Antibody-Dependent Enhancement of SARS-CoV-2 Infection of Human Immune Cells: In Vitro Assessment Provides Insight in COVID-19 Pathogenesis.

Patients with COVID-19 generally raise antibodies against SARS-CoV-2 following infection, and the antibody level is positively correlated to the severity of disease. Whether the viral antibodies exacerbate COVID-19 through antibody-dependent enhancement (ADE) is still not fully understood. Here, we conducted in vitro assessment of whether convalescent serum enhanced SARS-CoV-2 infection or induced excessive immune responses in immune cells. Our data revealed that SARS-CoV-2 infection of primary B cells, macrophages and monocytes, which express variable levels of FcγR, could be enhanced by convalescent serum from COVID-19 patients. We also determined the factors associated with ADE, and found which showed a time-dependent but not viral-dose dependent manner. Furthermore, the ADE effect is not associated with the neutralizing titer or RBD antibody level when testing serum samples collected from different patients. However, it is higher in a medium level than low or high dilutions in a given sample that showed ADE effect, which is similar to dengue. Finally, we demonstrated more viral genes or dysregulated host immune gene expression under ADE conditions compared to the no-serum infection group. Collectively, our study provides insight into the understanding of an association of high viral antibody titer and severe lung pathology in severe patients with COVID-19.

The interferon landscape along the respiratory tract impacts the severity of COVID-19.

Severe coronavirus disease 2019 (COVID-19) is characterized by overproduction of immune mediators, but the role of interferons (IFNs) of the type I (IFN-I) or type III (IFN-III) families remains debated. We scrutinized the production of IFNs along the respiratory tract of COVID-19 patients and found that high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity. Production of specific IFN-III, but not IFN-I, members denotes patients with a mild pathology and efficiently drives the transcription of genes that protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In contrast, compared to subjects with other infectious or noninfectious lung pathologies, IFNs are overrepresented in the lower airways of patients with severe COVID-19 that exhibit gene pathways associated with increased apoptosis and decreased proliferation. Our data demonstrate a dynamic production of IFNs in SARS-CoV-2-infected patients and show IFNs play opposing roles at distinct anatomical sites.

Genome-wide screening of SARS-CoV-2 infection-related genes based on the blood leukocytes sequencing data set of patients with COVID-19.

Coronavirus disease 2019 (COVID-19) is a global epidemic disease caused by a novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing serious adverse effects on human health. In this study, we obtained a blood leukocytes sequencing data set of COVID-19 patients from the GEO database and obtained differentially expressed genes (DEGs). We further analyzed these DEGs by protein-protein interaction analysis and Gene Ontology enrichment analysis and identified the DEGs closely related to SARS-CoV-2 infection. Then, we constructed a six-gene model (comprising IFIT3, OASL, USP18, XAF1, IFI27, and EPSTI1) by logistic regression analysis and calculated the area under the ROC curve (AUC) for the diagnosis of COVID-19. The AUC values of the training group, testing group, and entire group were 0.930, 0.914, and 0.921, respectively. The six genes were highly expressed in patients with COVID-19 and positively correlated with the expression of SARS-CoV-2 invasion-related genes (ACE2, TMPRSS2, CTSB, and CTSL). The risk score calculated by this model was also positively correlated with the expression of TMPRSS2, CTSB, and CTSL, indicating that the six genes were closely related to SARS-CoV-2 infection. In conclusion, we comprehensively analyzed the functions of DEGs in the blood leukocytes of patients with COVID-19 and constructed a six-gene model that may contribute to the development of new diagnostic and therapeutic ideas for COVID-19. Moreover, these six genes may be therapeutic targets for COVID-19.

SARS-CoV-2 Spike Protein Induces Paracrine Senescence and Leukocyte Adhesion in Endothelial Cells.

