Comparison of rhesus and cynomolgus macaques as an infection model for COVID-19
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Abstract
A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.
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SciScore for 10.1101/2020.09.17.301093: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All experimental work was conducted under the authority of a UK Home Office approved project license (PDC57C033) that had been subject to local ethical review at PHE Porton Down by the Animal Welfare and Ethical Review Body (AWERB) and approved as required by the Home Office Animals (Scientific Procedures) Act 1986. Randomization Before the start of the experiment, socially compatible animals were randomly assigned to challenge groups, to minimise bias. Blinding Scans were evaluated by an expert thoracic radiologist, blinded to the animal’s clinical status, for the presence of: disease features characteristic of COVID-19 in humans (ground glass opacity (GGO), … SciScore for 10.1101/2020.09.17.301093: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All experimental work was conducted under the authority of a UK Home Office approved project license (PDC57C033) that had been subject to local ethical review at PHE Porton Down by the Animal Welfare and Ethical Review Body (AWERB) and approved as required by the Home Office Animals (Scientific Procedures) Act 1986. Randomization Before the start of the experiment, socially compatible animals were randomly assigned to challenge groups, to minimise bias. Blinding Scans were evaluated by an expert thoracic radiologist, blinded to the animal’s clinical status, for the presence of: disease features characteristic of COVID-19 in humans (ground glass opacity (GGO), consolidation, crazy paving, nodules, peri-lobular consolidation; distribution: upper, middle, lower, central 2/3, bronchocentric); pulmonary embolus and the extent of any abnormalities estimated (<25%, 25-50%, 51-75%, 76-100%). Power Analysis not detected. Sex as a biological variable Study groups comprised three males and three females of each species and all were adults aged 2 to 4 years and weighing between 2.89 and 4.85kg at time of challenge. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Following incubation, cells were washed with FACS buffer consisting of PBS + 1% FCS and incubated for 30 minutes at room temperature with optimal dilutions of the amine-reactive Live/Dead Fixable Red viability cell stain (Life Technologies) and the antibodies CD4 PerCP-Cy5.5, CD8 APC-Fire750, CD69-BV510, (all from BD Biosciences) and CD20-Pe-Dazzle-594, γδ-TCR-BV421 (Biolegend, London, UK) prepared in BD Biosciences Brilliant stain buffer (BD Biosciences, Oxford, UK). CD69-BV510suggested: NoneCD20-Pe-Dazzle-594suggested: Noneγδ-TCR-BV421suggested: NoneIntracellular antigen staining was applied by incubation at room temperature for 30 minutes with the antibodies CD3-AF700, IFN-γ-PeCy7, TNF-α-BUV395, GM-SCF-PE (all from BD Biosciences, Oxford, United Kingdom), IL-2-APC (Miltenyi Biotech Ltd), IL-17-BV711 (Biolegend, London, UK) prepared in brilliant stain buffer. IL-17-BV711suggested: NoneCells were then incubated for 30 minutes at room temperature with optimal dilutions of the following antibodies: anti-CD4-PerCP-Cy5.5, anti-CD8-APC-Fire750 anti-CD11c-PE. anti-CD14-APC, anti-CD16-BV786, anti-CD20-PE-Dazzle (all from BioLegend); anti-CD3-AF700, anti-CD56-BV605, anti-HLA-DR-BUV395 (all from BD Biosciences); anti-CD159a-PC7 anti-CD4-PerCP-Cy5.5suggested: Noneanti-CD8-APC-Fire750suggested: Noneanti-CD11c-PEsuggested: Noneanti-CD14-APCsuggested: (Sigma-Aldrich Cat# SAB4700104, RRID:AB_10898543)anti-CD16-BV786suggested: Noneanti-CD20-PE-Dazzlesuggested: Noneanti-CD3-AF700suggested: Noneanti-CD56-BV605suggested: Noneanti-HLA-DR-BUV395suggested: NoneExperimental Models: Cell Lines Sentences Resources Viruses and Cells: SARS-CoV-2 Victoria/01/202040 was generously provided by The Doherty Institute, Melbourne, Australia at P1 after primary growth in Vero/hSLAM cells and subsequently passaged twice at PHE Porton in Vero/hSLAM cells [ECACC 04091501]. Vero/hSLAMsuggested: ECACC Cat# 04091501, RRID:CVCL_L037)Virus titre of the challenge stocks was determined by plaque assay on Vero/E6 cells [ECACC 85020206]. Vero/E6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Plaque assay: Samples were incubated in 24-well plates (Nunc, ThermoFisher Scientific, Loughborough, UK) containing twice washed with Dulbecco’s PBS (DPBS) monolayers of Vero E6 cells seeded the previous day at 1.5 x 105 cells/well under Overlay media consisting of MEM (Life Technologies) containing 1.5% carboxymethylcellulose (Sigma), 4% (v/v Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Whole genome sequencing was performed, on the P3 challenge stock, using both Nanopore and Illumina as described previously41. Nanoporesuggested: NoneELISPOT plates were analysed using the CTL scanner and software (CTL, Germany) and further analysis carried out using GraphPad Prism (version 8.0.1) GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPad Software, USA) Immunophenotyping and Intracellular cytokine staining assays. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Cells were then incubated for 30 minutes at room temperature with optimal dilutions of the following antibodies: anti-CD4-PerCP-Cy5.5, anti-CD8-APC-Fire750 anti-CD11c-PE. anti-CD14-APC, anti-CD16-BV786, anti-CD20-PE-Dazzle (all from BioLegend); anti-CD3-AF700, anti-CD56-BV605, anti-HLA-DR-BUV395 (all from BD Biosciences); anti-CD159a-PC7 BD Biosciences)suggested: NoneFlow cytometric acquisition and analysis: Cells were analysed using a five laser LSRII Fortessa instrument (BD Biosciences) and data were analysed using FlowJo (version 9.7.6, BD Biosciences) 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: We detected the following sentences addressing limitations in the study:Given the limitations in reproducing the range of underlying health conditions in humans that link to poorer infection outcomes (e.g. diabetes, obesity and age) in macaques, other strategies of disease enhancement such as challenge, route, dose, strain, or manipulation of the host immunity, will be required. The limited supply of rhesus macaques is now impacting on future COVID-19 studies to support the development of vaccines and therapeutic products 37. The potential offered by cynomolgus macaques as an appropriate model will greatly increase the international community’s ability to perform these critical studies in support of pre-clinical evaluation and product licensure. Moreover, cynomolgus macaques with a Mauritian genotype have more restricted genetic variability and more limited and better-defined MHC, providing an advantage in the battle to elucidate correlates of protective immunity. These features have been of particular value in HIV vaccine research where models are established in both rhesus macaques (Indian genotype) and cynomolgus macaques (Mauritian genotype) 38. The MHC homogeneity associated with the Mauritian cynomolgus macaque has reduced the variability between animals after vaccination and has enhanced comparisons of vaccine regimens. In addition, the improved consistency in outcome facilitates the use of fewer animals to obtain statistically significant results than would be required if more genetically diverse species were to be used.
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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