key: cord-0754782-nbq3pnbj
authors: Joaquín Cáceres, C.; Cardenas-Garcia, Stivalis; Carnaccini, Silvia; Seibert, Brittany; Rajao, Daniela S.; Wang, Jun; Perez, Daniel R.
title: Efficacy of GC-376 against SARS-CoV-2 virus infection in the K18 hACE2 transgenic mouse model
date: 2021-01-27
journal: bioRxiv
DOI: 10.1101/2021.01.27.428428
sha: 6bb002988180fd6c83ebb2496a45ec1387e7097c
doc_id: 754782
cord_uid: nbq3pnbj

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the defining global health emergency of this century. GC-376 is a Mpro inhibitor with antiviral activity against SARS-CoV-2 in vitro. Using the K18-hACE2 mouse model, the in vivo antiviral efficacy of GC-376 against SARS-CoV-2 was evaluated. GC-376 treatment was not toxic in K18-hACE2 mice and produced milder tissue lesions, reduced viral loads, fewer presence of viral antigen, and reduced inflammation in comparison to vehicle-treated controls, most notably in the brain in mice challenged with a low virus dose. Although GC-376 was not sufficient to improve neither clinical symptoms nor survival, it did show a positive effect against SARS-CoV-2 in vivo. This study supports the notion that the K18-hACE2 mouse model is suitable to study antiviral therapies against SARS-CoV-2, and GC-376 represents a promising lead candidate for further development to treat SARS-CoV-2 infection.

The coronavirus disease pandemic caused by the Severe Acute 39

Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the defining global health crisis 40 of our time reaching over 45 million cases worldwide by October 2020 1 . SARS-CoV-2 41 belongs to the Coronaviruses family, which are RNA viruses commonly associated with 42 mild upper respiratory illness. In the past few years, novel coronaviruses have emerged 43 from animal reservoirs crossing the species barrier causing sporadic outbreaks in 44 humans such as SARS-CoV and the Middle Eastern Respiratory Syndrome (MERS). 45

The current global health emergency prompted a race to develop resources to combat 46 the pandemic. Several vaccines against SARS-CoV-2 are currently in different stages of 47 control group (Fig 1D) . In mice challenged with the high virus dose (G3 and G4), there 189 was a period of relatively normal activity (response to the environment and stimulus 190

given by the personnel) and physical appearance (Fig 2A and B ) followed by rapid 191 weight loss and presentation of clinical signs (Fig 2C) . By 6 dpc, mice in G3 (high virus 192 dose/vehicle, white symbols) showed about 20% weight loss and by 8 dpc there were 193 no survivors because they have either succumbed to the infection or had to be 194 humanely euthanized due to severe clinical signs (lethargy, ruffled fur, labored 195 breathing, and/or ≥25% body weight loss, Fig 2D) . These observations are consistent 196 with previous reports 20, 21, 24, 25 . G4 mice (high virus dose/GC-376) showed similar clinical 197 signs but such progression was delayed by about 24 h (~15% weight loss by 6 dpc). In 198 terms of body weight loss, statistically significant differences were established between 199 G3 and G4 from 4 to 6 dpc. Of note, the G4 mice (high virus dose/GC-376) showed 200 20% survival (n=1 out of 5) but such difference was not statistically significant. between 5 to 7 dpc in all mice regardless of group at the low virus challenge dose 208 compared to mock-challenged/vehicle treated mice, ultimately leading to 60% survival 209 ( Fig 2G and H) . These observations are significant because it suggests that disease 210 progression due to SARS-CoV-2 infection in the k18-hACE2 mouse model is virus dose 211 dependent adding more value to the system to better study SARS-CoV-2 pathogenesis 212 and host responses. Of note, a trend towards increased weight loss but faster body 213 weight recovery was observed in G6 mice (low virus dose/GC-376) compared to G5 214 mice (low virus dose/vehicle). However, and despite the 100 times lower virus challenge 215 dose, we did not notice an improvement in the clinical outcome of mice treated with GC-216 376 compared to mice in vehicle control group. Taken together, these results suggest 217 that GC-376 has modest benefit in K18-hACE2 mice infected with SARS-CoV-2 in 218 terms of clinical signs outcome, weight changes, and survival.

A trend towards reduced SARS-CoV-2 vRNA loads in GC-376-treated mice, 220 particularly in the brain. 221

To better characterize the effect of GC-376 treatment against SARS-CoV-2, 222 sections of NT, lungs, brain, liver, small intestine (SI) and spleen were collected at 2 223 and 5 dpc and each tissue split into two to assess vRNA viral loads and for 224 histopathological analyses, respectively. Tissue homogenates were prepared, vRNA 225 extracted and then vRNA loads and vRNA tissue distribution were evaluated by RT-226 qPCR. High vRNA content consistent with active virus replication was observed in 227 tissue homogenates from the NT, lungs, and brain of mice in G3 (high virus 228 dose/vehicle). RT-qPCR results were consistent with the clinical observations in G3 229 mice, with average higher vRNA signals at 2 dpc than at 5 dpc in samples from the 230 lungs and NT, but the opposite in other tissues (brain, liver, SI and spleen) indicative of 231 systemic virus spread ( Fig 3A) . Likewise, vRNA loads in G5 mice (low virus 232 dose/vehicle) followed disease progression with higher average signals on 5 dpc 233 compared to 2 dpc, particularly in the brain where these differences were statistically 234 significant ( Fig 3A) . Also consistent with the clinical outcome of the disease, reduced 235 vRNA load averages were observed in NT, lungs and brain samples from mice in G4 236 (high virus dose/GC-376) compared to homologous samples from mice in G3 (high virus 237 dose/vehicle). Samples from mice in G4 showed more individual variations in vRNA 238 loads, several at levels below limit of detection, than the counterparts in G3 (Fig 3B) . 239

