Integrated longitudinal multi-omics study identifies immune programs associated with COVID-19 severity and mortality in 1152 hospitalized participants (preprint)

Hospitalized COVID-19 patients exhibit diverse clinical outcomes, with some individuals diverging over time even though their initial disease severity appears similar. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity. In this study, we carried out deep immunophenotyping and conducted longitudinal multi-omics modeling integrating ten distinct assays on a total of 1,152 IMPACC participants and identified several immune cascades that were significant drivers of differential clinical outcomes. Increasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, NETosis, and T-cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma immunoglobulins and B cells, as well as dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to the failure of viral clearance in patients with fatal illness. Our longitudinal multi-omics profiling study revealed novel temporal coordination across diverse omics that potentially explain disease progression, providing insights that inform the targeted development of therapies for hospitalized COVID-19 patients, especially those critically ill.

Systems biological assessment of the temporal dynamics of immunity to a viral infection in the first weeks and months of life (preprint)

The dynamics of innate and adaptive immunity to infection in infants remain obscure. Here, we used a multi-omics approach to perform a longitudinal analysis of immunity to SARS-CoV-2 infection in infants and young children in the first weeks and months of life by analyzing blood samples collected before, during, and after infection with Omicron and Non-Omicron variants. Infection stimulated robust antibody titers that, unlike in adults, were stably maintained for >300 days. Antigen-specific memory B cell (MCB) responses were durable for 150 days but waned thereafter. Somatic hypermutation of V-genes in MCB accumulated progressively over 9 months. The innate response was characterized by upregulation of activation markers on blood innate cells, and a plasma cytokine profile distinct from that seen in adults, with no inflammatory cytokines, but an early and transient accumulation of chemokines (CXCL10, IL8, IL-18R1, CSF-1, CX3CL1), and type I IFN. The latter was strongly correlated with viral load, and expression of interferon-stimulated genes (ISGs) in myeloid cells measured by single-cell transcriptomics. Consistent with this, single-cell ATAC-seq revealed enhanced accessibility of chromatic loci targeted by interferon regulatory factors (IRFs) and reduced accessibility of AP-1 targeted loci, as well as traces of epigenetic imprinting in monocytes, during convalescence. Together, these data provide the first snapshot of immunity to infection during the initial weeks and months of life.

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques (preprint)

Type-I interferons (IFN-I) are critical mediators of innate control of viral infections, but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, and for the first time, IFN-I signaling was modulated in rhesus macaques (RMs) prior to and during acute SARS-CoV-2 infection using a mutated IFN2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. In SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. Notably, IFNmod treatment resulted in a potent reduction in (i) SARS-CoV-2 viral load in Bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes; (ii) inflammatory cytokines, chemokines, and CD163+MRC1- inflammatory macrophages in BAL; and (iii) expression of Siglec-1, which enhances SARS-CoV-2 infection and predicts disease severity, on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. This study, using an intervention targeting both IFN- and IFN-{beta} pathways, shows that excessive inflammation driven by type 1 IFN critically contributes to SARS-CoV-2 pathogenesis in RMs, and demonstrates the potential of IFNmod to limit viral replication, SARS-CoV-2 induced inflammation, and COVID-19 severity.

The Arabidopsis Framework Model version 2 predicts the organism-level effects of circadian clock gene mis-regulation (preprint)

Predicting a multicellular organism's phenotype quantitatively from its genotype is challenging, as genetic effects must propagate across scales. Circadian clocks are intracellular regulators that control temporal gene expression patterns and hence metabolism, physiology and behaviour. Here we explain and predict canonical phenotypes of circadian timing in a multicellular, model organism. We used diverse metabolic and physiological data to combine and extend mathematical models of rhythmic gene expression, photoperiod-dependent flowering, elongation growth and starch metabolism within a Framework Model for the vegetative growth of Arabidopsis thaliana, sharing the model and data files in a structured, public resource. The calibrated model predicted the effect of altered circadian timing upon each particular phenotype in clock-mutant plants under standard laboratory conditions. Altered night-time metabolism of stored starch accounted for most of the decrease in whole-plant biomass, as previously proposed. Mobilisation of a secondary store of malate and fumarate was also mis-regulated, accounting for any remaining biomass defect. We test three candidate mechanisms for the accumulation of these organic acids. Our results link genotype through specific processes to higher-level phenotypes, formalising our understanding of a subtle, pleiotropic syndrome at the whole-organism level, and validating the systems approach to understand complex traits starting from intracellular circuits. This work updates the first biorXiv version, February 2017, https://doi.org/10.1101/105437, with an expanded description and additional analysis of the same core data sets and the same FMv2 model, summary tables and supporting, follow-on data from three further studies with further collaborators. This biorXiv revision constitutes the second version of this report.

