Targeting C5aR1 signaling reduced neutrophil extracellular traps and ameliorates COVID-19 pathology (preprint)

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions, and plays immunopathological roles in inflammatory diseases, we investigated whether C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill COVID-19 patients compared to patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular trap (NET)s-dependent immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonist of C5aR1 could be useful for COVID-19 treatment. Keywords: COVID-19, C5aR1, C5a, SARS-CoV-2, Myeloid cells, Neutrophils, NETs

Increased mTOR signaling, impaired autophagic flux and cell-to-cell viral transmission are hallmarks of SARS-CoV-2 infection. (preprint)

The COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has two characteristics that distinguish it from other viral infections. It affects more severely people with pre-existing comorbidities and viral load peaks prior to the onset of the symptoms. Investigating factors that could contribute to these characteristics, we found increased mTOR signaling and suppressed genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2. Transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients revealed that COVID-19 severity is associated with increased expression of genes related to mTOR signaling and decreased expression of genes related to au-tophagy, lysosome function, and vesicle fusion. SARS-CoV-2 infection in Vero E6 cells also re-sulted in virus retention inside the cells and trafficking of virus-bearing vesicles between neighboring cells. Our findings support a scenario where SARS-CoV-2 benefits from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling, which might relate to undetectable proliferation and evasion of the immune system.

Multi-layered transcriptomic analyses reveal an immunological overlap between COVID-19 and hemophagocytic lymphohistiocytosis associated with disease severity (preprint)

Clinical and hyperinflammatory overlap between COVID-19 and hemophagocytic lymphohistiocytosis (HLH) has been reported. However, the underlying mechanisms are unclear. Here we show that COVID-19 and HLH have an overlap of signaling pathways and gene signatures commonly dysregulated, which were defined by investigating the transcriptomes of 1253 subjects (controls, COVID-19, and HLH patients) using microarray, bulk RNA-sequencing (RNAseq), and single-cell RNAseq (scRNAseq). COVID-19 and HLH share pathways involved in cytokine and chemokine signaling as well as neutrophil-mediated immune responses that associate with COVID-19 severity. These genes are dysregulated at protein level across several COVID-19 studies and form an interconnected network with differentially expressed plasma proteins which converge to neutrophil hyperactivation in COVID-19 patients admitted to intensive care unit. scRNAseq analysis indicated that these genes are specifically upregulated across different leukocyte populations, including lymphocyte subsets and immature neutrophils. Artificial intelligence modeling confirmed the strong association of these genes with COVID-19 severity. Thus, our work indicates putative therapeutic pathways for intervention.

Immunodominant B cell epitope in SARS-CoV-2 RBD comprises a B.1.351 and P.1 mutation hotspot: implications for viral spread and antibody escape (preprint)

Recent SARS-CoV-2 variants pose important concerns due to their higher transmissibility (1) and escape (2) from previous infections or vaccine-induced neutralizing antibodies (nAb). The receptor binding domain (RBD) of the Spike protein is a major nAb target (3), but data on its B cell epitopes are still lacking. Using a peptide microarray, we identified an immunodominant epitope (S415-429) recognized by 68% of sera from 71 convalescent Brazilians infected with the ancestral variant. In contrast with previous studies, we have identified a linear IgG and IgA antibody binding epitope within the RBD. IgG and IgA antibody levels for this epitope positively correlated with nAb titers, suggesting a potential target of antibody neutralizing activity. Interestingly, this immunodominant RBD region harbors the mutation hotspot site K417 present in P.1 (K417T) and B.1.351 (K417N) variants. In silico simulation analyses indicate impaired RBD binding to nAb in both variants and that glycosylation in the B.1.351 417N could further hinder antibody binding as compared to the K417T mutation in P.1. This is in line with published data showing that nAb from either convalescents or anti-CoV-2 vaccinees are less effective towards B.1.351 than for P.1. Our data support the occurrence of immune pressure and selection involving this immunodominant epitope that may have critically contributed to the recent COVID-19 marked rise in Brazil and South Africa, and pinpoint a potential additional immune escape mechanism for SARS-CoV-2.

Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory programming and continual clearance of apoptotic cells (preprint)

COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV2-infected apoptotic cells (AC) exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence that monocytes from severe COVID-19 patients express reduced levels of efferocytic receptors and fail to uptake AC. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppress macrophage anti-inflammation and efficient tissue repair programs and provide mechanistic insights for the pathogenesis of the hyperinflammation and extensive tissue damage associated with COVID-19.

SARS-CoV-2 Uses CD4 to Infect T Helper Lymphocytes (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as coronavirus disease-2019 (COVID-19). SARS-CoV-2 infects the lungs and may cause several immune-related complications such as lymphocytopenia and cytokine storm which are associated with the severity of the disease and predict mortality . The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is not fully understood. Here we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS- CoV-2 in T helper cells in a mechanism that also requires ACE2 and TMPRSS2. Once inside T helper cells, SARS-CoV-2 assembles viral factories, impairs cell function and may cause cell death. SARS-CoV-2 infected T helper cells express higher amounts of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may explain the poor adaptive immune response of many COVID- 19 patients.

