N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global public health crisis. The causative agent, the SARS-CoV-2 virus, enters host cells via molecular interactions between the viral spike protein and the host cell ACE2 surface protein. The SARS-CoV-2 spike protein is extensively decorated with up to 66 N-linked glycans. Glycosylation of viral proteins is known to function in immune evasion strategies but may also function in the molecular events of viral entry into host cells. Here, we show that N-glycosylation at Asn331 and Asn343 of SARS-CoV-2 spike protein is required for it to bind to ACE2 and for the entry of pseudovirus harboring the SARS-CoV-2 spike protein into cells. Interestingly, high-content glycan binding screening data have shown that N-glycosylation of Asn331 and Asn343 of the RBD is important for binding to the specific glycan molecule G4GN (Galß-1,4 GlcNAc), which is critical for spike-RBD-ACE2 binding. Furthermore, IL-6 was identified through antibody array analysis of conditioned media of the corresponding pseudovirus assay. Mutation of N-glycosylation of Asn331 and Asn343 sites of the spike receptor-binding domain (RBD) significantly reduced the transcriptional upregulation of pro-inflammatory signaling molecule IL-6. In addition, IL-6 levels correlated with spike protein levels in COVID-19 patients' serum. These findings establish the importance of RBD glycosylation in SARS-CoV-2 pathogenesis, which can be exploited for the development of novel therapeutics for COVID-19.

Identification and validation of circulating biomarkers for detection of liver cancer with antibody array.

The aim of this study was to find new protein biomarkers that could be used to detect hepatocellular carcinoma (HCC) in the serum. We identified 11 proteins in the tissue that could be used to classify samples from HCC and control subjects. The 11 identified tissue biomarkers were combined with 10 commonly used serum HCC biomarkers for further verification in a large number of serum samples from HCC patients and healthy controls. 17 of the 21 prospective serum biomarkers were determined to be differentially expressed through collinearity and significance analysis. Through the method of supervised learning, a random forest model was constructed to reduce the dimensionality of the number of differentially expressed proteins, and finally, 4 differentially expressed proteins were identified: AFP, GDF15, CEACAM-1, and MMP-9, and suggested to have potential application in clinical diagnosis of HCC.

Development and evaluation of time-resolved fluorescent immunochromatographic assay for quantitative detection of SARS-CoV-2 spike antigen.

BACKGROUND: The spread of COVID-19 worldwide caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has necessitated efficient, sensitive diagnostic methods to identify infected people. We report on the development of a rapid 15-minute time-resolved fluorescent (TRF) lateral flow immunochromatographic assay for the quantitative detection of the SARS-CoV-2 spike protein receptor-binding domain (S1-RBD). OBJECTIVES: Our objective was to develop an efficient method of detecting SARS-CoV-2 within 15 min of sample collection. METHODS: We constructed and evaluated a portable, disposable lateral flow device, which detected the S1-RBD protein directly in nasopharyngeal swab samples. The device emits a fluorescent signal in the presence of S1-RBD, which can be captured by an automated TRF instrument. RESULTS: The TRF lateral flow assay signal was linear from 0 to 20 ng/ml and demonstrated high accuracy and reproducibility. When evaluated with clinical nasopharyngeal swabs, the assay was performed at >80% sensitivity, >84% specificity, and > 82% accuracy for detection of the S1-RBD antigen. CONCLUSION: The new S1-RBD antigen test is a rapid (15 min), sensitive, and specific assay that requires minimal sample preparation. Critically, the assay correlated closely with PCR-based methodology in nasopharyngeal swab samples, showing that the detected S1-RBD antigen levels correlate with SARS-CoV-2 virus load. Therefore, the new TRF lateral flow test for S1-RBD has potential application in point-of-care settings.

Induction of a cytokine storm involves suppression of the Osteopontin-dependent TH1 response.

