Hydroxychloroquine in the treatment and prophylaxis of SARS-CoV-2 infection in non- human primates CURRENT STATUS:

COVID-19 has become a pandemic that has caused over 200,000 deaths worldwide, with no antiviral drug or vaccine yet available. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro . Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals worldwide but definitive evidence for HCQ efficacy in treatment of COVID-19 is still missing. We evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in monkey African green monkey kidney (VeroE6) cells but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to placebo, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor HCQ+AZTH showed a significant effect on the viral load levels in any of the tested compartments. When the drug was used as a pre-exposure prophylaxis (PrEP), HCQ did not confer protection against acquisition of infection. Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral treatment for COVID-19 in humans. establishes in model of COVID in and for the preclinical evaluation of antiviral candidate molecules 2,4,5,26,19,20. Our showed no antiviral activity nor clinical efficacy of HCQ the timing of treatment after (after load in and and illustrate

4 Abstract COVID-19 has become a pandemic that has caused over 200,000 deaths worldwide, with no antiviral drug or vaccine yet available. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals worldwide but definitive evidence for HCQ efficacy in treatment of COVID-19 is still missing.
We evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in monkey African green monkey kidney (VeroE6) cells but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to placebo, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor HCQ+AZTH showed a significant effect on the viral load levels in any of the tested compartments. When the drug was used as a pre-exposure prophylaxis (PrEP), HCQ did not confer protection against acquisition of infection.
Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral treatment for COVID-19 in humans.

Main Text
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent for COVID-19 and originated in Wuhan, China, in 2019, has developed quickly into a global pandemic that has already caused over 200,000 deaths worldwide. COVID-19 is characterized by initial mild disease associated with respiratory symptoms at the peak of viral replication 1. Nasal and tracheal peaks of viral load occur close to the time of mild symptom onset, and viral load declines afterwards 2. In some patients, a late severe immunological syndrome occurs 6 to 14 days after onset of symptoms, characterized by high levels of inflammatory proteins (a "cytokine storm") 3. This may require intensive care and is responsible for most of the fatalities 2,4,5. Since no cure or prophylaxis is yet available against COVID-19, the World Health Organization (WHO) has provided recommendations for using investigational anti-COVID therapeutics in approved, randomized, Hydroxychloroquine sulfate (HCQ) has well documented in vitro activity against various viruses 6 and has emerged as an active compound against SARS-CoV-2 from different screening programs, including a 1,520 library of FDA-approved compounds 7. In VeroE6 cells, HCQ has a 50% maximal effective concentration (EC50) varying between 0.7 and 4 µM, as recently reported [7][8][9] . HCQ may inhibit viral transport in endosomes by alkalinizing the intra-organelle compartment 9,10. In addition, it was suggested that chloroquine and its derivatives might interfere with glycosylation mechanisms, as reported for other viruses 11, and this activity on the angiotensin convertase enzyme 2 (ACE2) and the virus spike protein may potentially affect infectivity. Moreover, the drug may also act as an immunomodulatory agent 12,13. In lupus, HCQ decreases the level of inflammatory cytokines such as IL-1β, IL-6 and TNF-α 10,14,15, which may be relevant for COVID-19 3. Furthermore, it has been proposed that azithromycin (AZTH), which displays in vitro antiviral activity against SARS-COV-2 7,16, could potentiate the efficacy of HCQ 17 .
Based on these properties, HCQ has been considered for COVID-19 treatment, alone or in combination with AZTH, but the results of clinical studies are still inconclusive 17,18 or pending.
Based on previous studies of SARS-COV and MERS-COV infection, we and others have set up experimental models of non-human primates (NHP) infected with SARS-CoV-2 that reproduce human infection and make possible the evaluation of drug efficacy in well-controlled settings 19,20. Here we used our model of cynomolgus macaque (Macaca fascicularis) to test different treatment strategies with HCQ, alone or in combination with AZTH, before or after the peak of viral replication. We also tested HCQ administration as pre-exposure prophylaxis (PrEP) against SARS-CoV-2 infection.

