Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity

  1. Antonella Scaglione
  2. Silvana Opp
  3. Alicia Hurtado
  4. Ziyan Lin
  5. Christine Pampeno
  6. Maria G. Noval
  7. Sara A. Thannickal
  8. Kenneth A. Stapleford
  9. Daniel Meruelo

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Abstract

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world’s population at record speeds. However, there is still a demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus αOX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical, and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles, and metabolic analysis indicate a reprogramming of T cells in vaccinated mice. Activated T cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus αOX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response, which can be used as a new candidate to combat SARS-CoV-2. Given the T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.

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  1. Version published to 10.3389/fimmu.2021.719077
  2. ScreenIT

    SciScore for 10.1101/2021.05.28.446009: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: 4.5 In vivo experiments: All experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of New York University Grossman School of Medicine.
    Field Sample Permit: high-containment research facility under the responsibility of the Office of Science & Research and its Director of High-Containment Laboratories.
    Sex as a biological variableSix to 12-week old female C57BL/6J albino mice (B6(Cg)-Tyr/J,Cat#000058) and Hemizygous (B6(Cg)-Tg(K18-ACE2)2Prlmn/J; Cat#034860) (hACE2-Tg) mice expressing the human ACE2 receptor or non-carrier controls were purchased from Jackson Laboratory.
    RandomizationAfter incubation, five fields were randomly selected in each well to count the number of fused and unfused cells under an inverted fluorescence microscope (Nikon Eclipse Ti-S)
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ) and anti-rabbit hACE2 (Thermo Fisher,1:100) were applied overnight at 4 °C followed by incubation of appropriate secondary antibodies conjugated with fluorophores.
    anti-rabbit hACE2
    suggested: None
    Fluorochrome-conjugated antibodies against mouse CD3, CD4, CD44, CD38, ICOS, OX40, CD62L, Perforin, Granzyme B and Tbet, CXCR5 were purchased from Biolegend.
    CD3
    suggested: None
    CD4
    suggested: (RayBiotech Cat# CS-11-0133, RRID:AB_1228052)
    CD44
    suggested: None
    CD38
    suggested: None
    ICOS
    suggested: None
    CD62L
    suggested: None
    CXCR5
    suggested: None
    Fluorochrome-conjugated antibodies against mouse CD8a were purchased from BD Biosciences.
    CD8a
    suggested: None
    Fluorochrome-conjugated antibodies against CXCR3 and Ki67 were purchased from Thermofisher.
    CXCR3
    suggested: None
    Ki67
    suggested: None
    Primary antibodies to SARS-CoV-2 spike (GTX, 1:1000) and p24 (Abcam, 1:1000) were added overnight at 4 °C.
    p24
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    . 293T/ACE2 cell line was obtained from BEI Resources.
    293T/ACE2
    suggested: RRID:CVCL_YZ65)
    Helper and replicon RNAs were then electroporated into BHK cells and incubated at 37°C in αMEM supplemented with 10% FCS.
    BHK
    suggested: None
    For preparing effector cells expressing SARS-CoV-2 spike, 293T cells were transiently co-transfected with pCDNA3.1-Spike and pMAX-GFP or with pMAX-GFP only as control, and applied onto 293T/ACE2 cells after 48 h.
    293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    4.8 In vivo delivery of nLacZ-SARS-CoV-2 pseudotype and X-Gal histochemistry: Isoflurane-anesthetized 4-week-old young adult hACE2-Tg mice were dosed intranasally with a 70-μl volume of nLacZ-encoding lentiviral vector (titer 5.18×103 TU/ml).
    hACE2-Tg
    suggested: None
    Purified T-cells from C57BL/6J immunized or control mice were plated at 6×105 cells/well in a Seahorse XF24 cell culture microplate.
    C57BL/6J
    suggested: None
    Recombinant DNA
    SentencesResources
    Briefly, plasmids carrying the replicon (pT7-SV-Spike) orT7-DMHelper RNAs were linearized with XhoI.
    pT7-SV-Spike
    suggested: None
    4.3 Pseudotyped Lentivirus Production: SARS CoV-2 pseudotyped lentiviruses were produced by transfecting the 293T cells with the pLenti-Puro vectors (Addgene) expressing Luciferase or β-Galactosidase, with pcDNa3.1 vector expressing SARS-CoV-2 spike (BEI repository) and the helper plasmid pSPAX2 (Addgene).
    pLenti-Puro
    suggested: RRID:Addgene_39481)
    pcDNa3.1
    suggested: RRID:Addgene_79663)
    pSPAX2
    suggested: RRID:Addgene_12260)
    The VSV-G and empty lentiviruses were produced by replacing pCDNA3.1-Spike with pcDNA3.1-VSV-G or pCDNA3.1 empty vector, respectively (Addgene).
    pcDNA3.1-VSV-G
    suggested: None
    For preparing effector cells expressing SARS-CoV-2 spike, 293T cells were transiently co-transfected with pCDNA3.1-Spike and pMAX-GFP or with pMAX-GFP only as control, and applied onto 293T/ACE2 cells after 48 h.
    pCDNA3.1-Spike
    suggested: None
    pMAX-GFP
    suggested: RRID:Addgene_16007)
    Software and Algorithms
    SentencesResources
    Flow cytometry analysis was performed on a LSR II machine (BD Bioscience) and data were analyzed using FlowJo (Tree Star).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Sequencing reads were mapped to the reference genome (mm10) using the STAR aligner (v2.6.1d)[89].
    STAR
    suggested: (STAR, RRID:SCR_004463)
    The mean read insert sizes and their standard deviations were calculated using Picard tools (v.2.18.20) (http://broadinstitute.github.io/picard).
    Picard
    suggested: (Picard, RRID:SCR_006525)
    The read count tables were generated using subread (v1.6.3)[90], (normalized based on their library size factors using DEseq2[91], and differential expression analysis was performed.
    DEseq2
    suggested: (DESeq2, RRID:SCR_015687)
    All the downstream statistical analyses and generating plots were performed in R environment (v4.0.3) (https://www.r-project.org/).
    https://www.r-project.org/
    suggested: (R Project for Statistical Computing, RRID:SCR_001905)
    The results of gene set enrichment analysis were generated by GSEA software[92; 93].
    GSEA
    suggested: (SeqGSEA, RRID:SCR_005724)
    The network of Gene Ontology terms was generated by Enrichment Map in Cytoscape.
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    Additional protein–protein functional associations used in this study for bar graphs were retrieved from STRING (http://www.string-db.org/, version 11)[94], a well-known public database on several collected associations between proteins from various organisms.
    STRING
    suggested: (STRING, RRID:SCR_005223)
    Figures were prepared using GraphPad Prism 7, Adobe Photoshop and ImageJ Software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Adobe Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    (GraphPad Software) to naïve mice.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 38, 44, 46, 47 and 49. 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.
    • No protocol registration statement was detected.

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

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  3. Version published to 10.1101/2021.05.28.446009v2 on bioRxiv
  4. Version published to 10.1101/2021.05.28.446009v1 on bioRxiv