Single-cell RNA sequencing reveals in vivo signatures of SARS-CoV-2-reactive T cells through 'reverse phenotyping' (preprint)

The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we used single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induced transcriptional shifts by antigenic stimulation in vitro and took advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for reverse phenotyping. This allowed identification of SARS-CoV-2-reactive TCRs and revealed phenotypic effects introduced by antigen-specific stimulation. We characterized transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and showed correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

Distant Residues Modulate the Conformational Opening in SARS-CoV-2 Spike Protein (preprint)

Infection by SARS-CoV-2 involves the attachment of the receptor binding domain (RBD) of its spike proteins to the ACE2 receptors on the peripheral membrane of host cells. Binding is initiated by a down to up conformational change in the spike protein, an opening which presents the RBD to the receptor. To date, computational and experimental studies for therapeutics have concentrated, for good reason, on the RBD. However, the RBD region is highly prone to mutations, and therefore will possibly arise drug resistance. In contrast, we here focus on the correlations between the RBD and residues distant to it in the spike protein. We thereby provide a deeper understanding of the role of distant residues in the molecular mechanism of infection. Predictions of key mutations in distant allosteric binding sites are provided, with implications for therapeutics. Identifying these emerging mutants can also go a long way towards pre-designing vaccines for future outbreaks. The model we use, based on time-independent component analysis (tICA) and protein graph connectivity network, is able to identify multiple residues that exhibit long-distance coupling with the RBD opening. Mutation on these residues can lead to new strains of coronavirus with different degrees of transmissibility and virulence. The most ubiquitous D614G mutation and the A570D mutation of the highly contageous UK SARS-CoV-2 variant are predicted ab-initio from our model. Conversely, broad spectrum therapeutics like drugs and monoclonal antibodies can be generated targeting these key distant but conserved regions of the spike protein. Significance statementThe novel coronavirus SARS-CoV-2 has created the largest pandemic of recent times, resulting in economic and public health crises. Significant research effort to design drugs against COVID-19 is focused on the receptor binding domain of the spike protein, although this region is prone to mutations that can cause resistance against therapeutics. We applied deep data analysis methods on all-atom molecular dynamics simulations of the spike protein to identify key non-RBD residues that play a crucial role in spike-receptor binding and infection of human cells. These residues can not only be targeted by broad spectrum antibodies and drugs, but can also offer predictive insights into the mutations with the potential to generate new strains that might appear during future epidemics.

Extended in vitro inactivation of SARS-CoV-2 by titanium dioxide surface coating (preprint)

SARS-CoV-2 transmission occurs via airborne droplets and surface contamination. We show tiles coated with TiO2 120 days previously can inactivate SARS-CoV-2 under ambient indoor lighting with 87% reduction in titres at 1h and complete loss by 5h exposure. TiO2 coatings could be an important tool in containing SARS-CoV-2.

Engineered receptor binding domain immunogens elicit pan-coronavirus neutralizing antibodies (preprint)

Effective countermeasures are needed against emerging coronaviruses of pandemic potential, similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Designing immunogens that elicit broadly neutralizing antibodies to conserved viral epitopes on the major surface glycoprotein, spike, such as the receptor binding domain (RBD) is one potential approach. Here, we report the generation of homotrimeric RBD immunogens from different sarbecoviruses using a stabilized, immune-silent trimerization tag. We find that that a cocktail of homotrimeric sarbecovirus RBDs can elicit a neutralizing response to all components even in context of prior SARS-CoV-2 imprinting. Importantly, the cross-neutralizing antibody responses are focused towards conserved RBD epitopes outside of the ACE-2 receptor-binding motif. This may be an effective strategy for eliciting broadly neutralizing responses leading to a pan-sarbecovirus vaccine.

Circular RNA profiling reveals abundant and diverse circRNAs of SARS-CoV-2, SARS-CoV and MERS-CoV origin (preprint)

Circular RNAs (circRNAs) encoded by DNA genomes have been identified across host and pathogen species as parts of the transcriptome. Accumulating evidences indicate that circRNAs play critical roles in autoimmune diseases and viral pathogenesis. Here we report that RNA viruses of the Betacoronavirus genus of Coronaviridae, SARS-CoV-2, SARS-CoV and MERS-CoV, encode a novel type of circRNAs. Through de novo circRNA analyses of publicly available coronavirus-infection related deep RNA-Sequencing data, we identified 351, 224 and 2,764 circRNAs derived from SARS-CoV-2, SARS-CoV and MERS-CoV, respectively, and characterized two major back-splice events shared by these viruses. Coronavirus-derived circRNAs are more abundant and longer compared to host genome-derived circRNAs. Using a systematic strategy to amplify and identify back-splice junction sequences, we experimentally identified over 100 viral circRNAs from SARS-CoV-2 infected Vero E6 cells. This collection of circRNAs provided the first line of evidence for the abundance and diversity of coronavirus-derived circRNAs and suggested possible mechanisms driving circRNA biogenesis from RNA genomes. Our findings highlight circRNAs as an important component of the coronavirus transcriptome. SummaryWe report for the first time that abundant and diverse circRNAs are generated by SARS-CoV-2, SARS-CoV and MERS-CoV and represent a novel type of circRNAs that differ from circRNAs encoded by DNA genomes.

Binding of SARS-CoV-2 spike protein to ACE2 is disabled by thiol-based drugs; evidence from in vitro SARS-CoV-2 infection studies. (preprint)

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the SARS-CoV-2 spike protein is an envelope glycoprotein that binds angiotensin converting enzyme 2 as an entry receptor. The capacity of enveloped viruses to infect host cells depends on a precise thiol/disulfide balance in their surface glycoprotein complexes. To determine if cystines in the SARS-CoV-2 spike protein maintain a native binding interface that can be disrupted by drugs that cleave cystines, we tested if thiol-based drugs have efficacy in receptor binding and cell infection assays. We found that thiol-based drugs, cysteamine and WR-1065 (the active metabolite of amifostine) in particular, decrease binding of SARS-CoV-2 spike protein to its receptor, decrease the entry efficiency of SARS-CoV-2 spike pseudotyped virus, and inhibit SARS-CoV-2 live virus infection. Our findings uncover a vulnerability of SARS-CoV-2 to thiol-based drugs and provide rationale to test thiol-based drugs, especially cysteamine and amifostine, as novel treatments for COVID-19. One Sentence SummaryThiol-based drugs decrease binding of SARS-CoV-2 spike protein to its receptor and inhibit SARS-CoV-2 cell entry.