Egg-Derived Anti-SARS-CoV-2 Immunoglobulin Y (IgY) With Broad Variant Activity as Intranasal Prophylaxis Against COVID-19

  1. Lyn R. Frumkin
  2. Michaela Lucas
  3. Curtis L. Scribner
  4. Nastassja Ortega-Heinly
  5. Jayden Rogers
  6. Gang Yin
  7. Trevor J. Hallam
  8. Alice Yam
  9. Kristin Bedard
  10. Rebecca Begley
  11. Courtney A. Cohen
  12. Catherine V. Badger
  13. Shawn A. Abbasi
  14. John M. Dye
  15. Brian McMillan
  16. Michael Wallach
  17. Traci L. Bricker
  18. Astha Joshi
  19. Adrianus C. M. Boon
  20. Suman Pokhrel
  21. Benjamin R. Kraemer
  22. Lucia Lee
  23. Stephen Kargotich
  24. Mahima Agochiya
  25. Tom St. John
  26. Daria Mochly-Rosen

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Abstract

COVID-19 emergency use authorizations and approvals for vaccines were achieved in record time. However, there remains a need to develop additional safe, effective, easy-to-produce, and inexpensive prevention to reduce the risk of acquiring SARS-CoV-2 infection. This need is due to difficulties in vaccine manufacturing and distribution, vaccine hesitancy, and, critically, the increased prevalence of SARS-CoV-2 variants with greater contagiousness or reduced sensitivity to immunity. Antibodies from eggs of hens (immunoglobulin Y; IgY) that were administered the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were developed for use as nasal drops to capture the virus on the nasal mucosa. Although initially raised against the 2019 novel coronavirus index strain (2019-nCoV), these anti-SARS-CoV-2 RBD IgY surprisingly had indistinguishable enzyme-linked immunosorbent assay binding against variants of concern that have emerged, including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529). This is different from sera of immunized or convalescent patients. Culture neutralization titers against available Alpha, Beta, and Delta were also indistinguishable from the index SARS-CoV-2 strain. Efforts to develop these IgY for clinical use demonstrated that the intranasal anti-SARS-CoV-2 RBD IgY preparation showed no binding (cross-reactivity) to a variety of human tissues and had an excellent safety profile in rats following 28-day intranasal delivery of the formulated IgY. A double-blind, randomized, placebo-controlled phase 1 study evaluating single-ascending and multiple doses of anti-SARS-CoV-2 RBD IgY administered intranasally for 14 days in 48 healthy adults also demonstrated an excellent safety and tolerability profile, and no evidence of systemic absorption. As these antiviral IgY have broad selectivity against many variants of concern, are fast to produce, and are a low-cost product, their use as prophylaxis to reduce SARS-CoV-2 viral transmission warrants further evaluation.

https://www.clinicaltrials.gov/ct2/show/NCT04567810 , identifier NCT04567810.

