Monoclonal Antibodies as SARS-CoV-2 Serology Standards: Experimental Validation and Broader Implications for Correlates of Protection.

COVID-19 has highlighted challenges in the measurement quality and comparability of serological binding and neutralization assays. Due to many different assay formats and reagents, these measurements are known to be highly variable with large uncertainties. The development of the WHO international standard (WHO IS) and other pool standards have facilitated assay comparability through normalization to a common material but does not provide assay harmonization nor uncertainty quantification. In this paper, we present the results from an interlaboratory study that led to the development of (1) a novel hierarchy of data analyses based on the thermodynamics of antibody binding and (2) a modeling framework that quantifies the probability of neutralization potential for a given binding measurement. Importantly, we introduced a precise, mathematical definition of harmonization that separates the sources of quantitative uncertainties, some of which can be corrected to enable, for the first time, assay comparability. Both the theory and experimental data confirmed that mAbs and WHO IS performed identically as a primary standard for establishing traceability and bridging across different assay platforms. The metrological anchoring of complex serological binding and neuralization assays and fast turn-around production of an mAb reference control can enable the unprecedented comparability and traceability of serological binding assay results for new variants of SARS-CoV-2 and immune responses to other viruses.

Development of a Cell-Based SARS-CoV-2 Pseudovirus Neutralization Assay Using Imaging and Flow Cytometry Analysis.

COVID-19 is an ongoing, global pandemic caused by the novel, highly infectious SARS-CoV-2 virus. Efforts to mitigate the effects of SARS-CoV-2, such as mass vaccination and development of monoclonal therapeutics, require precise measurements of correlative, functional neutralizing antibodies that block virus infection. The development of rapid, safe, and easy-to-use neutralization assays is essential for faster diagnosis and treatment. Here, we developed a vesicular stomatitis virus (VSV)-based neutralization assay with two readout methods, imaging and flow cytometry, that were capable of quantifying varying degrees of neutralization in patient serum samples. We tested two different spike-pseudoviruses and conducted a time-course assay at multiple multiplicities of infection (MOIs) to optimize the assay workflow. The results of this assay correlate with the results of previously developed serology and surrogate neutralization assays. The two pseudovirus readout methods produced similar values of 50% neutralization titer values. Harvest-free in situ readouts for live-cell imaging and high-throughput analysis results for flow cytometry can provide unique capabilities for fast evaluation of neutralization, which is critical for the mitigation of future pandemics.

Number Concentration Measurements of Polystyrene Submicrometer Particles.

The number of techniques to measure number concentrations and size distributions of submicrometer particles has recently increased. Submicrometer particle standards are needed to improve the accuracy and reproducibility of these techniques. The number concentrations of fluorescently labeled polystyrene submicrometer sphere suspensions with nominal 100 nm, 200 nm and 500 nm diameters were measured using seven different techniques. Diameter values were also measured where possible. The diameter values were found to agree within 20%, but the number concentration values differed by as much as a factor of two. Accuracy and reproducibility related with the different techniques are discussed with the goal of using number concentration standards for instrument calibration. Three of the techniques were used to determine SI-traceable number concentration values, and the three independent values were averaged to give consensus values. This consensus approach is proposed as a protocol for certifying SI-traceable number concentration standards.

Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19.

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.

Expanding NIST Calibration of Fluorescent Microspheres for Flow Cytometry to More Fluorescence Channels and Smaller Particles.

The National Institute of Standards and Technology (NIST), the National Institutes of Health (NIH) and other industry stakeholders have been working together to enable fluorescence intensities of flow cytometer calibration beads to be assigned quantitative equivalent reference fluorophore (ERF) values with high accuracy and precision. The ultimate goal of this effort is to accurately quantify the number of antibodies bound to individual living cells. The expansion of this effort to assign ERF values to more than 50 fluorescence channels and particles with diameters ranging from 10 µm down to 80 nm is reported here.

Measurement and standardization challenges for extracellular vesicle therapeutic delivery vectors.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are nonreplicating lipid bilayer particles shed by most cell types which have the potential to revolutionize the development and efficient delivery of clinical therapeutics. This article provides an introduction to the landscape of EV-based vectors under development for the delivery of protein- and nucleic acid-based therapeutics. We highlight some of the most pressing measurement and standardization challenges that limit the translation of EVs to the clinic. Current challenges limiting development of EVs for drug delivery are the lack of: standardized cell-based platforms for the production of EV-based therapeutics; EV reference materials that allow researchers/manufacturers to validate EV measurements and standardized measurement systems for determining the molecular composition of EVs.

