Rapid quantitative electrochemical detection of SARS-CoV-2 antibodies in plasma and dried blood spot samples

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is a highly contagious disease with several variants, continues to spread as part of the global pandemic. With the roll-out of vaccines and development of new therapeutics that may be targeted to distinct viral molecules, there is a need to screen populations for viral antigen-specific SARS-CoV-2 antibodies. Here, we describe a rapid, multiplexed, electrochemical (EC) platform with on-chip control that enables detection of SARS-CoV-2 antibodies in less than 10 min using 1.5 {micro}L of a patient sample. The EC biosensor demonstrated 100% sensitivity and specificity, and an area under the receiver operating characteristic curve of 1, when evaluated using 93 clinical samples, including plasma and dried blood spot samples from 54 SARS-CoV-2 positive and 39 negative patients. This EC biosensor platform enables simple, cost-effective, sensitive, and rapid detection of anti-SARS-CoV-2 antibodies in complex clinical samples, which is convenient for monitoring host humoral responses to vaccination or viral infection in broad population testing, including applications in low-resource settings. We also demonstrate the feasibility of using dried blood spot samples that can be collected locally and transported to distant clinical laboratories at ambient temperature for detection of anti-SARS-CoV-2 antibodies which can be used for serological surveillance and demonstrate the utility of remote sampling.

Different HMGCR-inhibiting statins vary in their association with increased survival in patients with COVID-19

ImportanceDrug repurposing requires distinguishing established drug class targets from novel molecule-specific mechanisms and rapidly derisking their therapeutic potential in a time-critical manner, particularly in a pandemic scenario. In response to the challenge to rapidly identify treatment options for COVID-19, several studies reported that statins, as a drug class, reduce mortality in these patients. However, it is unknown if different statins exhibit consistent function or may have varying therapeutic benefit. ObjectivesTo test if different statins differ in their ability to exert protective effects based on molecular computational predictions and electronic medical record analysis. Main Outcomes and MeasuresA Bayesian network tool was used to predict drugs that shift the host transcriptomic response to SARS-CoV-2 infection towards a healthy state. Drugs were predicted using 14 RNA-sequencing datasets from 72 autopsy tissues and 465 COVID-19 patient samples or from cultured human cells and organoids infected with SARS-CoV-2, with a total of 2,436 drugs investigated. Top drug predictions included statins, which were then assessed using electronic medical records containing over 4,000 COVID-19 patients on statins to determine mortality risk in patients prescribed specific statins versus untreated matched controls. The same drugs were tested in Vero E6 cells infected with SARS-CoV-2 and human endothelial cells infected with a related OC43 coronavirus. ResultsSimvastatin was among the most highly predicted compounds (14/14 datasets) and five other statins, including atorvastatin, were predicted to be active in > 50% of analyses. Analysis of the clinical database revealed that reduced mortality risk was only observed in COVID-19 patients prescribed a subset of statins, including simvastatin and atorvastatin. In vitro testing of SARS-CoV-2 infected cells revealed simvastatin to be a potent direct inhibitor whereas most other statins were less effective. Simvastatin also inhibited OC43 infection and reduced cytokine production in endothelial cells. Conclusions and RelevanceDifferent statins may differ in their ability to sustain the lives of COVID-19 patients despite having a shared drug target and lipid-modifying mechanism of action. These findings highlight the value of target-agnostic drug prediction coupled with patient databases to identify and clinically evaluate non-obvious mechanisms and derisk and accelerate drug repurposing opportunities.

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.

Enteric coronavirus infection and treatment modeled with an immunocompetent human intestine-on-a-chip

Many patients infected with coronaviruses, such as SARS-CoV-2 and NL63 that use ACE2 receptors to infect cells, exhibit gastrointestinal symptoms and viral proteins are found in the human gastrointestinal tract, yet little is known about the inflammatory and pathological effects of coronavirus infection on the human intestine. Here, we used a human intestine-on-a-chip (Intestine Chip) microfluidic culture device lined by patient organoid-derived intestinal epithelium interfaced with human vascular endothelium to study host cellular and inflammatory responses to infection with NL63 coronavirus. These organoid-derived intestinal epithelial cells dramatically increased their ACE2 protein levels when cultured under flow in the presence of peristalsis-like mechanical deformations in the Intestine Chips compared to when cultured statically as organoids or in Transwell inserts. Infection of the intestinal epithelium with NL63 on-chip led to inflammation of the endothelium as demonstrated by loss of barrier function, increased cytokine production, and recruitment of circulating peripheral blood mononuclear cells (PMBCs). Treatment of NL63 infected chips with the approved protease inhibitor drug, nafamostat, inhibited viral entry and resulted in a reduction in both viral load and cytokine secretion, whereas remdesivir, one of the few drugs approved for COVID19 patients, was not found to be effective and it also was toxic to the endothelium. This model of intestinal infection was also used to test the effects of other drugs that have been proposed for potential repurposing against SARS-CoV-2. Taken together, these data suggest that the human Intestine Chip might be useful as a human preclinical model for studying coronavirus related pathology as well as for testing of potential anti-viral or anti-inflammatory therapeutics.

A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID19 therapeutics in viral pandemics

The rising threat of pandemic viruses, such as SARS-CoV-2, requires development of new preclinical discovery platforms that can more rapidly identify therapeutics that are active in vitro and also translate in vivo. Here we show that human organ-on-a-chip (Organ Chip) microfluidic culture devices lined by highly differentiated human primary lung airway epithelium and endothelium can be used to model virus entry, replication, strain-dependent virulence, host cytokine production, and recruitment of circulating immune cells in response to infection by respiratory viruses with great pandemic potential. We provide a first demonstration of drug repurposing by using oseltamivir in influenza A virus-infected organ chip cultures and show that co-administration of the approved anticoagulant drug, nafamostat, can double oseltamivirs therapeutic time window. With the emergence of the COVID-19 pandemic, the Airway Chips were used to assess the inhibitory activities of approved drugs that showed inhibition in traditional cell culture assays only to find that most failed when tested in the Organ Chip platform. When administered in human Airway Chips under flow at a clinically relevant dose, one drug - amodiaquine - significantly inhibited infection by a pseudotyped SARS-CoV-2 virus. Proof of concept was provided by showing that amodiaquine and its active metabolite (desethylamodiaquine) also significantly reduce viral load in both direct infection and animal-to-animal transmission models of native SARS-CoV-2 infection in hamsters. These data highlight the value of Organ Chip technology as a more stringent and physiologically relevant platform for drug repurposing, and suggest that amodiaquine should be considered for future clinical testing.