Hypoxia inducible factors regulate infectious SARS-CoV-2, epithelial damage and respiratory symptoms in a hamster COVID-19 model.

Understanding the host pathways that define susceptibility to SARS-CoV-2 infection and disease are essential for the design of new therapies. Oxygen levels in the microenvironment define the transcriptional landscape, however the influence of hypoxia on virus replication and disease in animal models is not well understood. In this study, we identify a role for the hypoxic inducible factor (HIF) signalling axis to inhibit SARS-CoV-2 infection, epithelial damage and respiratory symptoms in Syrian hamsters. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced the levels of infectious virus in the upper and lower respiratory tract. Nasal and lung epithelia showed a reduction in SARS-CoV-2 RNA and nucleocapsid expression in treated animals. Transcriptomic and pathological analysis showed reduced epithelial damage and increased expression of ciliated cells. Our study provides new insights on the intrinsic antiviral properties of the HIF signalling pathway in SARS-CoV-2 replication that may be applicable to other respiratory pathogens and identifies new therapeutic opportunities.

Receptor binding and escape from Beta antibody responses drive Omicron-B.1.1.529 evolution

On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses.

Antibody evasion by the Brazilian P.1 strain of SARS-CoV-2

Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations: P.1 from Brazil, B.1.351 from South Africa and B.1.1.7 from the UK (12, 10 and 9 changes in the spike respectively). All have mutations in the ACE2 binding site with P.1 and B.1.351 having a virtually identical triplet: E484K, K417N/T and N501Y, which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine induced antibody responses than B.1.351 suggesting that changes outside the RBD impact neutralisation. Monoclonal antibody 222 neutralises all three variants despite interacting with two of the ACE2 binding site mutations, we explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.

Naturally-acquired immunity in Syrian Golden Hamsters provides protection from re-exposure to emerging heterosubtypic SARS-CoV-2 variants B.1.1.7 and B.1.351

The ability of acquired immune responses against SARS-CoV-2 to protect after subsequent exposure to emerging variants of concern (VOC) such as B1.1.7 and B1.351 is currently of high significance. Here, we use a hamster model of COVID-19 to show that prior infection with a strain representative of the original circulating lineage B of SARS-CoV-2 induces protection from clinical signs upon subsequent challenge with either B1.1.7 or B1.351 viruses, which recently emerged in the UK and South Africa, respectively. The results indicate that these emergent VOC may be unlikely to cause disease in individuals that are already immune due to prior infection, and this has positive implications for overall levels of infection and COVID-19 disease.

Identification and quantification of SARS-CoV-2 leader subgenomic mRNA gene junctions in nasopharyngeal samples shows phasic transcription in animal models of COVID-19 and aberrant pattens in humans

IntroductionSARS-CoV-2 has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgRNAs has a unique 5 sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS-junction), that can be identified using sequencing. ResultsHigh resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS-junctions and be used as a proxy to quantify sgmRNAs for understanding virus biology. This was tested on published datasets and clinical samples from patients and longitudinal samples from animal models with COVID-19. DiscussionLeTRS identified known leader-TRS-junctions and identified novel species that were common across different species. The data indicated multi-phasic abundance of sgmRNAs in two different animal models, with spikes in sgmRNA abundance reflected in human samples, and therefore has implications for transmission models and nucleic acid-based diagnostics.

Sequential delivery of LAIV and SARS-CoV-2 in the ferret model can reduce SARS-CoV-2 shedding and does not result in enhanced lung pathology.

Co-circulation of SARS-CoV-2 and influenza viruses could pose unpredictable risks to health systems globally, with recent studies suggesting more severe disease outcomes in co-infected patients. The lack of a readily available COVID-19 vaccine has reinforced the importance of influenza vaccine programmes during the COVID-19 pandemic. Live Attenuated Influenza Vaccine (LAIV) is an important tool in protecting against influenza, particularly in children. However, it is unknown whether LAIV administration might influence the outcomes of acute SARS-CoV-2 infection or disease. To investigate this, quadrivalent LAIV (QLAIV) was administered to ferrets 3 days pre- or post-SARS-CoV-2 infection. LAIV administration did not exacerbate SARS-CoV-2 disease course or lung pathology with either regimen. Additionally, LAIV administered prior to SARS-CoV-2 infection significantly reduced SARS-CoV-2 replication and shedding in the upper respiratory tract (URT). We conclude that LAIV administration in close proximity to SARS-CoV-2 infection does not exacerbate mild disease and can reduce SARS-CoV-2 shedding.

mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection

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 g 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.

Diagnosis of SARS-CoV-2 infection with LamPORE, a high-throughput platform combining loop-mediated isothermal amplification and nanopore sequencing

LamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA that combines loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and from stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 7-10 genome copies/{micro}l of extracted RNA. This is above the limit achievable by RT-PCR but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among these LamPORE had a diagnostic sensitivity of 99.1% (226/228 [95% CI 96.9-99.9%]). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279 [98.0-100.0%]). Overall, 1.4% (7/514 [0.5-2.9]) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494 [94.8-98.1]). This indicates that LamPORE has a similar performance to RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients, and offers a promising approach to high-throughput testing.

