key: cord-338543-q6cl5kjp
authors: Salguero, Francisco J.; White, Andrew D.; Slack, Gillian S.; Fotheringham, Susan A.; Bewley, Kevin R.; Gooch, Karen E.; Longet, Stephanie; Humphries, Holly E.; Watson, Robert J.; Hunter, Laura; Ryan, Kathryn A.; Hall, Yper; Sibley, Laura; Sarfas, Charlotte; Allen, Lauren; Aram, Marilyn; Brunt, Emily; Brown, Phillip; Buttigieg, Karen R.; Cavell, Breeze E.; Cobb, Rebecca; Coombes, Naomi S.; Daykin-Pont, Owen; Elmore, Michael J.; Gkolfinos, Konstantinos; Godwin, Kerry J.; Gouriet, Jade; Halkerston, Rachel; Harris, Debbie J.; Hender, Thomas; Ho, Catherine M.K.; Kennard, Chelsea L.; Knott, Daniel; Leung, Stephanie; Lucas, Vanessa; Mabbutt, Adam; Morrison, Alexandra L.; Ngabo, Didier; Paterson, Jemma; Penn, Elizabeth J.; Pullan, Steve; Taylor, Irene; Tipton, Tom; Thomas, Stephen; Tree, Julia A.; Turner, Carrie; Wand, Nadina; Wiblin, Nathan R.; Charlton, Sue; Hallis, Bassam; Pearson, Geoffrey; Rayner, Emma L.; Nicholson, Andrew G.; Funnell, Simon G.; Dennis, Mike J.; Gleeson, Fergus V.; Sharpe, Sally; Carroll, Miles W.
title: Comparison of Rhesus and Cynomolgus macaques as an authentic model for COVID-19
date: 2020-09-17
journal: bioRxiv
DOI: 10.1101/2020.09.17.301093
sha: 
doc_id: 338543
cord_uid: q6cl5kjp

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques, resembling the mild clinical cases of COVID-19 in humans. Immune responses against SARS-CoV-2 were also similar in both species and equivalent to those reported in milder infections and convalescent human patients. Importantly, we have devised a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the optimal study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of novel and repurposed interventions against SARS-CoV-2. Accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

A novel acute respiratory syndrome, now called Coronavirus disease-19 (COVID-19) 51 was first reported in Wuhan, China in December 2019. The genetic sequence of the 52 causative agent was found to have similarity with two highly pathogenic respiratory 53 beta Coronaviruses, SARS 1 and MERS 2 , and was later called SARS-CoV-2 3 . It has 54 currently infected >21 million individuals resulting in >750,000 deaths 4 . Among the 55 clinical and pathological signs of SARS-CoV-2 infection in humans, pneumonia 56 accompanied by respiratory distress seem to be the most clinically relevant 5,6 . (Figure 2 ). High levels of viral dpc before falling and remaining below the assay's lower limit of detection (LLOD) 146 from eleven dpc to 18 dpc. Throat swabs from cynomolgus macaques contained 147 higher levels of viral RNA early in infection (one to three dpc) and remained ≥4.5 x 148 10 4 copies/ml for all animals between four and nine dpc. Virus shedding from the gastrointestinal tract was assessed by RT-qPCR performed 156 on rectal swab samples. In rhesus macaques, low levels of viral RNA were detected 157 from one dpc to nine dpc. In cynomolgus macaques, viral RNA was similarly 158 detected at a low level in rectal swabs from one dpc to nine dpc. However viral RNA 159 levels above the LLOQ were detected at both three dpc and five dpc in cynomolgus 160 macaques in comparison to two dpc and three dpc in rhesus macaques ( Figure 2D ). 161 Viral RNA was detected at only two timepoints after challenge in whole blood 162 samples and remained below the LLOQ throughout the study ( Figure 2E ). In rhesus 163 macaques, viral RNA was detected in one animal at three dpc, whilst in cynomolgus 164 macaques, viral RNA was detected in two animals at six dpc. were seen ( Figure 3I ). Mononuclear cells, primarily lymphocytes also were noted 238 surrounding and infiltrating the walls of blood vessels and airways ( Figure 3J ). An 239 increased prominence of bronchial-associated lymphoid tissue (BALT) was noted. In 240 the lungs of rhesus macaques, changes in the alveoli and BALT were similar in 241 appearance and frequency to those described in the cynomolgus macaques, and 242 perivascular lymphocytic cuffing of small vessels, characterised by concentric 243 infiltrates of mononuclear cells, was also seen occasionally ( Figure 3K and 3L). In summary, using the histopathology scoring system developed here, the scores 251 were higher in both macaque species at 4/5 dpc compared to 14/15 and 18/19dpc, 252 mostly due to higher scores in the alveolar damage parameters observed at the early 253 time point ( Figure 4A ). In the liver, microvesicular, centrilobular vacuolation, consistent with glycogen, 267 together with, small, random, foci of lymphoplasmacytic cell infiltration were noted 268 rarely (data not shown). This is considered to represent a mild, frequently observed In rhesus macaques, the IFN-γ SFU measured following stimulation with spike 300 protein peptide megapools (MP) 1-3 did not differ significantly between animals 301 euthanised at either the day 4-5 (early) or the day 14-19 (late) post-infection time-302 point in comparison to SFU frequencies measured in the naïve control animals.

However, comparison of the summed MP 1-3-specific response indicated that 304 significantly higher SFU frequencies were present in the animals euthanised at the In general, there was a trend for spike protein peptide-specific IFN-γ SFU 311 frequencies measured in PBMC samples collected from cynomolgus macaques to 312 be greater than those detected in rhesus macaques, although these differences did 313 not reach statistical significance. 

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MNCs. Stacked bars show the group median with 95% confidence intervals. PBMC: 1048 Naïve rhesus n=8, early rhesus n= 1, late rhesus n=2, naïve cyno = 7, early cyno 1049 n=2, late cyno n=2. Lung: early rhesus n= 2, late rhesus n=3, early cyno n=2, late