according to manufacturer’s instructions, and followed incubated at 37°C in a 5% CO2 incubator for 24 h.
    HEK-293
    suggested: None
    Pseudovirus–serum mixtures were transferred onto confluent Vero E6 cell monolayers in white 96-well plates and incubated for 24 h at 37°C in a 5% CO2 incubator.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    In one experiment, two groups of BALB/c or C57BL/6J mice (10 per group) were intramuscularly immunized via needle injection with 3 doses of 100 μg of either VIU-1005 or control plasmid at 2-week interval and blood samples were collected for serological testing every 2 weeks starting from day 0 (pre-bleed) until week 8.
    BALB/c
    suggested: None
    C57BL/6J
    suggested: None
    Software and Algorithms
    SentencesResources
    In silico design of codon-optimized synthetic consensus S protein: All
    All
    suggested: None
    The final dataset was multiply aligned using CLUSTALW and the Shannon entropy for each amino acid position were determined and the consensus protein sequence was then obtained for the full-length S glycoprotein.
    CLUSTALW
    suggested: (ClustalW, RRID:SCR_017277)
    Images were captured using Olympus BX51 Fluorescence Microscope and were analyzed using Image-Pro Plus software.
    Image-Pro Plus
    suggested: (Image-Pro Plus, RRID:SCR_007369)
    The median inhibitory concentration (IC50) of neutralizing antibodies (nAbs) was determined using four-parameter logistic (4PL) curve in GraphPad Prism V8 software (GraphPad Co.) and calculated as the reciprocal of the serum dilution at which RLU was reduced by 50% compared with the virus control wells after subtraction of the background RLUs in the control groups with cells only.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    All data were collected using a 15 laser Aria flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo v10 software (Tree Star).
    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: 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

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  2. Version published to 10.1101/2021.02.01.429219 on bioRxiv