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  1. Version published to 10.3389/fimmu.2022.899617
  2. Version published to 10.1101/2022.01.07.22268914v2 on medRxiv
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    SciScore for 10.1101/2022.01.07.22268914: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Written informed consent was obtained from all participants.
    IACUC: Animal welfare assurance and standards The protocols and any amendments or procedures involving the care and use of animals hen immunization and rat tolerability studies were reviewed and approved by Charles River Laboratories Institutional Animal Care and Use Committee before conduct.
    IRB: Regulatory and ethics considerations Ethical review of the clinical trial protocol and any amendments was obtained by Bellberry Human Research Ethics Committee the Australian National Review Board and the clinical trial was conducted solely at Linear Clinical Research, Perth Australia.
    Sex as a biological variableGLP rat toxicity and safety study Thirtyeight female and 38 male 8weekold Sprague Dawley rats were used in a GLP study conducted at Charles River Laboratories Spencerville
    RandomizationAnimals were randomly assigned to groups males and females were randomized separately and housed in accordance with the USDA Animal Welfare Act 9 CFR, Parts 1, 2 and 3 and as described in the Guide for the Care and Use of Laboratory Animals.
    BlindingCytokine level measurement and analysis This nonGLP blinded assay was performed on serum by the Immunoassay Team at the Human Immune Monitoring Center at Stanford University Stanford, CA. Assay kits RECYMAG65K27PMX Rat were purchased from EMD Millipore and used according to the manufacturers recommendations, with modifications described as follows.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    To this end, an antihuman Fc antibody Jackson ImmunoResearch Labs was immobilized on all flow cells of a CM5 chip GE Healthcare.
    antihuman Fc
    suggested: None
    Rabbit anti chicken IgY HL secondary antibody, HRP Invitrogen diluted 13000 was added to each strip and incubated at room temperature for 12 hours.
    anti chicken IgY HL
    suggested: None
    Detection of infection was accomplished using an antiSARSCoV2 nucleocapsid protein detection antibody Sino Biological, and a goat rabbit secondary antibody conjugated to AlexaFluor488.
    antiSARSCoV2 nucleocapsid protein
    suggested: None
    rabbit
    suggested: None
    After PBS washes, the secondary antibody peroxidase HRPconjugated rabbit anti chicken IgY Testing Facility antibody tracking No. A45764 of 2 gmL was added for 30 minutes.
    anti chicken IgY
    suggested: None
    For the 2microglobulin antibody positive control staining 1 gmL of antibodies were incubated with the slides for 1 hour and bound antibodies were detected with biotinylated secondary antibody goat antirabbit IgG 2 gmL for 30 minutes, as above.
    2microglobulin
    suggested: None
    antirabbit IgG
    suggested: None
    Evaluation of immunoglobulin E IgE and antiIgE antibodies was an exploratory objective.
    antiIgE
    suggested: None
    In Part 1, participants were randomly assigned to receive a single dose of antiSARS CoV2 RBD IgY antibodies or placebo in a sequential escalating manner.
    antiSARS CoV2 RBD IgY
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The mixture was then added to 293ThACE2 cells and infection was allowed for 24 hours.
    293ThACE2
    suggested: None
    Vero76 cells were inoculated with SARSCoV2Was1 MT020880.1 at a multiplicity of infection of 0.01 and incubated at 37C with 5 CO2 and 80 humidity.
    Vero76
    suggested: None
    Vero E6 cells were maintained in DMEM with 10 FBS.
    Vero E6
    suggested: None
    SARSCoV2 strain 2019nCoVUSAWA12020 was propagated on VeroTMPRSS2 cells, and the virus titer was determined by plaque assays on VerohACE2 and VerohACE2TMPRSS2 cells.
    VeroTMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    VerohACE2TMPRSS2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    GLP rat toxicity and safety study Thirtyeight female and 38 male 8weekold Sprague Dawley rats were used in a GLP study conducted at Charles River Laboratories Spencerville
    Sprague Dawley
    suggested: RRID:RGD_737903)
    Software and Algorithms
    SentencesResources
    The halfmaximal inhibitory concentration IC50 and 50 neutralization titer NT50 values for the IgY and positive control human serum were determined using GraphPad Prism software GraphPad Software.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Regression analysis employed linear mixed models 21 in SASSTAT SAS Institute with a separate model fit for each treatment and cytokine, and with separate sets of models with and without gender as a covariate, for 108 models in total.
    SAS Institute
    suggested: (Statistical Analysis System, RRID:SCR_008567)
    Statistical Analysis Data were analyzed with GraphPad Prism 9.0 and statistical significance was assigned when P values were 0.05.
    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:
    Limitations to current vaccines include global vaccine availability and affordability, vaccine hesitancy, and rapidly emerging highly infective viral strains that escape vaccine-induced immunity. This has been particularly apparent following the emergence of Delta and Omicron. The latter variant was first detected in specimens collected on November 8, 2021 [32], and within a few weeks became the dominant SARS-CoV-2 variant in the United States [33]. Both convalescent sera from early strain-infected patients and fully vaccinated individuals exhibited a low neutralization capacity against Omicron [9–12]; a reduction of 30 to 40-fold in neutralization titers was reported. Furthermore, of eight currently authorized or approved monoclonal antibodies, seven did not neutralize the Omicron variant and one had a 3-fold reduction in neutralization titer [11]. These data highlight the need for alternative and complementary approaches to curb COVID-19. Here, we describe the production of the first chicken egg yolk-derived anti-index SARS- CoV-2 RBD IgY polyclonal antibodies as an intranasal drop product for humans with equal in vitro activity against all variants of concern. These IgY were raised in SPF hens and showed an excellent safety profile when given intranasally by drops to rats for 28 days (4 mg/day). No toxicity, innate inflammatory response, or systemic exposure to IgY were noted in this GLP study. In 48 healthy adult participants, anti-SARS-CoV-2 RBD IgY given intranasally at...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04567810CompletedSafety, Tolerability, and Pharmacokinetics of Anti-Severe Ac…

    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

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  4. Version published to 10.1101/2022.01.07.22268914v1 on medRxiv