Ethics and biosafety statement
Cynomolgus macaques (Macaca fascicularis), aged 37-40 months and originating from Mauritian AAALAC certified breeding centers were used in this study. All animals were housed in IDMIT infrastructure facilities (CEA, Fontenay-aux-roses), under BSL-2 and BSL-3 containment when necessary (Animal facility authorization #D92-032-02, Prefecture des Hauts de Seine, France) and in compliance with European Directive 2010/63/EU, the French regulations and the Standards for Human Care and Use of Laboratory Animals, of the Office for Laboratory Animal Welfare (OLAW, assurance number #A5826-01, US). The protocols were approved by the institutional ethical committee “Comité d'Ethique en Expérimentation Animale du Commissariat à l’Energie Atomique et aux Energies Alternatives” (CEtEA #44) under statement number A20-011. The study was authorized by the “Research, Innovation and Education Ministry” under registration number APAFIS#24434- 2020030216532863v1.
Hydroxychloroquine and azithromycin
Hydroxychloroquine sulfate was manufactured for Sanofi by the Chinoin Pharmaceutical and Chemical works (Budapest, Hungary) under Good Manufacturing Practice (GMP) conditions and provided as the base powder. Batch number DU017 was solubilized extemporaneously in water at 5, 10 or 15 mg/mL depending on the group and the dose. Azithromycin 250 mg tablets (Sandoz, France; batch number KH5525) were crushed and suspended extemporaneously at 12 mg of AZTH per mL in water.
Animals and study design
To evaluate the efficacy of HCQ and HCQ+AZTH treatments, the animals were randomly assigned in sex balanced experimental groups. Challenged animals were exposed to a total dose of 106 pfu of SARS- CoV-2 via the combination of intranasal and intra-tracheal routes (Day 0), using atropine (0.04 mg/kg) for pre-medication and ketamine (5mg/kg) with medetomidine (0.042 mg/kg) for anesthesia. The “high dose” regimen in group “Hi D1” (n=5) consisted of a loading dose of 90 mg/kg at 1 dpi and a daily maintenance dose of 45 mg/kg, for a total of 10 days. The “Hi D1+AZTH” group (n=5) regimen consisted of the same HCQ regimen as for the Hi D1 group combined with one loading dose of 36 mg/kg of AZTH at 1 dpi, followed by a daily maintenance dose of 18 mg/kg AZTH, for 10 days. The “low dose” (Lo) regimen consisted of a HCQ loading dose of 30 mg/kg and a daily maintenance dose of 15 mg/kg for 12 days. The low dose treatment of the “Lo D1” group (n=4) was initiated at day 1 pi and the low dose treatment of the “Lo D5” group (n=4) was initiated at 5 dpi. The PrEP group (n=5) regimen consisted of a loading dose of 30 mg/kg seven days before challenge, followed by a daily dose of 15 mg/kg for four days and the 45 mg/kg for three days before virus challenge, and then until day 6 pi. Treatments were delivered by gavage. Placebo animals received water, which was the vehicle for HCQ. Animals were observed daily and clinical exams were performed at baseline, daily for one week, and then twice weekly, on anaesthetized animals using ketamine (5 mg/kg) and metedomidine (0.042 mg/kg). Body weight, rectal temperature, respiration, heart rates and oxygen saturation were recorded and blood, as well as nasal, tracheal and rectal swabs, were collected. Broncho-alveolar lavages (BAL) were performed using 30 mL sterile saline on 6, 14, 21 and 28 dpi. Chest CT was performed at baseline and on 2, 5 and 11/13 dpi in anesthetized animals using tiletamine (4 mg/kg) and zolazepam (4 mg/kg). Blood cell counts, haemoglobin and haematocrit were determined from EDTA blood using a HMX A/L analyzer (Beckman Coulter). Biochemistry parameters (ALAT, ASAT, albumin, haptoglobin, creatinine, creatine kinase, LDH and total protein) were analyzed with standard kits (Siemens) and C- reactive protein with a canine kit (Randox) in lithium heparin plasma, inactivated with Triton X-100, using ADVIA1800 analyzer (Siemens).
The pharmacokinetics of HCQ was assessed using the same administration procedure in 6 uninfected animals, randomly assigned by pairs in 3 experimental groups as described in Extended Data Fig. 4. The “PK Lo” group received a low loading dose (30 mg/kg) at day 0 and a low daily maintenance dose (15 mg/kg) for 5 days. The “PK Hi” and “PK Hi + AZTH” groups received a high loading HCQ dose (90 mg/kg) on day 0 and a high daily maintenance dose (45 mg/kg) for 6 days, along with AZTH for the second group (loading dose of 36 mg/kg and maintenance of 18 mg/kg). Blood samples were taken at 0, 2, 4, 6 hours post-treatment (hpt) on day 0, and before treatment on the following days. For the “PK Hi” and “PK Hi + AZTH” groups, blood samples were also collected at 0, 2, 4 and 6 hpt after treatment administration on day 5. Animals were humanly euthanized 24 h after the last dose administration using 18.2 mg/kg of pentobarbital sodium intravenously under tiletamine (4 mg/kg) and zolazepam (4 mg/kg) anesthesia. Samples of lung were collected at necropsy for HCQ quantification.
