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.

Determinants of SARS-CoV-2 receptor gene expression in upper and lower airways (preprint)

The recent outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has led to a worldwide pandemic. One week after initial symptoms develop, a subset of patients progresses to severe disease, with high mortality and limited treatment options. To design novel interventions aimed at preventing spread of the virus and reducing progression to severe disease, detailed knowledge of the cell types and regulating factors driving cellular entry is urgently needed. Here we assess the expression patterns in genes required for COVID-19 entry into cells and replication, and their regulation by genetic, epigenetic and environmental factors, throughout the respiratory tract using samples collected from the upper (nasal) and lower airways (bronchi). Matched samples from the upper and lower airways show a clear increased expression of these genes in the nose compared to the bronchi and parenchyma. Cellular deconvolution indicates a clear association of these genes with the proportion of secretory epithelial cells. Smoking status was found to increase the majority of COVID-19 related genes including ACE2 and TMPRSS2 but only in the lower airways, which was associated with a significant increase in the predicted proportion of goblet cells in bronchial samples of current smokers. Both acute and second hand smoke were found to increase ACE2 expression in the bronchus. Inhaled corticosteroids decrease ACE2 expression in the lower airways. No significant effect of genetics on ACE2 expression was observed, but a strong association of DNA- methylation with ACE2 and TMPRSS2- mRNA expression was identified in the bronchus.

Prime-boost protein subunit vaccines against SARS-CoV-2 are highly immunogenic in mice and macaques (preprint)

SARS-CoV-2 vaccines are advancing into human clinical trials, with emphasis on eliciting high titres of neutralising antibodies against the viral spike (S). However, the merits of broadly targeting S versus focusing antibody onto the smaller receptor binding domain (RBD) are unclear. Here we assessed prototypic S and RBD subunit vaccines in homologous or heterologous prime-boost regimens in mice and non-human primates. We find S is highly immunogenic in mice, while the comparatively poor immunogenicity of RBD was associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augmented neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines were comparably immunogenic in macaques, eliciting serological neutralising activity that generally exceed levels in convalescent humans. These studies confirm recombinant S proteins as promising vaccine candidates and highlight multiple pathways to achieving potent serological neutralisation.

An intestinal cell type in zebrafish is the nexus for the SARS-CoV-2 receptor and the Renin-Angiotensin-Aldosterone System that contributes to COVID-19 comorbidities (preprint)

People with underlying conditions, including hypertension, obesity, and diabetes, are especially susceptible to negative outcomes after infection with the coronavirus SARS-CoV-2. These COVID-19 comorbidities are exacerbated by the Renin-Angiotensin-Aldosterone System (RAAS), which normally protects from rapidly dropping blood pressure or dehydration via the peptide Angiotensin II (Ang II) produced by the enzyme Ace. The Ace paralog Ace2 degrades Ang II, thus counteracting its chronic effects. Ace2 is also the SARS-CoV-2 receptor. Ace, the coronavirus, and COVID-19 comorbidities all regulate Ace2, but we dont yet understand how. To exploit zebrafish (Danio rerio) as a disease model to understand mechanisms regulating the RAAS and its relationship to COVID-19 comorbidities, we must first identify zebrafish orthologs and co-orthologs of human RAAS genes, and second, understand where and when these genes are expressed in specific cells in zebrafish development. To achieve these goals, we conducted genomic analyses and investigated single cell transcriptomes. Results showed that most human RAAS genes have an ortholog in zebrafish and some have two or more co-orthologs. Results further identified a specific intestinal cell type in zebrafish larvae as the site of expression for key RAAS components, including Ace, Ace2, the coronavirus co-receptor Slc6a19, and the Angiotensin-related peptide cleaving enzymes Anpep and Enpep. Results also identified specific vascular cell subtypes as expressing Ang II receptors, apelin, and apelin receptor genes. These results identify specific genes and cell types to exploit zebrafish as a disease model for understanding the mechanisms leading to COVID-19 comorbidities. SUMMARY STATEMENTGenomic analyses identify zebrafish orthologs of the Renin-Angiotensin-Aldosterone System that contribute to COVID-19 comorbidities and single-cell transcriptomics show that they act in a specialized intestinal cell type.

