key: cord-0774762-oe43mx2m
authors: Lieber, Carolin M.; Cox, Robert M; Sourimant, Julien; Wolf, Josef D.; Juergens, Kate; Phung, Quynh; Saindane, Manohar T; Natchus, Michael G; Painter, George R; Sakamoto, Kaori; Greninger, Alexander L.; Plemper, Richard K
title: SARS-CoV-2 variant of concern type and biological sex affect efficacy of molnupiravir in dwarf hamster model of severe COVID-19
date: 2022-02-07
journal: bioRxiv
DOI: 10.1101/2022.02.04.479171
sha: d144dc6dba73ab8e582bbb4343112f7f50a1ddde
doc_id: 774762
cord_uid: oe43mx2m

SARS-CoV-2 variants of concern (VOC) have triggered distinct infection waves in the coronavirus disease 2019 (COVID-19) pandemic, culminating in currently all-time high incidence rates of VOC omicron. Orally available direct-acting antivirals such as molnupiravir promise to improve disease management and limit SARS-CoV-2 spread. However, molnupiravir efficacy against VOC delta was questioned based on clinical trial results and its potency against omicron is unknown. This study evaluates molnupiravir against a panel of relevant VOC in three efficacy models: primary human airway epithelium organoids, the ferret model of upper respiratory disease, and a lethal Roborovski dwarf hamster efficacy model of severe COVID-19-like acute lung injury. All VOC were equally efficiently inhibited by molnupiravir in cultured cells and organoids. Treatment consistently reduced upper respiratory VOC shedding in ferrets and prevented viral transmission. Pathogenicity in the dwarf hamsters was VOC-dependent and highest for gamma, omicron, and delta with fulminant lung histopathology. Oral molnupiravir started 12 hours after infection resulted in complete survival of treated dwarf hamsters independent of challenge VOC. However, reduction in lung virus differed VOC-dependently, ranging from one (delta) to four (gamma) orders of magnitude compared to vehicle-treated animals. Dwarf hamsters infected with VOC omicron showed significant individual variation in response to treatment. Virus load reduction was significant in treated males, but not females. The dwarf hamster model recapitulates mixed efficacy of molnupiravir seen in human trials and alerts that therapeutic benefit of approved antivirals must be continuously reassessed in vivo as new VOC emerge.

in November 2021, VOC omicron has rapidly replaced delta as the dominant circulating strain in most 27 geographical regions 11 , propelled by sharply reduced sensitivity to neutralizing antibodies directed against earlier 28 lineages and greatly increased infectivity 12 . Although clinical signs associated with VOC omicron are typically 29 milder than those of its predecessors, record-high daily infection rates have driven high absolute hospitalization 30 numbers, creating an urgent need for therapeutics to improve disease management. 31

Molnupiravir was the first orally available SARS-CoV-2 inhibitor approved for clinical use against COVID-32 19 13 . Intermediate results of the early months of a large efficacy trial revealed an encouraging 50% reduction of 33 hospitalizations in the treatment group, but later analysis of the full dataset reduced efficacy to a 30% lower 34 hospitalization rate overall 14 . Based on geographical location of trial participants and VOC prevalence in the 35 earlier versus later phase of the trial, an advisory board to the FDA considered lower efficacy of molnupiravir 36 against VOC delta as a possible explanation for the mixed results 15 . However, VOC delta was efficiently inhibited 37 by the molnupiravir parent metabolite N 4 -hydroxycytidine (NHC) in ex vivo studies 16 , suggesting unchanged 38 sensitivity to the drug. 39

Human airway epithelium organoids, ferrets, mice, and Syrian golden hamsters have emerged as preclinical 40 models to assess efficacy of anti-SARS-CoV-2 drug candidates. Ferrets recapitulate the predominant clinical 41 presentation of SARS-CoV-2 in younger patients, characterized by high viral load in the upper respiratory tract, 42 strong viral shedding, and efficient airborne transmission [17] [18] [19] . By contrast, Syrian golden hamsters infected with 43 SARS-CoV-2 develop transient pneumonia but do not recapitulate hallmark features of life-threatening severe COVID-19. Disease typically remains mild-to-asymptomatic in golden hamsters and animals fully recover within 45 two weeks 20 . Lethal disease with severe histopathology affecting lung, liver, and kidney can be induced in 46 transgenic K18-hACE2 mice expressing human ACE2, but organ distribution of the receptor is non-physiological, 47 of high lung virus loads (Fig. 3j ) already 12 hours after infection. Comparison analysis of VOC gamma 24 hours 122 after infection confirmed that rapid viral invasion of the dwarf hamsters was not limited to VOC delta. 123