Increased mortality in COVID-19 cases is often associated with microvascular complications. We have recently shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein promotes an inflammatory cytokine interleukin 6 (IL-6)/IL-6R-induced trans signaling response and alarmin secretion. Virus-infected or spike-transfected human epithelial cells exhibited an increase in senescence, with a release of senescence-associated secretory phenotype (SASP)-related inflammatory molecules. Introduction of the bromodomain-containing protein 4 (BRD4) inhibitor AZD5153 to senescent epithelial cells reversed this effect and reduced SASP-related inflammatory molecule release in TMNK-1 or EAhy926 (representative human endothelial cell lines), when cells were exposed to cell culture medium (CM) derived from A549 cells expressing SARS-CoV-2 spike protein. Cells also exhibited a senescence phenotype with enhanced p16, p21, and senescence-associated ß-galactosidase (SA-ß-Gal) expression and triggered SASP pathways. Inhibition of IL-6 trans signaling by tocilizumab and inhibition of inflammatory receptor signaling by the Bruton's tyrosine kinase (BTK) inhibitor zanubrutinib, prior to exposure of CM to endothelial cells, inhibited p21 and p16 induction. We also observed an increase in reactive oxygen species (ROS) in A549 spike-transfected and endothelial cells exposed to spike-transfected CM. ROS generation in endothelial cell lines was reduced after treatment with tocilizumab and zanubrutinib. Cellular senescence was associated with an increased level of the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), which have in vitro leukocyte attachment potential. Inhibition of senescence or SASP function prevented VCAM-1/ICAM-1 expression and leukocyte attachment. Taken together, we identified that human endothelial cells exposed to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers, leading to enhanced leukocyte adhesion. IMPORTANCE The present study was aimed at examining the underlying mechanism of extrapulmonary manifestations of SARS-CoV-2 spike protein-associated pathogenesis, with the notion that infection of the pulmonary epithelium can lead to mediators that drive endothelial dysfunction. We utilized SARS-CoV-2 spike protein expression in cultured human hepatocytes (Huh7.5) and pneumocytes (A549) to generate conditioned culture medium (CM). Endothelial cell lines (TMNK-1 or EAhy926) treated with CM exhibited an increase in cellular senescence markers by a paracrine mode and led to leukocyte adhesion. Overall, the link between these responses in endothelial cell senescence and a potential contribution to microvascular complication in productively SARS-CoV-2-infected humans is implicated. Furthermore, the use of inhibitors (BTK, IL-6, and BRD4) showed a reverse effect in the senescent cells. These results may support the selection of potential adjunct therapeutic modalities to impede SARS-CoV-2-associated pathogenesis.

Cell Analysis from Dried Blood Spots: New Opportunities in Immunology, Hematology, and Infectious Diseases.

Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50-70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS-CoV-2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide.

A streamlined whole blood CyTOF workflow defines a circulating immune cell signature of COVID-19.

Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 parameters at single-cell resolution. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining, and data acquisition protocols can all introduce technical variation that can confound integrative analyses of large data-sets of samples processed across multiple labs. Here, we present a streamlined whole blood CyTOF workflow which addresses many of these sources of experimental variation and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample-processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA), a dry tube 30-marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We apply this workflow to characterize 184 whole blood samples collected longitudinally from a cohort of 72 hospitalized COVID-19 patients and healthy controls, highlighting dynamic disease-associated changes in circulating immune cell frequency and phenotype.

SARS-CoV-2 infection is associated with a pro-thrombotic platelet phenotype.