The levels of monoamine neurotransmitters and measures of mental and emotional health in HCV patients treated with ledipasvir (LDV) and sofosbuvir (SOF) with or without ribavirin (RBV).

Mental and emotional health (MEH) impairment is commonly encountered in hepatitis C patients. Although the exact mechanism remains unknown, alterations in neurotransmitter and cytokine levels maybe associated with hepatitis C virus (HCV)-related MEH issues.The aim of the study was to assess association of serum biomarkers with self-reports of MEH in HCV patients before treatment and after achieving sustained virologic response (SVR).The HCV genotype-1-infected patients who achieved SVR at 12 weeks after treatment with ledipasvir (LDV)/sofosbuvir (SOF) ±â€Šribavirin (RBV) were selected. Frozen serum samples from baseline, end of treatment (EOT), and posttreatment week 4 (PTW4) were used to assay 16 cytokines and monoamine neurotransmitters. Validated self-reports were used to assess MEH.Hundred patients were evaluated. Mean age was 53 years (57% male, 86% white). Compared with baseline, emotional well-being and emotional health significantly increased by EOT, and role emotional, emotional well-being, and emotional health significantly increased at PTW4 in the RBV-containing arm (P < 0.05). In patients taking LDV/SOF + RBV, serotonin levels were significantly decreased at PTW4 compared with baseline (P = 0.046). Compared with baseline, there were significant decreases in interleukin (IL)-10 levels at EOT and PTW4 in both treatment groups. The changes in IL-8 also differed significantly between LDV/SOF + RBV and LDV/SOF groups (P < 0.05). Changes in dopamine and tryptophan levels at EOT correlated with increasing emotional health scores, whereas changes in monocyte chemoattractant protein-1 at EOT and IL-8 at PTW4 correlated with increasing mental health scores. The neurotransmitters and cytokines were found to be independent predictors of MEH scores in multiple regression analysis.Cytokine and neurotransmitter changes are associated with mental and emotional health. Patient-reported outcome scores change during and after treatment.

Current management of patients with nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) and its progressive form, nonalcoholic steatohepatitis (NASH) are the most common causes of chronic liver disease in industrialized countries. NAFLD has also been strongly associated with type II diabetes and cardiovascular diseases. This study was a multipurposed review, which included discussion of recent studies investigating the cellular and genetic basis of these diseases, the pathogenesis of NAFLD and the current treatment and management of nonalcoholic steatohepatitis. Currently, maintaining a healthy weight through dietary changes and exercise, the use of insulin-modulating pharmacologic agents for diabetes control and the use of lipid-lowering, anti-oxidants have been the most widely recommended treatments. Inclusion of pathogenic mechanisms in treatment design will allow future therapies to target-specific pathways involved in NAFLD pathogenesis.

Ischemia-reperfusion injury and pregnancy initiate time-dependent and robust signs of up-regulation of cardiac progenitor cells.

To explore how cardiac regeneration and cell turnover adapts to disease, different forms of stress were studied for their effects on the cardiac progenitor cell markers c-Kit and Isl1, the early cardiomyocyte marker Nkx2.5, and mast cells. Adult female rats were examined during pregnancy, after myocardial infarction and ischemia-reperfusion injury with/out insulin like growth factor-1(IGF-1) and hepatocyte growth factor (HGF). Different cardiac sub-domains were analyzed at one and two weeks post-intervention, both at the mRNA and protein levels. While pregnancy and myocardial infarction up-regulated Nkx2.5 and c-Kit (adjusted for mast cell activation), ischemia-reperfusion injury induced the strongest up-regulation which occurred globally throughout the entire heart and not just around the site of injury. This response seems to be partly mediated by increased endogenous production of IGF-1 and HGF. Contrary to c-Kit, Isl1 was not up-regulated by pregnancy or myocardial infarction while ischemia-reperfusion injury induced not a global but a focal up-regulation in the outflow tract and also in the peri-ischemic region, correlating with the up-regulation of endogenous IGF-1. The addition of IGF-1 and HGF did boost the endogenous expression of IGF and HGF correlating to focal up-regulation of Isl1. c-Kit expression was not further influenced by the exogenous growth factors. This indicates that there is a spatial mismatch between on one hand c-Kit and Nkx2.5 expression and on the other hand Isl1 expression. In conclusion, ischemia-reperfusion injury was the strongest stimulus with both global and focal cardiomyocyte progenitor cell marker up-regulations, correlating to the endogenous up-regulation of the growth factors IGF-1 and HGF. Also pregnancy induced a general up-regulation of c-Kit and early Nkx2.5+ cardiomyocytes throughout the heart. Utilization of these pathways could provide new strategies for the treatment of cardiac disease.