Cytokine release syndromes represent a severe turn in certain disease states, which may be caused by several infections, including those with the virus SARS-CoV-2. This inefficient, even harmful, immune response has been associated with a broad release of chemokines. Although a cellular (type I) immune reaction is efficacious against viral infections, we noted a type I deficit in the cytokine patterns produced by cytokine storms of all reported etiologies. Agents including lipopolysaccharide (LPS, bacterial), anti-CD3 (antibody) and a version of the prominent SARS-CoV-2 viral surface molecule, Spike Glycoprotein, were individually sufficient to induce IL-6 and multiple chemokines in mice. They failed to upregulate the TH1 inducer cytokine Osteopontin, and the pathophysiologic triggers actually suppressed the PMA-induced Osteopontin secretion from monocytic cells. Osteopontin administration partially reversed the chemokine elevation, more effectively so in a mouse strain with TH1 bias. Corroboration was obtained from the inverse correlation in the levels of IL-6 and Osteopontin in plasma samples from acute COVID-19 patients. We hypothesize that the inhibition of Osteopontin by SARS-CoV-2 Spike Glycoprotein or LPS represents an immune evasion mechanism employed by the pathogens of origin. The ensuing dysfunctional inflammatory response promotes a vicious cycle of amplification, resulting in a cytokine storm.

Developing an Effective Peptide-Based Vaccine for COVID-19: Preliminary Studies in Mice Models.

Coronavirus disease 2019 (COVID-19) has caused massive health and economic disasters worldwide. Although several vaccines have effectively slowed the spread of the virus, their long-term protection and effectiveness against viral variants are still uncertain. To address these potential shortcomings, this study proposes a peptide-based vaccine to prevent COVID-19. A total of 15 B cell epitopes of the wild-type severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein were selected, and their HLA affinities predicted in silico. Peptides were divided into two groups and tested in C57BL/6 mice with either QS21 or Al(OH)3 as the adjuvant. Our results demonstrated that the peptide-based vaccine stimulated high and durable antibody responses in mice, with the T and B cell responses differing based on the type of adjuvant employed. Using epitope mapping, we showed that our peptide-based vaccine produced antibody patterns similar to those in COVID-19 convalescent individuals. Moreover, plasma from vaccinated mice and recovered COVID-19 humans had the same neutralizing activity when tested with a pseudo particle assay. Our data indicate that this adjuvant peptide-based vaccine can generate sustainable and effective B and T cell responses. Thus, we believe that our peptide-based vaccine can be a safe and effective vaccine against COVID-19, particularly because of the flexibility of including new peptides to prevent emerging SARS-CoV-2 variants and avoiding unwanted autoimmune responses.

Serum molecular biomarkers in neuromyelitis optica and multiple sclerosis.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is a rare and severe inflammatory demyelinating disorder of the central nervous system (CNS), which mainly affects the optic nerves and spinal cord. The aims of this study were to determine whether the expression levels of serological cytokines could distinguish 1) NMOSD from healthy controls (HCs); and 2) NMOSD patients with and without the aquaporin-4 (AQP4) antibody biomarker from each other; and 3) NMOSD patients without the antibody to AQP4 from MS patients. METHODS: The expression levels of 200 proteins in serum from 41 NMOSD (32 with antibodies to AQP4, 9 without antibodies to AQP4), 12 MS patients, and 34 HCs were measured using glass-based antibody arrays. None of the patients received any immunosuppressive treatment. In parallel, the correlation between protein expression in NMOSD/MS patients and clinical traits was determined with Weighted Gene Co-expression Network Analysis (WGCNA). RESULTS: Thirty-nine serological proteins were differentially expressed in NMOSD patients compared to HCs, with 29 of these proteins not observed in MS patients. In addition, the data reveal 15 differentially-expression proteins (DEPs) between AQP4-IgG seronegative and AQP4-IgG seropositive NMOSD patients, and 9 DEPs between NMOSD and MS patients who did not have AQP4-IgG. CONCLUSION: Serological IL-17B is significantly upregulated in both NMOSD and MS patients compared to HCs, and could be a key biomarker of NMOSD and MS. Serological VEGF, MPIF-1 and NrCAM were positively associated with AQP4-IgG titer. We also demonstrate that EGF may be involved in the breakdown of the BBB by downregulating Claudin-5.

A Case-Control Study of Follicular Fluid Cytokine Profiles in Women with Diminished Ovarian Reserve.

