Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model

  1. Santosh Dhakal
  2. Camilo A. Ruiz-Bedoya
  3. Ruifeng Zhou
  4. Patrick S. Creisher
  5. Jason S. Villano
  6. Kirsten Littlefield
  7. Jennie Ruelas Castillo
  8. Paula Marinho
  9. Anne Jedlicka
  10. Alvaro A. Ordonez
  11. Natalia Majewski
  12. Michael J. Betenbaugh
  13. Kelly Flavahan
  14. Alice L. Mueller
  15. Monika M. Looney
  16. Darla Quijada
  17. Filipa Mota
  18. Sarah E. Beck
  19. Jacqueline Brockhurst
  20. Alicia Braxton
  21. Natalie Castell
  22. Franco R. D’Alessio
  23. Kelly A. Metcalf Pate
  24. Petros C. Karakousis
  25. Joseph L. Mankowski
  26. Andrew Pekosz
  27. Sanjay K. Jain
  28. Sabra L. Klein
  29. for the Johns Hopkins COVID-19 Hamster Study Group

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Abstract

In the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males compared with females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8-10 weeks of age) were inoculated intranasally with 10 5 TCID 50 of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developing more extensive pneumonia as noted on chest computed tomography, and recovering more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including IFNβ and TNFα, were comparable between the sexes. However, during the recovery phase of infection, females mounted two-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole inactivated SARS-CoV-2 and mutant S-RBDs, as well as virus neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2 associated sex differences seen in the human population.

Importance

Men experience more severe outcomes from COVID-19 than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human clinical isolate of SARS-CoV-2. After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in females than male hamsters. Greater lung pathology during the acute phase combined with reduced antiviral antibody responses during the recovery phase of infection in males than females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection.

One Sentence Summary

Following SARS-CoV-2 infection, male hamsters experience worse clinical disease and have lower antiviral antibody responses than females.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.02.438292: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationAnimals were randomly assigned to be euthanized at 2, 4, 7, 14, or 28-days post infection (dpi).
    BlindingThe investigators were blinded to the group assignments.
    Power Analysisnot detected.
    Sex as a biological variableAnimal experiments: Male and female golden Syrian hamsters (7-8 weeks of age) were purchased from Envigo (Haslett, MI).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibody ELISAs: Hamster antibody ELISA protocol was modified from human COVID-19 antibody ELISA protocol described previously (14).
    COVID-19
    suggested: None
    After washing plates 3 times, HRP-conjugated secondary IgG (1:10000, Abcam, MA, USA), IgA (1:250, Brookwood Biomedical, AL, USA) or IgM (1:250, Brookwood Biomedical, AL, USA) antibodies were added.
    HRP-conjugated secondary IgG
    suggested: (Creative Diagnostics Cat# LAB-22, RRID:AB_2489589)
    IgA
    suggested: None
    IgM
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To obtain whole inactivated SARS-CoV-2, VeroTMPRSS2 cells were infected at a MOI of 0.01 and the infected cell culture supernatant was collected at 72 hours post infection.
    VeroTMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    Briefly, tissue homogenates were 10-fold serially diluted in infection media (CM with 2.5% instead of 10% FBS), transferred in sextuplicate into the 96-well plates confluent with Vero-E6-TMPRSS2 cells, incubated at 370C for 4 days, and stained with naphthol blue-black solution for visualization.
    Vero-E6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    Statistical Analyses: Statistical analyses were done in GraphPad Prism 9.
    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: 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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04359329RecruitingEstrogen Patch for COVID-19 Symptoms
    NCT04539626RecruitingEstrogen Therapy in Non-severe COVID-19 Patients

    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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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.

    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 10.1101/2021.04.02.438292 on bioRxiv