Recovery from Acute SARS-CoV-2 Infection and Development of Anamnestic Immune Responses in T Cell-Depleted Rhesus Macaques
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Abstract
Patients with severe COVID-19 often have decreased numbers of T cells, a cell type important in fighting most viral infections. However, it is not known whether the loss of T cells contributes to severe COVID-19 or is a consequence of it.
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SciScore for 10.1101/2021.04.02.438262: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Ethics and biosafety statement: All in vivo experiments were performed in accordance with Animal Study Proposal RML 2020-046-E approved by the Institutional Animal Care and Use Committee of Rocky Mountain Laboratories (National Institutes of Health (NIH)) and carried out by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility, according to the institution’s guidelines for animal use, following the guidelines and basic principles in the NIH Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, and the United States Department of Agriculture and the United States … SciScore for 10.1101/2021.04.02.438262: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Ethics and biosafety statement: All in vivo experiments were performed in accordance with Animal Study Proposal RML 2020-046-E approved by the Institutional Animal Care and Use Committee of Rocky Mountain Laboratories (National Institutes of Health (NIH)) and carried out by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility, according to the institution’s guidelines for animal use, following the guidelines and basic principles in the NIH Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, and the United States Department of Agriculture and the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals. Randomization were randomly divided into four groups of three. Blinding Chest radiographs were interpreted in a blinded manner by a board-certified clinical veterinarian. Power Analysis not detected. Sex as a biological variable Animals: In the first experiment, 12 adult male rhesus macaques aged 2 1/2 to 5 yrs. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources mouse/rhesus CDR-grafted form of the depleting anti-CD8α antibody, M-T807 (each at 10mg/ml prepared in pharmaceutical grade by the NIH Nonhuman Primate Reagent Resource), 3) both CD4R1 and M-T807 or 4) physiological saline. anti-CD8αsuggested: NoneSpecific staining was detected using SARS-CoV/SARS-CoV-2 nucleocapsid antibody (Sino Biological cat#40143-MM05) at a 1:1000 dilution, CD4 antibody (abcam cat#ab133616) at a 1:100 dilution, CD8 antibody (Sino Biological cat#10980-T24) at a 1:500 dilution, and CD20 (Thermo Scientific cat#RB-9013) at a 1:250 dilution. CD4suggested: (Abcam Cat# ab133616, RRID:AB_2750883)CD8suggested: NoneCD20suggested: (Thermo Fisher Scientific Cat# MA1-22864, RRID:AB_558114)Meso Scale Discovery V-PLEX SARS-CoV-2 Panel 1 Kit (Rockville, MD) was used for determining antibody binding for IgG (K15359U) and IgM (K15360U) specific for SARS-CoV-2 S1 RBD. K15359Usuggested: NoneIgM ( K15360Usuggested: NoneCells were incubated for 30 min with the following cell surface antibodies: BUV661-anti-CD45 (D058-128, BD Biosciences 741657 Lot 0198214), PE-Cy7-anti-CD20 (2H7, BD Biosciences 560735 Lot 0170481), BV786-anti-CD3 (SP34-2, BD Biosciences 563918 Lot 0133645), FITC-anti-CD8 (DK25, Millipore FCMAB176F Lot 3398058), and Pacific Blue-anti-CD4 (OKT4, BioLegend 317424 Lot B258189) in 2% FBS PBS supplemented with Brilliant Stain Buffer (BD Biosciences). PE-Cy7-anti-CD20suggested: NoneBV786-anti-CD3suggested: NoneOKT4suggested: (BioLegend Cat# 317424, RRID:AB_571953)Cells were incubated for 30 min with the following antibodies: PE-anti-Foxp3 (259D, BioLegend 3202080 Lot B293618) and AF700-anti-Ki-67 (B56, BD Biosciences 561277 Lot 9315354) PE-anti-Foxp3suggested: NoneAF700-anti-Ki-67 ( B56suggested: NoneExperimental Models: Cell Lines Sentences Resources Pseudovirions were prepared in HEK293T cells by co-transfecting 60 μg of HIV-1NLΔEnv-NanoLuc and CMV-SARS-CoV-2 SΔ19 at a 3:2 ratio in T-175 flasks using the calcium phosphate method, then were concentrated by ultracentrifugation in a sucrose cushion 31. HEK293Tsuggested: NoneWe utilized A549-ACE2 cells 32 as target cells as these cells attached better to the culture plate compared to 293T-ACE2 cells 30, allowing for extensive washes given that the nanoluciferase reporter yielded high backgrounds. A549-ACE2suggested: None293T-ACE2suggested: RRID:CVCL_YZ65)Software and Algorithms Sentences Resources Morphometric analysis: CD4 and CD8 IHC stained sections were scanned with an Aperio ScanScope XT (Aperio Technologies, Inc., Vista, CA) and analyzed using the ImageScope Positive Pixel Count algorithm (version 9.1). ImageScopesuggested: (ImageScope, RRID:SCR_014311)Graphpad Prism 8 was used for preparation of graphs and statistical analysis. Graphpad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Best-fit nonlinear regression curves were constructed based on a two-phase decay equation (GraphPad Prism 8), and 80% inhibition values (20% infection) were interpolated. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)The multi-parameter data were collected within the BSL-4 using a Cytoflex LX (Beckman Coulter) and analyzed using FlowJo software (version 10.7.1; TreeStar, Inc). FlowJosuggested: (FlowJo, RRID:SCR_008520)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 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.
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