A Tethered Ligand Assay to Probe the SARS-CoV-2 ACE2 Interaction under Constant Force (preprint)

The current COVID-19 pandemic has a devastating global impact and is caused by the SARS-CoV-2 virus. SARS-CoV-2 attaches to human host cells through interaction of its receptor binding domain (RBD) located on the viral Spike (S) glycoprotein with angiotensin converting enzyme-2 (ACE2) on the surface of host cells. RBD binding to ACE2 is a critical first step in SARS-CoV-2 infection. Viral attachment occurs in dynamic environments where forces act on the binding partners and multivalent interactions play central roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using specific molecular handles, we tether the fusion proteins between a functionalized flow cell surface and magnetic beads in magnetic tweezers. We observe repeated interactions of RBD and ACE2 under constant loads and can fully quantify the force dependence and kinetics of the binding interaction. Our results suggest that the SARS-CoV-2 ACE2 interaction has higher mechanical stability, a larger free energy of binding, and a lower off-rate than that of SARS-CoV-1, the causative agents of the 2002-2004 SARS outbreak. In the absence of force, the SARS-CoV-2 RBD rapidly (within [≤]1 ms) engages the ACE2 receptor if held in close proximity and remains bound to ACE2 for 400-800 s, much longer than what has been reported for other viruses engaging their cellular receptors. We anticipate that our assay will be a powerful tool investigate the roles of mutations in the RBD that might alter the infectivity of the virus and to test the modes of action of neutralizing antibodies and other agents designed to block RBD binding to ACE2 that are currently developed as potential COVID-19 therapeutics.

Discovery and Development of Human SARS-CoV-2 Neutralizing Antibodies using an Unbiased Phage Display Library Approach (preprint)

SARS-CoV-2 neutralizing antibodies represent an important component of the ongoing search for effective treatment of and protection against COVID-19. We report here on the use of a naive phage display antibody library to identify a panel of fully human SARS-CoV-2 neutralizing antibodies. Following functional profiling in vitro against an early pandemic isolate as well as a recently emerged isolate bearing the D614G Spike mutation, the clinical candidate antibody, STI-1499, and the affinity-engineered variant, STI-2020, were evaluated for in vivo efficacy in the Syrian golden hamster model of COVID-19. Both antibodies demonstrated potent protection against the pathogenic effects of the disease and a dose-dependent reduction of virus load in the lungs, reaching undetectable levels following a single dose of 500 micrograms of STI-2020. These data support continued development of these antibodies as therapeutics against COVID-19 and future use of this approach to address novel emerging pandemic disease threats.

No evidence that plasmablasts transdifferentiate into developing neutrophils in severe COVID-19 disease (preprint)

A recent study by Wilk et al. of the transcriptome of peripheral blood mononuclear cells (PBMCs) in seven patients hospitalized with COVID-19 described a population of 'developing neutrophils' that were 'phenotypically related by dimensionality reduction' to plasmablasts, and that these two cell populations represent a 'linear continuum of cellular phenotype'. The authors suggest that, in the setting of acute respiratory distress syndrome (ARDS) secondary to severe COVID-19, a 'differentiation bridge from plasmablasts to developing neutrophils' connected these distantly related cell types. This conclusion is controversial as it appears to violate several basic principles in cell biology relating to cell lineage identity and fidelity. Correctly classifying cells and their developmental history is an important issue in cell biology and we suggest that this conclusion is not supported by the data as we show here that: (1) regressing out covariates such as unique molecular identifiers (UMIs) can lead to overfitting; and (2) that UMAP embeddings may reflect the expression of similar genes but not necessarily direct cell lineage relationships.

Identification of TMEM106B as proviral host factor for SARS-CoV-2 (preprint)

The ongoing COVID-19 pandemic is responsible for worldwide economic damage and nearly one million deaths. Potent drugs for the treatment of severe SARS-CoV-2 infections are not yet available. To identify host factors that support coronavirus infection, we performed genome-wide functional genetic screens with SARS-CoV-2 and the common cold virus HCoV-229E in non-transgenic human cells. These screens identified PI3K type 3 as a potential drug target against multiple coronaviruses. We discovered that the lysosomal protein TMEM106B is an important host factor for SARS-CoV-2 infection. Furthermore, we show that TMEM106B is required for replication in multiple human cell lines derived from liver and lung and is expressed in relevant cell types in the human airways. Our results identify new coronavirus host factors that may potentially serve as drug targets against SARS-CoV-2 or to quickly combat future zoonotic coronavirus outbreaks.

Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins (preprint)

Membrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.

