Animals Twelve rhesus macaques of Indian origin (Macaca mulatta) were used in this study. Study groups comprised three males and three females and all were adults aged 4 years and weighing between 4.30 and 8.24kg at time of challenge. Before the start of the experiment, socially compatible animals were randomly assigned to challenge groups, to minimise bias.
Animals were housed in compatible social groups, in cages in accordance with the UK Home Office Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Procedures (2014) and National Committee for Refinement, Reduction and Replacement (NC3Rs) Guidelines on Primate Accommodation, Care and Use, August 2006. Prior to challenge, the animals were housed at Advisory Committee on Dangerous Pathogens (ACDP) level two in cages approximately 2.5M high by 4M long by 2M deep, constructed with high level observation balconies and with a floor of deep litter to allow foraging. Following challenge, animals were transferred to ACDP Level three and housed in banks of cages of similar construction placed in directional airflow containment systems that allowed group housing and environmental control whilst providing a continuous, standardised inward flow of fully conditioned fresh air identical for all groups. Additional environmental enrichment was afforded by the provision of toys, swings, feeding puzzles and DVDs for visual stimulation. In addition to ad libitum access to water and standard old-world primate pellets, diet was supplemented with a selection of fresh vegetables and fruit. All experimental work was conducted under the authority of a UK Home Office approved project license 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. Animals were sedated by intramuscular (IM) injection with ketamine hydrochloride (Ketaset, 100mg/ml, Fort Dodge Animal Health Ltd, Southampton, UK; 10mg/kg) for procedures requiring removal from their housing. None of the animals had been used previously for experimental procedures. Twenty-eight healthy, female ferrets (Mustela putorius furo) aged 5-7 months were obtained from a UK Home Office accredited supplier (Highgate Farm, UK). The mean weight at the time of challenge was 973 g/ferret (range 825 to 1129g). Animals were housed as described previously (bioRxiv 2020.05.29.123810; doi: https://doi.org/10.1101/2020.05.29.123810). All experimental work was conducted under the authority of a UK Home Office approved project licence that had been subject to local ethical review at PHE Porton Down by the Animal Welfare and Ethical Review Body (AWERB). One animal in the ChAdOx1 nCoV-19 prime only group steadily lost weight from arrival at the facility (5 days prior to vaccination) and throughout the post-vaccination follow-up and was sacrificed on welfare grounds at day 14 of the study. As the weight loss was observed from arrival it was not deemed vaccine related, therefore all immunological data from this animal has been excluded from the analysis.
Vaccinations Rhesus macaques received 2.5x1010vp ChAdOx1 nCoV-19 administered in 100ml intramuscularly or received 100ml of phosphate buffered saline intramuscularly and were challenged with SARS-CoV-2 twenty-seven days later. Ferrets were randomly assigned to ChAdOx1 nCoV-19 and ChAdOx1 GFP vaccinated groups. An identifier chip (Bio-Thermo Identichip, Animalcare Ltd, UK) was inserted subcutaneously into the dorsal cervical region of each animal. Ferrets were immunised with 2.5 x 1010 virus particles of ChAdOx1 nCoV-19 or ChAdOx1 GFP intramuscularly administered as a 100ml volume into the hind leg. Twenty-eight days after vaccination, half of the vaccinated animals were challenged with SARS-CoV-2, while the other half received a booster dose of ChAdOx1 nCoV-19 of ChAdOx1 GFP and were challenged with SARS-CoV-2 a further twenty-eight days later.