In vitro efficacy of hydroxychloroquine against SARS-CoV-2 infection
We first evaluated the in vitro antiviral activity of HCQ against a SARS-CoV-2 virus isolated from one of the first COVID-19 patients in France. Post-infection treatment of Vero E6 cells with HCQ resulted in a dose-dependent antiviral effect, with 50% inhibitory concentration (IC50) values of 2.2 µM (0.7 µg/mL) and 4.4 µM (1.4 µg/mL) at 48 and 72 hours pi, respectively, which is in the range of 0.7 to 4 µM previously published 21 (Extended Data Fig. 1a). We next studied infection in the reconstituted 6 human airway epithelium MucilAirTM model (HAE) developed from primary nasal or bronchial cells differentiated and cultivated in the air/liquid interphase 22. Unlike our previous observations for remdesivir 23, the antiviral activity of HCQ in Vero E6 cells did not translate to the HAE, with doses of 1 µM or 10 µM failing to reduce significantly SARS-CoV-2 apical viral titers at 48 hpi (Extended Data   Fig. 1b). HCQ also did not protect the epithelial integrity during infection, as evidenced by transepithelial electrical resistance (TEER) values comparable to untreated cells and significantly lower than those of the mock-infected controls.

Infection of macaques with SARS-CoV-2
Cynomolgus macaques were infected on day 0 with a total dose of 106 pfu of a primary SARS-CoV-2 isolate (BetaCoV/France/IDF/0372/2020) (passaged twice in VeroE6 cells) by combined intra-nasal and intra-tracheal routes. Control animals (CTRL, n=8) had high viral load levels in nasopharyngeal and tracheal samples (swabs), as estimated by RT-qPCR, as early as day 1 post infection (1 dpi). In tracheal samples, the viral load peaked at 2 dpi (Fig. 1b, Extended Data Fig. 2a), with a median (minmax) peak value of 7.9log10 copies/mL. Afterward, viral loads progressively decreased and most animals had undetectable viral load by 10 dpi. Similar profiles were observed for nasal shedding (Extended Data Fig. 2b), whereas low viral loads were detected for more than 3 weeks in rectal samples and broncho-alveolar lavages (Extended Data Fig. 2c,d). Animals exhibited mild clinical signs, as reported in the majority of human cases during the early infection period, including early lymphopenia (2 dpi) and coughing or sneezing without dyspnea (Extended Data Fig. 5). No major changes were observed regarding heart rate, respiratory rate, and oximetry. Interestingly, typical focal ground glass opacities associated with pleural thickening 24,25 were observed by CT scan with variable degrees of severity (Fig. 2, Extended Data Fig. 3). Lesions were detectable as early as 2 dpi and persisted up to 13 dpi in some animals. None of the control animals developed an immunological syndrome similar to what is observed in the late stages of the severe forms of the human disease.

Treatment with hydroxychloroquine
To assess the anti-viral efficacy of HCQ, macaques received HCQ daily by gavage for ten days or more. A dosing regimen of 90 mg/kg on day 1 pi (loading dose) followed by a daily maintenance dose 7 of 45 mg/kg was identified in a group of uninfected animals as capable of generating clinically relevant drug exposure (Extended Data Fig. 4b).
In parallel, we also tested a lower dosing regimen, with a loading dose of 30 mg/kg and a maintenance dose of 15 mg/kg. Overall, 9 animals were infected at day 0 and treated at 1 dpi using the high dosing regimen (Hi D1, n=5) or the low dosing regimen (Lo D1, n=4). We also examined the effect of a late treatment starting at 5 dpi, when viral loads are 3 to 4 logs lower as compared with peak values, to evaluate the benefit of HCQ in accelerating the virus clearance (Lo D5, n=4).
All treated animals had tracheal viral load kinetics similar to those of untreated animals, with median peak viral load of 7.1 and 7.5 log10 copies/mL in Hi D1 and Lo D1, respectively, compared with 7.9 log10 copies/mL in the CTRL group. Likewise, the areas under the curve (AUC) of viral load were similar, with values of 36.9 and 39.7 log10 copies.day/mL, as compared with 40.3 log10 copies.day/mL in CTRL animals (p=0.62 and 0.37, respectively). Similar results were obtained in nasal swabs, and there were no differences in the levels of viral replication in broncho-alveolar lavages (Fig.   1d, Extended Data Fig. 2). In animals treated from 1 dpi or 5 dpi, HCQ did not accelerate the time to viral clearance, and the median times to first unquantifiable viral load were 4.5, 7.0, 7.0, and 7.0 days in the CTRL, Lo D1, Hi D1, and Lo D5 groups, respectively.
Next we evaluated the (HCQ + AZTH) combination therapy administered from day 1 pi (Hi D1 + AZTH, n=5), with AZTH given at a loading dose of 36 mg/kg followed by a daily dose of 18 mg/kg to mimic human exposure. No impact of treatment was observed on either viral load in the different compartments or clinical scores. Clinical signs were comparable to controls, with some animals exhibiting high CT scores in the Hi D1 + AZTH group (Fig 2). In parallel, we also treated animals with a high dose of HCQ, starting 7 days before viral challenge as pre-exposure prophylaxis (PrEP, n=5).
Again, the kinetics of viral loads were similar to those of the control group with no reduction in terms of AUC, peak viral load or time to first unquantifiable viral load ( Fig. 1, Extended Data Fig. 2).