Prophylactic intranasal administration of a TLR2 agonist reduces upper respiratory tract viral shedding in a SARS-CoV-2 challenge ferret model

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.

Comparison of Rhesus and Cynomolgus macaques as an authentic model for COVID-19.

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.

Pixatimod (PG545), a clinical-stage heparan sulfate mimetic, is a potent inhibitor of the SARS-CoV-2 virus

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a co-receptor with the ACE2 protein for recognition of the S1 spike protein on SARS-CoV-2 virus, providing a tractable new target for therapeutic intervention. Clinically-used heparins demonstrate inhibitory activity, but world supplies are limited, necessitating alternative solutions. Synthetic HS mimetic pixatimod is a drug candidate for cancer with immunomodulatory and heparanase-inhibiting properties. Here we show that pixatimod binds to and destabilizes the SARS-CoV-2 spike protein receptor binding domain (S1-RBD), and directly inhibits its binding to human ACE2, consistent with molecular modelling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of live SARS-CoV-2 virus show that pixatimod potently inhibits infection of monkey Vero E6 and human bronchial epithelial cells at concentrations within its safe therapeutic dose range. Furthermore, in a K18-hACE2 mouse model pixatimod demonstrates that pixatimod markedly attenuates SARS-CoV-2 viral titer and COVID-19-like symptoms. This demonstration of potent anti-SARS-CoV-2 activity establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics. Together with other known activities of pixatimod our data provides a strong rationale for its clinical investigation as a potential multimodal therapeutic to address the COVID-19 pandemic.

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient

The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6[A] crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

Dose-dependent response to infection with SARS-CoV-2 in the ferret model: evidence of protection to re-challenge

In December 2019 an outbreak of coronavirus disease (COVID-19) emerged in Wuhan, China. The causative agent was subsequently identified and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread worldwide causing a pandemic. Currently there are no licensed vaccines or therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in development and repurposed drugs are being tested in the clinic. There is a vital need for authentic COVID-19 animal models to further our understanding of pathogenesis and viral spread in addition to pre-clinical evaluation of candidate interventions. Here we report a dose titration study of SARS-CoV-2 to determine the most suitable infectious dose to use in the ferret model. We show that a high (5x106 pfu) and medium (5x104 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral RNA shedding in both groups of six challenged animals, whilst a low dose (5x102 pfu) resulted in only one of six displaying signs of URT viral RNA replication. The URT shedding lasted up to 21 days in the high dose animals with intermittent positive signal from day 14. Sequential culls revealed distinct pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were observed although animals did present with a consistent post-viral fatigue lasting from day 9-14 in the medium and high dose groups. After virus shedding ceased, re-challenged ferrets were shown to be fully protected from acute lung pathology. The endpoints of URT viral RNA replication in addition to distinct lung pathology and post viral fatigue were observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 14-21 parallel observations reported in a minority of clinical cases.

Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs from patients with COVID-19

COVID-19 is a complex disease phenotype where the underlying microbiome could influence morbidity and mortality. Amplicon and metagenomic MinION based sequencing was used to rapidly (within 8 hours) identify SARS-CoV-2 and assess the microbiome in nasopharyngeal swabs obtained from patients with COVID-19 by the ISARIC 4C consortium.

Characterisation of the transcriptome and proteome of SARS-CoV-2 using direct RNA sequencing and tandem mass spectrometry reveals evidence for a cell passage induced in-frame deletion in the spike glycoprotein that removes the furin-like cleavage site.

Direct RNA sequencing using an Oxford Nanopore MinION characterised the transcriptome of SARS-CoV-2 grown in Vero E6 cells. This cell line is being widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. This revealed the pattern of viral transcripts, (i.e. subgenomic mRNAs), generally fitted the predicted replication and transcription model for coronaviruses. A 24 nt in-frame deletion was detected in subgenomic mRNAs encoding the spike (S) glycoprotein. This feature was identified in over half of the mapped transcripts and was predicted to remove a proposed furin cleavage site from the S glycoprotein. This motif directs cleavage of the S glycoprotein into functional subunits during virus entry or exit. Cleavage of the S glycoprotein can be a barrier to zoonotic coronavirus transmission and affect viral pathogenicity. Allied to this transcriptome analysis, tandem mass spectrometry was used to identify over 500 viral peptides and 44 phosphopeptides, covering almost all of the proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein. Detection of an apparently viable deletion in the furin cleavage site of the S glycoprotein reinforces the point that this and other regions of SARS-CoV-2 proteins may readily mutate. This is of clear significance given the interest in the S glycoprotein as a potential vaccine target and the observation that the furin cleavage site likely contributes strongly to the pathogenesis and zoonosis of this virus. The viral genome sequence should be carefully monitored during the growth of viral stocks for research, animal challenge models and, potentially, in clinical samples. Such variations may result in different levels of virulence, morbidity and mortality.