Enzyme-linked immunosorbent assay Maxisorp plates (Nunc) were coated overnight at 4°C with 250 ng/well spike protein in PBS, prior to blocking with 100 µl of casein in PBS (Thermo Fisher) for 1hr at RT. NHP serum was serially diluted 2x in casein in PBS was incubated at RT for 1hr. Antibodies were detected using affinity-purified polyclonal antibody alkaline phosphatase-labelled goat-anti-monkey IgG (Rocklands Laboratories), anti-monkey IgM (Rockland Laboratories) or anti-monkey IgA (Rockland Laboratories) in casein and developed with NPP-substrate (Sigma) and read at 405 nm. All wells were washed at least 3x with PBST 0.05% tween in between steps. Endpoint titers were calculated as follows: the log10 OD against log10 sample dilution was plotted and a regression analysis of the linear part of this curve allowed calculation of the endpoint titer with an OD of three times the background. Ferret serum was diluted in casein and incubated at RT for 2hr. Antibodies were detected using affinity-purified polyclonal antibody HRP-labelled goat-anti-ferret IgG (Abcam) in casein and TMB highest sensitivity (Abcam), developed for 12 minutes, and reaction was stopped using H2SO4 and read at 450 nm. Anti-spike IgM or IgA antibodies were detected with alkaline Phosphatase conjugated anti-ferret IgM (Rockland Laboratories) or anti-ferret IgA (Sigma), development with NPP-substrate and read at 405 nm. All wells were washed at least 3x with PBST 0.05% tween in between steps. Ferret samples were run against a standard positive pool of serum generated from ChAdOx1 nCoV-19 vaccinated ferrets with high endpoint titre. Due to high levels of non-specific responses, background was defined as the mean + 2x stdev of all animals at day 0.
Plaque Reduction Neutralisation Assay Heat-inactivated (56°C for 30 min) serum samples were serially diluted and incubated with approximately 60 PFU of wild type SARS-CoV-2 (2019-nCoV/Victoria/1/2020), for 1 h at 37°C in 5% CO2. Samples were then incubated with Vero E6 [Vero 76, clone E6 (ECACC 85020206), European Collection of Authenticated Cell Cultures, UK] monolayers in 24-well plates (Nunc, ThermoFisher Scientific, Loughborough, UK) under MEM (Life Technologies, California, USA) containing 1.5% carboxymethylcellulose (Sigma), 5% (v/v) foetal calf serum (Life Technologies) and 25mM HEPES buffer (Sigma). After incubation, at 37°C for 96 hours, plates were fixed overnight with 20% (w/v) formalin/PBS, washed with tap water and stained with methyl crystal violet solution (0.2% v/v) (Sigma). The neutralising antibody titres were defined as the serum dilutions resulting in a 50% reduction relative to the total number of plaques counted without antibody by using Probit analysis written in R programming language for statistical computing and graphics. An internal positive control for the PRNT assay was run using a sample of human MERS convalescent serum known to neutralise SARS-CoV-2 (National Institute for Biological Standards and Control, UK)
Micro neutralisation test (mVNT) using lentiviral-based pseudotypes bearing the SARS-CoV-2 Spike. Lentiviral-based SARS-CoV-2 pseudotyped viruses were generated in HEK293T cells incubated at 37 °C, 5% CO2 as previously described (npj Vaccines (2020) 5:69; https://doi.org/10.1038/s41541-020-00221-3). Briefly, cells were seeded at a density of 7.5 x 105 in 6 well dishes, before being transfected with plasmids as follows: 500 ng of SARS-CoV-2 spike, 600 ng p8.91 (encoding for HIV-1 gag-pol), 600 ng CSFLW (lentivirus backbone expressing a firefly luciferase reporter gene), in Opti-MEM (Gibco) along with 10 µL PEI (1 µg/mL) transfection reagent. A ‘no glycoprotein’ control was also set up using the pcDNA3.1 vector instead