key: cord-0696321-z53a8xfu
authors: Cox, Robert M.; Wolf, Josef D.; Plemper, Richard K.
title: Therapeutic MK-4482/EIDD-2801 Blocks SARS-CoV-2 Transmission in Ferrets
date: 2020-10-12
journal: Res Sq
DOI: 10.21203/rs.3.rs-89433/v1
sha: 8c38a0e996cd531d45c245bf15a67b464e8f570b
doc_id: 696321
cord_uid: z53a8xfu

The COVID-19 pandemic is having a catastrophic impact on human health. Widespread community transmission has triggered stringent distancing measures with severe socioeconomic consequences. Gaining control of the pandemic will depend on interruption of transmission chains until protective herd immunity arises. Ferrets and related members of the weasel genus transmit SARS-CoV-2 efficiently with minimal clinical signs, resembling spread in the young-adult population. We previously reported an orally efficacious nucleoside analog inhibitor of influenza viruses, EIDD-2801 (or MK-4482), that was repurposed against SARS-CoV-2 and is in phase II/III clinical trials. Employing the ferret model, we demonstrate in this study high SARS-CoV-2 burden in nasal tissues and secretions that coincides with efficient direct-contact transmission. Therapeutic treatment of infected animals with twice-daily MK-4482/EIDD-2801 significantly reduced upper respiratory tract SARS-CoV-2 load and completely suppressed spread to untreated contact animals. This study identifies oral MK-4482/EIDD-2801 as a promising antiviral countermeasure to break SARS-CoV-2 community transmission chains.

The COVID-19 pandemic is having a catastrophic impact on human health. Widespread 2 community transmission has triggered stringent distancing measures with severe socioeconomic 3 consequences. Gaining control of the pandemic will depend on interruption of transmission chains 4 until protective herd immunity arises. Ferrets and related members of the weasel genus transmit 5 SARS-CoV-2 efficiently with minimal clinical signs, resembling spread in the young-adult 6 population. We previously reported an orally efficacious nucleoside analog inhibitor of influenza 7 viruses, EIDD-2801 (or MK-4482), that was repurposed against SARS-CoV-2 and is in phase II/III 8 clinical trials. Employing the ferret model, we demonstrate in this study high SARS-CoV-2 burden 9 in nasal tissues and secretions that coincides with efficient direct-contact transmission. 10

reduced upper respiratory tract SARS-CoV-2 load and completely suppressed spread to untreated 12 contact animals. This study identifies oral MK-4482/EIDD-2801 as a promising antiviral 13 countermeasure to break SARS-CoV-2 community transmission chains. 14

The coronavirus disease (COVID)-19 pandemic is exerting a global impact on human health not 16 experienced from a single pathogen since the Spanish flu outbreak of 1918. The etiologic agent, CoV-2, has spread to over 35.5 million people to date, causing over 1 million deaths and substantial 18 morbidity, and having an unprecedented catastrophic effect on societies and the global economy 1 . 19

Interrupting widespread community transmission is paramount to establishing pandemic control and 20 relaxing social-distancing measures. However, no vaccine prophylaxis is yet available and approved 21 antiviral treatments such as remdesivir and reconvalescent serum cannot be delivered orally 2,3 , making 22 them poorly suitable for transmission control. 23

We recently reported the development of MK-4482/EIDD-2801 4,5 , the orally available pro-drug of 24 the nucleoside analog N 4 -hydroxycytidine (NHC), which has shown potent anti-influenza virus activity 25 in mice, guinea pigs, ferrets, and human airway epithelium organoids 4,6,7 . Acting through induction of 26 error catastrophe in virus replication 4,8 , NHC has broad-spectrum anti-RNA virus activity and is 27 currently being tested in advanced clinical trials (NCT04405570 and NCT04405739) for the treatment 28 of SARS-CoV-2 infection. In addition to ameliorating acute disease, we have demonstrated in a guinea 29 pig transmission model that NHC effectively blocks influenza virus spread from infected animals to 30 untreated contact animals 7 . 31

Several mouse models of SARS-CoV-2 infection have been developed, some of which were 32 employed to confirm in vivo efficacy of MK-4482/EIDD-2801 also against beta-coronaviruses 9 . 33 However, human SARS-CoV-2 cannot productively infect mice without extensive viral adaptation or 34 introduction of human ACE2 into transgenic animals, and none of the mouse models supports 35 transmission to uninfected mice 10 . Spillover of SARS-CoV-2 to farmed minks, subsequent large-scale 36 mink-to-mink transmission and, in some cases, zoonotic transmission back to humans revealed efficient 37 viral spread among members of the weasel genus without prior adaptation [11] [12] [13] [14] . Although mink farms 38 reported elevated animal mortality and gastrointestinal and respiratory clinical signs 15 , outbreak follow-39 up revealed continued intra-colony spread for extended periods of time 14 , suggesting that acute clinical 40 signs in the majority of infected animals may be mild or absent. These mink field reports corroborated 41 results obtained with experimentally infected ferrets showing that mustelids of the weasel genus transmit 42 SARS-CoV-2 efficiently without strong clinical disease manifestation 16, 17 . This presentation of SARS-43

