Influenza Virus-like Particle-Based Hybrid Vaccine Containing RBD Induces Immunity against Influenza and SARS-CoV-2 Viruses

  1. Ramireddy Bommireddy
  2. Shannon Stone
  3. Noopur Bhatnagar
  4. Pratima Kumari
  5. Luis Munoz
  6. Judy Oh
  7. Ki-Hye Kim
  8. Jameson Berry
  9. Kristen Jacobsen
  10. Lahcen Jaafar
  11. Swe-Htet Naing
  12. Allison Blackerby
  13. Tori Gaag
  14. Chloe Wright
  15. Lilin Lai
  16. Christopher Pack
  17. Sampath Ramachandiran
  18. Mehul Suthar
  19. Sang-Moo Kang
  20. Mukesh Kumar
  21. Shaker Reddy
  22. Periasamy Selvaraj

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Abstract

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since millions of people are exposed to influenza virus and SARS-CoV-2, it is of great interest to develop a two-in-one vaccine that will be able to protect against infection of both viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that a hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.

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  1. Version published to 10.3390/vaccines10060944
  2. ScreenIT

    SciScore for 10.1101/2022.02.01.478657: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Mice: BALB/c mice (Taconic Biosciences Inc.) 2-3 months age (female) were purchased and housed in the Emory University Division of Animal Resources (DAR) facility and used according to the University IACUC guidelines.
    Sex as a biological variableMice: BALB/c mice (Taconic Biosciences Inc.) 2-3 months age (female) were purchased and housed in the Emory University Division of Animal Resources (DAR) facility and used according to the University IACUC guidelines.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and proteins: Purified anti-mouse GM-CSF (clone MP1-22E9) and anti-mouse IL-12 (clone C17.8) were from BioXcell and used for affinity chromatography purification of GPI-RBD-GM-CSF fusion protein and GPI-IL-12, respectively.
    anti-mouse GM-CSF
    suggested: None
    GPI-IL-12
    suggested: None
    Anti-spike RBD antibody (clone MM57) obtained from Sino Biologicals (Cat#40592).
    Anti-spike RBD antibody
    suggested: None
    Anti-spike RBD
    suggested: None
    FITC-conjugated goat secondary antibody against mouse IgG/IgM was purchased from BD Pharmingen (Cat# 555988).
    mouse IgG/IgM
    suggested: (Novus Cat# NBP1-75215, RRID:AB_11005727)
    Peroxidase (HRP)-conjugated goat anti-mouse IgG F(ab’)2 specific antibody was from ThermoFisher Scientific/Pierce (Cat#31436).
    anti-mouse IgG
    suggested: (Thermo Fisher Scientific Cat# 31436, RRID:AB_228313)
    HRP-conjugated donkey anti-human IgG antibody was obtained from Jackson Immunoresearch (Cat#709-036-098).
    HRP-conjugated donkey anti-human IgG antibody
    suggested: None
    anti-human IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 709-036-098, RRID:AB_2340497)
    Expression of both the S1 RBD and GM-CSF on the surface of transfected CHO-S cells was confirmed by flow cytometry using fluorophore conjugated antibodies against RBD, Clone MM57 (Sino Biologicals) and GM-CSF, Clone MP1-22E9 (BioLegend).
    GM-CSF
    suggested: None
    After transfer, the membranes with the proteins were blocked for 1 hr at room temperature with 5 % milk in phosphate buffered saline and 0.2 % Tween 20 (PBS-T) and incubated with a primary antibody (anti-RBD, anti-mouse GM-CSF or anti-mouse IL-12) overnight at 4 °C in PBS-T with on a shaker at low speed.
    anti-RBD
    suggested: None
    Next day, membranes were washed three times with PBS-T and then incubated with appropriate secondary antibody conjugated with alkaline phosphatase that provides a visual color change upon addition of the chromogenic substrate (mixture of BCIP (5-bromo-4chloro-3-indolyl phosphate-catalog# 34040) and NBT (nitro-blue tetrazolium chloride, catalog# 34035 from Thermo Scientific)).
    5-bromo-4chloro-3-indolyl phosphate-catalog# 34040
    suggested: None
    Resulting incorporation was detected by western blot and flow cytometry analysis using anti-RBD mAb, clone MM57 (Sino Biologicals), anti-mouse GM-CSF (clone MP1-22E9, BioLegend), and anti-mouse IL-12 (clone C17.8, Invitrogen) antibodies.
    anti-mouse IL-12
    suggested: None