of the SARS-CoV-2 S expressing plasmid. The following day, the transfection mix was replaced with 3 mL DMEM with 10% FBS (DMEM-10%) and incubated for 48 and 72 hours, after which supernatants containing pseudotyped SARS-CoV-2 (SARS-CoV-2 pps) were harvested, pooled and centrifuged at 1,300 x g for 10 minutes at 4 °C to remove cellular debris. Target HEK293T cells, previously transfected with 500 ng of a human ACE2 expression plasmid (Addgene, Cambridge, MA, USA) were seeded at a density of 2 × 104 in 100 µL DMEM-10% in a white flat-bottomed 96-well plate one day prior to harvesting SARS-CoV-2 pps. The following day, SARS-CoV-2 pps were titrated 10-fold on target cells, and the remainder stored at -80 °C. For mVNTs, NHP plasma was diluted 1:10 and ferret plasma diluted 1:20 in serum-free media and 50 µL was added to a 96-well plate in triplicate and titrated 2-fold. A fixed titred volume of SARS-CoV-2 pps was added at a dilution equivalent to 105 signal luciferase units in 50 µL DMEM-10% and incubated with sera for 1 hour at 37 °C, 5% CO2 (giving a final sera dilution of 1:40). Target cells expressing human ACE2 were then added at a density of 2 x 104 in 100 µL and incubated at 37 °C, 5% CO2 for 72 hours. Firefly luciferase activity was then measured with BrightGlo luciferase reagent and a Glomax-Multi+ Detection System (Promega, Southampton, UK). Pseudotyped virus neutralisation titres were expressed as a 50% neutralisation dose (ND50) using a Spearman and Karber formula.
ELISpot PBMCs from rhesus macaques and ferrets were isolated from whole blood by layering over Lymphoprep (density 1.077g) and centrifugation for 30 minutes at 1000g. PBMCs were collected from the interface, washed with Hanks Balanced Salt Solution (HBSS) prior to resuspension in complete media (RPMI supplemented with 10% FCS, Pent-Strep, L-Glut and Hepes). IFNg ELISpot assay was performed using NHP IFNg (Mabtech) or Ferret IFNg ELISpotBASIC Kit according to the manufacturer’s protocol (MABtech). PBMCs were plated at a concentration of 250 000 cells per well (NHPs) or 100 000 cells per well (Ferrets) and were stimulated overnight (18 to 20 hours) with four contiguous peptide pools spanning the length of the SARS-CoV-2 spike protein sequence at a concentration of 2µg/mL per peptide (Mimotopes) (Table S7). Spots were counted and analysed on an AID ELISpot Reader (AID). Spot forming units (SFU) per 1.0x106 PBMCs were summed across the 4 peptide pools for each animal after subtraction of background response (media and PBMC only wells). Simultaneous production of IFNg, IL13 and IL5 was detected with a custom FLUROspotFLEX kit (Mabtech) using anti-monkey IFNg FluroSpot set 490, anti-monkey IL13 FluroSpot set 550 and anti-human IL5 FluroSpot set 640. ELISpot was performed with the same stimulation conditions as above (200 000 cells and 4 peptide pools), with plates developed according to the manufacturer's instructions. Spot were enumerated using Mabtech IRISTM reader and analysed with SpotReader software (Mabtech). Post-challenge NHP ELISpot were performed on PBMCs isolated over a Ficoll-Paque Plus (GE Healthcare, USA) density gradient and anti-human/simian IFNg kit (Mabtech). 200 000 cells per well were stimulated with 3 pools of SARS-CoV-2 peptides (Table S7, Pool 1 peptides 1 to 96, Pool 2 peptides 97 to 192, Pool 3 peptides 193 to 316) at a final concentration of 1.7µg/ml, Phorbol 12-myristate (Sigma-Aldrich Dorset, UK) (100 ng/ml) and ionomycin (CN Biosciences, Nottingham, UK) (1 mg/ml) were used as a positive control. ELISpot plates were analysed using the CTL scanner and software (CTL, Germany) and further analysis carried out using GraphPad Prism (version 8.0.1) (GraphPad Software, USA).