Relation between HCQ concentration levels and viral kinetics
In the animals of Hi D1, Hi D1+AZTH and PrEP groups, the plasma exposures were comparable to those observed in routine clinical practice 3-5 days after HCQ initiation using a 200 mg three times 8 daily dose (C Solas, data from the Pharmacokinetics and Toxicology Laboratory) (Fig. 3a). Drug trough concentrations were lower in both the Lo D1 and Lo D5 groups. When we assessed whether the higher drug exposure could generate more rapid virus clearance, neither the time to attain the viral load limit of quantification, nor the peak viral load were significantly associated with plasma HCQ concentrations ( Fig. 3b-d). Finally, in an additional group of uninfected macaques, we characterized the HCQ pharmacokinetic profile in blood and plasma as the drug accumulation in lung 6 days after treatment initiation ( Fig. 3e-f, Extended Data Fig. 4). The blood concentrations in Hi HCQ were higher than 1.4 μg/mL, showing that the drug concentrations in blood remained above the drug EC50 identified in VeroE6 cells during the dosing period (see above). Drug concentrations were even higher in lung tissues with a lung to plasma ratio ranging from 27 to 177 (Fig. 3f), allowing lung tissues to achieve concentrations above the drug EC50 in VeroE6 cells in all animals during the dosing period.
Immunopathogenesis and host response to hydroxychloroquine treatment High alanine aminotransferase (ALAT) and creatinine kinase levels were observed in animals treated with the high HCQ and particularly the HCQ+AZTH regimen compared with controls (Extended Data Interestingly, when compared with controls, TNF-α was significantly increased and IL-1RA was significantly reduced at day 2 pi (Fig. 4, Extended Data Fig. 6) in the groups that received the high dose of HCQ alone (p=0.032 and p= 0.028, respectively) or with AZTH (p=0.037 and p=0.045, respectively). This may reflect the immunomodulatory properties of these drugs. 9

Conclusions
Our study establishes that SARS-CoV-2 infection in cynomolgus macaques provides a relevant model for studying the early stages of COVID infection in humans and is appropriate for the preclinical evaluation of antiviral candidate molecules 2,4,5,26,19,20. Our experiments showed no antiviral activity nor clinical efficacy of HCQ treatment, regardless of the timing of treatment initiation, either before infection, early after infection (before viral load peak) or late after infection (after viral load peak). This was in spite of high drug concentration in blood and lung and plasma exposure similar to that observed in COVID patients treated with HCQ. Thus, treatment with HCQ is unlikely to have antiviral activity in respiratory compartments in humans. Our results illustrate the frequent discrepancy between results from in vitro classic assays and in vivo experiments, as reported for other viral infections such as influenza, dengue or chikungunya, where clinical trials failed to demonstrate efficacy of chloroquine or HCQ 6,27.
In conclusion, our results evaluation of HCQ in a non-human primate model of COVID-19 does not support its use as an antiviral agent for the treatment of COVID-19 in humans.

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.
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. 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.

Viruses and cells
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-ofsars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2).

Plasma cytokine analysis
Cytokines were quantified in EDTA plasma using NHP ProcartaPlex immunoassay (ThermoFisher   Scientific)  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.

Statistical analysis
The following viral kinetic parameters were calculated in each experimental group as medians (min-     Pharmacokinetics and viral kinetics parameters in cynomolgus macaques a, Individual mean 25 plasma trough concentration of HCQ in macaques during treatment and internal patients data (n=25). b, Time to the first measurement below the limit of quantification in macaques having mean plasma trough concentration <0.1 µg/mL (black) and >0.1 µg/mL (grey). c, Peak viral load according to mean HCQ plasma trough concentration. d, Area under the curve (AUC) or viral kinetic curves between 1 and 9 days pi. e, Correlation between HCQ lung and plasma concentrations. f, Correlation between HCQ lung and blood concentrations.

Figure 4
Cytokines and chemokines in the plasma of SARS-CoV-2 exposed cynomolgus macaques treated with hydroxychloroquine (a) Heatmap of plasma concentrations of eotaxin/CCL11, MCP-1/CCL2, IFN-α, IL-1RA, IL-2 and IL-15 at days 0, 2, 5, 7 and 9 pi. Scales are in pg/mL. Asterisks (*) represent a significant difference at one time-point between the CTRL group and the Hi D1 and/or Hi D1 + AZTH groups. Statistical significance was determined using a two-sided Mann-Whitney U-test.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. ExtendedData.pdf