Ovarian reserve is an important determinant of a woman's reproductive potential, and women with diminished ovarian reserve (DOR) often seek in vitro fertilization (IVF). The underlying etiology of DOR is unknown, but follicular fluid cytokine concentrations likely play a role in follicular development and maturation. The present study seeks to investigate the expression of cytokines in follicular fluid (FF) of women with DOR undergoing IVF and explore correlated functional pathways. One hundred ninety-four women undergoing ovarian stimulation were recruited at the time of oocyte retrieval. Women were classified as having DOR if they met one or more of the following criteria: AMH < 1 ng/ml, FSH > 10 mIU/ml, and/or AFC < 10. Controls included women undergoing IVF for male factor, tubal factor due to tubal ligation, or planned oocyte cryopreservation (non-oncologic). The concentrations of 480 cytokines and related growth factors in follicular fluid were determined using a multiplex immunoassay. Fifty-nine cytokines had significantly different concentrations (53 higher and 6 lower) in the DOR relative to the control group after adjusting for age and body mass index (BMI) (false discovery rate; FDR < 0.1). Using the most informative 44 biomarkers as indicated by a random forest (RF) model, an area under the curve (AUC) of 0.78 was obtained. Thus, follicular microenvironment differs between women with DOR and normal ovarian reserve. The differentially expressed cytokines belong to diverse processes that are primarily involved in follicular maturation and ovulation. These changes may play an important role in treatment outcomes in women with DOR.

Quantitative Detection of Anti-SARS-CoV-2 Antibodies Using Indirect ELISA.

OBJECTIVE: Real-time reverse transcription-polymerase chain reaction is the gold standard for the diagnosis of COVID-19, but it is necessary to utilize other tests to determine the burden of the disease and the spread of the outbreak such as IgG-, IgM-, and IgA-based antibody detection using enzyme-linked immunosorbent assay (ELISA). MATERIALS AND METHODS: We developed an indirect ELISA assay to quantitatively measure the amount of COVID-19 IgG, IgM, and IgA antibodies present in patient serum, dried blood, and plasma. RESULTS: The population cutoff values for positivity were determined by receiver operating characteristic curves to be 1.23 U/mL, 23.09 U/mL, and 6.36 U/mL for IgG, IgM, and IgA, respectively. After albumin subtraction, the specificity remained >98% and the sensitivity was 95.72%, 83.47%, and 82.60%, respectively, for IgG, IgM, and IgA antibodies to the combined spike subunit 1 receptor binding domain and N proteins in serum. Plasma and dried blood spot specimens were also validated on this assay. CONCLUSION: This assay may be used for determining the seroprevalence of SARS-CoV-2 in a population exposed to the virus or in vaccinated individuals.

Dried blood sample analysis by antibody array across the total testing process.

Dried blood samples (DBSs) have many advantages; yet, impediments have limited the clinical utilization of DBSs. We developed a novel volumetric sampling device that collects a precise volume of blood, which overcomes the heterogeneity and hematocrit issues commonly encountered in a traditional DBS card collection as well as allowing for more efficient extraction and processing procedures and thus, more efficient quantitation, by using the entire sample. We also provided a thorough procedure validation using this volumetric DBS collection device with an established quantitative proteomics analysis method, and then analyzed 1000 proteins using this approach in DBSs concomitantly with serum for future consideration of utility in clinical applications. Our data provide a first step in the establishment of a DBS database for the broad application of this sample type for widespread use in clinical proteomic and other analyses applications.

Three subtypes of lung cancer fibroblasts define distinct therapeutic paradigms.

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-ß signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.

Comprehensive Profiling of Inflammatory Factors Revealed That Growth Differentiation Factor-15 Is an Indicator of Disease Severity in COVID-19 Patients.

To systematically explore potential biomarkers which can predict disease severity in COVID-19 patients and prevent the occurrence or development of severe COVID-19, the levels of 440 factors were analyzed in patients categorized according to COVID-19 disease severity; including asymptomatic, mild, moderate, severe, convalescent and healthy control groups. Factor candidates were validated by ELISA and functional relevance was uncovered by bioinformatics analysis. To identify potential biomarkers of occurrence or development of COVID-19, patient sera from three different severity groups (moderate, severe, and critical) at three time points (admission, remission, and discharge) and the expression levels of candidate biomarkers were measured. Eleven differential factors associated with disease severity were pinpointed from 440 factors across 111 patients of differing disease severity. The dynamic changes of GDF15 reflect the progression of the disease, while the other differential factors include TRAIL R1, IGFBP-1, IGFBP-4, VCAM-1, sFRP-3, FABP2, Transferrin, GDF15, IL-1F7, IL-5Rα, and CD200. Elevation of white blood cell count, neutrophil count, neutrophil-lymphocyte ratio (NLR), Alanine aminotransferase and Aspartate aminotransferase, low lymphocyte and eosinophil counts in the severe group were associated with the severity of COVID-19. GDF15 levels were observed to be associated with the severity of COVID-19 and the dynamic change of GDF15 levels was closely associated with the COVID-19 disease progression. Therefore, GDF15 might serve as an indicator of disease severity in COVID-19 patients.

