key: cord-0966261-jzekvix4
authors: Abdelnabi, Rana; Foo, Caroline S.; Kaptein, Suzanne J. F.; Zhang, Xin; Langendries, Lana; Vangeel, Laura; Breuer, Judith; Pang, Juanita; Williams, Rachel; Vergote, Valentijn; Heylen, Elisabeth; Leyssen, Pieter; Dallmeier, Kai; Coelmont, Lotte; Jochmans, Dirk; Chatterjee, Arnab K.; De Jonghe, Steven; Weynand, Birgit; Neyts, Johan
title: The combined treatment of Molnupiravir and Favipiravir results in a marked potentiation of antiviral efficacy in a SARS-CoV-2 hamster infection model
date: 2021-03-10
journal: bioRxiv
DOI: 10.1101/2020.12.10.419242
sha: 584316b57c0822a97192bc2fe051e6dc6f3420ae
doc_id: 966261
cord_uid: jzekvix4

Favipiravir and Molnupiravir, orally available antivirals, have been reported to exert antiviral activity against SARS-CoV2. In recent days preliminary efficacy data have been reported in COVID-19 patients. We here studied the combined antiviral effect of the drugs in the SARS-CoV2 hamster infection model. We first demonstrate that Molnupiravir can reduce infectious virus titers in lungs of infected animals in a dose-dependent manner by up to 3.5 log10 which is associated with a marked improvement of virus-induced lung pathology. When animals are treated with a combination of suboptimal doses of Molnupiravir and Favipiravir (that each alone result in respectively a 1.3 log10 and 1.1 log10 reduction of infectious virus titers in the lungs), a marked combined potency is observed. Infectious virus titers in the lungs of animals treated with the combo are on average reduced by 4.5 log10 and infectious virus are no longer detected in the lungs of 60% of treated infected animals. Both drugs result in an increased mutation frequency of the remaining viral RNA recovered from the lungs. In the combo-treated hamsters an increased frequency of C-to-T and G-to-A mutations in the viral RNA is observed as compared to the single treatment groups which may explain the pronounced antiviral potency of the combination. Our findings may lay the basis for the design of clinical studies to test the efficacy of the combination of Molnupiravir and Favipiravir in the treatment of COVID-19.

CoV-2. At day four post-infection (pi), the animals were euthanized and lungs were collected for 89 quantification of viral RNA, infectious virus titers and lung histopathology as described before 4 (Fig. 90 1A). Molnupiravir treatment resulted in a dose-dependent reduction in the viral RNA copies per mg of 91 lung tissue with 1.3 (P=0.002), 1.9 (P<0.0001) and a 3.3 log10 (P<0.0001) reduction was noted in the 92 groups that had been treated BID with 75, 150 or 200 mg/kg, respectively (Fig. 1B) 1D ). All the doses studied were well tolerated without significant weight 99 loss or any obvious adverse effects (Fig. 1E) . and was formulated as 50 or 100 mg/ml (for groups with the highest dose) stocks in a vehicle 178 containing 10%PEG400 and 2.5% Kolliphor-EL in water. Favipiravir was purchased from BOC Sciences 179 (USA) and was formulated as a 50 mg/mL stock in 3% sodium bicarbonate. 180 RT-qPCR was performed on a LightCycler96 platform (Roche) as described before 4 . 199

Lung tissues were homogenized using bead disruption (Precellys) in 350 µL minimal essential medium 201 and centrifuged (10,000 rpm, 5min, 4°C) to pellet the cell debris. To quantify infectious SARS-CoV-2 202 particles, endpoint titrations were performed as described before 4 . 203

For histological examination, the lungs were fixed overnight in 4% formaldehyde and embedded in 205 paraffin. Tissue sections (5 μm) were analyzed after staining with hematoxylin and eosin and scored 206 blindly for lung damage by an expert pathologist. The scored parameters, to which a cumulative score 207 of 1 to 3 was attributed, were the following: congestion, intra-alveolar hemorrhagic, apoptotic bodies 208 in bronchus wall, necrotizing bronchiolitis, perivascular edema, bronchopneumonia, perivascular 209 inflammation, peribronchial inflammation and vasculitis. 210

Genomic sequences from all samples were obtained using SureSelect XT target enrichment and Illumina 212 sequencing.

Reads generated were trimmed with Trim Galore 213 (https://github.com/FelixKrueger/TrimGalore). Duplicated reads were removed using Picard 214 (http://broadinstitute.github.io/picard). Reads from the inoculation sample were mapped to the SARS-215

CoV-2 reference genome (NC_045512) from GenBank using BWA-MEM 14 . The mapping quality was 216 checked using Qualimap and the consensus whole genome sequence was generated using QUASR 15, 16 . 217

Reads from the lung samples were mapped to this unique reference sequence. Genomes with less than 218 less than a 100 read depth were excluded. Variants above 1% and with a minimum of 2 supporting 219 reads per strand were identified at sites with a read depth of ≥ 10 using VarScan 17 . 220

GraphPad Prism (GraphPad Software, Inc.) was used to perform statistical analysis. Statistical 222 significance was determined using the non-parametric Mann Whitney U-test. P-values of ≤0.05 were 223 considered significant. 224

All of the data generated or analysed during this study are included in this published article. 226

We thank Carolien De Keyzer, Lindsey Bervoets, Thibault Francken, Elke Maas, Jasper Rymenants, Birgit 228

Voeten, Dagmar Buyst, Niels Cremers, Bo Corbeels and Kathleen Van den Eynde for excellent technical 229 assistance. We are grateful to Piet Maes for kindly providing the SARS-CoV-2 strain used in this study. 

