Immunization with SARS-CoV-2 nucleocapsid protein triggers a pulmonary immune response in rats

  1. Everidiene K. V. B. Silva
  2. Camila G. Bomfim
  3. Ana P. Barbosa
  4. Paloma Noda
  5. Irene L. Noronha
  6. Bianca H V. Fernandes
  7. Rafael R. G. Machado
  8. Edison L. Durigon
  9. Sergio Catanozi
  10. Letícia G. Rodrigues
  11. Fabiana Pieroni
  12. Sérgio G. Lima
  13. Zelita A. J. Queiroz
  14. Ives Charlie-Silva
  15. Lizandre K. R. Silveira
  16. Walcy R. Teodoro
  17. Vera L. Capelozzi
  18. Cristiane R. Guzzo
  19. Camilla Fanelli

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Abstract

The SARS-CoV-2 pandemic have been affecting millions of people worldwide, since the beginning of 2020. COVID-19 can cause a wide range of clinical symptoms, which varies from asymptomatic presentation to severe respiratory insufficiency, exacerbation of immune response, disseminated microthrombosis and multiple organ failure, which may lead to dead. Due to the rapid spread of SARS-CoV-2, the development of vaccines to minimize COVID-19 severity in the world population is imperious. One of the employed techniques to produce vaccines against emerging viruses is the synthesis of recombinant proteins, which can be used as immunizing agents. Based on the exposed, the aim of the present study was to verify the systemic and immunological effects of IM administration of recombinant Nucleocapsid protein (NP), derived from SARS-CoV-2 and produced by this research group, in 2 different strains of rats ( Rattus norvegicus ); Wistar and Lewis. For this purpose, experimental animals received 4 injections of NP, once a week, and were submitted to biochemical and histological analysis. Our results showed that NP inoculations were safe for the animals, which presented no clinical symptoms of worrying side effects, nor laboratorial alterations in the main biochemical and histological parameters, suggesting the absence of toxicity induced by NP. Moreover, NP injections successfully triggered the production of specific anti-SARS-CoV-2 IgG antibodies by both Wistar and Lewis rats, showing the sensitization to have been well sufficient for the immunization of these strains of rats. Additionally, we observed the local lung activation of the Bronchus-Associated Lymphoid Tissue (BALT) of rats in the NP groups, suggesting that NP elicits specific lung immune response. Although pre-clinical and clinical studies are still required, our data support the recombinant NP produced by this research group as a potential immunizing agent for massive vaccination, and may represent advantages upon other recombinant proteins, since it seems to induce specific pulmonary protection.

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    SciScore for 10.1101/2021.08.24.457520: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Animals and Experimental Protocol: All experimental procedures employed in the present study were approved by the Research Ethics Committee for the Use of Experimental Animals of the University of São Paulo Medical School (
    Euthanasia Agents: By the end of the study, after 4 weeks of follow up, rats were subjected to isoflurane inhalation anaesthesia and submitted to a xipho-pubic laparotomy.
    Sex as a biological variableAdult male rats from Wistar and Lewis strains, weighing 250-300 g, were obtained from an established colony at the University of São Paulo.
    RandomizationTo access uniform and proportional lung samples, 10 fields (five non-overlapping fields in two different sections) were randomly analyzed in proximal and distal lung parenchyma.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Production of specific anti-SARS-CoV-2 antibodies by immunized animals: To verify the success of NP sensitization, we analyzed the presence of specific IgG anti-NP in the serum of inoculated animals by Western Immunoblotting (WB).
    anti-SARS-CoV-2
    suggested: None
    anti-NP
    suggested: None
    v) Tween 20 solution, and then incubated with a secondary anti-Rat IgG peroxidase-conjugated antibody (Sigma-Aldrich), diluted 1:5000 in 5% skim milk, for 2 hours at 4°C.
    anti-Rat IgG
    suggested: (LSBio (LifeSpan Cat# LS-C70055-5000, RRID:AB_1653203)
    Specific immunostaining for macrophages was obtained by means of a streptavidin-biotin-alkaline-phosphatase IHC technique, using a primary monoclonal mouse anti-rat ED1 antibody (Serotec, #MCA341R, Oxford, UK), followed by a secondary biotinylated horse anti-mouse IgG antibody, rat adsorbed (Vector #BA2001, California, USA).
    anti-rat ED1
    suggested: (Bio-Rad Cat# MCA341R, RRID:AB_2291300)
    anti-mouse IgG
    suggested: (Vector Laboratories Cat# BA-2001, RRID:AB_2336180)
    The specific primary antibody polyclonal rabbit anti-human CD3 (Dako, #A4052, California, USA) was used, followed by polyclonal goat anti-rabbit IgG + HRP polymer (Spring Laboratories, #DHRR, California, USA).
    anti-human CD3
    suggested: None
    anti-rabbit IgG
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Ten Wistar and 7 Lewis rats were subjected to intramuscular (IM) injections of 100μL of recombinant SARS-CoV-2 Nucleocapsid Protein (NP) diluted at 1,5 μg/μL in protein buffer, weekly, during 4 consecutive
    Wistar
    suggested: None
    Recombinant DNA
    SentencesResources
    The pET-28a containing the nucleocapsid DNA fragment was used for the expression of the recombinant protein in E. coli BL21 STAR (DE3) strain.
    pET-28a
    suggested: RRID:Addgene_44242)
    Software and Algorithms
    SentencesResources
    The pET-28a containing the nucleocapsid DNA fragment was used for the expression of the recombinant protein in E. coli BL21 STAR (DE3) strain.
    STAR
    suggested: (STAR, RRID:SCR_004463)
    All analyses were performed using the GraphPad Prism 7® software.
    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:
    However, one of the limitations of using experimental animals are the inherent difficulties due to anatomic and physiological differences between other mammalian species and the human beings. It is of note that the lower respiratory tract of mammals is constantly exposed to multiple airborne pathogens, and a prompt and effective immune response against such inhaled invaders is crucial for the survival of species [26, PABST 2010]. In this context, during the evolutionary process, a mucosal secondary lymphoid tissue, embedded in the walls of the large airways, the Bronchus-Associated Lymphoid Tissue (BALT) became part of the immune defense arsenal of many mammals. BALT is constitutive during all life phases in some species, including rats and rabbits, but it is absent in healthy adult mice and humans [Sminia et al., 1989, Troy D. Randall 2010]. Although more studies with different species, more similar to the human beings, regarding the specific pulmonary immune response are still required, we can suppose that, interestingly, the IM immunization with recombinant NP was able to activate the BALT of the rats, as a response of the systemic immune response triggered by the immunization, reflecting in the overactivity of immune cells observed in the lung of NP rats. Our findings suggest that, in spite of the IM administration, our immunizing agent was able to trigger an immune response specifically in the lungs of the injected rats. This is clinically relevant: the immunomodulation ...

    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

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  2. Version published to 10.1101/2021.08.24.457520v1 on bioRxiv