Similarly, more individual variations in vRNA levels were observed in NT, lungs and 240 brain samples from G6 mice (low virus dose/GC-376) than in G5 mice (low virus 241 dose/vehicle) ( Fig 3C) . Interestingly, brain samples from G6 mice showed statistically 242 significant lower vRNA levels than those from G5 mice. Overall, these results suggest 243 high levels of SARS-CoV-2 replication in the NT, lungs, and brain of K18-hACE2 mice 244 and detectable vRNA in liver, SI and spleen. GC-376 treatment leads towards reduced 245 virus load averages with the caveat of noticeable individual variations among treated 246 animals, and the inability to properly prevent the burden and mortality caused by SARS-247

CoV-2 in K18-hACE2 mice. At the low dose virus challenge, GC-376 prevented to great 248 extent the virus' ability to reach the brain, but such effect was not correlated with 249

increased survival compared to vehicle-treated controls. 250

Histopathological analysis of lungs, NT, and brains at 2, 5, and 14 dpc (Table 1 ) 252

showed overall milder lesions in mice treated with GC-376 and challenged with the low 253 dose virus (Fig. 4) . At 2 dpc, no differences were observed in the two groups 254 challenged with the high dose (G3 and G4). Lesions at 2 dpc consisted of mild-255 moderate interstitial lymphoplasmacytic and histiocytic inflammation especially centered 256 around vessels and peribronchiolar spaces (Fig 4 A- in the G6 (low dose/GC-376) group presented brain lesions (Fig 4V) , as opposed to 299 mice in G5 (low dose/vehicle) which presented lesions similar to the high dose 300 challenge groups (Fig. 4W) . 301 Viral antigen in lungs was readily detected in lungs of G3 and G4 (high 302 dose/vehicle and high dose/GC376 respectively) with little variations in the distribution, 303 prevalence in the section and intensity of the staining (Fig 5, A-B ). In the case of G6 and 304 G5 (low dose/GC-376 and low dose/vehicle), viral antigen was present, but lower in G6 305 compared to G5 (Fig.5 C-D) . By 5 dpc, amount of viral antigen increased with no 306 appreciable difference between high dose or low dose challenge groups (Fig 5 F-I) . In 307 lungs, viral antigen staining was observed within the cytoplasm of pneumocytes type I, 308

II, and alveolar macrophages. (Fig 3C) . 328 IHC using anti-CD3 or anti-Iba-1 antibodies was performed to better characterize 329 the type and extent of the cellular inflammatory infiltrate, T lymphocytes and cells of the 330 monocyte/macrophage lineage, respectively (Table 2) . No significant differences in 331 numbers of CD3+ cells in any organ were noticed between vehicle-treated mice or GC-332 376 treated mice challenged with the high dose SARS-CoV-2 at the given timepoints 333 (Fig 6, panels 1-2, 6-7, 11-12, 16-17) . On the contrary, a clearly lower number of CD3+ 334 cells were observed at both 2 and 5 dpc in the lungs, NT and brain of G6 mice (low 335 virus dose/GC-376) compared to G5 mice (low virus dose/vehicle) (Fig. 6, panels 3-4, 8-336   9, 13-14, 18-19 ). This suggests a potential implication of GC-376 in preventing more 337 severe inflammation and virus-induced tissue damage (Fig 6; Table 2 ). Similar results 338

were observed with Iba-1 IHC, showing similar staining patterns in the organs from the 339 high dose challenge groups but lower staining overall in G6 (low dose/GC-376) when 340 compared against its control G5 (low dose/vehicle) (Fig 6, panels 21-40) . Findings were 341 overall consistent with the extent of inflammatory reaction and lesions observed by HE.

Taken together, these results suggest that GC-376 is able to prevent to some extent the The toxicity of GC-376 (7-day course at 40 mg/kg/day) was evaluated through 402 changes in body weight and survival (Fig 2) and histopathological changes in different 403 tissues at 7 days post-treatment (14 dpc). These analyses indicated lack of obvious acute toxicity of GC-376 in K18-hACE-2 mice. Although previous studies in cats showed 405 no GC-376 associated toxicity those were performed using lower doses than in our 406

Balb/c mice no specific toxicity evaluations were performed 6,7,23 . 408

We observed a modest although clearly beneficial effect of GC-376 against Mice were humanely euthanized at 14 dpc and lungs, brain, liver, spleen, heart and SI 594 were collected. HE slides were generated and analyzed in comparison with the negative 595 control group. Representative pictures were taken at 20X magnification for all tissues 596 except brain samples that were taken at 4X. 597 

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We thank the personnel from the Animal Health Research Center and the 468