mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2
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Abstract
The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic. Critical to the rapid evaluation of vaccines and antivirals against SARS-CoV-2 is the development of tractable animal models to understand the adaptive immune response to the virus. To this end, the use of common laboratory strains of mice is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Importantly, using this system, we functionally identified the CD4+ and CD8+ structural peptide epitopes targeted during SARS-CoV-2 infection in H2 b restricted mice and confirmed their existence in an established model of SARS-CoV-2 pathogenesis. We demonstrated that, identical to what has been seen in humans, the antigen-specific CD8+ T cells in mice primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. As the focus of the immune response in mice is highly similar to that of the humans, the identification of functional murine SARS-CoV-2-specific T cell epitopes provided in this study will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2 infection.
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SciScore for 10.1101/2020.08.07.241877: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Ethics statement-: All animal studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Saint Louis University Animal Care and Use Committee (IACUC; protocol 2771). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Horseradish peroxidase conjugated goat-anti-human IgG secondary antibody was added and allowed to incubate for 1 hour at room temperature prior to being washed. IgGsuggested: NoneExperimental Models: Cell Lines Sen… SciScore for 10.1101/2020.08.07.241877: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Ethics statement-: All animal studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Saint Louis University Animal Care and Use Committee (IACUC; protocol 2771). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Horseradish peroxidase conjugated goat-anti-human IgG secondary antibody was added and allowed to incubate for 1 hour at room temperature prior to being washed. IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources A p2 stock was then grown from this p1 stock by infecting Vero-E6 cells at an MOI of 0.01 in complete DMEM and harvested at 96 hours post infection. Vero-E6suggested: NoneIn vitro transfection and infection experiments were completed using murine fibroblast 3T3 cells cultured in complete DMEM and were obtained from American Type Culture Collection (ATCC CRL-1658). 3T3suggested: NoneThe antibody-virus complex was then added to each well of a 96-well flat bottom plate containing a monolayer of Vero WHO cells. Vero WHOsuggested: ECACC Cat# 88020401, RRID:CVCL_JF53)Foci of infected Vero cells were stained with anti-SARS polyclonal guinea pig sera (BEI) overnight at 4°C and washed 3 times with 0.05% Triton-X in PBS. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Experimental Models: Organisms/Strains Sentences Resources Eight-week-old Ifnar1-/- mice were transfected with 10 μg of RNA using Polyplus in vivo-jet RNA in vivo transfection reagent prepared according to the manufacturer’s instructions and administered via intravenous (IV) and intranasal (IN) combination route (100 μl and 20 μl, respectively). 24 hours following transfections, mice were infected with 5×104 focus forming units (FFU) of SARS-CoV-2 via IV and IN combination route (100 μl and 20 μl, respectively). Ifnar1-/-suggested: NoneResults 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.
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SciScore for 10.1101/2020.08.07.241877: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Materials and Methods: Ethics statementAll animal studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Saint Louis University Animal Care and Use Committee (IACUC; protocol 2771). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Horseradish peroxidase conjugated goat-antihuman IgG secondary antibody was added and allowed to incubate for 1 hour at room temperature prior to being washed. …SciScore for 10.1101/2020.08.07.241877: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Materials and Methods: Ethics statementAll animal studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Saint Louis University Animal Care and Use Committee (IACUC; protocol 2771). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Horseradish peroxidase conjugated goat-antihuman IgG secondary antibody was added and allowed to incubate for 1 hour at room temperature prior to being washed. IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Briefly, serum from each mouse was serially diluted and combined with ~100 FFU of SARS-CoV-2 for one hour before the antibody-virus complex was added to a monolayer of Vero cells. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)A p2 stock was then grown from this p1 stock by infecting Vero- E6 cells at an MOI of 0.01 in complete DMEM and harvested at 96 hours post infection. Vero- E6suggested: NoneIn vitro transfection and infection experiments were completed using murine fibroblast 3T3 cells cultured in complete DMEM and were obtained from American Type Culture Collection (ATCC CRL-1658). 3T3suggested: NoneThe antibody-virus complex was then added to each well of a 96-well flat bottom plate containing a monolayer of Vero WHO cells. Vero WHOsuggested: ECACC Cat# 88020401, RRID:CVCL_JF53)Experimental Models: Organisms/Strains Sentences Resources After day 21 post primary infection, the Ifnar1-/- mice received a second hACE2 or GFP mRNA transfection, followed 24 hours later by a boost with 5x104 FFU per mouse of SARS-CoV-2, administered both IV and IN route. Ifnar1-/-suggested: NoneResults 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 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.
About SciScore
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