CoV-2 infection resembles the experience of frequently asymptomatic or mildly symptomatic SARS-44

CoV-2 spread in the human young-adult population 18 . 45

In this study, we have explored the efficacy of oral MK-4482/EIDD-2801 against SARS-CoV-2 in 46 the ferret model. We demonstrate significant reduction of upper respiratory tract virus load in animals 47 treated therapeutically with MK-4482/EIDD-2801. Whereas SARS-CoV-2 efficiently spread to all 48 contacts of vehicle-treated source animals, MK-4482/EIDD-2801 treatment blocked all SARS-CoV-2 49 transmission. These results support the administration of MK-4482/EIDD-2801 to asymptomatic or 50 mildly symptomatic SARS-CoV-2 positives to rapidly block community transmission chains in addition 51 to the treatment of patients with advanced clinical signs or severe disease. 52

To validate host invasion and tissue tropism of SARS-CoV-2 in ferrets, we inoculated animals 54 intranasally with 1´10 4 or 1´10 5 plaque-forming units (pfu) of SARS-CoV-2 clinical isolate 2019-55 nCoV/USA-WA1/2020 per animal. Shed virus burden was monitored daily over a 10-day period and 56 virus load in the upper and lower respiratory tract determined on days four and ten after infection. In 57 animals of the high inoculum group, virus release from the upper respiratory tract peaked three days 58 after infection and was undetectable by day seven (Fig. 1a) . No efficient infection was noted in the low 59 inoculum group. Shedding profiles closely correlated with infectious particle load in nasal turbinates; a 60 heavy virus tissue burden in the high inoculum group was present on day 4, which greatly decreased by 61 approximately four orders of magnitude by day 10 (Fig. 1b) . 62

Low inoculum resulted in light virus load in the turbinates on day 4 and undetectable burden 63 thereafter. However, qPCR-based quantitation of viral RNA copy numbers in the turbinates revealed 64 continued presence of a moderate (approx. 10 4 copies/g tissue) to high (³10 7 copies/g tissue) virus load 65 after low and high inoculum, respectively (Fig. 1c) . Independent of inoculum amount, no infectious 66 particles were detected in bronchoalveolar lavages or lung tissue samples (extended data Fig. 1 ). At both 67 days 4 and 10, several organ samples (lung, heart, kidney, liver) were also qPCR-negative ( Fig. 1d) , 68 confirming inefficient infection of the ferret lower respiratory tract and limited systemic host invasion. 69

Only small and large intestine samples were PCR-positive on day 4 after infection, and rectal swabs 70 showed continued low-grade shedding of viral genetic material (Fig. 1e) . 71

Animals in the high-inoculum group experienced a transient drop in body weight that reached a low 72 plateau on days 5-6 after infection, but fully recovered by the end of study (Fig. 1f) . No other clinical 73 signs such as fever or respiratory discharge were noted. Complete blood counts taken every second day 74 revealed no significant deterioration from the normal range in either inoculum group in overall white 75 blood cells counts and lymphocyte, neutrophil, and platelet populations (Fig. 1g ). Relative expression 76 levels of type I and II interferon and IL-6 in ferret peripheral blood mononuclear cells (PBMCs) sampled 77 in 48-hour intervals reached a plateau approximately 3 days after infection and stayed moderately 78 elevated until the end of the study (Fig. 1h ). Selected interferon-stimulated genes (ISGs) with antiviral 79 effector function (MX1 and ISG15) showed a prominent expression peak four days after infection, 80 followed by return to baseline expression by study end. 81

Informed by these results, ferrets were infected in subsequent MK-4482/EIDD-2801 efficacy tests 83 with 1´10 5 pfu/animal and infectious virions in nasal lavages determined twice daily (Fig. 2a) SARS-CoV-2 RNA was still detectable in nasal tissues extracted from animals of all groups, albeit 98 significantly reduced (p=0.0089 and p=0.0081 for the 5 mg/kg and 15 mg/kg MK-4482/EIDD-2801 99 groups, respectively) in treated animals versus the vehicle controls ( Fig. 2d) . Animals of the 12-hour 100 therapeutic groups showed a significant reduction (p£0.044) in effector ISG expression compared to 101 vehicle-treated animals, although no significant differences in relative interferon and IL-6 induction 102 were observed (extended data Fig. 2) . 103

