A Time-Series Metabolomic Analysis of SARS-CoV-2 Infection in a Ferret Model.

The global threat of COVID-19 has led to an increased use of metabolomics to study SARS-CoV-2 infections in animals and humans. In spite of these efforts, however, understanding the metabolome of SARS-CoV-2 during an infection remains difficult and incomplete. In this study, metabolic responses to a SAS-CoV-2 challenge experiment were studied in nasal washes collected from an asymptomatic ferret model (n = 20) at different time points before and after infection using an LC-MS-based metabolomics approach. A multivariate analysis of the nasal wash metabolome data revealed several statistically significant features. Despite no effects of sex or interaction between sex and time on the time course of SARS-CoV-2 infection, 16 metabolites were significantly different at all time points post-infection. Among these altered metabolites, the relative abundance of taurine was elevated post-infection, which could be an indication of hepatotoxicity, while the accumulation of sialic acids could indicate SARS-CoV-2 invasion. Enrichment analysis identified several pathways influenced by SARS-CoV-2 infection. Of these, sugar, glycan, and amino acid metabolisms were the key altered pathways in the upper respiratory channel during infection. These findings provide some new insights into the progression of SARS-CoV-2 infection in ferrets at the metabolic level, which could be useful for the development of early clinical diagnosis tools and new or repurposed drug therapies.

CoviRx: A User-Friendly Interface for Systematic Down-Selection of Repurposed Drug Candidates for COVID-19

Although various vaccines are now commercially available, they have not been able to stop the spread of COVID-19 infection completely. An excellent strategy to get safe, effective, and affordable COVID-19 treatments quickly is to repurpose drugs that are already approved for other diseases. The process of developing an accurate and standardized drug repurposing dataset requires considerable resources and expertise due to numerous commercially available drugs that could be potentially used to address the SARS-CoV-2 infection. To address this bottleneck, we created the CoviRx.org platform. CoviRx is a user-friendly interface that allows analysis and filtering of large quantities of data, which is onerous to curate manually for COVID-19 drug repurposing. Through CoviRx, the curated data have been made open source to help combat the ongoing pandemic and encourage users to submit their findings on the drugs they have evaluated, in a uniform format that can be validated and checked for integrity by authenticated volunteers. This article discusses the various features of CoviRx, its design principles, and how its functionality is independent of the data it displays. Thus, in the future, this platform can be extended to include any other disease beyond COVID-19.

Use of Human Lung Tissue Models for Screening of Drugs against SARS-CoV-2 Infection.

The repurposing of licenced drugs for use against COVID-19 is one of the most rapid ways to develop new and alternative therapeutic options to manage the ongoing pandemic. Given circa 7817 licenced compounds available from Compounds Australia that can be screened, this paper demonstrates the utility of commercially available ex vivo/3D airway and alveolar tissue models. These models are a closer representation of in vivo studies than in vitro models, but retain the benefits of rapid in vitro screening for drug efficacy. We demonstrate that several existing drugs appear to show anti-SARS-CoV-2 activity against both SARS-CoV-2 Delta and Omicron Variants of Concern in the airway model. In particular, fluvoxamine, as well as aprepitant, everolimus, and sirolimus, has virus reduction efficacy comparable to the current standard of care (remdesivir, molnupiravir, nirmatrelvir). Whilst these results are encouraging, further testing and efficacy studies are required before clinical use can be considered.

Systematic Down-Selection of Repurposed Drug Candidates for COVID-19.