Determination of HCQ concentrations
Quantification of HCQ in plasma, blood and lung tissues was performed by a sensitive and selective validated high-performance liquid chromatography coupled with tandem mass spectrometry method (Quattro Premier XE LC-MS/MS, Waters, USA) as previously described 1, with lower limits of quantification of respectively 0.015 µg/mL for plasma and 0.05 µg/mL for blood and lung tissue. Blood samples were centrifuged within 1-hour to collect plasma samples. Lung biopsies collected after euthanasia were thoroughly rinsed with cold 0.9% NaCl to remove blood contamination and blotted with filter paper. Then, each lung biopsy was weighed and homogenized with 1 ml of 0.9% NaCl using a Mixer mill MM200 (Retsch, Germany). Cellular debris was removed by centrifugation, and the supernatant was stored at -80°C.
HCQ was extracted by a simple protein precipitation method, using methanol for plasma and ice-cold acetonitrile for blood and tissue homogenates. Briefly, 100 µL of samples matrix was spiked with 10 µL of internal standard working solution (HCQ-d5, Alsachim), then vortexed for 2 minutes followed by centrifugation for 10 minutes at 4°C. The supernatant was evaporated for blood and tissue homogenate samples. Dry residues or plasma supernatants were then transferred to 96-well plates and 5 µL was injected. To assess the selectivity and specificity of the method and matrix effect, blank plasma, blood and tissues from control animals were processed and compared with that of HCQ and IS-spiked plasma, blood or tissue homogenate samples. Furthermore, each baseline sample (H0) of treated animals was processed in duplicate, including one spiked with HCQ prepared equivalent to quality control samples (QCs).
Concentrations in blood (µg/mL), plasma (µg/mL) and lung (µg/g) were determined for each uninfected animal, and in plasma only for infected animals. Drug accumulation in lung was assessed by calculating a lung to blood and a lung to plasma concentration ratio as recently.
HCQ plasma trough concentrations determined within the context of routine therapeutic drug monitoring using the same method, 3 to 5 days after initiation of HCQ at 200 mg three times daily were provided for comparison.
Viruses and cells
For the in vivo studies, SARS-CoV-2 virus (hCoV-19/France/lDF0372/2020 strain) was isolated by the National Reference Center for Respiratory Viruses (Institut Pasteur, Paris, France) as described in Lescure et al.5. Virus stocks used in vivo were produced by two passages on VeroE6 cells in DMEM (Dulbecco's Modified Eagles Medium) without FBS, supplemented with 1% PS (penicillin at 10,000 U/ml and streptomycin at 10,000 µg/mL) and 1 µg/mL TPCK-trypsin at 37°C in a humidified CO2 incubator and titrated on Vero E6 cells.
For the in vitro studies, the viral strain hCoV-19/France/IDF0571/2020 was provided by Dr. X. Lescure and Prof. Y. Yazdanpanah from the Bichat Hospital, Paris, France, where the isolate was obtained from another patient returning from Jichang (China) and passaged three times. For the virus used in the in vivo experiments, whole genome sequencing was performed as described in Lescure et al. with no modifications observed compared with the initial specimen5. For sequencing of the virus used in vitro, viral RNA extraction was done using the QiAmp viral RNA Kit (Qiagen). The complete viral genome sequence was obtained using Illumina MiSeq sequencing technology. Sequences were deposited after assembly on the GISAID EpiCoV platform under accession numbers ID EPI_ISL_406596 for hCoV- 19/France/lDF0372/2020 and EPI_ISL_411218 for hCoV-19/France/IDF0571/2020.
Viral replication kinetics and antiviral treatment in VeroE6 cells
VeroE6 cells were seeded 24 h in advance in multi-well 6 plates, washed twice with PBS and then infected with SARS-CoV-2 at the indicated MOIs. For HCQ treatment, the inoculum of infected VeroE6 was removed 1 hpi and cells were immediately treated with solutions in DMEM of HCQ. Supernatants were collected at 48 and 72 hpi and stored at -80°C for RNA extraction and viral quantification.
Viral quantification in VeroE6 cells
Viral stocks and collected samples were titrated by tissue culture infectious dose 50% (TCID50/ml) in VeroE6 cells, using the Reed & Muench statistical method. Relative quantification of viral genome was performed by one-step real-time quantitative reverse transcriptase and polymerase chain reaction (RT- qPCR) from viral RNA extracted using QiAmp viral RNA Kit (Qiagen) in the case of supernatants/apical washings. Primer and probe sequences were selected from those designed by the School of Public Health/University of Hong Kong (Leo Poon, Daniel Chu and Malik Peiris) and synthetized by Eurogentec. Real-time one-step RT-qPCR was performed using the EXPRESS One-Step Superscript™ qRT-PCR Kit (Invitrogen, reference 1178101K) Thermal cycling was performed in a StepOnePlus™ Real-Time PCR System (Applied Biosystems) in MicroAmp™ Fast Optical 96-well reaction plates (Applied Biosystems, reference 4346907), as described in Pizzorno et al 2.
