Long-term serial passaging of SARS-CoV-2 reveals signatures of convergent evolution (preprint)

Measures to control the COVID-19 pandemic such as antiviral therapy and vaccination have been challenged by ongoing virus evolution under antiviral and immune pressures. Understanding viral evolutionary dynamics is crucial for responding to SARS-CoV-2, and preparing for the next pandemic, by informing prediction of virus adaptation, public health strategies, and design of broadly effective therapies. Whole-genome sequencing (WGS) of SARS-CoV-2 during the pandemic enabled fine-grained studies of virus evolution in the human population. Serial passaging in vitro offers a controlled environment to investigate the emergence and persistence of genetic variants that may confer selective advantage. Nine virus lineages, including four "variants of concern" and three former "variants under investigation" as designated by the World Health Organisation, were chosen to investigate intra- and inter-lineage evolution through long-term serial passaging in Vero E6 cells. Viruses were sampled over at least 33 passages (range 33-100) and analysed using WGS to examine evolutionary dynamics and identify key mutations with implications for virus fitness, transmissibility, and immune evasion. All passages continued to replicate in culture, despite regular accumulation of mutations. There was evidence of convergent acquisition of mutations both across passage lines and compared with contemporaneous SARS CoV-2 clinical sequences from population studies. Some of these convergent mutations are hypothesised to be important in proliferation of SARS-CoV-2 lineages, such as by evading host immune responses (e.g. S:A67V, S:H655Y). Given these mutations arose in vitro, in the absence of a multicellular host immune response, this suggests virus genome mutation resulted from stochastic events, rather than immune-driven mutation. There was a regular gain and loss of low-frequency variants during serial passaging, but some became fixed in subsequent multiple passages, suggesting either a benefit of the mutation in vitro, or at least a lack of deleterious effect. Our findings reveal valuable insights into the evolution of SARS-CoV-2 by quantitatively investigating evolutionary dynamics of the virus over the greatest number of serial passages to date. Knowledge of these evolutionary trends will be useful for public health and the development of antiviral and vaccine measures to reduce the effects of SARS CoV-2 infection on the human population.

TMPRSS2 activation of Omicron lineage Spike glycoproteins is regulated by TMPRSS2 cleavage of ACE2 (preprint)

Continued high levels spread of SARS-CoV-2 globally enabled accumulation of changes within the Spike glycoprotein, leading to resistance to neutralising antibodies and concomitant changes to entry requirements that increased viral transmission fitness. Herein, we demonstrate a significant change in ACE2 and TMPRSS2 use by primary SARS-CoV-2 isolates that occurred upon arrival of Omicron lineages. Mechanistically we show this shift to be a function of two distinct ACE2 pools based on cleavage or non-cleavage of ACE2 by TMPRSS2 activity. In engineered cells overexpressing ACE2 and TMPRSS2, ACE2 was cleaved by TMPRSS2 and this led to either augmentation or progressive attenuation of pre-Omicron and Omicron lineages, respectfully. In contrast, TMPRSS2 resistant ACE2 restored infectivity across all Omicron lineages through enabling ACE2 binding that facilitated TMPRSS2 spike activation. Therefore, our data support the tropism shift of Omicron lineages to be a function of evolution towards the use of uncleaved pools of ACE2 with the latter consistent with its role as a chaperone for many tissue specific amino acid transport proteins.

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein (preprint)

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used mRNA display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 Wuhan strain infection and also pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor binding domain and other domains, distal to the ACE2 receptor-interaction site. Collectively, our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that can be targeted by peptides and potentially other drug-like molecules.

Discovery of Antiviral Cyclic Peptides Targeting the Main Protease of SARS-CoV-2 via mRNA Display (preprint)

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.

Potent in vitro anti-SARS-CoV-2 activity by gallinamide A and analogues via inhibition of cathepsin L (preprint)

The emergence of SARS-CoV-2 in late 2019, and the subsequent COVID-19 pandemic, has led to substantial mortality, together with mass global disruption. There is an urgent need for novel antiviral drugs for therapeutic or prophylactic application. Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is recognized as a promising drug target. The marine natural product, gallinamide A and several synthetic analogues, were identified as potent inhibitors of cathepsin L activity with IC50 values in the picomolar range. Lead molecules possessed selectivity over cathepsin B and other related human cathepsin proteases and did not exhibit inhibitory activity against viral proteases Mpro and PLpro. We demonstrate that gallinamide A and two lead analogues potently inhibit SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range, thus further highlighting the potential of cathepsin L as a COVID-19 antiviral drug target.

