Design and Development of an Antigen Test for SARS-CoV-2 Nucleocapsid Protein to Validate the Viral Quality Assurance Panels.

The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for the calibration and quality assurance of inactivated SARS-CoV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. A heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)-C-terminal domain (CTD) across all SARS-CoV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprised of UV-inactivated SARS-CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (limit of detection: 1.72-8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a 20 amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust 'pan-variant' antigen-based assays for viruses prone to high mutational rates.

In Vitro Diagnostic Assay to Detect SARS-CoV-2-Neutralizing Antibody in Patient Sera Using Engineered ACE-2 Mini-Protein.

The recent development and mass administration of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines allowed for disease control, reducing hospitalizations and mortality. Most of these vaccines target the SARS-CoV-2 Spike (S) protein antigens, culminating with the production of neutralizing antibodies (NAbs) that disrupt the attachment of the virus to ACE2 receptors on the host cells. However, several studies demonstrated that the NAbs typically rise within a few weeks after vaccination but quickly reduce months later. Thus, multiple booster administration is recommended, leading to vaccination hesitancy in many populations. Detecting serum anti-SARS-CoV-2 NAbs can instruct patients and healthcare providers on correct booster strategies. Several in vitro diagnostics kits are available; however, their high cost impairs the mass NAbs diagnostic testing. Recently, we engineered an ACE2 mimetic that interacts with the Receptor Binding Domain (RBD) of the SARS-2 S protein. Here we present the use of this engineered mini-protein (p-deface2 mut) to develop a detection assay to measure NAbs in patient sera using a competitive ELISA assay. Serum samples from twenty-one patients were tested. Nine samples (42.8%) tested positive, and twelve (57.1%) tested negative for neutralizing sera. The data correlated with the result from the standard commercial assay that uses human ACE2 protein. This confirmed that p-deface2 mut could replace human ACE2 in ELISA assays. Using bacterially expressed p-deface2 mut protein is cost-effective and may allow mass SARS-CoV-2 NAbs detection, especially in low-income countries where economical diagnostic testing is crucial. Such information will help providers decide when a booster is required, reducing risks of reinfection and preventing the administration before it is medically necessary.

Increased Production of LIGHT by T Cells in Eosinophilic Esophagitis Promotes Differentiation of Esophageal Fibroblasts Toward an Inflammatory Phenotype.

BACKGROUND & AIMS: Eosinophilic esophagitis (EoE) is an antigen-mediated eosinophilic disease of the esophagus that involves fibroblast activation and progression to fibrostenosis. Cytokines produced by T-helper type 2 cells and transforming growth factor beta 1 (TGFß1) contribute to the development of EoE, but other cytokines involved in pathogenesis are unknown. We investigate the effects of tumor necrosis factor superfamily member 14 (TNFSF14, also called LIGHT) on fibroblasts in EoE. METHODS: We analyzed publicly available esophageal CD3+ T-cell single-cell sequencing data for expression of LIGHT. Esophageal tissues were obtained from pediatric patients with EoE or control individuals and analyzed by immunostaining. Human primary esophageal fibroblasts were isolated from esophageal biopsy samples of healthy donors or patients with active EoE. Fibroblasts were cultured; incubated with TGFß1 and/or LIGHT; and analyzed by RNA sequencing, flow cytometry, immunoblots, immunofluorescence, or reverse transcription polymerase chain reaction. Eosinophils were purified from peripheral blood of healthy donors, incubated with interleukin 5, cocultured with fibroblasts, and analyzed by immunohistochemistry. RESULTS: LIGHT was up-regulated in the esophageal tissues from patients with EoE, compared with control individuals, and expressed by several T-cell populations, including T-helper type 2 cells. TNF receptor superfamily member 14 (TNFRSF14, also called HVEM) and lymphotoxin beta receptor are receptors for LIGHT that were expressed by fibroblasts from healthy donors or patients with active EoE. Stimulation of esophageal fibroblasts with LIGHT induced inflammatory gene transcription, whereas stimulation with TGFß1 induced transcription of genes associated with a myofibroblast phenotype. Stimulation of fibroblasts with TGFß1 increased expression of HVEM; subsequent stimulation with LIGHT resulted in their differentiation into cells that express markers of myofibroblasts and inflammatory chemokines and cytokines. Eosinophils tethered to esophageal fibroblasts after LIGHT stimulation via intercellular adhesion molecule-1. CONCLUSIONS: T cells in esophageal tissues from patients with EoE express increased levels of LIGHT compared with control individuals, which induces differentiation of fibroblasts into cells with inflammatory characteristics. TGFß1 increases fibroblast expression of HVEM, a receptor for LIGHT. LIGHT mediates interactions between esophageal fibroblasts and eosinophils via ICAM1. This pathway might be targeted for the treatment of EoE.