Viral infection and treatment in reconstituted human airway epithelia (HAE)
MucilAirTM HAE reconstituted from human primary cells obtained from nasal or bronchial biopsies were provided by Epithelix SARL (Geneva, Switzerland) and maintained in air-liquid interphase with specific culture medium in Costar Transwell inserts (Corning, NY, USA) according to the manufacturer’s instructions. For infection experiments, apical poles were gently washed twice with warm OptiMEM medium (Gibco, ThermoFisher Scientific) and then infected directly with a 150 μl dilution of virus in OptiMEM medium, at a multiplicity of infection (MOI) of 0.1. For mock infection, the same procedure was performed using OptiMEM as inoculum. Samples collected from apical washes or basolateral medium at different time-points were separated into 2 tubes: one for TCID50 viral titration and one RT-qPCR. HAE cells were harvested in RLT buffer (Qiagen) and total ARN was extracted using the RNeasy Mini Kit (Qiagen) for subsequent RT-qPCR and Nanostring assays. Treatments with HCQ were applied through basolateral poles. All treatments were initiated on day 0 (1h after viral infection) and continued once daily. Samples were collected at 48 hpi. Variations in transepithelial electrical resistance (Δ TEER) were measured using a dedicated volt-ohm meter (EVOM2, Epithelial Volt/Ohm Meter for TEER) and expressed as Ohm/cm2.
Virus quantification in NHP samples
Upper respiratory (nasal and tracheal) and rectal specimens were collected with swabs (Universal transport medium, Copan, Italy or Viral Transport Medium, CDC, DSR-052-01). All specimens were stored between 2°C and 8°C until analysis with a plasmid standard concentration range containing an RdRp gene fragment including the RdRp-IP4 RT-PCR target sequence. The protocol describing the procedure for the detection of SARS-CoV-2 is available on the WHO website (https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcr-assays-for-the-detection-of- sars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2).
Plasma cytokine analysis
Cytokines were quantified in EDTA plasma using NHP ProcartaPlex immunoassay (ThermoFisher Scientific) for IFN-α, IL-1RA, IL-1β, CCL-2/MCP-1 CCL-11/eotaxin, CXCL-11/ITAC, CXCL- 13/BLC, granzyme B and PDGF-BB, using NHP Milliplex (Millipore) for CD40L, G-CSF, GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-8/CXCL-8, IL-10, IL-13, IL-15, IL-17A, CCL-3/MIP-1 α, CCL-4/MIP-1β, TNF-α, VEGF and a Bioplex 200 analyzer (Bio-Rad) according to manufacturer’s instructions.
Chest computed tomography and image analysis
Acquisition was done using a computed tomography (CT) system (Vereos-Ingenuity, Philips) in BSL- 3 containment on anaesthetized animals placed in a supine position and monitored for heart rate, oxygen saturation and body temperature. A bolus of iodine contrast agent (Vizipaque 320 mg I/mL, GE Heathcare, 3mL/kg) was injected (Medrad CT Stellant® injector, Bayer) in the saphenous vein seconds prior to the initiation of CT acquisition. The CT detector collimation was 64 × 0.6 mm, the tube voltage was 120 kV and intensity of about 120mAs. Automatic dose optimization tools (Dose Right, Z- DOM, 3D-DOM by Philips Healthcare) regulated the intensity. CT Images were reconstructed with a slice thickness of 1.25 mm and an interval of 0.25 mm.
Images were analyzed using INTELLISPACE PORTAL 8 software (Philips healthcare). All images had the same window level of -300 and window width of 1600. Lesions were defined as ground glass opacitiy, crazy-paving pattern, or consolidation or pleural thickening as previously described 3,4. Lesions and scoring were assessed independently in each lung lobe by two persons, and final results were made by consensus. Overall CT score includes lesion type (scored from 0 to 3) and lesion volume (scored from 0 to 4) summed for each lobe as detailed in Extended Data Fig. 3.
The following viral kinetic parameters were calculated in each experimental group as medians (min- max): viral load peak, area under the curve (AUC) of the log10 viral load, time to first unquantifiable viral load. Each viral kinetic parameter was compared to untreated animals using Wilcoxon or Log-rank tests. To evaluate a potential effect of drug exposure on viral dynamics, we further evaluated the correlation of the viral kinetic parameters with the plasma concentrations of HCQ, taking the mean trough concentrations observed in each infected animal between 1 and up to day 15 post treatment as a marker for drug exposure during treatment period (Spearman test, without adjusting for tests multiplicity).
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