There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5–15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.

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 assess 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. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe 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 preferred 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 interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

41In December 2019 an outbreak of coronavirus disease emerged in 42 Wuhan, China. The causative agent was subsequently identified and named severe 43 acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread 44 worldwide causing a pandemic. Currently there are no licensed vaccines or 45 therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in 46 development and repurposed drugs are being tested in the clinic. There is a vital need 47 for authentic COVID-19 animal models to further our understanding of pathogenesis 48 and viral spread in addition to pre-clinical evaluation of candidate interventions. 49 50Here we report a dose titration study of SARS-CoV-2 to determine the most suitable 51 infectious dose to use in the ferret model. We show that a high (5x10 6 pfu) and medium 52 (5x10 4 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral 53 RNA shedding in both groups of six challenged animals, whilst a low dose (5x10 2 pfu) 54 resulted in only one of six displaying signs of URT viral RNA replication. The URT 55 shedding lasted up to 21 days in the high dose animals with intermittent positive signal 56 from day 14. Sequential culls revealed distinct pathological signs of mild multifocal 57 bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and 58 medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 59 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were 60 observed although animals did present with a consistent post-viral fatigue lasting from 61 day 9-14 in the medium and high dose groups. After virus shedding ceased, re-62 challenged ferrets were shown to be fully protected from acute lung pathology. The 63Page 4 of 39 endpoints of URT viral RNA replication in addition to distinct lung pathology and post 64 viral fatigue were observed most consistently in the high dose group. This ferret model 65 of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of 66 patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 67 14-21 parallel observations reported in a minority of clinical cases. 68 69 70 71 Word count: 327 72 Introduction 73 74 Coronaviruses are positive sense, single stranded RNA viruses belonging to the family 75 Coronaviridae 1 . These viruses can infect a range of animals, including humans and 76 usually cause a mild respiratory infection, much like the common cold. Two highly 77 pathogenic coronaviruses have emerged in the human population in the last 20 years; 78 severe acute respiratory syndrome (SARS) CoV and middle eastern respiratory 79 syndrome (MERS) CoV. SARS-CoV infected approximately 8,000 people worldwide with 80 a case fatality rate (CFR) of 10%, while MERS-CoV has infected approximately 2,500 81 people with a CFR of 36% 2 .82 83 In December 2019 several pneumonia cases of unknown cause emerged in Wuhan, 84 Hubei, China. Deep sequencing analysis from lower respiratory tract samples from ...

Ferrets were experimentally inoculated with SARS-CoV-2 (severe acute respiratory syndrome (SARS)-related coronavirus 2) to assess infection dynamics and host response. During the resulting subclinical infection, viral RNA was monitored between 2 and 21 days post-inoculation (dpi), and reached a peak in the upper respiratory cavity between 4 and 6 dpi. Viral genomic sequence analysis in samples from three animals identified the Y453F nucleotide substitution relative to the inoculum. Viral RNA was also detected in environmental samples, specifically in swabs of ferret fur. Microscopy analysis revealed viral protein and RNA in upper respiratory tract tissues, notably in cells of the respiratory and olfactory mucosae of the nasal turbinates, including olfactory neuronal cells. Antibody responses to the spike and nucleoprotein were detected from 21 dpi, but virus-neutralizing activity was low. A second intranasal inoculation (re-exposure) of two ferrets after a 17-day interval did not produce re-initiation of viral RNA shedding, but did amplify the humoral response in one animal. Therefore, ferrets can be experimentally infected with SARS-CoV-2 to model human asymptomatic infection.

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The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.

Background The novel human coronavirus SARS-CoV-2 is a major ongoing global threat with huge economic burden. Like all respiratory viruses, 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. Methods SARS-CoV-2 Victoria/01/2020 was passaged in Vero/hSLAM cells and virus titre determined by plaque assay. Challenge virus was delivered by intranasal instillation to female ferrets at 5.0 × 10 6 pfu/ml. Treatment groups received intranasal INNA-051, developed by Ena Respiratory. SARS-CoV-2 RNA was detected using the 2019-nCoV CDC RUO Kit and QuantStudio™ 7 Flex Real-Time PCR System. Histopathological analysis was performed using cut tissues stained with haematoxylin and eosin (H&E). Findings 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. After 5 days post-exposure to SARS-CoV-2, INNA-051 significantly reduced virus in throat swabs ( p =<0.0001) by up to a 24 fold (96% reduction) and in nasal wash ( p =0.0107) up to a 15 fold (93% reduction) in comparison to untreated animals. Interpretation 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. Funding This work was funded by Ena Respiratory, Melbourne, Australia.

The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic necessitates the fast development of vaccines to meet a worldwide need. mRNA-based vaccines are the most promising technology for rapid and safe SARS-CoV-2 vaccine development and production. We have designed CVnCoV, a lipid-nanoparticle (LNP) encapsulated, sequence optimised mRNA-based SARS-CoV-2 vaccine that encodes for full length, pre-fusion stabilised Spike protein. Unlike other mRNA-based approaches, CVnCoV exclusively consists of non-chemically modified nucleotides and can be applied at comparatively low doses. Here we demonstrate that CVnCoV induces robust humoral and cellular responses in non-human primates (NHPs). Animals vaccinated with 8 μg of CVnCoV were protected from challenge infection with SARS-CoV-2. Comprehensive analyses of pathological changes in challenged animals via lung histopathology and Computed Tomography (CT) scans gave no indication of enhanced disease upon CVnCoV vaccination. These results demonstrate safety, immunogenicity, and protective efficacy of CVnCoV in NHPs that extend our previously published preclinical data and provide strong support for further clinical testing in ongoing phase 2b/3 efficacy studies.