    Enzyme-linked immunosorbent assay (ELISA): SARS-CoV-2 S protein RBD specific antibodies of different subtypes (IgG, IgG1, IgG2a) were determined in sera by enzyme-linked immunosorbent assay (ELISA) using an approach similar to one previously described using PR8 or WSN as targets (44, 45).
    IgG1, IgG2a
    suggested: None
    Plates were washed and diluted secondary antibody (HRP-conjugated) against mouse total IgG or immunoglobulin isotypes (IgG1, IgG2a) were added and incubated for 30 minutes at room temperature.
    mouse total IgG
    suggested: None
    To measure influenza antigen-specific antibody levels in immune sera, inactivated A/PR8 H1N1 virus (200 ng/well) was coated onto ELISA plates, followed by addition of diluted immune sera.
    antigen-specific
    suggested: None
    IgG isotypes were measured using goat anti-mouse immunoglobulin (Ig) G, IgG1 and IgG2a, and horse-radish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotechnology).
    anti-mouse immunoglobulin (Ig) G, IgG1
    suggested: None
    Plaque Reduction Neutralization Test (PRNT): The titers of anti-SARS-CoV-2 neutralizing antibodies were measured in the serum of BALB/c mice using PRNT assay as described previously (41).
    anti-SARS-CoV-2 neutralizing
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    For mouse sera, CHO-S cells were incubated with diluted serum samples (100-100,000 times diluted in FACS buffer).
    CHO-S
    suggested: None
    The antibody-virus mixture was then added to Vero cells and incubated at 37° C for 1 hour.
    Vero
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: BALB/c mice (Taconic Biosciences Inc.) 2-3 months age (female) were purchased and housed in the Emory University Division of Animal Resources (DAR) facility and used according to the University IACUC guidelines.
    BALB/c
    suggested: None
    Recombinant DNA
    SentencesResources
    This construct was cloned into the pCHO 1.0 vector using the AvrII and BstZ17l sites (Invitrogen).
    pCHO 1.0
    suggested: None
    Software and Algorithms
    SentencesResources
    Peroxidase (HRP)-conjugated goat anti-mouse IgG F(ab’)2 specific antibody was from ThermoFisher Scientific/Pierce (Cat#31436).
    ThermoFisher Scientific/Pierce
    suggested: None
    After incubation with secondary antibody, cells were washed with FACS buffer and resuspended in FACS buffer and acquired in a FACSCalibur (BD Biosciences) flow cytometer.
    FACSCalibur
    suggested: None
    Data analyzed using FlowJo software (FlowJo LLC).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The FRNT-mNG50 titers were interpolated using a 4-parameter nonlinear regression in GraphPad Prism 8.4.3.
    GraphPad Prism
    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: We detected the following sentences addressing limitations in the study:
    The limitation of the current study is that a comparative analysis of GPI-RBD-GM-CSF and GPI-RBD was not carried out to demonstrate the contribution of GM-CSF as an adjuvant in VLP vaccine. Attempts to make GPI-RBD in CHO cells were not successful. Further, our study did not investigate whether incorporating VLPs with different amounts of GPI-anchored fusion protein results in stronger immune response to SARS-CoV-2. However, the comparison of antibody response induced by purified GPI-RBD-GM-CSF molecule with RBD-His-Tag suggests that GM-CSF stimulated antibody production against RBD. Further, our study focused only on the RBD domain of SARS-CoV-2 S protein but not the full-length S protein which may limit the breadth of protective immune response. In summary, our results demonstrate that influenza VLP-based delivery of SARS-CoV-2 RBD protein in combination with cytokine adjuvants can be used as a platform to develop multivalent vaccines targeting the variant strains of viruses which are currently observed in ongoing SARS-CoV-2 pandemic. Our fusion protein vaccine design also allows for creation of fusion proteins with new variant sequences and quickly purify using anti-GM-CSF mAb affinity chromatography. Further, use of immobilized cytokines as adjuvants will provide a safer way to induce anti-viral immunity with minimal side-effects.

    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.

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  3. Version published to 10.1101/2022.02.01.478657v1 on bioRxiv