Simultaneous detection of SARS-CoV-2 RNA and host antibodies enabled by a multiplexed electrochemical sensor platform

The current COVID-19 pandemic highlights the continued need for rapid, accurate, and cost-effective point-of-care (POC) diagnostics that can accurately assess an individuals infection and immunity status for SARS-CoV-2. As the virus continues to spread and vaccines become more widely available, detecting viral RNA and serological biomarkers can provide critical insights into the status of infectious, previously infectious, and vaccinated individuals over time. Here, we describe an integrated, low-cost, 3D printed, lab-on-a-chip device that extracts, concentrates, and amplifies viral RNA from unprocessed patient saliva and simultaneously detects RNA and multiple host anti-SARS-CoV-2 antibodies via multiplexed electrochemical (EC) outputs in two hours. The EC sensor platform enables single-molecule CRISPR/Cas-based molecular detection of SARS-CoV-2 viral RNA as well as serological detection of antibodies against the three immunodominant SARS-CoV-2 viral antigens. This study demonstrates that microfluidic EC sensors can enable multiplexed POC diagnostics that perform on par with traditional laboratory-based techniques, enabling cheaper and more widespread monitoring of infection and immunity over time.

Tie2 activation protects against prothrombotic endothelial dysfunction in COVID-19

Profound endothelial dysfunction accompanies the microvascular thrombosis commonly observed in severe COVID-19. In the quiescent state, the endothelial surface is anticoagulant, a property maintained at least in part via constitutive signaling through the Tie2 receptor. During inflammation, the Tie2 antagonist angiopoietin-2 (Angpt-2) is released from activated endothelial cells and inhibits Tie2, promoting a prothrombotic phenotypic shift. We sought to assess whether severe COVID-19 is associated with procoagulant dysfunction of the endothelium and alterations in the Tie2-angiopoietin axis. Primary human endothelial cells treated with plasma from patients with severe COVID-19 upregulated the expression of thromboinflammatory genes, inhibited expression of antithrombotic genes, and promoted coagulation on the endothelial surface. Pharmacologic activation of Tie2 with the small molecule AKB-9778 reversed the prothrombotic state induced by COVID-19 plasma in primary endothelial cells. On lung autopsy specimens from COVID-19 patients, we found a prothrombotic endothelial signature as evidenced by increased von Willebrand Factor and loss of anticoagulant proteins. Assessment of circulating endothelial markers in a cohort of 98 patients with mild, moderate, or severe COVID-19 revealed profound endothelial dysfunction indicative of a prothrombotic state. Angpt-2 concentrations rose with increasing disease severity and highest levels were associated with worse survival. These data highlight the disruption of Tie2-angiopoietin signaling and procoagulant changes in endothelial cells in severe COVID-19. Moreover, our findings provide novel rationale for current trials of Tie2 activating therapy with AKB-9778 in severe COVID-19 disease.

Ultrasensitive measurement of both SARS-CoV2 RNA and serology from saliva

Tests for COVID-19 generally measure SARS-CoV2 viral RNA from nasal swabs or antibodies against the virus from blood. It has been shown, however, that both viral particles and antibodies against those particles are present in saliva, which is more accessible than both swabs and blood. We present methods for highly sensitive measurements of both viral RNA and serology from the same saliva sample. We developed an efficient saliva RNA extraction method and combined it with an ultrasensitive serology test based on Single Molecule Array (Simoa) technology. We apply our test to the saliva of patients who presented to the hospital with COVID-19 symptoms, some of whom tested positive with a conventional RT-qPCR nasopharyngeal swab test. We demonstrate that combining viral RNA detection by RT-qPCR with serology identifies more patients as infected than either method alone. Our results suggest the utility of combining viral RNA and serology testing from saliva, a single easily accessible biofluid.

Innate lymphoid cells and disease tolerance in SARS-CoV-2 infection

Risk of severe COVID-19 increases with age, is greater in males, and is associated with lymphopenia, but not with higher burden of SARS-CoV-2. It is unknown whether effects of age and sex on abundance of specific lymphoid subsets explain these correlations. This study found that the abundance of innate lymphoid cells (ILCs) decreases more than 7-fold over the human lifespan -- T cell subsets decrease less than 2-fold -- and is lower in males than in females. After accounting for effects of age and sex, ILCs, but not T cells, were lower in adults hospitalized with COVID-19, independent of lymphopenia. Among SARS-CoV-2-infected adults, the abundance of ILCs, but not of T cells, correlated inversely with odds and duration of hospitalization, and with severity of inflammation. ILCs were also uniquely decreased in pediatric COVID-19 and the numbers of these cells did not recover during follow-up. In contrast, children with MIS-C had depletion of both ILCs and T cells, and both cell types increased during follow-up. In both pediatric COVID-19 and MIS-C, ILC abundance correlated inversely with inflammation. Blood ILC mRNA and phenotype tracked closely with ILCs from lung. Importantly, blood ILCs produced amphiregulin, a protein implicated in disease tolerance and tissue homeostasis, and the percentage of amphiregulin-producing ILCs was higher in females than in males. These results suggest that, by promoting disease tolerance, homeostatic ILCs decrease morbidity and mortality associated with SARS-CoV-2 infection, and that lower ILC abundance accounts for increased COVID-19 severity with age and in males.

Profound Treg perturbations correlate with COVID-19 severity

The hallmark of severe COVID-19 disease has been an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We explored the hypothesis that perturbations in FoxP3+ T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in both Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, which correlated with poor outcomes. Accordingly, these Tregs over-expressed a range of suppressive effectors, but also pro-inflammatory molecules like IL32. Most strikingly, they acquired similarity to tumor-infiltrating Tregs, known to suppress local anti-tumor responses. These traits were most marked in acute patients with severe disease, but persisted somewhat in convalescent patients. These results suggest that Tregs may play nefarious roles in COVID-19, via suppressing anti-viral T cell responses during the severe phase of the disease, and/or via a direct pro-inflammatory role.

Rapid 'mix and read' assay for scalable detection of SARS-CoV-2 antibodies in patient plasma

The human beta coronavirus SARS-CoV-2, causative virus of COVID-19, has infected more than 15 million people globally and continues to spread. Widespread, population level testing to detect active and past infections is critical to curb the COVID-19 pandemic. Antibody (serological) testing is the only option for detecting past infections outside the narrow window accessible to nucleic acid-based tests. However, currently available serological assays commonly lack scalability. Here, we describe the development of a rapid homogenous serological assay for the detection of antibodies to SARS-CoV-2 in patient plasma. We show that the fluorescence-based assay accurately detects seroconversion in COVID-19 patients from less than 1 L of plasma. Using a cohort of samples from COVID-19 infected or healthy individuals, we demonstrate detection with 100% sensitivity and specificity. This assay addresses an important need for a robust, low barrier to implementation, and scalable serological assay with complementary strengths to currently available serological platforms.