VOC gamma, delta, and omicron were selected for an efficacy study assessing mitigation of viral pneumonia 125 and acute lung injury with molnupiravir. Animals were inoculated with 1 × 10 4 pfu intranasally to prevent 126 premature death, followed by initiation of treatment (250 mg/kg orally b.i.d.) 12 hours after infection (Fig. 4a) , 127 when lung virus load was high and first lung lesions became detectable (Fig. 3i,j) . The higher molnupiravir dose 128 compared to that administered to ferrets was used to compensate for high metabolic activity of the dwarf 129 hamsters 27 and is consistent with the dose level administered to other rodent species 28,29 . Oral molnupiravir 130 alleviated clinical signs (Extended Data 5) and ensured complete survival of all treated animals independent of 131 challenge VOC, whereas approximately 50% (gamma) and 90% (delta, omicron) of animals in the different 132 vehicle groups succumbed to the infection within 2 to 7 days (Fig. 4b) . Lung virus load assessed three days after 133 infection was consistently high (approximately 10 7 -10 8 pfu/g lung tissue) in all vehicle-treated groups (Fig. 4c) . 134

Treatment significantly lowered lung titers independent of challenge VOC, albeit effect size varied greatly from 135 approximately 1 (delta) to over 4 (gamma) orders of magnitude. Although likewise statistically significant 136 compared to vehicle-treated animals, impact of molnupiravir on omicron lung load showed major variation 137 between individual animals, creating low, high, and super responder groups characterized by lung titer reductions 138 of approximately 1, 4, and >5 orders of magnitude, respectively (Fig. 4c ). Infectious titers in lung were closely 139 mirrored by viral RNA copies present in lung and tracheas (Extended Data 6). 140

Having powered this study with approximately equal numbers of male and female animals in each group, we 141 queried the dataset for a possible impact of biological sex on outcome (Fig. 4d) . No significant differences in lung 142 virus load between males and females were detected in any of the three vehicle groups, which was consistent with 143 absence of a correlation between biological sex and probability of survival of the vehicle-treated animals in our 144

survival study (Extended Data 7). Comparison of lung virus burden of males and females in the molnupiravir-145 treated groups revealed no statistically significant differences in effect size in animals infected with VOC gamma 146 or delta, but biological sex had a statistically significant influence on molnupiravir benefit of animals infected 147 with omicron (Fig. 4d , Extended Data 8). Whereas lung titer reductions in treated males were highly significant 148 compared to vehicle-treated males, females, or all vehicle-treated animals combined, no significant change in 149 lung virus load against any of these vehicle groups was detected in females treated with molnupiravir. 150

In earlier studies with SARS-CoV-2 in the ferret model 19 , we noted rapid appearance of characteristic host 152 adaptation mutations such as an L260F substitution in nsp6 and a Y453F mutation in spike 26 in virus populations 153 extracted from ferret nasal turbinates. To probe for possible virus adaptations to the dwarf hamsters, we sequenced 154 whole genomes of virus populations recovered from the different vehicle or molnupiravir treatment groups. No 155 dwarf hamster-typical mutations were detected that were dominant across all VOC populations, but we detected 156 several VOC type-specific substitutions with >20% allele frequency compared to the respective virus inoculum. 157

Irrespective of treatment status, a spike D142G substitutions was present in nearly all VOC delta populations, but 158 no dominating mutations emerged in spike proteins of the different VOC gamma and omicron reisolates 159 Table 1 ). All recovered VOC gamma populations harbored an nsp6 V181F substitution and all 160 VOC omicron populations contained the nsp6 L260F mutation that was originally considered to be characteristic 161 for adaptation to weasels 19 . However, none of the recovered VOC delta populations contained substitutions in 162 nsp6. We found isolated additional substitutions in some virus populations recovered from individual animals in 163 the respective infection and treatment groups (Supplementary Table 1 , Supplementary Datasets 1-3), but detected 164 no correlation to relative viral fitness in vehicle-treated dwarf hamsters or link to molnupiravir treatment success. 165