Measurement of NHP serum cytokines Rhesus macaque PBMCs were stimulated for 16 hours with 2 pools of SARS-CoV-2 peptides (S1 and S2) and cytokine measured using MescoScaleDiscovery (MSD) Technology V-PLEX Proinflammaotry Panel 1 NHP kit according to the manufacturer’s instructions. Log10 Fold Change (Log10FC) was calculated by dividing concentration detected in stimulated wells by unstimulated wells, baseline detectable level of each cytokine was set at 0.01mg.
Intracellular cytokine staining Ferret PBMCs were stimulated for 18-20 hours with 2 pools of SARS-CoV-2 spike peptides (S1-pool 1 and pool 2 or S2-pool 3 and pool 4) at a final concentration of 2µg/ml or ConA in the presence of golgi-stop (BD) and golgi-plug (BD). Cells were surface stained with anti-mouse/rat/human CD3 Alexa 405 (Clone PC3/188A) (Santa Cruz Biotechnology), anti-human CD8 APCCy7 (Clone OKT8) (Thermofisher) and live-dead aqua (Thermofisher), fixed with Fix-Perm solution prior to intracellular staining with anti-bovine IFNg PE (Clone CC302) (Abserotec) and anti-mouse TNFa A647 (Clone MP6-XT22) (D28 samples). Data was acquired on a BD Fortessa and analysed in FlowJo version 9 or above. Data is presented total spike response, by summing together the frequency of cytokine positive cells detected in S1 and S2 stimulated wells after background subtraction of media stimulated cells.
Challenge Animals were challenged with SARS-CoV-2 (VERO/hSLAM cell passage 3 (Victoria/1/2020)) at a final challenge dose of 5x106 pfu. NHPs received 2ml intratracheally followed by 1ml intranasally, ferrets received a total of 1ml split equally between both nares.
Prior to challenge ferrets were sedated by intramuscular injection of ketamine/xylazine (17.9 mg/kg and 3.6 mg/kg bodyweight). Challenge virus prepared as described previously (bioRxiv 2, 2020.05.29.123810 (2020) and doi: https://doi.org/10.1101/2020.09.17.301093) was delivered by intranasal instillation (1.0 mL total, 0.5 mL per nostril) diluted in phosphate buffered saline (PBS). Nasal washes were obtained by flushing ferret nasal cavities with 2 mL PBS. Throat swabs were collected using a standard swab (Sigma Virocult®) gently stroked across the back of the pharynx in the tonsillar area. Throat swabs were processed, and aliquots stored in viral transport media (VTM) and AVL at ≤-60°C until assay. Clinical signs of disease were monitored as described previously bioRxiv 2, 2020.05.29.123810 (2020). The necropsy procedures were also as described previously bioRxiv 2, 2020.05.29.123810 (2020).
Computed Tomography (CT) Radiology of NHPs CT scans were collected from sedated macaques using a 16 slice Lightspeed CT scanner (General Electric Healthcare, Milwaukee, WI, USA) in the prone and supine position. The change in position assists differentiation between pulmonary changes due to gravity dependant atelectasis from ground glass opacity at the lung bases caused by COVID. All axial scans were performed at 120KVp, with Auto mA (ranging between 10 and 120) and were acquired using a small scan field of view. Rotation speed was 0.8s. Images were displayed as an 11cm field of view. To facilitate full examination of the cardiac / pulmonary vasculature, lymph nodes and extrapulmonary tissues, Niopam 300 (Bracco, Milan, Italy), a non-ionic, iodinated contrast medium, was administered intravenously (IV) at 2ml/kg body weight and scans collected immediately after injection and ninety seconds from the mid-point of injection. Scans were evaluated by an expert thoracic radiologist, blinded to the animal’s treatment and clinical status for the presence of COVID disease features: ground glass opacity (GGO), consolidation, crazy paving, nodules, peri-lobular consolidation; distribution - upper, middle, lower, central 2/3, peripheral, bronchocentric) and for pulmonary embolus.