TREM2+ and interstitial macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques (preprint)

The COVID-19 pandemic remains a global health crisis, yet, the immunopathological mechanisms driving the development of severe disease remain poorly defined. Here, we utilize a rhesus macaque (RM) model of SARS-CoV-2 infection to delineate perturbations in the innate immune system during acute infection using an integrated systems analysis. We found that SARS-CoV-2 initiated a rapid infiltration (two days post infection) of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and induction of interferon-stimulated genes. At this early interval, we also observed a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generated a novel compendium of RM-specific lung macrophage gene expression using a combination of sc-RNA-Seq data and bulk RNA-Seq of purified populations under steady state conditions. Using these tools, we generated a longitudinal sc-RNA-seq dataset of airway cells in SARS-CoV-2-infected RMs. We identified that SARS-CoV-2 infection elicited a rapid recruitment of two subsets of macrophages into the airway: a C206+MRC1- population resembling murine interstitial macrophages, and a TREM2+ population consistent with CCR2+ infiltrating monocytes, into the alveolar space. These subsets were the predominant source of inflammatory cytokines, accounting for ~75% of IL6 and TNF production, and >90% of IL10 production, whereas the contribution of CD206+MRC+ alveolar macrophages was significantly lower. Treatment of SARS-CoV-2 infected RMs with baricitinib (Olumiant(R)), a novel JAK1/2 inhibitor that recently received Emergency Use Authorization for the treatment of hospitalized COVID-19 patients, was remarkably effective in eliminating the influx of infiltrating, non-alveolar macrophages in the alveolar space, with a concomitant reduction of inflammatory cytokines. This study has delineated the major subsets of lung macrophages driving inflammatory and anti-inflammatory cytokine production within the alveolar space during SARS-CoV-2 infection.

CCR2-dependent monocyte-derived cells restrict SARS-CoV-2 infection (preprint)

SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19) and current evidence suggests severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts viral burden in the lung. We find that a recently developed mouse-adapted MA-SARS-CoV-2 strain, as well as the emerging B.1.351 variant, trigger an inflammatory response in the lung characterized by expression of pro-inflammatory cytokines and interferon-stimulated genes. scRNA-seq analysis of lung homogenates identified a hyper-inflammatory monocyte profile. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes infiltration of classical monocytes into the lung and expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

Bromelain Inhibits SARS-CoV-2 Infection in VeroE6 Cells (preprint)

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The initial interaction between Transmembrane Serine Protease 2 (TMPRSS2) primed SARS-CoV-2 spike (S) protein and host cell receptor angiotensin-converting enzyme 2 (ACE-2) is a pre-requisite step for this novel coronavirus pathogenesis. Here, we expressed a GFP-tagged SARS-CoV-2 S-Ectodomain in Tni insect cells. That contained sialic acid-enriched N- and O-glycans. Surface resonance plasmon (SPR) and Luminex assay showed that the purified S-Ectodomain binding to human ACE-2 and immunoreactivity with COVID-19 positive samples. We demonstrate that bromelain (isolated from pineapple stem and used as a dietary supplement) treatment diminishes the expression of ACE-2 and TMPRSS2 in VeroE6 cells and dramatically lowers the expression of S-Ectodomain. Importantly, bromelain treatment reduced the interaction between S-Ectodomain and VeroE6 cells. Most importantly, bromelain treatment significantly diminished the SARS-CoV-2 infection in VeroE6 cells. Altogether, our results suggest that bromelain or bromelain rich pineapple stem may be used as an antiviral against COVID-19.

Evaluation of nafamostat mesylate safety and inhibition of SARS-CoV-2 replication using a 3-dimensional human airway epithelia model (preprint)

In the current COVID-19 pandemic context, Ensysce and its subsidiary Covistat have been working to repurpose nafamostat mesylate as an effective oral and inhalation treatment against SARS-CoV-2 infection. Prior reports used cell lines to demonstrate the antiviral potential of nafamostat against coronaviral infections and determined its mechanism of action through inhibition of transmembrane protease serine 2 (TMPRSS2). We selected a biologically relevant pre-clinical experimental model of SARS-CoV-2 lung infection using a 3D human reconstituted airway epithelial model of nasal origin to characterize the effects of nafamostat on tissue-level cellular ultrastructure and viral infection kinetics. Our results confirm the not only the relevance of this model for the preclinical evaluation of safety and efficacy of antiviral candidates, but also the highly potent nature of nafamostat SARS-CoV-2 antiviral activity. The studies described herein provided evidence demonstrating the therapeutic potential of nafamostat against COVID-19, as well as its safety upon exposure to lung airway cellular.