Macroscopic assessment of the lungs extracted three days after infection revealed severe tissue damage with 167 large lesions covering approximately 30% (omicron) to 50% (gamma, delta) of the lung surface area of vehicle-168 treated animals (Fig. 4e, Supplementary Fig. 6 ). Molnupiravir treatment significantly reduced macroscopic tissue 169 damage independent of challenge VOC (Fig. 4e , Extended Data 9). Histological examination of lungs extracted 170 from animals infected with VOC gamma and delta revealed markers of severe viral infection in vehicle-treated 171 animals, including perivascular cuffing, alveolitis, hyalinization of blood vessels, interstitial pneumonia, and 172 leucocyte infiltration (Fig. 4f , Extended Data 9). One of the VOC gamma-infected animals developed pronounced peribronchiolar metaplasia. Due to high lethality of VOC delta, only one animal of the vehicle group reached the 174 predefined endpoint for tissue harvest in this study, whereas the others died prematurely and could not be 175 examined. Molnupiravir alleviated histopathology associated with either VOC, decreasing immune cell 176 infiltration and reducing signs of inflammation. Greater residual damage was detected in treated animals infected 177 with VOC delta compared to gamma, which was consistent with the significantly greater molnupiravir-mediated 178 reduction in gamma lung load detected in the efficacy study (Fig. 4c) . present we cannot conclusively address, however, whether the difference in virus load reduction between VOC 221 gamma and delta in dwarf hamsters recapitulates the variable clinical success of molnupiravir because of a 222 common mechanism or due to a phenocopy effect. Although VOC delta and gamma replicated to similar lung 223 titers in vehicle-treated animals, delta was associated with the shortest time-to-death. Individual VOC could 224 indirectly modulate molnupiravir pharmacokinetic properties differentially by spreading to organs other than lung 225 with distinct kinetics. However, our analysis of viral organ distribution in the dwarf hamsters revealed very low 226 viral RNA burden in liver, the primary site of drug metabolism 40 . Alternatively, the rapid-onset lung 227 histopathology seen with VOC delta may directly contribute to lower effect size of therapy. 228 iv) Unexpectedly, molnupiravir efficacy against VOC omicron was variable between individual dwarf 229 hamsters. Biological sex of the animals emerged as a correlate for therapeutic benefit of molnupiravir use against 230 omicron, with treated males faring overall better than females. By contrast, biological sex had no effect on 231 treatment benefit when dwarf hamster were infected with VOC gamma or delta, which matched human trial data 232 reported for these VOC 14 . Dwarf hamsters are outbred and animals used in this study were not raised under 233 controlled conditions, introducing individual differences in body weight, age, microbiome, drug metabolism, 234 and/or prior disease history as additional variables, which certainly are all equally present also in human patients. 235

However, dwarf hamsters were randomly assigned to the different study groups and these factors, if indeed of 236 importance, should have resulted in equal individual variation in viral load in the vehicle group or in animals 237 infected with VOC gamma or delta. Whole genome sequence analysis of VOC omicron populations recovered 238 from the hamsters at the end of infection revealed furthermore no correlation between potential differential VOC 239 omicron adaptation to the dwarf hamster host and effect size of molnupiravir therapy, pointing overall to high 240 variability of omicron disease dynamics in treated dwarf hamsters. 241

In the absence of controlled clinical data assessing molnupiravir efficacy against omicron, it is currently 242 unclear to what degree the dwarf hamster-derived results extend to human therapy. Our study demonstrates, 243 however, that pharmacological mitigation of severe COVID-19 is complex and that attempts to predict drug 244 efficacy based on unchanged ex vivo inhibitory concentrations alone 16 may be premature. temperature (c) of infected dwarf hamsters. d, Survival curves of infected dwarf hamsters from (a). e, Viral RNA 372 copies in select organs extracted from infected hamsters 3 days after infection. f, Images of lungs from hamsters 373 mock infected or inoculated with 1 × 10 5 pfu WA1, VOC omicron, or VOC gamma 3 days after infection. g, 374

Infectious titers from the lungs of hamsters shown in (f). h, Schematic of the dwarf hamster pathogenesis study 375 utilizing 1 × 10 4 pfu VOC delta and gamma. i, Images of lungs extracted from hamsters mock infected or 376 inoculated with 1 × 10 4 pfu VOC omicron or VOC gamma 0.5 (delta) and 1 day (delta and gamma) after infection. 377 j, Infectious titers from the lungs of hamsters shown in (i). Symbols represent independent biological repeats (e, 378 g, j), lines intersect group medians (b-c), columns show group medians (e, g, j), and error bars represent 95% 379 confidence intervals. LoD, limit of detection; LoQ, limit of quantitation. 380 