Novel coronavirus disease 2019 (COVID-19) is associated with a hypercoagulable state, characterized by abnormal coagulation parameters and by increased incidence of cardiovascular complications. With this study, we aimed to investigate the activation state and the expression of transmembrane proteins in platelets of hospitalized COVID-19 patients. We investigated transmembrane proteins expression with a customized mass cytometry panel of 21 antibodies. Platelets of 8 hospitalized COVID-19 patients not requiring intensive care support and without pre-existing conditions were compared to platelets of healthy controls (11 donors) with and without in vitro stimulation with thrombin receptor-activating peptide (TRAP). Mass cytometry of non-stimulated platelets detected an increased surface expression of activation markers P-Selectin (0.67 vs. 1.87 median signal intensity for controls vs. patients, p = 0.0015) and LAMP-3 (CD63, 0.37 vs. 0.81, p = 0.0004), the GPIIb/IIIa complex (4.58 vs. 5.03, p < 0.0001) and other adhesion molecules involved in platelet activation and platelet-leukocyte interactions. Upon TRAP stimulation, mass cytometry detected a higher expression of P-selectin in COVID-19 samples compared to controls (p < 0.0001). However, we observed a significantly reduced capacity of COVID-19 platelets to increase the expression of activation markers LAMP-3 and P-Selectin upon stimulation with TRAP. We detected a hyperactivated phenotype in platelets during SARS-CoV-2 infection, consisting of highly expressed platelet activation markers, which might contribute to the hypercoagulopathy observed in COVID-19. In addition, several transmembrane proteins were more highly expressed compared to healthy controls. These findings support research projects investigating antithrombotic and antiplatelet treatment regimes in COVID-19 patients, and provide new insights on the phenotypical platelet expression during SARS-CoV-2 infection.

COVID-19 and hematology findings based on the current evidences: A puzzle with many missing pieces.

In December 2019, a new type of coronavirus was detected for the first time in Wuhan, Hubei Province, China. According to the reported data, the emerging coronavirus has spread worldwide, infecting more than fifty-seven million individuals, leading to more than one million deaths. The current study aimed to review and discuss the hematological findings of COVID-19. Laboratory changes and hematologic abnormalities have been reported repeatedly in COVID-19 patients. WBC count and peripheral blood lymphocytes are normal or slightly reduced while these indicators may change with the progression of the disease. In addition, several studies demonstrated that decreased hemoglobin levels in COVID-19 patients were associated with the severity of the disease. Moreover, thrombocytopenia, which is reported in 5%-40% of patients, is known to be associated with poor prognosis of the disease. COVID-19 can present with various hematologic manifestations. In this regard, accurate evaluation of laboratory indicators at the beginning and during COVID-19 can help physicians to adjust appropriate treatment and provide special and prompt care for those in need.

Ifit2 deficiency restricts microglial activation and leukocyte migration following murine coronavirus (m-CoV) CNS infection.

The interferon-induced tetratricopeptide repeat protein (Ifit2) protects mice from lethal neurotropic viruses. Neurotropic coronavirus MHV-RSA59 infection of Ifit2-/- mice caused pronounced morbidity and mortality accompanied by rampant virus replication and spread throughout the brain. In spite of the higher virus load, induction of many cytokines and chemokines in the brains of infected Ifit2-/- mice were similar to that in wild-type mice. In contrast, infected Ifit2-/- mice revealed significantly impaired microglial activation as well as reduced recruitment of NK1.1 T cells and CD4 T cells to the brain, possibly contributing to the lack of viral clearance. These two deficiencies were associated with a lower level of microglial expression of CX3CR1, the receptor of the CX3CL1 (Fractalkine) chemokine, which plays a critical role in both microglial activation and leukocyte recruitment. The above results uncovered a new potential role of an interferon-induced protein in immune protection.

An Antioxidant Enzyme Therapeutic for COVID-19.

The COVID-19 pandemic has taken a significant toll on people worldwide, and there are currently no specific antivirus drugs or vaccines. Herein it is a therapeutic based on catalase, an antioxidant enzyme that can effectively breakdown hydrogen peroxide and minimize the downstream reactive oxygen species, which are excessively produced resulting from the infection and inflammatory process, is reported. Catalase assists to regulate production of cytokines, protect oxidative injury, and repress replication of SARS-CoV-2, as demonstrated in human leukocytes and alveolar epithelial cells, and rhesus macaques, without noticeable toxicity. Such a therapeutic can be readily manufactured at low cost as a potential treatment for COVID-19.

The role of IgG Fc receptors in antibody-dependent enhancement.