The extent of lung involvement was estimated (<25%, 25-50%, 51-75%, 76-100%) and quantified using a scoring system developed for COVID disease, as follows:
COVID disease pattern: Nodule(s): Score 1 for 1, 2 for 2 or 3, 3 for 4 or more. GGO: Score 1 if measures < 1 cm, 2 if 1 to 2 cm, 3 if 2 -3 cm, 4 if > 3 cm. Consolidation Score: 2 if measures < 1 cm, 4 if 1 to 2 cm, 6 if 2 -3 cm, 8 if > 3 cm. Zone classification: Each side of the lung was divided (from top to bottom) into three zones: The upper zone (above the carina), the middle zone (from the carina to the inferior pulmonary vein), and the lower zone (below the inferior pulmonary vein). Each zone was then divided into two areas: the anterior area (the area before the vertical line of the midpoint of the diaphragm in the sagittal position) and the posterior area (the area after the vertical line of the mid-point of the diaphragm in the sagittal position). This results in 12 zones in total. Measures: COVID pattern score = Nodule score + GGO score + consolidation score. Distribution (Zone) score = number of zones with disease, maximum score 12. Total CT score = COVID pattern score + Distribution (zone) score
Whole Blood Immunophenotyping Assays were performed using 50µl of heparinised blood incubated for 30 minutes at room temperature with optimal dilutions of the following antibodies: anti-CD3-AF700, anti-CD4-APC-H7, anti-CD8-PerCP-Cy5.5, anti-CD95-Pe-Cy7, anti-CD14-PE, anti-HLA-DR-BUV395, anti-CD25-FITC (all from BD Biosciences, Oxford, UK); anti-γδ-TCR-BV421, anti-CD16-BV786, anti-CD20-PE-Dazzle (all from BioLegend); and amine reactive fixable viability stain red (Life Technologies); all prepared in brilliant stain buffer (BD Biosciences). Red blood cell contamination was removed using a Utilyse reagent kit as per the manufacturer’s instructions (Agilent). BD Compbeads (BD Biosciences) were labelled with the above fluorochromes for use as compensation controls. Following antibody labelling, cells and beads were fixed in a final concentration of 4% paraformaldehyde solution (Sigma Aldrich, Gillingham, UK) prior to flow cytometric acquisition. Cells were analysed using a five laser LSRII Fortessa instrument (BD Biosciences) and data were analysed using FlowJo (version 9.7.6, BD Biosciences). Immediately prior to flow cytometric acquisition, 50 µl of Truecount bead solution (Beckman Coulter) was added to each sample. Leukocyte populations were identified using a forward scatter-height (FSC-H) versus side scatter-area (SSC-A) dot plot to identify the lymphocyte, monocyte and granulocyte populations, to which appropriate gating strategies were applied to exclude doublet events and non-viable cells. Lymphocyte sub populations including T-cells, NK-cells, NKT-cells and B-cells were delineated by the expression pattern of CD3, CD20, CD95, CD4, CD8, CD127, CD25, CD16 and the activation and inhibitory markers HLA-DR and PD-1. Classical- and non-classical-monocytes were identified by expression pattern of HLA-DR, CD14 and CD16. Granulocyte populations were delineated into neutrophils and eosinophils by expression of HLA-DR and CD14.