Lopinavir-ritonavir is not an effective inhibitor of the main protease activity of SARS-CoV-2 in vitro (preprint)

COVID-19 has caused over 900,000 deaths worldwide as of September 2020, and effective medicines are urgently needed. Lopinavir was identified as an inhibitor of the HIV protease, and a lopinavir-ritonavir combination therapy was reported to be beneficial for the treatment of SARS and MERS. However, recent clinical tests could not prove that lopinavir-ritonavir therapy was an effective treatment for COVID-19. In this report, we examined the effect of lopinavir and ritonavir to the activity of the purified main protease (Mpro) protein of SARS-CoV-2, the causative virus of COVID-19. Unexpectedly, lopinavir and ritonavir did not inhibit Mpro activity. These results will aid the drug candidate selection for ongoing and future COVID-19 clinical trials.

SARS-CoV-2 protein Nsp1 alters actomyosin cytoskeleton and phenocopies arrhythmogenic cardiomyopathy-related PKP2 mutant (preprint)

Mutations in desmosomal Plakophilin-2 (PKP2) are the most prevalent drivers of arrhythmogenic-cardiomyopathy (ACM) and a common cause of sudden death in young athletes. However, partner proteins that elucidate PKP2 cellular mechanism behind cardiac dysfunction in ACM are mostly unknown. Here we identify the actin-based motor proteins Myh9 and Myh10 as key PKP2 interactors and demonstrate that expression of the ACM-related PKP2 mutant R735X alters actin fiber organization and cell mechanical stiffness. We also show that SARS-CoV-2 Nsp1 protein acts similarly to this known pathogenic R735X mutant, altering the actomyosin component distribution on cardiac cells. Our data reveal that Nsp1 hijacks PKP2 into the cytoplasm and mimics the effect of delocalized R735X mutant. These results demonstrate that cytoplasmic PKP2 drives actomyosin deregulation and structural collapse, validating a critical role of PKP2 localization in the regulation of actomyosin architecture. The fact that Nsp1 and R735X share similar phenotypes also suggests that direct SARS-CoV-2 heart infection could induce a transient ACM-like disease in COVID-19 patients, which may contribute to right ventricle dysfunction, observed in patients with poor prognosis.

High affinity modified ACE2 receptors prevent SARS-CoV-2 infection (preprint)

The SARS-CoV-2 spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor via receptor binding domain (RBD) to enter into the cell. Inhibiting this interaction is a main approach to block SARS-CoV-2 infection and it is required to have high affinity to RBD independently of viral mutation for effective protection. To this end, we engineered ACE2 to enhance the affinity with directed evolution in human cells. Three cycles of random mutation and cell sorting achieved more than 100-fold higher affinity to RBD than wild-type ACE2. The extracellular domain of modified ACE2 fused to the Fc region of the human immunoglobulin IgG1 had stable structure and neutralized SARS-CoV-2 pseudotyped lentivirus and authentic virus with more than 100-fold lower concentration than wild-type. Engineering ACE2 decoy receptors with directed evolution is a promising approach to develop a SARS-CoV-2 neutralizing drug that has affinity comparable to monoclonal antibodies yet displaying resistance to escape mutations of virus.

Baricitinib treatment resolves lower airway inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques (preprint)

Effective therapeutics aimed at mitigating COVID-19 symptoms are urgently needed. SARS-CoV- 2 induced hypercytokinemia and systemic inflammation are associated with disease severity. Baricitinib, a clinically approved JAK1/2 inhibitor with potent anti-inflammatory properties is currently being investigated in COVID-19 human clinical trials. Recent reports suggest that baricitinib may also have antiviral activity in limiting viral endocytosis. Here, we investigated the immunologic and virologic efficacy of baricitinib in a rhesus macaque model of SARS-CoV-2 infection. Viral shedding measured from nasal and throat swabs, bronchoalveolar lavages and tissues was not reduced with baricitinib. Type I IFN antiviral responses and SARS-CoV-2 specific T cell responses remained similar between the two groups. Importantly, however, animals treated with baricitinib showed reduced immune activation, decreased infiltration of neutrophils into the lung, reduced NETosis activity, and more limited lung pathology. Moreover, baricitinib treated animals had a rapid and remarkably potent suppression of alveolar macrophage derived production of cytokines and chemokines responsible for inflammation and neutrophil recruitment. These data support a beneficial role for, and elucidate the immunological mechanisms underlying, the use of baricitinib as a frontline treatment for severe inflammation induced by SARS-CoV-2 infection.

Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are non-specific promiscuous SARS-CoV-2 main protease inhibitors (preprint)

There is an urgent need for vaccines and antiviral drugs to combat the COVID-19 pandemic. Encouraging progress has been made in developing antivirals targeting SARS-CoV-2, the etiological agent of COVID-19. Among the drug targets being investigated, the viral main protease (Mpro) is one of the most extensively studied drug targets. Mpro is a cysteine protease that hydrolyzes the viral polyprotein at more than 11 sites and it is highly conserved among coronaviruses. In addition, Mpro has a unique substrate preference for glutamine in the P1 position. Taken together, it appears that Mpro inhibitors can achieve both broad-spectrum antiviral activity and a high selectivity index. Structurally diverse compounds have been reported as Mpro inhibitors, with several of which also showed antiviral activity in cell culture. In this study, we investigated the mechanism of action of six previously reported Mpro inhibitors, ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 using a consortium of techniques including FRET-based enzymatic assay, thermal shift assay, native mass spectrometry, cellular antiviral assays, and molecular dynamics simulations. Collectively, the results showed that the inhibition of Mpro by these six compounds is non-specific and the inhibition is abolished or greatly reduced with the addition of reducing reagent DTT. In the absence of DTT, these six compounds not only inhibit Mpro, but also a panel of viral cysteine proteases including SARS-CoV-2 papain-like protease, the 2Apro and 3Cpro from enterovirus A71 (EV-A71) and EV-D68. However, none of the compounds inhibits the viral replication of EV-A71 or EV-D68, suggesting that the enzymatic inhibition potency IC50 values obtained in the absence of DTT cannot be used to faithfully predict their cellular antiviral activity. Overall, we provide compelling evidence suggesting that ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 are non-specific SARS-CoV-2 Mpro inhibitors, and urge the scientific community to be stringent with hit validation.

Positive selection within the genomes of SARS-CoV-2 and other Coronaviruses independent of impact on protein function (preprint)

Background: The emergence of a novel coronavirus (SARS-CoV-2) associated with severe acute respiratory disease (COVID-19) has prompted efforts to understand the genetic basis for its unique characteristics and its jump from non-primate hosts to humans. Tests for positive selection can identify apparently nonrandom patterns of mutation accumulation within genomes, highlighting regions where molecular function may have changed during the origin of a species. Several recent studies of the SARS-CoV-2 genome have identified signals of conservation and positive selection within the gene encoding Spike protein based on the ratio of synonymous to nonsynonymous substitution. Such tests cannot, however, detect changes in the function of RNA molecules. Methods: Here we apply a test for branch-specific oversubstitution of mutations within narrow windows of the genome without reference to the genetic code. Results: We recapitulate the finding that the gene encoding Spike protein has been a target of both purifying and positive selection. In addition, we find other likely targets of positive selection within the genome of SARS-CoV-2, specifically within the genes encoding Nsp4 and Nsp16. Homology-directed modeling indicates no change in either Nsp4 or Nsp16 protein structure relative to the most recent common ancestor. Thermodynamic modeling of RNA stability and structure, however, indicates that RNA secondary structure within both genes in the SARS-CoV-2 genome differs from those of RaTG13, the reconstructed common ancestor, and Pan-CoV-GD (Guangdong). These SARS-CoV-2-specific mutations may affect molecular processes mediated by the positive or negative RNA molecules, including transcription, translation, RNA stability, and evasion of the host innate immune system. Our results highlight the importance of considering mutations in viral genomes not only from the perspective of their impact on protein structure, but also how they may impact other molecular processes critical to the viral life cycle.

Type I and Type III IFN Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures (preprint)

The newly emerged human coronavirus, SARS-CoV-2, has caused a pandemic of respiratory illness. The innate immune response is critical for protection against Coronaviruses. However, little is known about the interplay between the innate immune system and SARS-CoV-2. Here, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by pro-inflammatory cytokines and chemokine induction, including IL-6, TNF, CXCL8. We also identified NF-{kappa}B and ATF4 transcription factors as key drivers of this pro-inflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III IFN induction during SARS-CoV-2 infection. Pre-treatment or post-treatment with type I and III IFNs dramatically reduced virus replication in pHAE cultures and this corresponded with an upregulation of antiviral effector genes. Our findings demonstrate that SARS-CoV-2 induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients. IMPORTANCEThe current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines or antivirals approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. SARS-CoV-2 infection of primary human airway epithelial cultures induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients.