key: cord-258914-g6pv8zz9 authors: Proud, Pamela C.; Tsitoura, Daphne; Watson, Robert J.; Chua, Brendon Y; Aram, Marilyn J.; Bewley, Kevin R.; Cavell, Breeze E.; Cobb, Rebecca; Dowall, Stuart; Fotheringham, Susan A.; Ho, Catherine M. K.; Lucas, Vanessa; Ngabo, Didier; Rayner, Emma; Ryan, Kathryn A.; Slack, Gillian S.; Thomas, Stephen; Wand, Nadina I.; Yeates, Paul; Demaison, Christophe; Jackson, David C.; Bartlett, Nathan W.; Mercuri, Francesca; Carroll, Miles W. title: Prophylactic intranasal administration of a TLR2 agonist reduces upper respiratory tract viral shedding in a SARS-CoV-2 challenge ferret model date: 2020-09-25 journal: bioRxiv DOI: 10.1101/2020.09.25.309914 sha: doc_id: 258914 cord_uid: g6pv8zz9 Respiratory viruses such as coronaviruses represent major ongoing global threats, causing epidemics and pandemics with huge economic burden. Rapid spread of virus through populations poses an enormous challenge for outbreak control. Like all respiratory viruses, the most recent novel human coronavirus SARS-CoV-2, initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT to reduce SARS-CoV-2 transmission and provide protection against COVID-19. As with other respiratory CoVs, SARS-CoV-2 primarily spreads via the airborne route, 86 with respiratory droplets expelled by infected individuals 6 . Virus can be transmitted 87 from symptomatic, as well as pre-or asymptomatic individuals 7,8 , with asymptomatic 88 individuals being able to shed virus, and therefore being capable to transmit the disease, 89 for longer than those with symptoms 9 . As with other respiratory viruses such as influenza, 90 recent evidence suggests that, the epithelium of the upper respiratory tract (URT) is the 91 initial site of SARS-CoV-2 infection 10, 11 . This is consistent with the abundant nasal 92 epithelial cell expression of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 93 (ACE2) and its decreasing expression throughout the lower respiratory tract 11 . 94 A topical treatment of the URT that boosts anti-viral immunity and restricts viral replication 95 is a promising method to promote viral clearance, reduce viral shedding and 96 transmission. The TLRs are key microbe-recognition receptors with a crucial role in 97 activation of host defence and protection from infections and therefore attractive drug 98 targets against infectious diseases [12] [13] [14] To determine whether TLR2/6 agonists are also active against SARS-CoV-2, we used In life samples were taken at days 1, 3, 5, 7, 10 and 12, with scheduled culls at days 137 3 (n=6) and end of study days 12-14 (n=18) (Fig 1A) . comparison test, significant (>10 fold) reduction in nasal viral RNA was observed at 5 186 dpc (p=0.0057) and highly significant (p<0.0001), greater than 10-fold reduction in 187 throat viral RNA was apparent from 5 to 7 dpc following INNA-051 i.n. treatment 188 ( Figure S1 ). Group 2 (20ug/mL) appears to be the most optimal dosing in this study To assess SARS-CoV-2 detected beyond the URT, lung tissue samples were 196 collected, on scheduled cull day 3 (6/24 animals) and days 12-14 (18/24 animals) dpc 197 and analysed for viral RNA levels. On day 3 dpc, two culled ferrets from the control 198 vehicle group had detectable viral RNA levels (7.42x10 4 and 2.86x10 4 copies/ml) (Fig 199 2C ). There was one ferret in Group 1 showing detectable, but below the quantifiable Access to food and water was ad libitum and environmental enrichment was provided. Ferrets were anaesthetised with ketamine/xylazine (17.9 mg/kg and 3.6 mg/kg 401 bodyweight) and exsanguination was effected via cardiac puncture, followed by 402 injection of an anaesthetic overdose (sodium pentabarbitone Dolelethal, Vetquinol UK 403 Ltd, 140 mg/kg). A necropsy was performed immediately after confirmation of death. The left lung was dissected and used for subsequent virology procedures. x 10 6 pfu/ml SARS-CoV-2. Nasal wash and throat swabs were collected at days 1, 3, 586 5, 7, 10 & 12 post challenge (p.c.) for all treatment groups and control group. Scheduled culls were performed for 6/24 ferrets on day 3 p.c. and 18/24 ferrets on Origin and evolution of pathogenic coronaviruses Evolutionary history, potential intermediate animal host, and cross-462 species analyses of SARS-CoV-2 Epidemiology and cause of severe acute respiratory 465 syndrome (SARS) in Guangdong, People's Republic of China Isolation of a Novel Coronavirus from a Man with Pneumonia 470 in Saudi Arabia Genomic characterisation and epidemiology of 2019 novel 473 coronavirus: implications for virus origins and receptor binding The trinity of 476 COVID-19: immunity, inflammation and intervention Clinical characteristics of 24 asymptomatic infections with COVID-479 19 screened among close contacts in Nanjing, China. Science China. 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Expert opinion 560 on biological therapy ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-562 2 pneumonia in rhesus macaques. bioRxiv TLR1-and TLR6-independent recognition of 565 bacterial lipopeptides Transloading of tumor antigen-derived peptides into antigen-568 presenting cells The synthetic bacterial lipopeptide Pam3CSK4 modulates 571 respiratory syncytial virus infection independent of TLR activation Isolation and rapid sharing of the 2019 novel coronavirus CoV-2) from the first patient diagnosed with COVID-19 in Australia. The Medical 575 journal of Australia Metagenomic Nanopore Sequencing of Influenza Virus 577 Direct from Clinical Respiratory Samples Viral RNA was quantified by RT-qPCR. 600 (a) Nasal wash (b) Throat swab (c) Lung tissue. Geometric mean +/-standard 601 deviation are displayed on the graphs. Dashed horizontal lines denote the lower limit 602 of quantification (LLOQ) and lower limit of detection (LLOD) Dunnett's multiple comparisons test are displayed above the error bars (*)