Human Eosinophils Express a Distinct Gene Expression Program in Response to IL-3 Compared with Common ß-Chain Cytokines IL-5 and GM-CSF.

Despite recent advances in asthma management with anti-IL-5 therapies, many patients have eosinophilic asthma that remains poorly controlled. IL-3 shares a common ß subunit receptor with both IL-5 and GM-CSF but, through α-subunit-specific properties, uniquely influences eosinophil biology and may serve as a potential therapeutic target. We aimed to globally characterize the transcriptomic profiles of GM-CSF, IL-3, and IL-5 stimulation on human circulating eosinophils and identify differences in gene expression using advanced statistical modeling. Human eosinophils were isolated from the peripheral blood of healthy volunteers and stimulated with either GM-CSF, IL-3, or IL-5 for 48 h. RNA was then extracted and bulk sequencing performed. DESeq analysis identified differentially expressed genes and weighted gene coexpression network analysis independently defined modules of genes that are highly coexpressed. GM-CSF, IL-3, and IL-5 commonly upregulated 252 genes and downregulated 553 genes, producing a proinflammatory and survival phenotype that was predominantly mediated through TWEAK signaling. IL-3 stimulation yielded the most numbers of differentially expressed genes that were also highly coexpressed (n = 119). These genes were enriched in pathways involving JAK/STAT signaling. GM-CSF and IL-5 stimulation demonstrated redundancy in eosinophil gene expression. In conclusion, IL-3 produces a distinct eosinophil gene expression program among the ß-chain receptor cytokines. IL-3-upregulated genes may provide a foundation for research into therapeutics for patients with eosinophilic asthma who do not respond to anti-IL-5 therapies.

Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing.

Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a "renewable" capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings.

Yeast dual-affinity biobricks: Progress towards renewable whole-cell biosensors.

Point-of-care (POC) diagnostic biosensors offer a promising solution to improve healthcare, not only in developed parts of the world, but also in resource limited areas that lack adequate medical infrastructure and trained technicians. However, in remote and resource limited settings, cost and storage of traditional POC immunoassays often limit actual deployment. Synthetically engineered biological components ("BioBricks") provide an avenue to reduce costs and simplify assay procedures. In this article, the design and development of an ultra-low cost, whole-cell "renewable" capture reagent for use in POC diagnostic applications is described. Yeast cells were genetically modified to display both single chain variable fragment (scFv) antibodies and gold-binding peptide (GBP) on their surfaces for simple one step enrichment and surface functionalization. Electrochemical impedance spectroscopy (EIS) and fluorescent imaging were used to verify and characterize the binding of cells to gold electrodes. A complete electrochemical detection assay was then performed on screen-printed electrodes fixed with yeast displaying scFv directed to Salmonella outer membrane protein D (OmpD). Electrochemical assays were optimized and cross-validated with established fluorescence techniques. Nanomolar detection limits were observed for both formats.

WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility.

We previously identified Waf1 Cip1 stabilizing protein 39 (WISp39) as a binding partner for heat shock protein 90 (Hsp90). We now report that WISp39 has an essential function in the control of directed cell migration, which requires WISp39 interaction with Hsp90. WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge. WISp39 binds Coronin 1B, known to regulate the Arp2/3 complex and Cofilin at the leading edge. WISp39 preferentially interacts with phosphorylated Coronin 1B, allowing it to complex with Slingshot phosphatase (SSH) to dephosphorylate and activate Cofilin. WISp39 also regulates Arp2/3 complex localization at the leading edge. WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant. We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge.

Downregulating galectin-3 inhibits proinflammatory cytokine production by human monocyte-derived dendritic cells via RNA interference.

Galectin-3 (Gal-3), a ß-galactoside-binding lectin, serves as a pattern-recognition receptor (PRR) of dendritic cells (DCs) in regulating proinflammatory cytokine production. Galectin-3 (Gal-3) siRNA downregulates expression of IL-6, IL-1ß and IL-23 p19, while upregulates IL-10 and IL-12 p35 in TLR/NLR stimulated human MoDCs. Furthermore, Gal-3 siRNA-treated MoDCs enhanced IFN-γ production in SEB-stimulated CD45RO CD4 T-cells, but attenuated IL-17A and IL-5 production by CD4 T-cells. Addition of neutralizing antibodies against Gal-3, or recombinant Gal-3 did not differentially modulate IL-23 p19 versus IL-12 p35. The data indicate that intracellular Gal-3 acts as cytokine hub of human DCs in responding to innate immunity signals. Gal-3 downregulation reprograms proinflammatory cytokine production by MoDCs that inhibit Th2/Th17 development.

Mechanisms of sulindac-induced apoptosis and cell cycle arrest.