Structural Variants in SARS-CoV-2 Occur at Template-Switching Hotspots (preprint)

The evolutionary dynamics of SARS-CoV-2 have been carefully monitored since the COVID-19 pandemic began in December 2019, however, analysis has focused primarily on single nucleotide polymorphisms and largely ignored the role of structural variants (SVs) as well as recombination in SARS-CoV-2 evolution. Using sequences from the GISAID database, we catalogue over 100 insertions and deletions in the SARS-CoV-2 consensus sequences. We hypothesize that these indels are artifacts of imperfect homologous recombination between SARS-CoV-2 replicates, and provide four independent pieces of evidence. (1) The SVs from the GISAID consensus sequences are clustered at specific regions of the genome. (2) These regions are also enriched for 5 and 3 breakpoints in the transcription regulatory site (TRS) independent transcriptome, presumably sites of RNA-dependent RNA polymerase (RdRp) template-switching. (3) Within raw reads, these structural variant hotspots have cases of both high intra-host heterogeneity and intra-host homogeneity, suggesting that these structural variants are both consequences of de novo recombination events within a host and artifacts of previous recombination. (4) Within the RNA secondary structure, the indels occur in "arms" of the predicted folded RNA, suggesting that secondary structure may be a mechanism for TRS-independent template-switching in SARS-CoV-2 or other coronaviruses. These insights into the relationship between structural variation and recombination in SARS-CoV-2 can improve our reconstructions of the SARS-CoV-2 evolutionary history as well as our understanding of the process of RdRp template-switching in RNA viruses.

Modeling SARS-CoV-2 infection in vitro with a human intestine-on-chip device (preprint)

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) has given rise to a global pandemic. The gastrointestinal symptoms of some COVID-19 patients are underestimated. There is an urgent need to develop physiologically relevant model that can accurately reflect human response to viral infection. Here, we report the creation of a biomimetic human intestine infection model on a chip system that allows to recapitulate the intestinal injury and immune response induced by SARS-CoV-2, for the first time. The microengineered intestine-on-chip device contains human intestinal epithelium (co-cultured human intestinal epithelial Caco-2 cells and mucin secreting HT-29 cells) lined in upper channel and vascular endothelium (human umbilical vein endothelial cells, HUVECs) in a parallel lower channel under fluidic flow condition, sandwiched by a porous PDMS membrane coated with extracellular matrix (ECM). At day 3 post-infection of SARS-CoV-2, the intestine epithelium showed high susceptibility to viral infection and obvious morphological changes with destruction of intestinal villus, dispersed distribution of mucus secreting cells and reduced expression of tight junction (E-cadherin), indicating the destruction of mucous layer and the integrity of intestinal barrier caused by virus. Moreover, the endothelium exhibited abnormal cell morphology with disrupted expression of adherent junction protein (VE-cadherin). Transcriptional analysis revealed the abnormal RNA and protein metabolism, as well as activated immune responses in both epithelial and endothelial cells after viral infection (e.g., up-regulated cytokine genes, TNF signaling and NF-kappa B signaling-related genes). This bioengineered in vitro model system can mirror the human relevant pathophysiology and response to viral infection at the organ level, which is not possible in existing in vitro culture systems. It may provide a promising tool to accelerate our understanding of COVID-19 and devising novel therapies.

Spike mutation D614G alters SARS-CoV-2 fitness and neutralization susceptibility (preprint)

A spike protein mutation D614G became dominant in SARS-CoV-2 during the COVID-19 pandemic. However, the mutational impact on viral spread and vaccine efficacy remains to be defined. Here we engineer the D614G mutation in the SARS-CoV-2 USA-WA1/2020 strain and characterize its effect on viral replication, pathogenesis, and antibody neutralization. The D614G mutation significantly enhances SARS-CoV-2 replication on human lung epithelial cells and primary human airway tissues, through an improved infectivity of virions with the spike receptor-binding domain in an "up" conformation for binding to ACE2 receptor. Hamsters infected with D614 or G614 variants developed similar levels of weight loss. However, the G614 virus produced higher infectious titers in the nasal washes and trachea, but not lungs, than the D614 virus. The hamster results confirm clinical evidence that the D614G mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increases transmission. For antibody neutralization, sera from D614 virus-infected hamsters consistently exhibit higher neutralization titers against G614 virus than those against D614 virus, indicating that (i) the mutation may not reduce the ability of vaccines in clinical trials to protect against COVID-19 and (ii) therapeutic antibodies should be tested against the circulating G614 virus before clinical development. ImportanceUnderstanding the evolution of SARS-CoV-2 during the COVID-19 pandemic is essential for disease control and prevention. A spike protein mutation D614G emerged and became dominant soon after the pandemic started. By engineering the D614G mutation into an authentic wild-type SARS-CoV-2 strain, we demonstrate the importance of this mutation to (i) enhanced viral replication on human lung epithelial cells and primary human airway tissues, (ii) improved viral fitness in the upper airway of infected hamsters, and (iii) increased susceptibility to neutralization. Together with clinical findings, our work underscores the importance of this mutation in viral spread, vaccine efficacy, and antibody therapy.