Cyclic variability of stromal cell-derived factor-1 and endothelial progenitor cells during the menstrual cycle.

The endometrium goes through a unique cycle of physiological angiogenesis during the normal menstrual cycle (MC). We studied whether there is a correlation between endothelial progenitor cells (EPCs) and plasma and endometrial levels of angiogenic growth factors during the MC. Ten healthy, regularly menstruating women provided blood samples and another 16 supplied endometrial biopsies. Blood samples were obtained over a single MC: twice in the proliferative and once in the secretory phase and at ovulation. Endometrial biopsies were provided in the proliferative or in the secretory phase. We assessed plasma levels of vascular endothelial and fibroblast growth factors, granulocyte and granulocyte-macrophage colony-stimulating factors and stromal cell-derived factor-1 (SDF-1) by ELISA; EPCs by a colony-forming unit (CFU) assay; immunostaining for endometrial SDF-1 by computer-assisted software; and endothelial cell (EC) markers by flow cytometry. In the proliferative phase, SDF-1 levels were significantly higher than during the secretory phase. EPC-CFUs correlated negatively to SDF-1 levels. Endometrial SDF-1 expression tended to be higher in the secretory than in the proliferative phase. Furthermore, vascular endothelial growth factor receptors and Tie-2 EPCs showed a cyclic pattern over the MC. Our results point to SDF-1 as a novel mediator of EPC trafficking during the MC.

Anti-endothelial cell antibodies are increased in patients with previous myocardial infarction.

OBJECTIVES: Atherosclerosis is an inflammatory disease of multifactorial origin, in which immune cells together with metabolic risk factors may initiate, propagate, and activate lesions in the arterial tree. We investigated the role of auto-antibodies against endothelial cells in patients with previous myocardial infarction. DESIGN: One hundred and four patients were studied four to five years after acute myocardial infarction (aged 36-84 years) and 83 sex-matched healthy controls (aged 32-70 years). Anti-endothelial cells IgM and IgG auto-antibodies (AECA) were quantified in plasma using flow cytometry. RESULTS: IgM and IgG AECA were significantly higher (23.08 ± 0.81 and 10.63 ± 0.31 channel shifts, respectively; p < 0.001) in patients as compared to controls (13.40 ± 0.95 and 3.53 ± 0.54 channel shifts, respectively). Further, patients who got an invasive treatment had significantly higher levels of AECA as compared to patients with only medical treatment. Plasma concentration of IgG was positively (p < 0.05) correlated to the levels of high-sensitivity C-reactive protein (hsCRP). CONCLUSIONS: We report for the first time evidence that AECA are related to signs of inflammation and are increased in patients with atherosclerosis and previous myocardial infarction and with further increase with severity of disease.

Anti endothelial cell autoantibodies selectively activate SAPK/JNK signalling in Wegener's granulomatosis.

The pathogenic role of anti-endothelial cell antibodies (AECA) in vascular injury is debated. It was previously shown that many patients with Wegener's granulomatosis (WG) have AECA that react with human kidney microvascular endothelial cells (EC). In addition, during active disease, renal endothelium strongly expresses the inflammatory molecules vascular adhesion protein-1 (VAP-1) and MHC class I-related antigen A (MICA). This study sought to determine whether AECA mediates this upregulation of VAP-1 and MICA and to define better the signaling pathways that are activated by these autoantibodies upon binding to EC in the kidney. Stimulation of human kidney microvascular EC with AECA IgG upregulated surface expression of MICA and VAP-1, elicited a rapid Ca2+ flux, induced high levels of the chemokines monocyte chemoattractant protein-1 and granulocyte chemotactic protein-2, induced specific phosphorylation of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and the transcription factors c-Jun and activating transcription factor-2, and activated NF-kappaB. Specific inhibitors of SAPK/JNK significantly reduced AECA-induced chemokine production and phosphorylation of c-Jun and activating transcription factor-2 and abrogated protein expression of MICA but not VAP-1. In kidney sections from patients with WG, infiltrating cells that expressed the ligand for MICA (NKG2D+) were identified, as were CD8+ and 32 gamma delta+ T cells. In conclusion, AECA may be involved in the pathogenesis of WG, and the SAPK/JNK pathway and the endothelial inflammatory protein VAP-1 may be novel therapeutic targets for vasculitis.