Total Viral RNA detection by Polymerase Chain Reaction RNA was isolated from nasal wash, throat swabs and BAL. Samples were inactivated in AVL (Qiagen) and ethanol. Downstream extraction was then performed using the BioSprint™96 One-For-All vet kit (Indical) and Kingfisher Flex platform as per manufacturer’s instructions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) targeting a region of the SARS-CoV-2 nucleocapsid (N) gene was used to determine viral loads and was performed using TaqPath™ 1-Step RT-qPCR Master Mix, CG (Applied Biosystems™), 2019-nCoV CDC RUO Kit (Integrated DNA Technologies) and QuantStudio™ 7 Flex Real-Time PCR System. Sequences of the N1 primers and probe were: 2019-nCoV_N1-forward, 5’ GACCCCAAAATCAGCGAAAT 3’; 2019-nCoV_N1-reverse, 5’ TCTGGTTACTGCCAGTTGAATCTG 3’; 2019-nCoV_N1-probe, 5’ FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1 3’. The cycling conditions were: 25°C for 2 minutes, 50°C for 15 minutes, 95°C for 2 minutes, followed by 45 cycles of 95°C for 3 seconds, 55°C for 30 seconds. The quantification standard was in vitro transcribed RNA of the SARS-CoV-2 N ORF (accession number NC_045512.2) with quantification between 1 x 101 and 1 x 106 copies/µl. Positive samples detected below the limit of quantification (LOQ) were assigned the value of 5 copies/µl, whilst undetected samples were assigned the value of < 2.3 copies/µl, equivalent to the assay’s lower limit of detection (LLOD).
Histopathology NHPs: Each animal was assigned a histology number for blinding purposes. The following samples from each animal was fixed in 10% neutral-buffered formalin, processed to paraffin wax and 4 µm thick sections cut and stained with haematoxylin and eosin (H&E); respiratory tract (left cranial and caudal lung lobes), trachea, larynx, tonsil, liver, kidney, spleen, mediastinal lymph node, and small and large intestine. Tissue sections were examined by light microscopy and evaluated subjectively and semi-quantitatively using a scoring system. Pathologists were blinded to treatment and group details and the slides randomised prior to examination in order to prevent bias (blind evaluation). The slides were reviewed independently by three board-certified veterinary pathologists. For the lung, three sections from each left lung lobe were sampled from different locations: proximal, medial and distal to the primary lobar bronchus. The scoring system was applied using the following parameters and scores: Parameters: Bronchial epithelial degeneration/necrosis with presence of exudates and/or inflammatory cell infiltration. Bronchiolar (primarily terminal) epithelial degeneration/necrosis with presence of exudates and/or inflammatory cell infiltration. Perivascular inflammatory infiltrates (cuffing). Peribronchiolar inflammatory infiltrates (cuffing). Acute diffuse alveolar damage (necrosis of pneumocytes). Alveolar cellular exudate and oedema and/or fibrin. Alveolar septal inflammatory cells and cellularity. Scores: 0 = Normal 1 = Minimal 2 = Mild 3 = Moderate 4 = Severe. Ferrets: A semiquantitative scoring system was developed to compare the severity of the lung lesions for each individual animal and among groups. This scoring system was applied independently to the cranial and caudal lung lobe tissue sections using the following parameters and scores: Parameters: Bronchial inflammation with presence of exudates and/or inflammatory cell infiltration. Bronchiolar inflammation with presence of exudates and/or inflammatory cell infiltration. Perivascular inflammatory infiltrates (cuffing). Infiltration of alveolar walls and spaces by inflammatory cells, mainly mononuclear Scores: 0 = None 1 = Minimal 2 = Mild 3 = Moderate 4 = Severe
Detection of virus by RNAscope An in-situ hybridisation method used on formalin-fixed, paraffin-embedded tissues was used to identify the SARS-CoV-2 virus in both lung lobes of NHPs. Briefly, tissues were pre-treated with hydrogen peroxide for 10 mins (RT), target retrieval for 15 mins (98-101⁰C) and protease plus for 30 mins (40⁰C) (all Advanced Cell Diagnostics). A V-nCoV2019-S probe (Advanced Cell Diagnostics) targeting the S-protein gene was incubated on the tissues for 2 hours at 40⁰C. Amplification of the signal was carried out following the RNAscope protocol (RNAscope 2.5 HD Detection Reagent – Red) using the RNAscope 2.5 HD red kit (Advanced Cell Diagnostics). Digital image analysis (Nikon NIS-Ar software) was carried out in order to calculate the total area of the lung section positive for viral RNA.