Swarm Learning as a privacy-preserving machine learning approach for disease classification (preprint)

Identification of patients with life-threatening diseases including leukemias or infections such as tuberculosis and COVID-19 is an important goal of precision medicine. We recently illustrated that leukemia patients are identified by machine learning (ML) based on their blood transcriptomes. However, there is an increasing divide between what is technically possible and what is allowed because of privacy legislation. To facilitate integration of any omics data from any data owner world-wide without violating privacy laws, we here introduce Swarm Learning (SL), a decentralized machine learning approach uniting edge computing, blockchain-based peer-to-peer networking and coordination as well as privacy protection without the need for a central coordinator thereby going beyond federated learning. Using more than 14,000 blood transcriptomes derived from over 100 individual studies with non-uniform distribution of cases and controls and significant study biases, we illustrate the feasibility of SL to develop disease classifiers based on distributed data for COVID-19, tuberculosis or leukemias that outperform those developed at individual sites. Still, SL completely protects local privacy regulations by design. We propose this approach to noticeably accelerate the introduction of precision medicine.

Group testing for SARS-CoV-2 allows for up to 10-fold efficiency increase across realistic scenarios and testing strategies (preprint)

Background: Due to the ongoing COVID-19 pandemic, demand for diagnostic testing has increased drastically, resulting in shortages of necessary materials to conduct the tests and overwhelming the capacity of testing laboratories. The supply scarcity and capacity limits affect test administration: priority must be given to hospitalized patients and symptomatic individuals, which can prevent the identification of asymptomatic and presymptomatic individuals and hence effective tracking and tracing policies. We describe optimized group testing strategies applicable to SARS-CoV-2 tests in scenarios tailored to the current COVID-19 pandemic and assess significant gains compared to individual testing. Methods: We account for biochemically realistic scenarios in the context of dilution effects on SARS-CoV-2 samples and consider evidence on specificity and sensitivity of PCR-based tests for the novel coronavirus. Because of the current uncertainty and the temporal and spatial changes in the prevalence regime, we provide analysis for a number of realistic scenarios and propose fast and reliable strategies for massive testing procedures. Findings: We find significant efficiency gaps between different group testing strategies in realistic scenarios for SARS-CoV-2 testing, highlighting the need for an informed decision of the pooling protocol depending on estimated prevalence, target specificity, and high- vs. low-risk population. For example, using one of the presented methods, all 1.47 million inhabitants of Munich, Germany, could be tested using only around 141 thousand tests if the infection rate is below 0.4% is assumed. Using 1 million tests, the 6.69 million inhabitants from the city of Rio de Janeiro, Brazil, could be tested as long as the infection rate does not exceed 1%. Interpretation: Altogether this work may help provide a basis for efficient upscaling of current testing procedures, taking the population heterogeneity into account and fine grained towards the desired study populations, e.g. cross-sectional versus health-care workers and adapted mixtures thereof.

Protocol of a population-based prospective COVID-19 cohort study Munich, Germany (KoCo19) (preprint)

Background: The SARS-CoV-2 pandemic is leading to the global introduction of public health interventions to prevent the spread of the virus and avoid the overload of health care systems, especially for the most severely affected patients. Scientific studies to date have focused primarily on describing the clinical course of patients, identifying treatment options and developing vaccines. In Germany, as in many other regions, current tests for SARS-CoV2 are not being conducted on a representative basis and in a longitudinal design. Furthermore, knowledge about the immune status of the population is lacking. Yet these data are needed to understand the dynamics of the pandemic and to thus appropriately design and evaluate interventions. For this purpose, we recently started a prospective population-based cohort in Munich, Germany, with the aim to better understand the state and dynamics of the pandemic. Methods: In 100, randomly selected constituencies out of 755, 3,000 Munich households are identified via random route and offered enrollment into the study. All household members are asked to complete a baseline questionnaire and subjects [≥]14 years of age are asked to provide a venous blood sample of [≤]3 ml for the determination of SARS-CoV-2 IgG/IgA status. The residual plasma and the blood pellet are preserved for later genetic and molecular biological investigations. For twelve months, each household member is asked to keep a diary of daily symptoms, whereabouts and contacts via WebApp. If symptoms suggestive for COVID-19 are reported, family members, including children <14 years, are offered a pharyngeal swab taken at the Division of Infectious Diseases and Tropical Medicine, LMU University Hospital Munich, for molecular testing for SARS-CoV-2. In case of severe symptoms, participants will be transferred to a Munich hospital. For one year, the study teams re-visits the households for blood sampling every six weeks. Discussion: With the planned study we will establish a reliable epidemiological tool to improve the understanding of the spread of SARS-CoV-2 and to better assess the effectiveness of public health measures as well as their socio-economic effects. This will support policy makers in managing the epidemic based on scientific evidence.