These results demonstrate oral efficacy of therapeutically administered MK-4482/EIDD-2801 104 against acute SARS-CoV-2 infection in the ferret model. Consistent with our previous pharmacokinetic 105 (PK) and toxicology work-up of MK-4482/EIDD-2801 in ferrets, treatment did not cause any 106 phenotypically overt adverse effects and white blood cell and platelet counts of drug-experienced 107 animals remained in the normal range (extended data Fig. 3) . 108

SARS-CoV-2 shedding into the ferret upper respiratory tract establishes conditions for productive 110 spread from infected source to uninfected contact animals 16, 17 . To assess transmission efficiency, we co-111 housed intranasally infected source animals with two uninfected contact animals each for a 3-day period, 112 starting 30 hours after source animal inoculation (Fig. 3a) . Nasal lavages and rectal swabs were obtained 113 from all animals once daily and blood sampled at study start and on days four and eight after the original 114

infection. Viral burden and RNA copy numbers in respiratory tissues were determined at the end of the 115 co-housing phase (source animals) and at study end (contact animals). 116

Infectious particles first emerged in nasal lavages of some contact animals 24 hours after the start of 117 co-housing (Fig. 3b) . By the end of the co-housing phase, all contact animals were infected and 118 approached peak virus replication phase, demonstrating that SARS-CoV-2 transmission among ferrets is 119 rapid and highly efficient. 120

A second cohort of source animals inoculated in parallel with SARS-CoV-2 received oral MK-122 4482/EIDD-2801 at the 5 mg/kg body weight dose level, administered b.i.d. starting 12 hours after 123 infection. Productive infection of these animals was validated by SARS-CoV-2 titers in nasal lavages 124 one day after infection (Fig. 3b ) that very closely matched those seen in the initial efficacy tests (Fig.  125 2b). Although we also co-housed the treated source animals for nearly 3 days with two untreated 126 contacts each, no infectious SARS-CoV-2 particles were detected in any of the series of nasal lavages 127 obtained from these contacts or in any of the contact animal nasal turbinates sampled at study end ( Fig.  128 3c). 129 Nasal turbinates extracted from the contacts of vehicle-treated source animals contained high viral 130 RNA copy numbers, underscoring successful host invasion after transmission (Fig. 3d ). Consistent with 131 our earlier observations, turbinates of treated source animals harbored moderate to high (³10 5 copies/g 132 tissue) amounts of viral RNA although infectious particles could not be detected. In contrast, all 133 respiratory tissues of the contacts co-housed with MK-4482/EIDD-2801-treated source animals 134 remained SARS-CoV-2 genome free, indicating the absence of any low-grade virus replication that 135 could have hypothetically progressed in these animals below the detection level of infectious particles 136 Rodriguez Mega, E. COVID has killed more than one million people. How many more will die? 209

Nature, doi:10.1038/d41586-020-02762-y (2020 Viruses were administered to source animals through intranasal inoculation and virus load monitored 340 periodically in nasal lavages and rectal swabs, and 4 or 10 days after exposure in respiratory tissues and 341 a subset of organs. Virus titers were determined based on plaque assay and viral RNA copy numbers, 342 blood samples subjected to CBC analysis and RT-qPCR quantitation of selected cytokine and innate 343 antiviral effector expression levels. 344

Vero-E6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 346 7.5% heat inactivated fetal bovine serum (FBS) at 37°C with 5% CO2. SARS-CoV-2 (SARS-CoV-347 2/human/USA-WA1/2020) was propagated using Vero-E6 cells supplemented with 2% FBS. Virus 348 stocks were stored at -80°C and titers were determined by plaque assay. Vero-E6 cells were routinely 349 checked in 6-month intervals for bacterial and mycoplasma contamination. 350

Samples were serially diluted (10-fold starting at 1:10 initial dilution) in DMEM supplemented with 352 2% FBS containing antibiotics-antimycotics (Gibco). Serial dilutions were added to Vero-E6 cells 353 seeded in 12-well plates at 3´10 5 cells per well 24-hours prior. Virus was allowed to adsorb for 1 hour at 354 37°C. Subsequently, inoculum was removed, and cells were overlaid with 1.2% Avicel (FMC 355 biopolymer) in DMEM and incubated for three days at 37°C with 5% CO2. Avicel was removed and 356 cells were washed once with PBS, fixed with 10% neutral buffered formalin, and plaques were 357 visualized using 1% crystal violet. 358