Cell-type apoptosis in lung during SARS-CoV-2 infection (preprint)

The SARS-CoV-2 pandemic has inspired renewed interest in understanding the fundamental pathology of acute respiratory distress syndrome (ARDS) following infection because fatal COVID-19 cases are commonly linked to respiratory failure due to ARDS. The pathologic alteration known as diffuse alveolar damage in endothelial and epithelial cells is a critical feature of acute lung injury in ARDS. However, the pathogenesis of ARDS following SRAS-CoV-2 infection remains largely unknown. In the present study, we examined apoptosis in post-mortem lung sections from COVID-19 patients and lung tissues from a non-human primate model of SARS-CoV-2 infection, in a cell-type manner, including type 1 and 2 alveolar cells and vascular endothelial cells (ECs), macrophages, and T cells. Multiple-target immunofluorescence (IF) assays and western blotting suggest both intrinsic and extrinsic apoptotic pathways are activated during SARS-CoV-2 infection. Furthermore, we observed that SARS-CoV-2 fails to induce apoptosis in human bronchial epithelial cells (i.e., BEAS2B cells) and primary human umbilical vein endothelial cells (HUVECs), which are refractory to SARS-CoV-2 infection. However, infection of co-cultured Vero cells and HUVECs or Vero cells and BEAS2B cells with SARS-CoV-2 induced apoptosis in both Vero cells and HUVECs/BEAS2B cells, but did not alter the permissiveness of HUVECs or BEAS2B cells to the virus. Post-exposure treatment of the co-culture of Vero cells and HUVECs with an EPAC1-specific activator ameliorated apoptosis in HUVECs. These findings may help to delineate a novel insight into the pathogenesis of ARDS following SARS-CoV-2 infection.

Detection and molecular characterisation of SARS-CoV-2 in farmed mink (Neovision vision) in Poland (preprint)

SARS-CoV-2 is the aetiological agent of COVID-19 disease and has been spreading worldwide since December 2019. The virus has been shown to infect different animal species under experimental conditions. Also, minks have been found to be susceptible to SARS-CoV-2 infection in fur farms in Europe and the USA. Here we investigated 91 individual minks from a farm located in Northern Poland. Using RT-PCR, antigen detection and NGS, we confirmed 15 animals positive for SARS-CoV-2. The result was verified by sequencing of full viral genomes, confirming SARS-CoV-2 infection in Polish mink. Country-scale monitoring conducted by veterinary inspection so far has not detected the presence of SARS-CoV-2 on other mink farms. Taking into consideration that Poland has a high level of positive diagnostic tests among its population, there is a high risk that more Polish mink farms become a source for SARS-CoV-2. Findings reported here and from other fur producing countries urge the assessment of SARS-CoV-2 prevalence in animals bred in Polish fur farms.

Berberine and obatoclax inhibit SARS-CoV-2 replication in primary human nasal epithelial cells in vitro (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and has infected an estimated 10% of the global population in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of a specific set of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.

Experimental SARS-CoV-2 infection of bank voles - general susceptibility but lack of direct transmission (preprint)

After experimental inoculation, SARS-CoV-2 infection was proven for bank voles by seroconversion within eight days and detection of viral RNA in nasal tissue for up to 21 days. However, transmission to contact animals was not detected. Therefore, bank voles are unlikely to establish effective SARS-CoV-2 transmission cycles in nature.

Exploring epitope and functional diversity of anti-SARS-CoV2 antibodies using AI-based methods (preprint)

Since the beginning of the COVID19 pandemics, an unprecedented research effort has been conducted to analyze the antibody responses in patients, and many trials based on passive immunotherapy -notably monoclonal antibodies - are ongoing. Twenty-one antibodies have entered clinical trials, 6 having reached phase 2/3, phase 3 or having received emergency authorization. These represent only the tip of the iceberg, since many more antibodies have been discovered and represent opportunities either for diagnosis purposes or as drug candidates. The main problem facing laboratories willing to develop such antibodies is the huge task of analyzing them and choosing the best candidate for exhaustive experimental validation. In this work we show how artificial intelligence-based methods can help in analyzing large sets of antibodies in order to determine in a few hours the best candidates in few hours. The MAbCluster method, which only requires knowledge of the amino acid sequences of the antibodies, allows to group the antibodies having the same epitope, considering only their amino acid sequences and their 3D structures (actual or predicted), and to infer some of their functional properties. We then use MAbTope to predict the epitopes for all antibodies for which they are not already known. This allows an exhaustive comparison of the available epitopes, but also gives a synthetic view of the possible combinations. Finally, we show how these results can be used to predict which antibodies might be affected by the different mutations arising in the circulating strains of the virus, such as the N501Y mutation that has started to spread in Great-Britain.

Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein using mRNA Display (preprint)

The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (KD) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect spike protein in solution by ELISA, and the co-crystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a {beta}-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD and the ligands discovered in this work may find future use as reagents for diagnostic applications.