Optimization of Non-Coding Regions Improves Protective Efficacy of an mRNA SARS-CoV-2 Vaccine in Nonhuman Primates

  1. Makda S. Gebre
  2. Susanne Rauch
  3. Nicole Roth
  4. Jingyou Yu
  5. Abishek Chandrashekar
  6. Noe B. Mercado
  7. Xuan He
  8. Jinyan Liu
  9. Katherine McMahan
  10. Amanda Martinot
  11. Tori Giffin
  12. David Hope
  13. Shivani Patel
  14. Daniel Sellers
  15. Owen Sanborn
  16. Julia Barrett
  17. Xiaowen Liu
  18. Andrew C. Cole
  19. Laurent Pessaint
  20. Daniel Valentin
  21. Zack Flinchbaugh
  22. Jake Yalley-Ogunro
  23. Jeanne Muench
  24. Renita Brown
  25. Anthony Cook
  26. Elyse Teow
  27. Hanne Andersen
  28. Mark G. Lewis
  29. Stefan O. Mueller
  30. Benjamin Petsch
  31. Dan H. Barouch

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Abstract

The CVnCoV (CureVac) mRNA vaccine for SARS-CoV-2 has recently been evaluated in a phase 2b/3 efficacy trial in humans. CV2CoV is a second-generation mRNA vaccine with optimized non-coding regions and enhanced antigen expression. Here we report a head-to-head study of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in nonhuman primates. We immunized 18 cynomolgus macaques with two doses of 12 ug of lipid nanoparticle formulated CVnCoV, CV2CoV, or sham (N=6/group). CV2CoV induced substantially higher binding and neutralizing antibodies, memory B cell responses, and T cell responses as compared with CVnCoV. CV2CoV also induced more potent neutralizing antibody responses against SARS-CoV-2 variants, including B.1.351 (beta), B.1.617.2 (delta), and C.37 (lambda). While CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded robust protection with markedly lower viral loads in the upper and lower respiratory tract. Antibody responses correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of an mRNA SARS-CoV-2 vaccine in nonhuman primates.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.13.456316: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: All animal 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).
    IACUC: All animal 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).
    Sex as a biological variablenot detected.
    RandomizationAnimals and study design: 18 cynomolgus macaques were randomly assigned to three groups.
    BlindingImmunologic and virologic assays were performed blinded.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    B cell immunophenotyping: Fresh PBMCs were stained with Aqua live/dead dye (Invitrogen) for 20 min, washed with 2% FBS/DPBS buffer, and suspended in 2% FBS/DPBS buffer with Fc Block (BD) for 10 min, followed by staining with monoclonal antibodies against CD45 (clone D058-1283, BUV805), CD3 (clone SP34.2, APC-Cy7), CD7 (clone M-T701, Alexa700), CD123 (clone 6H6, Alexa700), CD11c (clone 3.9, Alexa700), CD20 (clone 2H7, PE-Cy5), IgA (goat polyclonal antibodies, APC), IgG (clone G18-145, BUV737), IgM (clone G20-127, BUV396), IgD (goat polyclonal antibodies, PE), CD80 (clone L307.4, BV786), CD95 (clone DX2, BV711), CD27 (clone M-T271, BUV563), CD21 (clone B-ly4, BV605), CD14 (clone M5E2, BV570) and CD138 (clone DL-101, PE-CF594).
    CD45
    suggested: (BD Biosciences Cat# 564914, RRID:AB_2744401)
    CD3
    suggested: (GenWay Biotech Inc. Cat# GWB-220B7B, RRID:AB_10511043)
    CD7
    suggested: (BD Biosciences Cat# 741824, RRID:AB_2871159)
    CD123
    suggested: (BD Biosciences Cat# 751837, RRID:AB_2875808)
    CD11c
    suggested: (BioLegend Cat# 117331, RRID:AB_10900261)
    CD20
    suggested: (BD Biosciences Cat# 743611, RRID:AB_2741622)
    PE-Cy5), IgA
    suggested: (Bioss Cat# bs-0645R-PE-Cy5, RRID:AB_11071475)
    IgM
    suggested: (Creative Diagnostics Cat# DCABY-1319, RRID:AB_2493413)
    CD80
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    CD95
    suggested: (BD Biosciences Cat# 741292, RRID:AB_2870823)
    CD27
    suggested: (BD Biosciences Cat# 748705, RRID:AB_2873109)
    CD21
    suggested: (BD Biosciences Cat# 740395, RRID:AB_2740125)
    CD14
    suggested: (BioLegend Cat# 301831, RRID:AB_10897803)
    CD138
    suggested: None
    IFN-γ enzyme-linked immunospot (ELISPOT) assay: ELISPOT plates were coated with mouse anti-human IFN-γ monoclonal antibody from BD Pharmingen at a concentration of 5 μg/well overnight at 4°C.
    anti-human IFN-γ
    suggested: None
    The plates are washed a second time and incubated for 2 h with Streptavidin-alkaline phosphatase antibody from Southern Biotechnology (1 μg/mL).
    Streptavidin-alkaline phosphatase
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, the packaging plasmid psPAX2 (AIDS Resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and Spike protein expressing pcDNA3.1-SARS CoV-2 SΔCT of variants were co-transfected into HEK293T cells by lipofectamine 2000 (ThermoFisher).
    HEK293T
    suggested: None
    To determine the neutralization activity of the plasma or serum samples from participants, HEK293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 1.75 x 104 cells/well overnight.
    HEK293T-hACE2
    suggested: RRID:CVCL_A7UK)
    Recombinant DNA
    SentencesResources
    Briefly, the packaging plasmid psPAX2 (AIDS Resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and Spike protein expressing pcDNA3.1-SARS CoV-2 SΔCT of variants were co-transfected into HEK293T cells by lipofectamine 2000 (ThermoFisher).
    psPAX2
    suggested: RRID:Addgene_12260)
    pLenti-CMV
    suggested: None
    pcDNA3.1-SARS
    suggested: None
    The gene fragment was subsequently cloned into a pcDNA3.1+ expression plasmid using restriction site cloning (Integrated DNA Technologies).
    pcDNA3.1+
    suggested: RRID:Addgene_117272)
    Software and Algorithms
    SentencesResources
    Subsequent analyses were performed using FlowJo software (Treestar, v.9.9.6).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analyses: Statistical analyses were performed using GraphPad Prism (version 9.0) software (GraphPad Software) and comparison between groups was performed using a two-tailed nonparametric Mann-Whitney U t test.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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 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.

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

    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 10.1101/2021.08.13.456316v1 on bioRxiv