The influence of pH on SARS-CoV-2 infection and COVID-19 severity (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect a broad range of human tissues by using the host receptor angiotensin-converting enzyme 2 (ACE2). Individuals with comorbidities associated with severe COVID-19 display higher levels of ACE2 in the lungs compared to those without comorbidities, and conditions such as cell stress, elevated glucose levels and hypoxia may also increase the expression of ACE2. Here we showed that patients with Barrett's esophagus (BE) have a higher expression of ACE2 in BE tissues compared to normal squamous esophagus, and that the lower pH associated with BE may drive this increase in expression. Human primary monocytes cultured in reduced pH displayed increased ACE2 expression and viral load upon SARS-CoV-2 infection. We also showed in two independent cohorts of COVID-19 patients that previous use of proton pump inhibitors is associated with 2- to 3-fold higher risk of death compared to those not using the drugs. Our work suggests that pH has a great influence on SARS-CoV-2 Infection and COVID-19 severity.

In-depth Analysis of Laboratory Parameters Reveals the Interplay Between Sex, Age and Systemic Inflammation in Individuals with COVID-19 (preprint)

Introduction: The progression and severity of the coronavirus disease 2019 (COVID-19), an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), varies significantly in the population. While the hallmarks of SARS-CoV-2 and severe COVID-19 within routine laboratory parameters are emerging, little is known about the impact of sex and age on these profiles. Methods: We performed multidimensional analysis of millions of records of laboratory parameters and diagnostic tests for 178,887 individuals, of which 33,266 tested positive for SARS-CoV-2. These included complete blood cell count, electrolytes, metabolites, arterial blood gases, enzymes, hormones, cancer biomarkers, and others. Results: COVID-19 induced more alterations in the laboratory parameters in males compared to females between 13 and 60 years old, in contrast to older individuals, where several parameters were altered by COVID-19 in both men and women. Biomarkers of inflammation, such as C-reactive protein (CRP) and ferritin, were increased especially in older men with COVID-19, whereas other markers such as abnormal liver function tests were common across several age groups, except for young women. Low peripheral blood basophils and eosinophils were also more common in the elderly with COVID-19. Both male and female COVID-19 patients admitted to the intensive care unit (ICU) displayed alterations in the coagulation system, and higher levels of neutrophils, CRP, lactate dehydrogenase (LDH), among others. Discussion: Our study uncovers the laboratory profile of a large cohort of COVID-19 patients that underly discrepancies influenced by aging and biological sex. These profiles directly link COVID-19 disease presentation to an intricate interplay between sex, age and the immune response.

Monitoring social distancing and SARS-CoV-2 transmission in Brazil using cell phone mobility data (preprint)

Social distancing measures have emerged as the predominant intervention for containing the spread of COVID-19, but evaluating adherence and effectiveness remains a challenge. We assessed the relationship between aggregated mobility data collected from mobile phone users and the time-dependent reproduction number R(t), using severe acute respiratory illness (SARI) cases reported by Sao Paulo and Rio de Janeiro. We found that the proportion of individuals staying home all day (isolation index) had a strong inverse correlation with R(t) (rho

OUTBREAK: A user-friendly georeferencing online tool for disease surveillance (preprint)

The current COVID-19 pandemic has already claimed more than 100,000 victims and it will cause more deaths in the coming months. Tools that can track the number and locations of cases are critical for surveillance and can help in making policy decisions for controlling the outbreak. The current surveillance web-based dashboards run on proprietary platforms, which are often expensive and require specific computational knowledge. We present a new tool (OUTBREAK) for studying and visualizing epidemiological data. It permits even non-specialist users to input data most conveniently and track outbreaks in real-time. This tool has the potential to guide and help health authorities to intervene and minimize the effects of the outbreaks. It is freely available at http://outbreak.sysbio.tools/.

ACE2 Expression is Increased in the Lungs of Patients with Comorbidities Associated with Severe COVID-19 (preprint)

The pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in several thousand deaths worldwide in just a few months. Patients who died from Coronavirus disease 2019 (COVID-19) often had comorbidities, such as hypertension, diabetes, and chronic obstructive lung disease. The angiotensin-converting enzyme 2 (ACE2) was identified as a crucial factor that facilitates SARS-CoV2 to bind and enter host cells. To date, no study has assessed the ACE2 expression in the lungs of patients with these diseases. Here, we analyzed over 700 lung transcriptome samples of patients with comorbidities associated with severe COVID-19 and found that ACE2 was highly expressed in these patients, compared to control individuals. This finding suggests that patients with such comorbidities may have higher chances of developing severe COVID-19. We also found other genes, such as RAB1A, that can be important for SARS-CoV-2 infection in the lung. Correlation and network analyses revealed many potential regulators of ACE2 in the human lung, including genes related to histone modifications, such as HAT1, HDAC2, and KDM5B. In fact, epigenetic marks found in ACE2 locus were compatible to with those promoted by KDM5B. Our systems biology approach offers a possible explanation for increase of COVID-19 severity in patients with certain comorbidities.