Differential Cytokine Signatures of SARS-CoV-2 and Influenza Infection Highlight Key Differences in Pathobiology

BackgroundSeveral inflammatory cytokines are upregulated in severe COVID-19. We compared cytokines in COVID-19 versus influenza in order to define differentiating features of the inflammatory response to these pathogens and their association with severe disease. Because elevated body mass index (BMI) is a known risk factor for severe COVID-19, we examined the relationship of BMI to cytokines associated with severe disease. MethodsThirty-seven cytokines and chemokines were measured in plasma from 145 patients with COVID-19, 57 patients with influenza, and 30 healthy controls. Controlling for BMI, age, and sex, differences in cytokines between groups were determined by linear regression and random forest prediction was utilized to determine the cytokines most important in distinguishing severe COVID-19 and influenza. Mediation analysis was utilized to identify cytokines that mediate the effect of BMI on disease severity. ResultsIL-18, IL-1{beta}, IL-6, and TNF- were significantly increased in COVID-19 versus influenza patients while GM-CSF, IFN-{gamma}, IFN-{lambda}1, IL-10, IL-15, and MCP-2 were significantly elevated in the influenza group. In subgroup analysis based on disease severity, IL-18, IL-6, and TNF- were elevated in severe COVID-19, but not severe influenza. Random forest analysis identified high IL-6 and low IFN-{lambda}1 levels as the most distinct between severe COVID-19 and severe influenza. Finally, IL-1RA was identified as a potential mediator of the effects of BMI on COVID-19 severity. ConclusionsThese findings point to activation of fundamentally different innate immune pathways in SARS-CoV-2 and influenza infection, and emphasize drivers of severe COVID-19 to focus both mechanistic and therapeutic investigations. SummarySevere COVID-19 is marked by dysregulated inflammation and is associated with elevated BMI. By comparing cytokines and chemokines in patients with either COVID-19 or influenza, we identified distinct inflammatory pathways and a cytokine mediator of the effect of BMI.

Mitochondrial induced T cell apoptosis and aberrant myeloid metabolic programs define distinct immune cell subsets during acute and recovered SARS-CoV-2 infection

It remains unclear why some patients infected with SARS-CoV-2 readily resolve infection while others develop severe disease. To address this question, we employed a novel assay to interrogate immune-metabolic programs of T cells and myeloid cells in severe and recovered COVID-19 patients. Using this approach, we identified a unique population of T cells expressing high H3K27me3 and the mitochondrial membrane protein voltage-dependent anion channel (VDAC), which were expanded in acutely ill COVID-19 patients and distinct from T cells found in patients infected with hepatitis c or influenza and in recovered COVID-19. Increased VDAC was associated with gene programs linked to mitochondrial dysfunction and apoptosis. High-resolution fluorescence and electron microscopy imaging of the cells revealed dysmorphic mitochondria and release of cytochrome c into the cytoplasm, indicative of apoptosis activation. The percentage of these cells was markedly increased in elderly patients and correlated with lymphopenia. Importantly, T cell apoptosis could be inhibited in vitro by targeting the oligomerization of VDAC or blocking caspase activity. In addition to these T cell findings, we also observed a robust population of Hexokinase II+ polymorphonuclear-myeloid derived suppressor cells (PMN-MDSC), exclusively found in the acutely ill COVID-19 patients and not the other viral diseases. Finally, we revealed a unique population of monocytic MDSC (M-MDSC) expressing high levels of carnitine palmitoyltransferase 1a (CPT1a) and VDAC. The metabolic phenotype of these cells was not only highly specific to COVID-19 patients but the presence of these cells was able to distinguish severe from mild disease. Overall, the identification of these novel metabolic phenotypes not only provides insight into the dysfunctional immune response in acutely ill COVID-19 patients but also provide a means to predict and track disease severity as well as an opportunity to design and evaluate novel metabolic therapeutic regimens. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/20186064v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@89f509org.highwire.dtl.DTLVardef@1362640org.highwire.dtl.DTLVardef@940aeorg.highwire.dtl.DTLVardef@175792b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Evaluation of Serological SARS-CoV-2 Lateral Flow Assays for Rapid Point of Care Testing

BackgroundRapid point-of-care tests (POCTs) for SARS-CoV-2-specific antibodies vary in performance. A critical need exists to perform head-to-head comparison of these assays. MethodsPerformance of fifteen different lateral flow POCTs for the detection of SARS-CoV-2-specific antibodies was performed on a well characterized set of 100 samples. Of these, 40 samples from known SARS-CoV-2-infected, convalescent individuals (average of 45 days post symptom onset) were used to assess sensitivity. Sixty samples from the pre-pandemic era (negative control), that were known to have been infected with other respiratory viruses (rhinoviruses A, B, C and/or coronavirus 229E, HKU1, NL63 OC43) were used to assess specificity. The timing of seroconversion was assessed on five POCTs on a panel of 272 longitudinal samples from 47 patients of known time since symptom onset. ResultsFor the assays that were evaluated, the sensitivity and specificity for any reactive band ranged from 55%-97% and 78%-100%, respectively. When assessing the performance of the IgM and the IgG bands alone, sensitivity and specificity ranged from 0%-88% and 80%-100% for IgM and 25%-95% and 90%-100% for IgG. Longitudinal testing revealed that median time post symptom onset to a positive result was 7 days (IQR 5.4, 9.8) for IgM and 8.2 days (IQR 6.3 to 11.3). ConclusionThe testing performance varied widely among POCTs with most variation related to the sensitivity of the assays. The IgM band was most likely to misclassify pre-pandemic samples. The appearance of IgM and IgG bands occurred almost simultaneously.

Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping

The emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq (“cRASL-seq”), which enables analysis of any targeted pathogen-(and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to ∼1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ∼10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.Competing Interest StatementJ.J.C. and H.B.L. are listed as inventors on a patent describing the cRASL-seq method. H.B.L. has founded a company to license and commercialize oligonucleotide probe ligation related technologies.View Full Text