Antibody-dependent enhancement (ADE) is a mechanism by which the pathogenesis of certain viral infections is enhanced in the presence of sub-neutralizing or cross-reactive non-neutralizing antiviral antibodies. In vitro modelling of ADE has attributed enhanced pathogenesis to Fcγ receptor (FcγR)-mediated viral entry, rather than canonical viral receptor-mediated entry. However, the putative FcγR-dependent mechanisms of ADE overlap with the role of these receptors in mediating antiviral protection in various viral infections, necessitating a detailed understanding of how this diverse family of receptors functions in protection and pathogenesis. Here, we discuss the diversity of immune responses mediated upon FcγR engagement and review the available experimental evidence supporting the role of FcγRs in antiviral protection and pathogenesis through ADE. We explore FcγR engagement in the context of a range of different viral infections, including dengue virus and SARS-CoV, and consider ADE in the context of the ongoing SARS-CoV-2 pandemic.

Molecular profiling of immune cell-enriched Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) interacting protein USP13.

AIMS: Coronavirus disease 2019 (COVID-19), which is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is a major health concern worldwide. Due to the lack of specific medication and vaccination, drug-repurposing attempts has emerged as a promising approach and identified several human proteins interacting with the virus. This study aims to provide a comprehensive molecular profiling of the immune cell-enriched SARS-CoV-2 interacting protein USP13. MATERIALS AND METHODS: The list of immune cell-enriched proteins interacting with SARS-CoV-2 was retrieved from The Human Protein Atlas. Genomic alterations were identified using cBioPortal. Survival analysis was performed via Kaplan-Meier Plotter. Analyses of protein expression and tumor infiltration levels were carried out by TIMER. KEY FINDINGS: 14 human proteins that interact with SARS-CoV-2 were enriched in immune cells. Among these proteins, USP13 had the highest frequency of genomic alterations. Higher USP13 levels were correlated with improved survival in breast and lung cancers, while resulting in poor prognosis in ovarian and gastric cancers. Furthermore, copy number variations of USP13 significantly affected the infiltration levels of distinct subtypes of immune cells in head & neck, lung, ovarian and stomach cancers. Although our results suggested a tumor suppressor role for USP13 in lung cancer, in other cancers, its role seemed to be context-dependent. SIGNIFICANCE: It is critical to identify and characterize human proteins that interact with SARS-CoV-2 in order to have a better understanding of the disease and to develop better therapies/vaccines. Here, we provided a comprehensive molecular profiling the immune cell-enriched SARS-CoV-2 interacting protein USP13, which will be useful for future studies.

Clinical features in 52 patients with COVID-19 who have increased leukocyte count: a retrospective analysis.

Recent reports have showed that a proportion of patients with Coronavirus Disease 2019 (COVID-19) presented elevated leukocyte count. Clinical data about these patients is scarce. We aimed to evaluate the clinical findings of patients with COVID-19 who have increased leukocyte at admission. We retrospectively collected the clinical data on the 52 patients who have increased leukocyte count at admission from the 619 patients with confirmed COVID-19 who had pneumonia with abnormal features on chest CT scan in Renmin Hospital of Wuhan University in Wuhan, China, from February 3 to March 3, 2020. The mean age of the 52 patients with increased leukocyte count was 64.7 (SD 11.4) years, 32 (61.5%) were men and 47 (90.4%) had fever. Compared with the patients with non-increased leukocyte count, the patients with increased leukocyte count were significantly older (P < 0.01), were more likely to have underlying chronic diseases (P < 0.01), more likely to develop critically illness (P < 0.01), more likely to admit to an ICU (P < 0.01), more likely to receive mechanical ventilation (P < 0.01), had higher rate of death (P < 0.01) and the blood levels of neutrophil count and the serum concentrations of CRP and IL-6 were significantly increased, (P < 0.01). The older patients with COVID-19 who had underlying chronic disorders are more likely to develop leukocytosis. These patients are more likely to develop critical illness, with a high admission to an ICU and a high mortality rate.