Extended Data 5. Clinical signs during treatment with molnupiravir. Roborovski dwarf hamsters infected with 615

VOC delta, or VOC omicron and treated with molnupiravir or vehicle were infected 616 and monitored for clinical signs for 14 days. a-b, Body weight (a) and temperature were measured once daily

Symbols represent independent biological repeats (individual animals)

Histopathology of hamster lungs 3 days after infection. Staining with hematoxylin and eosin

Arrows highlight pleuritis. Br, bronchioles; Bl, blood vessel

Histopathology of hamster lungs 14 days after infection. Staining with hematoxylin and 643 eosin. Scale bar 50 μm

from (a). c, Infectious titers from the lungs of hamsters 3 days after infection as shown in (a). d, Role of biological 386 sex on antiviral efficacy shown in (c). e, Images of lungs from hamsters mock infected or inoculated with 1 × 10 4 387 pfu VOC delta, gamma, or omicron, treated with vehicle or molnupiravir, and harvested 3 days after infection. 388Quantitation of macroscopic lesions as a percent of total visible lung surface area for infected hamsters treated 389 with vehicle or molnupiravir are shown. Mock infected lungs (n=4) were included as a reference. f-g, 390Histopathology (f) and immunohistochemistry (g) of dwarf hamster lung slices from groups harvested 3 days 391 after infection. Staining with hematoxylin and eosin (f) or a-SARS-CoV-2 S (g). Br, bronchioles; Bl, blood 392 vessel; scale bar 50 μm. Symbols represent independent biological repeats, columns show a group medians (c, d, 393 e), and error bars represent 95% confidence intervals. Significance was determined using unpaired t-tests ( Reference laboratory and amplified on Calu-3 cells. All viruses were authenticated by whole genome next 434 generation sequencing prior to use. 435Virus yield reduction 436 12-well plates were seeded with 2 × 10 5 cells per well the day before infection. Each isolate was diluted in DMEM 437 to achieve a multiplicity of infection (MOI) of 0.1 pfu/cell, adsorbed on cells for 1 hour at 37°C following which 438 the inoculum was removed and replaced with DMEM with 2% heat-inactivated FBS. The media contained 439 additionally 0.1% DMSO (vehicle) and the indicated concentration of NHC (EIDD-1931). After 48 hours at 440 37 °C, the cell supernatant was harvested aliquoted and frozen at -80°C before titration by standard plaque assay. 441Log viral titers were normalized using the average top plateau of viral titers to define 100% and were analyzed 442 with a non-linear regression with variable slope to determine EC50 (Prism; GraphPad). 443

Eight-week differentiated HAE cells were infected (or mock-infected) with 3 × 10 4 pfu of SARS-CoV-2 VOC 445 and fixed at day 3 after infection. Zen Blue software, while keeping the same processing for the conditions that are directly compared (vehicle vs 459 treatment). 460

Samples were serially diluted (tenfold dilutions starting at a 1:10 initial dilution) in DMEM medium supplemented 462 with 2% FBS containing Antibiotic-Antimycotic (Gibco). The serial dilutions were added to Vero E6 cells seeded 463 in 12-well plates at 3 × 10 5 cells per well 24 hours previously. The virus was allowed to adsorb for 1 hour at 37 °C. 464Subsequently, the inoculum was removed, and the cells were overlaid with 1.2% Avicel (FMC BioPolymer) in 465 DMEM and incubated for 3 days at 37 °C with 5% CO2. After three days, the Avicel was removed, cells were 466 washed once with PBS, fixed with 10% neutral buffered formalin and plaques were visualized using 1% crystal 467 violet. For hamsters infected with VOC gamma, delta, and omicron, plaque assays were performed with VeroE6-468 TMPRSS2 cells. 469

Groups of ferrets were inoculated with 1 × 10 5 pfu of VOC alpha or delta (1ml; 0.5ml per nare). Twelve hours 471 after infection, groups of ferrets were treated twice daily (b.i.d.) with vehicle (1% methylcellulose) or 472 molnupiravir at a dose of 5 mg/kg in 1% methylcellulose. Treatments were administered by oral gavage and 473 continued every 12 hours until 4 days after infection. All ferrets were euthanized 4 days after infection and tissues 474 were harvested to determine SARS-CoV-2 titers and the presence of viral RNA. 475