A case of acute vascular rejection caused by endothelial-reactive non-HLA antibodies.

We describe a female patient who, despite negative conventional cross-matches, lost her first kidney graft in an acute humoral rejection. Prior to the second, AB0-incompatible (A1B to A1) living-donor kidney transplant, the patient had negative T- and B-cell cross-matches but had a positive donor-reactive endothelial cell cross-match. Following pre-transplant protein A and GlycoSorb-ABO immunoadsorptions to remove blood group B and anti-endothelial cell antibodies, Mabthera, and IVIG administrations, she was successfully transplanted. By the second post-operative day, creatinine levels were down to 96 micromol/L from 611 micromol/L pre-operatively. On day 9 creatinine rose again, and on the same day the endothelial cell crossmatch became positive for IgG, whereas the T-cell cross-match remained negative and the anti-A1B titers remained low. A kidney biopsy taken on day 10 post-transplant showed a picture of an acute vascular, antibody-mediated rejection. Following rejection treatment and repeated protein A and Glyco-Sorb-ABO immunoadsorptions, the patient's kidney function was again normalized. The use of a recently developed kit (XM-ONE) for the detection of anti-endothelial cell antibodies allowed us to identify a patient at risk for developing acute antibody-mediated rejection as well as to monitor treatment efficacy and post-transplant complications.

Circulating inflammatory endothelial cells contribute to endothelial progenitor cell dysfunction in patients with vasculitis and kidney involvement.

Impaired angiogenic function has been reported in patients with kidney failure. During vascular damage, endothelial cells may detach from the site of inflammation and be released into the peripheral blood. With the use of Wegener's granulomatosis as a study model, whether circulating inflammatory endothelial cells (IEC) can (1) be used as a disease activity marker and (2) contribute to sustained vascular damage by inducing endothelial progenitor cell (EPC) dysfunction were examined. IEC-defined as endothelial cells that express the two inflammatory-associated markers vascular-adhesion protein-1 (VAP-1) and MHC class I-related chain A (MICA)-were increased significantly in patients with active disease as compared with those in remission. IEC expressed high levels of inducible nitric oxide synthase and neutrophil-activating chemokines, such as macrophage inflammatory protein-1alpha, growth-related oncogene-alpha, epithelial neutrophil activating peptide-78, and IL-8, and induced increased neutrophil migration. IEC levels significantly correlated with C-reactive protein and extent of organ involvement. Patients with active disease had decreased numbers of EPC colony-forming units and a high expression of VAP-1 and MICA in kidney endothelium. EPC did not express VAP-1 or MICA. IEC significantly inhibited proliferation, migration, and endothelial nitric oxide synthase expression in EPC. Thus, apart from being a new disease activity marker, IEC may contribute to vascular damage by impairing the functional capacity for repair by EPC. IEC may provide a unique in vitro system to study pathogenesis of kidney and vascular diseases.

Only a specific subset of human peripheral-blood monocytes has endothelial-like functional capacity.

The monocyte population in blood is considered a possible source of endothelial precursors. Because endothelial-specific receptor tyrosine kinases act as regulators of endothelial cell function, we investigated whether expression of the vascular endothelial growth factor receptor-2 (VEGFR-2) on monocytes is important for their endothelial-like functional capacity. Peripheral-blood monocytes expressing vascular endothelial growth factor receptor-2 (VEGFR-2), or CD14+/VEGFR-2+, were isolated, and their phenotypic, morphologic, and functional capacities were compared with those of monocytes negative for this marker (CD14+/VEGFR-2-). CD14+/VEGFR-2+ cells constituted approximately 2% +/- 0.5% of the total population of monocytes and 0.08% +/- 0.04% of mononuclear cells in blood. CD14+/VEGFR-2+ cells exhibited the potential to differentiate in vitro into cells with endothelial characteristics. The cells were efficiently transduced by a lentiviral vector driving expression of the green fluorescence protein (GFP). Transplantation of GFP-transduced cells into balloon-injured femoral arteries of nude mice significantly contributed to efficient reendothelialization. CD14+/VEGFR-2- did not exhibit any of these characteristics. These data demonstrate that the expression of VEGFR-2 on peripheral blood monocytes is essential for their endothelial-like functional capacity and support the notion of a common precursor for monocytic and endothelial cell lineage. Our results help clarify which subpopulations may restore damaged endothelium and may participate in the maintenance of vascular homeostasis.