The mechanism underlying the chemopreventive effects of the non-steroidal anti-inflammatory drug sulindac remains unclear. Its active metabolite, sulindac sulfide, induces cell cycle arrest as well as apoptosis in mammalian cell lines. We now show that in murine thymocytes, sulindac sulfide-induced cell death is p53, bax, Fas, and FasL independent. In contrast, bcl2 transgenic thymocytes are resistant to sulindac sulfide-induced apoptosis. In addition, we demonstrate that sulindac sulfide-induced cell cycle arrest in mouse embryonic fibroblasts (MEFs) is partly mediated by the retinoblastoma tumor suppressor protein (Rb) and the cyclin kinase inhibitor p21waf1/cip1. Furthermore, MEFs deficient in p21 or Rb are more susceptible to sulindac sulfide-induced cell death. These results suggest that sulindac may selectively target premalignant cells with cell cycle checkpoint deficits.

Regulation of p21(WAF1/CIP1) stability by WISp39, a Hsp90 binding TPR protein.

p21(WAF1/CIP1), a cyclin-dependent kinase inhibitor and a critical regulator of cell cycle, is controlled transcriptionally by p53-dependent and -independent mechanisms and posttranslationally by the proteasome. We have identified WISp39, a tetratricopeptide repeat (TPR) protein that binds p21. WISp39 stabilizes newly synthesized p21 protein by preventing its proteasomal degradation. WISp39, p21, and hsp90 form a trimeric complex in vivo. The interaction of WISp39 with Hsp90 is abolished by point mutations within the C-terminal TPR domain of WISp39. Although this WISp39 TPR mutant binds p21 in vivo, it fails to stabilize p21. Our results suggest that WISp39 recruits Hsp90 to regulate p21 protein stability. WISp39 downregulation by siRNA prevents the accumulation of p21 and cell cycle arrest after ionizing radiation. The results demonstrate the importance of posttranslational stabilization of p21 protein by WISp39 in regulating cellular p21 activity.

UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair.

p53-mediated increase in cyclin-dependent kinase inhibitor p21(WAF1) protein is thought to be the major mediator of cell cycle arrest after DNA damage. Previously p21 protein levels have been reported to increase or to decrease after UV irradiation. We show that p21 protein is degraded after irradiation of a variety of cell types with low but not high doses of UV. Cell cycle arrest occurs despite p21 degradation via Tyr(15) inhibitory phosphorylation of cdk2 and differs from the classical p21-dependent checkpoint elicited by ionizing radiation. In contrast to the basal turnover of p21, degradation of p21 switches to ubiquitin/Skp2-dependent proteasome pathway following UV irradiation. ATR activation after UV irradiation is essential for signaling p21 degradation. Finally, UV-induced p21 degradation is essential for optimal DNA repair. These results provide novel insight into regulation of p21 protein and its role in the cellular response to DNA damage.

Cell cycle-dependent phosphorylation of the large subunit of replication factor C (RF-C) leads to its dissociation from the RF-C complex.

The five subunit replication factor C (RF-C) complex plays a critical role in DNA elongation. We find that the large subunit of RF-C (RF-Cp145) is phosphorylated in vivo whereas the smaller RF-C subunits are not phosphorylated. The phosphorylation of endogenous RFCp145 is modulated in a cell cycle-dependent manner. Phosphorylation is maximal in G2/M and is inhibited by an inhibitor of cyclin-dependent kinases. Phosphorylation of purified recombinant RF-C complex in vitro reveals that RF-Cp145 is preferentially phosphorylated by cdc2-cyclin B but not by cdk2-cyclin A or cdk2-cyclin E. In vitro phosphorylation of RF-C complex by cdc2-cyclin B kinases leads to dissociation of phosphorylated RFCp145 from the RF-C complex. Using different approaches we demonstrate that phosphorylated RFCp145 is indeed dissociated from RF-Cp40 and RF-Cp37 in vivo. These results suggest that destabilization of the RF-C complex by CDKs may inactivate the RF-C complex at the end of S phase.

Loss of p53 compensates for alpha v-integrin function in retinal neovascularization.

alpha(v)-Integrin antagonists block neovascularization in various species, whereas 20% of alpha(v)-integrin null mice are born with many normal looking blood vessels. Given that blockade of alpha(v)-integrins during angiogenesis induces p53 activity, we utilized p53 null mice to elucidate whether loss of p53 can compensate for alpha(v)-integrin function in neovascularization of the retina. Murine retinal vascularization was inhibited by systemic administration of an alpha(v)-integrin antagonist. In contrast, mice lacking p53 were refractory to this treatment, indicating that neovascularization in normal mice depends on alpha(v)-integrin-mediated suppression of p53. Blockade of alpha(v)-integrins during neovascularization resulted in an induction of p21(CIP1) in wild type and, surprisingly, in p53 null retinas, indicating that alpha(v)-integrin ligation regulates p21(CIP1) levels in a p53-independent manner. In conclusion, we demonstrate for the first time an in vivo intracellular mechanism for compensation of integrin function and that p53 and alpha(v)-integrins act in concert during retinal neovascularization.