SARS-CoV-2 is the cause of the COVID-19 pandemic which has claimed more than 6.5 million lives worldwide, devastating the economy and overwhelming healthcare systems globally. The development of new drug molecules and vaccines has played a critical role in managing the pandemic; however, new variants of concern still pose a significant threat as the current vaccines cannot prevent all infections. This situation calls for the collaboration of biomedical scientists and healthcare workers across the world. Repurposing approved drugs is an effective way of fast-tracking new treatments for recently emerged diseases. To this end, we have assembled and curated a database consisting of 7817 compounds from the Compounds Australia Open Drug collection. We developed a set of eight filters based on indicators of efficacy and safety that were applied sequentially to down-select drugs that showed promise for drug repurposing efforts against SARS-CoV-2. Considerable effort was made to evaluate approximately 14,000 assay data points for SARS-CoV-2 FDA/TGA-approved drugs and provide an average activity score for 3539 compounds. The filtering process identified 12 FDA-approved molecules with established safety profiles that have plausible mechanisms for treating COVID-19 disease. The methodology developed in our study provides a template for prioritising drug candidates that can be repurposed for the safe, efficacious, and cost-effective treatment of COVID-19, long COVID, or any other future disease. We present our database in an easy-to-use interactive interface (CoviRx that was also developed to enable the scientific community to access to the data of over 7000 potential drugs and to implement alternative prioritisation and down-selection strategies.

Tracking Co-Occurrence of N501Y, P681R, and Other Key Mutations in SARS-CoV-2 Spike for Surveillance

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has produced five variants of concern (VOC) to date. The important spike mutation 'N501Y' is common to Alpha, Beta, Gamma, and Omicron VOC, while the 'P681R' is key to Delta's spread. We have analysed circa 10 million SARS-CoV-2 genome sequences from the world's largest repository, 'Global Initiative on Sharing All Influenza Data (GISAID)', and demonstrated that these two mutations have co-occurred on the spike 'D614G' mutation background at least 5767 times from 12 May 2020 to 28 April 2022. In contrast, the Y501-H681 combination, which is common to Alpha and Omicron VOC, is present in circa 1.1 million entries. Over half of the 5767 co-occurrences were in France, Turkey, or US (East Coast), and the rest across 88 other countries;36.1%, 3.9%, and 4.1% of the co-occurrences were Alpha's Q.4, Gamma's P.1.8, and Omicron's BA.1.1 sub-lineages acquiring the P681R;4.6% and 3.0% were Delta's AY.5.7 sub-lineage and B.1.617.2 lineage acquiring the N501Y;the remaining 8.2% were in other variants. Despite the selective advantages individually conferred by N501Y and P681R, the Y501-R681 combination counterintuitively did not outcompete other variants in every instance we have examined. While this is a relief to worldwide public health efforts, in vitro and in vivo studies are urgently required in the absence of a strong in silico explanation for this phenomenon. This study demonstrates a pipeline to analyse combinations of key mutations from public domain information in a systematic manner and provide early warnings of spread. The study here demonstrates the usage of the pipeline using the key mutations N501Y, P681R, and D614G of SARS-CoV-2.

Characterisation and natural progression of SARS-CoV-2 infection in ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the infectious disease COVID-19, which has rapidly become an international pandemic with significant impact on healthcare systems and the global economy. To assist antiviral therapy and vaccine development efforts, we performed a natural history/time course study of SARS-CoV-2 infection in ferrets to characterise and assess the suitability of this animal model. Ten ferrets of each sex were challenged intranasally with 4.64 × 104 TCID50 of SARS-CoV-2 isolate Australia/VIC01/2020 and monitored for clinical disease signs, viral shedding, and tissues collected post-mortem for histopathological and virological assessment at set intervals. We found that SARS-CoV-2 replicated in the upper respiratory tract of ferrets with consistent viral shedding in nasal wash samples and oral swab samples up until day 9. Infectious SARS-CoV-2 was recovered from nasal washes, oral swabs, nasal turbinates, pharynx, and olfactory bulb samples within 3-7 days post-challenge; however, only viral RNA was detected by qRT-PCR in samples collected from the trachea, lung, and parts of the gastrointestinal tract. Viral antigen was seen exclusively in nasal epithelium and associated sloughed cells and draining lymph nodes upon immunohistochemical staining. Due to the absence of clinical signs after viral challenge, our ferret model is appropriate for studying asymptomatic SARS-CoV-2 infections and most suitable for use in vaccine efficacy studies.

