Clinical Validation of a Novel T-Cell Receptor Sequencing Assay for Identification of Recent or Prior Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: While diagnostic, therapeutic, and vaccine development in the coronavirus disease 2019 (COVID-19) pandemic has proceeded at unprecedented speed, critical gaps in our understanding of the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unaddressed by current diagnostic strategies. METHODS: A statistical classifier for identifying prior SARS-CoV-2 infection was trained using >4000 SARS-CoV-2-associated T-cell receptor (TCR) ß sequences identified by comparing 784 cases and 2447 controls from 5 independent cohorts. The T-Detect COVID (Adaptive Biotechnologies) assay applies this classifier to TCR repertoires sequenced from blood samples to yield a binary assessment of past infection. Assay performance was assessed in 2 retrospective (n = 346; n = 69) and 1 prospective cohort (n = 87) to determine positive percent agreement (PPA) and negative percent agreement (NPA). PPA was compared with 2 commercial serology assays, and pathogen cross-reactivity was evaluated. RESULTS: T-Detect COVID demonstrated high PPA in individuals with prior reverse transcription-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infection (97.1% 15+ days from diagnosis; 94.5% 15+ days from symptom onset), high NPA (∼100%) in presumed or confirmed SARS-CoV-2 negative cases, equivalent or higher PPA than 2 commercial serology tests, and no evidence of pathogen cross-reactivity. CONCLUSIONS: T-Detect COVID is a novel T-cell immunosequencing assay demonstrating high clinical performance for identification of recent or prior SARS-CoV-2 infection from blood samples, with implications for clinical management, risk stratification, surveillance, and understanding of protective immunity and long-term sequelae.

* Glioblastoma Exosomes for Therapeutic Angiogenesis in Peripheral Ischemia.

Peripheral ischemia as a result of occlusive vascular disease is a widespread problem in patients older than the age of 65. Angiogenic therapies that can induce microvascular growth have great potential for providing a long-lasting solution for patients with ischemia and would provide an appealing alternative to surgical and percutaneous interventions. However, many angiogenic therapies have seen poor efficacy in clinical trials, suggesting that patients with long-term peripheral ischemia have considerable therapeutic resistance to angiogenic stimuli. Glioblastoma is one of the most angiogenic tumor types, inducing robust vessel growth in the area surrounding the tumor. One major angiogenic mechanism used by the tumor cells to induce blood vessel growth is the production of exosomes and other extracellular vesicles that can carry pro-angiogenic and immunomodulatory signals. Here, we explored whether the pro-angiogenic aspects of glioblastoma-derived exosomes could be harnessed to promote angiogenesis and healing in the context of peripheral ischemic disease. We demonstrate that the exosomes derived from glioblastoma markedly enhance endothelial cell proliferation and increase endothelial tubule formation in vitro. An analysis of the microRNA expression using next generation sequencing identified that exosomes contained a high concentration of miR-221. In addition, we found that glioblastoma exosomes contained significant amounts of the proteoglycans glypican-1 and syndecan-4, which can serve as co-receptors for angiogenic factors, including fibroblast growth factor-2 (FGF-2). In a hindlimb ischemia model in mice, we found that the exosomes promoted enhanced revascularization in comparison to control alginate gels and FGF-2 treatment alone. Taken together, our results support the fact that glioblastoma-derived exosomes have powerful effects in increasing revascularization in the context of peripheral ischemia.

Glypican-1 nanoliposomes for potentiating growth factor activity in therapeutic angiogenesis.

Therapeutic angiogenesis is a highly appealing concept for treating tissues that become ischemic due to vascular disease. A major barrier to the clinical translation of angiogenic therapies is that the patients that are in the greatest need of these treatments often have long term disease states and co-morbidities, such as diabetes and obesity, that make them resistant to angiogenic stimuli. In this study, we identified that human patients with type 2 diabetes have reduced levels of glypican-1 in the blood vessels of their skin. The lack of this key co-receptor in the tissue may make the application of exogenous angiogenic growth factors or cell therapies ineffective. We created a novel therapeutic enhancer for growth factor activity consisting of glypican-1 delivered in a nanoliposomal carrier (a "glypisome"). Here, we demonstrate that glypisomes enhance FGF-2 mediated endothelial cell proliferation, migration and tube formation. In addition, glypisomes enhance FGF-2 trafficking by increasing both uptake and endosomal processing. We encapsulated FGF-2 or FGF-2 with glypisomes in alginate beads and used these to deliver localized growth factor therapy in a murine hind limb ischemia model. Co-delivery of glypisomes with FGF-2 markedly increased the recovery of perfusion and vessel formation in ischemic hind limbs of wild type and diabetic mice in comparison to mice treated with FGF-2 alone. Together, our findings support that glypisomes are effective means for enhancing growth factor activity and may improve the response to local angiogenic growth factor therapies for ischemia.

Syndecan-4 Enhances Therapeutic Angiogenesis after Hind Limb Ischemia in Mice with Type 2 Diabetes.

Delivering syndecan-4 with FGF-2 improves the effectiveness of FGF-2 therapy for ischemia in the diabetic disease state. The syndecan-4 proteoliposomes significantly enhance in vitro tubule formation as well as blood perfusion and vessel density in the ischemic hind limbs of diseased ob/ob mice. Syndecan-4 therapy also induces a marked immunomodulation in the tissues, increasing the polarization of macrophages toward the M2 phenotype.