Female ferrets (6-10 months of age) were purchased from Triple F Farms. Upon arrival, ferrets were 360 rested for one week, then randomly assigned to groups and housed individually in ventilated negative 361 pressure cages in an ABSL-3 facility. In order to establish a suitable inoculum for efficacy and 362 transmission studies, ferrets (n=4) were inoculated intranasally with 1´10 4 and 1´10 5 pfu of 2019-363 nCoV/USA-WA1/2020 in 1 ml (0.5 ml per nare). Prior to inoculation, ferrets were anesthetized with 364 dexmedetomidine/ketamine. Nasal lavages were performed once daily using 1 ml of PBS containing 2´ antibiotics-antimycotics (Gibco). For blood sampling, ferrets were anesthetized with dexmedetomidine 366 and approximately 0.5 ml blood was drawn from the anterior vena cava. Complete blood counts (CBC) 367 were performed using a Vetscan HM5 (Abaxis) in accordance with the manufacturer's protocol. Rectal 368 swabs were performed every two days. Groups of two ferrets were sacrificed 4-and 10-days post 369 infection and organs were harvested to determine virus titer and the presence of viral RNA in different 370 tissues. 371

Groups of ferrets (n=3 each) were inoculated with 1´10 5 pfu of 2019-nCoV/USA-WA1/2020 in 1 373 ml (0.5 ml per nare performed on all ferrets every 12 hours. Blood samples were obtained every two days after infection and 379 stored in K2-EDTA tubes (Sarstedt CB 300). CBC analysis was performed on each blood sample in 380 accordance with the manufacturer's protocols. After CBC analysis, red blood cells were lysed with ACK 381 buffer (150 mM NH4CL, 10mM KHCO3, 0.01 mM EDTA pH 7.4) and PBMCs were harvested and 382 stored at -80°C in RNAlater until further qPCR analysis was performed. Four days after infection, all 383 ferrets were euthanized and organs harvested to determine virus titers and the presence of viral RNA in 384 different tissues. 385

A group of 6 individually housed source ferrets were inoculated intranasally with 1´10 5 pfu of 387 2019-nCoV/USA-WA1/2020. Twelve hours after infection, source ferrets were split into two groups 388 (n=3 each) receiving vehicle or MK-4482/EIDD-2801 treatment at a dose of 5 mg/kg b.i.d. daily by oral 389 gavage. At 30 hours post infection, each source ferret was co-housed with two uninfected and untreated 390 contact ferrets. Ferrets were co-housed until 96 hours after infection, when source ferrets were 391 euthanized and contact animals housed individually. Contact animals were monitored for four days after 392 separation from source ferrets, then sacrificed. Nasal lavages and rectal swabs were performed every 24 393 hours on all ferrets. Blood samples were collected at 0, 4, and 8 days after source ferret infection. For all 394 ferrets, organs were harvested to determine virus titers and the presence of viral RNA in different 395 tissues. 396

For virus titration, organs were weighed and homogenized in PBS. Homogenates were centrifuged 398 for 5 minutes at 2,000´g at 4°C. Clarified supernatants were harvested and used in subsequent plaque 399 assays. For detection of viral RNA, harvested organs were stored in RNAlater at -80°C. Tissues were 400 ground and total RNA was extracted using a RNeasy mini kit (Qiagen). RNA was extracted from rectal 401 swabs using the ZR Viral RNA Kit (Zymo Research) in accordance with the manufacturer's protocols. 402

Detection of SARS-CoV-2 RNA was performed using the nCoV_IP2 primer-probe set (National 404 

Reference Center for Respiratory Viruses

Applied Biosystems 7500 Real-Time PCR System using the StepOnePlus Real-Time PCR 406

System was used to perform qPCR reactions

Scientific) was used in combination with the nCoV_IP2 primer-probe set to detect viral RNA

quantitate RNA copy numbers, a standard curve was created using a PCR fragment (nucleotides 12669-409 14146 of the SARS-CoV-2 genome) generated from viral cDNA using nCoV_IP2 forward primer and 410 the nCoV_IP4 reverse primer

We thank M. Kumar for providing an aliquot of 2019-nCoV/USA-WA1/2020 stock, members of 437 the GSU High Containment Core and the Department for Animal Research for support, and J. Sourimant 438 and A. L. Hammond for critical reading of the manuscript. This work was supported, in part, by Public 439Health Service grants AI071002 (to RKP) and AI141222 (to RKP), from the NIH/NIAID. The funders 440 had no role in study design, data collection and interpretation, or the decision to submit the work for 441 publication.