At Least Three Doses of Leading Vaccines Essential for Neutralisation of SARS-CoV-2 Omicron Variant.

Plasma samples taken at different time points from donors who received either AstraZeneca (Vaxzevria) or Pfizer (Comirnaty) or Moderna (Spikevax) coronavirus disease-19 (COVID-19) vaccine were assessed in virus neutralization assays against Delta and Omicron variants of concern and a reference isolate (VIC31). With the Pfizer vaccine there was 6-8-fold reduction in 50% neutralizing antibody titres (NT50) against Delta and VIC31 at 6 months compared to 2 weeks after the second dose; followed by 25-fold increase at 2 weeks after the third dose. Neutralisation of Omicron was only consistently observed 2 weeks after the third dose, with most samples having titres below the limit of detection at earlier timepoints. Moderna results were similar to Pfizer at 2 weeks after the second dose, while the titres for AstraZeneca samples derived from older donors were 7-fold lower against VIC31 and below the limit of detection against Delta and Omicron. Age and gender were not found to significantly impact our results. These findings indicate that vaccine matching may be needed, and that at least a third dose of these vaccines is necessary to generate sufficient neutralising antibodies against emerging variants of concern, especially Omicron, amidst the challenges of ensuring vaccine equity worldwide.

Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies against Delta and Omicron in Mice.

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We previously developed highly thermo-tolerant monomeric and trimeric receptor-binding domain derivatives that can withstand 100 °C for 90 min and 37 °C for four weeks and help eliminate cold-chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations and a 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for upcoming Phase I human clinical trials and that there is potential for increasing the efficacy with vaccine matching to improve the responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions, which show that, while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.

Advances and gaps in SARS-CoV-2 infection models.

The global response to Coronavirus Disease 2019 (COVID-19) is now facing new challenges such as vaccine inequity and the emergence of SARS-CoV-2 variants of concern (VOCs). Preclinical models of disease, in particular animal models, are essential to investigate VOC pathogenesis, vaccine correlates of protection and postexposure therapies. Here, we provide an update from the World Health Organization (WHO) COVID-19 modeling expert group (WHO-COM) assembled by WHO, regarding advances in preclinical models. In particular, we discuss how animal model research is playing a key role to evaluate VOC virulence, transmission and immune escape, and how animal models are being refined to recapitulate COVID-19 demographic variables such as comorbidities and age.

"But Mouse, You Are Not Alone": On Some Severe Acute Respiratory Syndrome Coronavirus 2 Variants Infecting Mice.

In silico predictions combined with in vitro, in vivo, and in situ observations collectively suggest that mouse adaptation of the severe acute respiratory syndrome 2 virus requires an aromatic substitution in position 501 or position 498 (but not both) of the spike protein's receptor binding domain. This effect could be enhanced by mutations in positions 417, 484, and 493 (especially K417N, E484K, Q493K, and Q493R), and to a lesser extent by mutations in positions 486 and 499 (such as F486L and P499T). Such enhancements, due to more favorable binding interactions with residues on the complementary angiotensin-converting enzyme 2 interface, are, however, unlikely to sustain mouse infectivity on their own based on theoretical and experimental evidence to date. Our current understanding thus points to the Alpha, Beta, Gamma, and Omicron variants of concern infecting mice, whereas Delta and "Delta Plus" lack a similar biomolecular basis to do so. This paper identifies 11 countries (Brazil, Chile, Djibouti, Haiti, Malawi, Mozambique, Reunion, Suriname, Trinidad and Tobago, Uruguay, and Venezuela) where targeted local field surveillance of mice is encouraged because they may have come in contact with humans who had the virus with adaptive mutation(s). It also provides a systematic methodology to analyze the potential for other animal reservoirs and their likely locations.