A Novel Coronavirus from Patients with Pneumonia in China

The trinity of COVID-19: immunity, 251 inflammation and intervention

Medical treatment options for COVID-19

Favipiravir at high doses has potent antiviral activity in SARS-CoV-255

2−infected hamsters, whereas hydroxychloroquine lacks activity

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in 258 human airway epithelial cell cultures and multiple coronaviruses in mice

β-D-N 4 -Hydroxycytidine Is a Potent Anti-alphavirus Compound That 261 Induces a High Level of Mutations in the Viral Genome

Favipiravir as a potential countermeasure against 263 neglected and emerging RNA viruses

Characterization of orally efficacious influenza drug with high resistance 265 barrier in ferrets and human airway epithelia

Orally delivered MK-4482 inhibits SARS-CoV-2 replication in the Syrian 267 hamster model

Acute SARS-CoV-2 Infection is Highly Cytopathic, Elicits a Robust Innate 269

Immune Response and is Efficiently Prevented by EIDD-2801

Therapeutically administered ribonucleoside analogue

MK-4482/EIDD-2801 blocks SARS-CoV-2 transmission in ferrets

Human Safety, Tolerability, and Pharmacokinetics of Molnupiravir

Broad-Spectrum Oral Antiviral Agent with Activity Against SARS-CoV-2

STAT2 signaling restricts viral dissemination but drives severe pneumonia 276 in SARS-CoV-2 infected hamsters

Fast and accurate short read alignment with Burrows-Wheeler transform

Qualimap 2: Advanced multi-sample quality 280 control for high-throughput sequencing data

Viral population analysis and minority-variant detection using short read 282 next-generation sequencing

VarScan 2: Somatic mutation and copy number alteration discovery in 284 cancer by exome sequencing

Dose-response efficacy of Molnupiravir (EIDD-2801) against SARS-CoV-2 in a hamster infection model

A) Set-up of the study. (B) Viral RNA levels in the lungs of control (vehicle-treated) and EIDD-2801-treated (75, 291 150 or 200 mg/kg, BID) SARS-CoV-2−infected hamsters at day 4 post-infection (pi) are expressed as

CoV-2 RNA copies per mg lung tissue. Individual data and median values are presented. (C) Infectious viral loads 293 in the lungs of control (vehicle-treated) and EIDD-2801-treated SARS-CoV-2−infected hamsters at day 4 pi are 294 expressed as log10 TCID50 per mg lung tissue Individual data and median values are presented. (D) Cumulative 295 severity score from H&E stained slides of lungs from control

Individual data and median values are presented and the dotted line represents the median 297 score of untreated non-infected hamsters. (E) Weight change at day 4 pi in percentage, normalized to the body 298 weight at the time of infection. Bars represent means ± SD

0001, ns=non-significant. All data (panels B, C, D, E) are from two 300 independent experiments except for the 75 mg/kg group. The number of animals were 15

EIDD-2801-treated (150 mg/kg, BID) and combination-treated (Favipiravir+EIDD-2801 at 306 300+150 mg/kg, BID, respectively) SARS-CoV-2−infected hamsters at day 4 post-infection (pi) are expressed as 307 log10 SARS-CoV-2 RNA copies per mg lung tissue. Individual data and median values are presented. (C) Infectious 308 viral loads in the lungs of control (vehicle-treated), Favipiravir-treated, EIDD-2801-treated and combination-309 treated (Favipiravir+EIDD-2801) SARS-CoV-2−infected hamsters at day 4 pi are expressed as log10 TCID50 per mg 310 lung tissue. Individual data and median values are presented. (D) Cumulative severity score from H&E stained 311 slides of lungs from control (vehicle-treated), Favipiravir-treated, EIDD-2801-treated and combination-treated 312 (Favipiravir+EIDD-2801) SARS-CoV-2−infected hamsters. Individual data and median values are presented and 313 the dotted line represents the median score of untreated non

EIDD-2801-treated (150 mg/kg, BID) and combination-treated (Favipiravir+EIDD-2801 at 300+150

BID, respectively) SARS-CoV-2−infected hamsters at day 4 post-infection (pi)

All data (panels B, C, D) are from two independent experiments with 15, 10, 319 10 and 10 animals for respectively the vehicle, Favipiravir 300 mg/kg, EIDD-2801 150 mg/kg and Favipiravir+EIDD-320 2801 condition