Two groups of six source ferrets were inoculated intranasally with 1 × 10 5 pfu of VOC alpha or gamma. After 477 twelve hours, groups of source ferrets were further divided into two groups, receiving vehicle (n=3 for VOC 478 alpha; n=4 for VOC gamma) or molnupiravir treatment (5 mg/kg b.i.d.) administered by oral gavage. At 54 hours 479 after infection, each source ferret was co-housed with two uninfected and untreated contact ferrets. Ferrets were 480 co-housed until 96 hours after infection, when sourced ferrets were euthanized and contact ferrets were housed 481 individually. All contact ferrets were monitored for 4 days after separation from source ferrets and then 482 euthanized. Nasal lavages were performed on source ferrets twice daily until cohousing started. After cohousing 483 started, nasal lavages were performed on all ferrets every 24 hours. Nasal turbinates were harvested for all ferrets 484 after euthanization to determine infectious titers and the presence of viral RNA. 485

Female and male hamsters (2-4 months of age) were purchased from Ron Van Der Vliet, Netherlands. The 487 hamsters were permanently quarantine housed under ABSL-2 conditions until study start. After a minimal resting 488 period of 2 weeks after arrival, animals were randomly assigned to groups for individual studies, transferred into 489 an ABSL-3 facility immediately prior to study start, and housed singly in ventilated negative-pressure cages 490 during the studies. To establish a pathogenicity profile, hamsters were inoculated intranasally with 1 × 10 5 pfu in 491 50 µl (25 µl per nare), unless otherwise stated (VOC delta 3 × 10 4 ). The hamsters were anaesthetized with 492 dexmedetomidine/ketamine before inoculation. Groups of hamsters were euthanized 14 days after infection and 493 their organs were harvested to determine the presence of viral RNA in different tissues (WA1 and VOC gamma 494 only). 495

Groups of hamsters were inoculated with 1 × 10 4 pfu in 50 µl (25 µl per nare). At 12 hours after infection, the 497 hamsters were treated b.i.d. with vehicle (1% methylcellulose) or molnupiravir at a dosage of 250 mg/kg body 498 weight, respectively. The compound was administered via oral gavage in 1% methylcellulose. After the start of 499 treatment, b.i.d. dosing was continued until 12 days after infection. Subgroups of hamsters were euthanized 3 500 days after infection. All studies were terminated 14 days after infection. Organs were harvested to determine virus 501 titers and presence of viral RNA in different tissues. 502

For virus titration, the organs were weighed and homogenized in PBS. The homogenates were centrifuged at 504 2,000g for 5 minutes at 4 °C. The clarified supernatants were harvested, frozen and used in subsequent plaque 505 assays. For detection of viral RNA, the harvested organs were stored in RNAlater at −80 °C. The tissues were 506 homogenized, and the total RNA was extracted using a RNeasy mini kit (Qiagen). 507Quantitation of SARS-CoV-2 RNA copy numbers 508 SARS-CoV-2 RNA was detected using the nCoV_IP2 primer-probe set (National Reference Center for 509Respiratory Viruses, Institute Pasteur) which targets the SARS-CoV-2 RdRP gene. RT-qPCR reactions were 510 performed using an Applied Biosystems 7500 real-time PCR system using the StepOnePlus real-time PCR 511 package. The nCoV_IP2 primer-probe set was used in combination with TaqMan fast virus 1-step master mix 512 (Thermo Fisher Scientific) to detect viral RNA. A standard curve was created using a PCR fragment (nt 12669-513 14146 of the SARS-CoV-2 genome) generated from viral complementary DNA using the nCoV_IP2 forward 514 primer and the nCoV_IP4 reverse primer to quantitate the RNA copy numbers. RNA copy numbers were 515 normalized to the weight of tissues used. 516 Extended Data 6. SARS-CoV-2 RNA copies in hamsters infected with different VOC. RNA copies were 621 determined in hamsters infected with 1 × 10 4 pfu VOC gamma, VOC delta, or VOC omicron treated with 622 molnupiravir or vehicle. a, SARS-CoV-2 RNA copies present in lungs (top) and trachea (bottom) of infected 623 hamsters. Symbols represent independent biological repeats (n, individual animals), columns show group 624 medians and error bars represent the 95% confidence intervals. Significance was determined using unpaired t-625