A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern.

Saturation suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temperature of ~7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An additional derivative with three additional mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temperature exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 derivative showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qualitatively improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or additional components, and can be stored at room temperature. They are a useful modality to combat COVID-19, especially in remote and low-resource settings.

Altered microRNA expression in COVID-19 patients enables identification of SARS-CoV-2 infection.

The host response to SARS-CoV-2 infection provide insights into both viral pathogenesis and patient management. The host-encoded microRNA (miRNA) response to SARS-CoV-2 infection, however, remains poorly defined. Here we profiled circulating miRNAs from ten COVID-19 patients sampled longitudinally and ten age and gender matched healthy donors. We observed 55 miRNAs that were altered in COVID-19 patients during early-stage disease, with the inflammatory miR-31-5p the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-423-5p, miR-23a-3p and miR-195-5p) independently classified COVID-19 cases with an accuracy of 99.9%. In a ferret COVID-19 model, the three-miRNA signature again detected SARS-CoV-2 infection with 99.7% accuracy, and distinguished SARS-CoV-2 infection from influenza A (H1N1) infection and healthy controls with 95% accuracy. Distinct miRNA profiles were also observed in COVID-19 patients requiring oxygenation. This study demonstrates that SARS-CoV-2 infection induces a robust host miRNA response that could improve COVID-19 detection and patient management.

Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric SARS-CoV-2 Receptor Binding Domain Derivative.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80-100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Live Virus Neutralisation of the 501Y.V1 and 501Y.V2 SARS-CoV-2 Variants following INO-4800 Vaccination of Ferrets.

The ongoing COVID-19 pandemic has resulted in significant global morbidity and mortality on a scale similar to the influenza pandemic of 1918. Over the course of the last few months, a number of SARS-CoV-2 variants have been identified against which vaccine-induced immune responses may be less effective. These "variants-of-concern" have garnered significant attention in the media, with discussion around their impact on the future of the pandemic and the ability of leading COVID-19 vaccines to protect against them effectively. To address concerns about emerging SARS-CoV-2 variants affecting vaccine-induced immunity, we investigated the neutralisation of representative 'G614', '501Y.V1' and '501Y.V2' virus isolates using sera from ferrets that had received prime-boost doses of the DNA vaccine, INO-4800. Neutralisation titres against G614 and 501Y.V1 were comparable, but titres against the 501Y.V2 variant were approximately 4-fold lower, similar to results reported with other nucleic acid vaccines and supported by in silico biomolecular modelling. The results confirm that the vaccine-induced neutralising antibodies generated by INO-4800 remain effective against current variants-of-concern, albeit with lower neutralisation titres against 501Y.V2 similar to other leading nucleic acid-based vaccines.

Type I Hypersensitivity in Ferrets Following Exposure to SARS-CoV-2 Inoculum: Lessons Learned.

This case report discusses Type I hypersensitivity in ferrets following exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inoculum, observed during a study investigating the efficacy of candidate COVID-19 vaccines. Following a comprehensive internal root-cause investigation, it was hypothesized that prior prime-boost immunization of ferrets with a commercial canine C3 vaccine to protect against the canine distemper virus had resulted in primary immune response to fetal bovine serum (FBS) in the C3 preparation. Upon intranasal exposure to SARS-CoV-2 virus cultured in medium containing FBS, an allergic airway response occurred in 6 out of 56 of the ferrets. The 6 impacted ferrets were randomly dispersed across study groups, including different COVID-19 vaccine candidates, routes of vaccine candidate administration, and controls (placebo). The root-cause investigation and subsequent analysis determined that the allergic reaction was unrelated to the COVID-19 vaccine candidates under evaluation. Histological assessment suggested that the allergic response was characterized by eosinophilic airway disease; increased serum immunoglobulin levels reactive to FBS further suggested this response was caused by immune priming to FBS present in the C3 vaccine. This was further supported by in vivo studies demonstrating ferrets administered diluted FBS also presented clinical signs consistent with a hyperallergic response, while clinical signs were absent in ferrets that received a serum-free SARS-CoV-2 inoculum. It is therefore recommended that vaccine studies in higher order animals should consider the impact of welfare vaccination and use serum-free inoculum whenever possible.

