Influenza viral particles harboring the SARS-CoV-2 spike RBD as a combination respiratory disease vaccine

  1. Ryan R. Chaparian
  2. Alfred T. Harding
  3. Kristina Riebe
  4. Amelia Karlsson
  5. Gregory D. Sempowski
  6. Nicholas S. Heaton
  7. Brook E. Heaton

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Abstract

Vaccines targeting SARS-CoV-2 have gained emergency FDA approval, however the breadth against emerging variants and the longevity of protection remains unknown. Post-immunization boosting may be required, perhaps on an annual basis if the virus becomes an endemic pathogen. Seasonal influenza virus vaccines are already developed every year, an undertaking made possible by a robust global vaccine production and distribution infrastructure. To create a seasonal combination vaccine targeting influenza viruses and SARS-CoV-2 that is also amenable to frequent reformulation, we have developed a recombinant influenza A virus (IAV) genetic platform that “reprograms” the virus to package an immunogenic domain of the SARS-CoV-2 spike (S) protein onto IAV particles. Vaccination with this combination vaccine elicits neutralizing antibodies and provides protection from lethal challenge with both pathogens. This technology may allow for leveraging of established influenza vaccine infrastructure to generate a cost-effective and scalable seasonal vaccine solution for both influenza and coronaviruses.

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    SciScore for 10.1101/2021.04.30.441968: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Animal infections/Live-attenuated vaccination: Animal infections were performed using age-matched C57BL/6J (Jackson Labs 000664) or B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (Jackson Labs 034860) in accordance with Duke University IACUC approved protocol numbers A189-18-08 and A081-20-04.
    Euthanasia Agents: Euthanasia was performed via a primary method of CO2 asphyxiation and bilateral thoracotomy as a secondary confirmation.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies were visualized using AlexFluor594-conjugated anti-mouse/anti-rabbit secondary antibodies (Thermo cat. no. A-11032/R37117) at a dilution of 1:500 for 1 hour at room temperature.
    anti-mouse/anti-rabbit
    suggested: (Vector Laboratories Cat# BA-1400, RRID:AB_2336187)
    The following antibodies were used for protein detection; PY102 (HA, 1 μg/mL), 4A5 (NA, 0.45 μg/mL), anti-matrix protein [E10] (M1 and M2, 1:1,000, Kerafast cat. no. EMS009), and anti-SARS-CoV-2 spike protein (RBD) polyclonal antibody (RBD, 1:1,000, Invitrogen cat. no. PA5-114451).
    HA, 1 μg/mL
    suggested: None
    anti-matrix protein
    suggested: (Kerafast Cat# EMS009, RRID:AB_2687434)
    M2
    suggested: None
    anti-SARS-CoV-2 spike protein (RBD
    suggested: None
    Anti-mouse (1:20,000, Thermo cat. no. A16072) and anti-rabbit (1:10,000, Thermo cat. no. A16104) secondary antibodies were diluted in PBST + 5% milk and applied to membranes for 60 minutes at room temperature.
    Anti-mouse
    suggested: None
    Primary antibodies were diluted using PBS + 3% BSA and incubated with immobilized proteins/viruses for ≥1 hour at room temperature (anti-RBD ProSci cat. no. 9087, anti-HA PY102 a kind gift from Dr. Tom Moran (Icahn School of Medicine at Mount Sinai), DH1041 and DH1044 kind gift from Drs.
    anti-RBD
    suggested: None
    anti-HA
    suggested: None
    Anti-human (1:10,000 Thermo cat. no. A18805), anti-mouse (1:5,000, Thermo cat. no. A16072), and anti-rabbit (1:5,000, Thermo cat. no. A16104) secondary antibodies were diluted in PBS + 3% BSA and incubated with immobilized proteins/virus for 1 hour at room temperature.
    Anti-human
    suggested: None
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and viruses: Human embryonic kidney cells (HEK293T, ATCC) were grown in Dulbecco’s Modified Eagle Medium supplemented with 5% fetal bovine serum, GlutaMAX (Gibco cat. no. 35050079), and penicillin/streptomycin.
    HEK293T
    suggested: None
    The allantoic fluid was then harvested from and titer determined via plaque assay on MDCK cells.
    MDCK
    suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
    Stocks were frozen at −80 °C and were titered by serially diluting virus in virus infection media then infecting a confluent monolayer of Vero E6 cells growing in 6-well, poly-L-lysine treated, plates for 1 hour.
    Vero E6
    suggested: None
    Plaque reduction neutralization assays: MDCK and Vero cells were used for influenza and SARS-CoV-2 plaque reduction assays, respectively.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Experimental Models: Organisms/Strains
    SentencesResources
    Primary antibodies were diluted using PBS + 3% BSA and incubated with immobilized proteins/viruses for ≥1 hour at room temperature (anti-RBD ProSci cat. no. 9087, anti-HA PY102 a kind gift from Dr. Tom Moran (Icahn School of Medicine at Mount Sinai), DH1041 and DH1044 kind gift from Drs.
    DH1041
    suggested: None
    Animal infections/Live-attenuated vaccination: Animal infections were performed using age-matched C57BL/6J (Jackson Labs 000664) or B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (Jackson Labs 034860) in accordance with Duke University IACUC approved protocol numbers A189-18-08 and A081-20-04.
    C57BL/6J
    suggested: None
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Recombinant DNA
    SentencesResources
    Once this was done, the codon optimized RBD was PCR amplified and cloned into the bicistronic pDZ rescue plasmid system for A/Puerto Rico/8/1934 using the NEBuider HiFi DNA assembly kit (NEB).
    pDZ
    suggested: RRID:Addgene_38952)
    Software and Algorithms
    SentencesResources
    Briefly, approximately 500 PFU of each plaque-purified stock was injected in 10-day old embryonated hen eggs purchased from Charles River Laboratories, Inc. and incubated for 72 hours at 37 °C.
    Charles River Laboratories
    suggested: (Charles River Laboratories, RRID:SCR_003792)
    Viral protein extracts were probed for M1 and normalized via densitometry (ImageJ).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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: 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

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  2. Version published to 10.1101/2021.04.30.441968v1 on bioRxiv