A. Provide supporting data showing that diabetic MPs contribute to the increases in the propagation of the virus compared to non-diabetic MP. A1. Demonstrate higher number of cellular internalizations of S-protein-bound DB MPs than that of S-protein-bound normal MPs. Experiments will be conducted in microvessels developed in microfluidics. Our preliminary data showed no significant differences in ACE2 expression and ACE2-mediated MP interaction with S-protein between normal and DB MPs. The main factors that contribute to DB MP-mediated increases in virus entry are their much higher quantity and more adhesive surface due to their largely externalized phosphatidylserine (PS) than that of normal MPs. GFPtagged S-protein will be incubated with MPs isolated from normal and DB plasma and flow cytometry will be used to confirm the S-protein-bound MPs. To demonstrate the quality differences between normal and DB MPs, equal amount of S-protein-bound DB and normal MPs will be perfused into the in vitro microvessels. Confocal images will be collected to compare the internalized S-protein-bound DB MPs with that of normal MPs by quantification of GFP fluorescence. To demonstrate the role of externalized PS on DB MPs in the MP adhesion and internalization, the S-protein-bound DB MPs will be pre-coated with a lipid binding protein, Annexin V, before perfusion into microvessels. We predict that precoating of DB MPs with Annexin V will prevent the DB MP adhesion to ECs and reduce their cellular internalizations. A2. Demonstrate DB MP-mediated increases in HCoV-NL63 infection and propagation when compared to normal MPs. HCoV-NL63 (BEI Resources) will be propagated in Caco-2 cells to produce a working virus stock. Cultured ECs will be incubated with HCoV-NL63 with serial dilutions (1:10-1000) in the presence of DB and normal plasma or equal number of isolated DB and normal MPs, respectively. The incubation of HCoV-NL63 alone serves as viral infection control. To demonstrate the specific role of DB MPs in facilitating viral infection from that of other components in DB plasma such as inflammatory mediators and cytokines, cultured ECs will be incubated with HCoV-NL63 in the presence of MP-free DB plasma and the results will be compared with DB plasma containing increased MPs. For rigor, efficiency of HCoVNL63 infection will be determined using three distinct methods. Total virus will be harvested from cells and supernatant at 96 hpi and quantified by both plaque assay and 50% tissue culture infectious dose (TCID 50) assay as previously published (PMID 19014487). Infection will also be quantified using a one-step, real-time, quantitative reverse transcription PCR assay on RNA harvested from cells at 96 hpi using a commercially available kit (Liferiver). Supernatant will be collected daily to examine the release of MPs and cytokines, serving as indicators of viral infection-induced EC activation. Results will be compared among groups (3-4 replicates per group for all proposed preliminary studies). B. Provide preliminary data to support that HCoV-NL63 virus could produce the illness that is similar to SARS-CoV-2. HCoV-NL63 has been reported to cause upper respiratory tract infections in children, and also to cause pneumonia, as well as airway inflammation in K18-ACE2 mice. In this preliminary study, we will develop a hamster model of HCoVNL63 infection. Golden Syrian hamsters (8-10 weeks old, Charles River) will be used for this study. Normal and STZinduced diabetic hamsters will be intranasally inoculated with serial dilutions of HCoV-NL63 (5 x 104, 5 x 105, or 5 x 106 TCID50) in 200 μL PBS to establish the ID50 in vivo. Oral and rectal swabs will be collected every 24 hours starting 1-day post-inoculation (dpi) for up to five days. Body weight and clinical signs of illness as indicated by labored breathing, hunched posture and immobility, and temperature will be monitored twice a day. Hamsters will be euthanized on 3 and 5 dpi to harvest lungs and peripheral tissues. Remaining animals are sacrificed at 7 dpi (n = 3 per group). Hamster lungs will be assessed for histology and viral replication (measure viral RNA levels by qPCR). Cytokines will be measured in bronchoalveolar lavage and lung homogenate. Plasma will be collected for MP and cytokine analysis (details see application experimental designs C4B2). Results will be compared among groups. We predict that HCoV-NL63-infected DB hamsters have more severe respiratory inflammatory responses than HCoV-NL63-infected normal hamsters. Our previous study showed that cross-transfusion of DB plasma to normal rats caused immediate vascular inflammation in the recipient rat, indicating a role of DB MPs in mediating propagation of inflammation in the vasculature. To evaluate the specific role of DB MPs in facilitation and propagation of HCoV-NL63 infection, 40% plasma volume of a HCoV-NL63- infected normal hamster will be replaced by MP-rich and MP-free DB plasma (normal plasma as sham control) or vice versa and the viral infection-induced illness and vascular pathology will be compared between groups. In case HCoVNL63 infection alone causes very mild clinical symptoms in hamsters, the alternative is to coinfect hamster with human influenza H1N1 and HCoV-NL63. Hamster is an established animal model for the study of human influenza infection and coinfection of SRAS-CoV-2 and influenza has been shown to enhance the severity of pneumonia in hamsters. C. Provide evidence for a novel therapeutic target to alleviate the adverse outcomes of COVID-19 patients with diabetes. The focus of this application is to reveal a novel mechanism by which diabetes exacerbates the COVID-19 outcomes. The proposed mechanistic studies will lead to the identification of a novel target for therapeutic development. Our previous study showed that carotid injection of Annexin V (1 ml, 100 μg/Kg) to a DB rat reduced plasma MPs from 465 x 104/μL to 22 x 104/μL 1h after the injection. We will apply the same approach to HCoV-NL63 infected DB hamsters, and the plasma MPs and viral infection outcomes will be evaluated accordingly. The drug development is out of scope of this application. However, these preliminary studies will provide mechanistic evidence supporting the development of a Annexin V-based therapeutic product in the future studies.

Project Narrative COVID-19, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has led to a global pandemic and a social-economic crisis. COVID-19 patients with pre-existing diseases such as diabetes have a greater risk of mortality compared to COVID-19 patients without these comorbidities. To date, the underlying mechanisms accounting for this disparity remain unknown. In response to an urgent demand for a better understanding of the pathogenesis of COVID-19 patients with pre-existing diseases to improve treatments and survival, this application aims to identify the mechanisms of higher risk and adverse outcomes of COVID-19 patients with diabetes and reveal the potential target for therapeutic development.