Metabolic Profiling from an Asymptomatic Ferret Model of SARS-CoV-2 Infection.

Coronavirus disease (COVID-19) is a contagious respiratory disease that is causing significant global morbidity and mortality. Understanding the impact of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection on the host metabolism is still in its infancy but of great importance. Herein, we investigated the metabolic response during viral shedding and post-shedding in an asymptomatic SARS-CoV-2 ferret model (n = 6) challenged with two SARS-CoV-2 isolates. Virological and metabolic analyses were performed on (minimally invasive) collected oral swabs, rectal swabs, and nasal washes. Fragments of SARS-CoV-2 RNA were only found in the nasal wash samples in four of the six ferrets, and in the samples collected 3 to 9 days post-infection (referred to as viral shedding). Central carbon metabolism metabolites were analyzed during viral shedding and post-shedding periods using a dynamic Multiple Reaction Monitoring (dMRM) database and method. Subsequent untargeted metabolomics and lipidomics of the same samples were performed using a Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS) methodology, building upon the identified differentiated central carbon metabolism metabolites. Multivariate analysis of the acquired data identified 29 significant metabolites and three lipids that were subjected to pathway enrichment and impact analysis. The presence of viral shedding coincided with the challenge dose administered and significant changes in the citric acid cycle, purine metabolism, and pentose phosphate pathways, amongst others, in the host nasal wash samples. An elevated immune response in the host was also observed between the two isolates studied. These results support other metabolomic-based findings in clinical observational studies and indicate the utility of metabolomics applied to ferrets for further COVID-19 research that advances early diagnosis of asymptomatic and mild clinical COVID-19 infections, in addition to assessing the effectiveness of new or repurposed drug therapies.

In vitro characterisation of SARS-CoV-2 and susceptibility of domestic ferrets (Mustela putorius furo).

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging virus that has caused significant human morbidity and mortality since its detection in late 2019. With the rapid emergence has come an unprecedented programme of vaccine development with at least 300 candidates under development. Ferrets have proven to be an appropriate animal model for testing safety and efficacy of SARS-CoV-2 vaccines due to quantifiable virus shedding in nasal washes and oral swabs. Here, we outline our efforts early in the SARS-CoV-2 outbreak to propagate and characterize an Australian isolate of the virus in vitro and in an ex vivo model of human airway epithelium, as well as to demonstrate the susceptibility of domestic ferrets (Mustela putorius furo) to SARS-CoV-2 infection following intranasal challenge.

Interoperable medical data: The missing link for understanding COVID-19.

Being able to link clinical outcomes to SARS-CoV-2 virus strains is a critical component of understanding COVID-19. Here, we discuss how current processes hamper sustainable data collection to enable meaningful analysis and insights. Following the 'Fast Healthcare Interoperable Resource' (FHIR) implementation guide, we introduce an ontology-based standard questionnaire to overcome these shortcomings and describe patient 'journeys' in coordination with the World Health Organization's recommendations. We identify steps in the clinical health data acquisition cycle and workflows that likely have the biggest impact in the data-driven understanding of this virus. Specifically, we recommend detailed symptoms and medical history using the FHIR standards. We have taken the first steps towards this by making patient status mandatory in GISAID ('Global Initiative on Sharing All Influenza Data'), immediately resulting in a measurable increase in the fraction of cases with useful patient information. The main remaining limitation is the lack of controlled vocabulary or a medical ontology.

Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates (k). The average T90 (time required for 1-log10 reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T90 for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58-43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.