Using Antibody Arrays for Biomarker Discovery.

Biomarkers for diseases are important for the development of clinical diagnostic tests and can provide early intervention for cancer or cardiovascular patients. Over the past decade, antibody array technology has achieved significant technological improvement in the quantitative measurement of more than a thousand proteins simultaneously and has been utilized to screen and identify unique proteins as disease biomarkers. However, few biomarkers have been translated into clinical application. This chapter will discuss the protocol for the screening and validation of unique proteins that create a new avenue for biomarker discovery.

Data Analysis for Antibody Arrays.

When obtaining high-throughput data from antibody arrays, researchers have to face a couple of questions: How and by what means can they get reasonable results significant to their research from these data? Similar to a gene microarray, the classical statistical pipeline of an antibody array includes data preprocessing transformation, differential expression analysis, classification, and biological annotation analysis. In this chapter, we will provide a pipeline of statistical approaches suitable for antibody arrays to facilitate better understanding of the results gained from each of these steps.

Sandwich-Based Antibody Arrays for Protein Detection.

Sandwich-based antibody arrays enable the detection of multiple proteins simultaneously, thus offering a time- and cost-effective alternative to single-plex platforms. The protein of interest is "sandwiched" between an antibody that captures it to the array and a second antibody that is used for detection. Here we describe a 1-day procedure to process samples, such as serum or cell lysates, with a quantitative sandwich-based antibody array on a glass substrate using fluorescence.

Global Detection of Proteins by Label-Based Antibody Array.

Because of narrow availability of antibody pairs and potential cross-reactivity between antibodies, the development of sandwich-based antibody arrays which need a pair of antibodies for each target has been restricted to higher density resulting in limited proteomic breadth of detection. Label-based array is one way to overcome this obstacle by directly labeling all targets in samples with fluorescent dyes such as Cy3 and Cy5. The labeled samples are then applied on the antibody array chip composed of capture antibodies. In this chapter, we will introduce this technology including array production and sample detection assay.

Cytometry Multiplex Bead Antibody Array.

The flow cytometry-based multiplex bead array is an advanced technology using antibody-conjugated multiplex beads to detect soluble targets in a liquid phase. This technology has been widely used for detection of soluble analytes like cytokines, chemokines, allergens, viral antigens, and cancer markers. RayPlex® Multiplex Beads Antibody Array series are developed by RayBiotech Life, Inc. to quantitatively detect a wide range of analytes with high sensitivity to meet increasing need of research and diagnosis.

Enhanced Protein Profiling Arrays for High-Throughput Quantitative Measurement of Cytokine Expression.

The antibody array has become a powerful technology in recent years and is widely used to detect the expression levels of various proteins such as cytokines, growth factors, chemokines, and angiogenic factors, some of which are involved in cancer progression. In this chapter, we describe a protein array technology called enhanced protein profiling array, which can simultaneously and quantitatively measure the expression levels of a few proteins in hundreds or thousands of samples, and an example of its use is presented.

Methods for Membranes and Other Absorbent Surfaces.

Membrane arrays are a unique array platform option for the detection of multiple analytes or materials simultaneously. Their naturally absorptive properties and near universal use in various laboratory methods make it an excellent source with which to probe multiple factors simultaneously. Any liquid sample type can be probed, from bacterial strains, tissue lysates, secreted proteins, to DNA aptamers. Below, we will describe some considerations in how to print a membrane array and then a specific usage of the membrane arrays as it relates to a sandwich-based antibody array technique for simultaneously detection of secreted proteins in a liquid sample.

Dried Blood-Based Protein Profiling Using Antibody Arrays.

Dried blood samples have been increasingly considered for clinical applications in recent years. The main disadvantages that limit DBS utility in clinical applications are the small sample volume collected, area bias and homogeneity issues, and sample preparation requirements for the necessary sensitivity and reproducibility required for clinical assessment. The recent advances in antibody array technology overcome the common disadvantages of immunoassay approaches by increasing the multiplex capabilities and decreasing the sample volume requirements as well as minimizing the expense and technical expertise required with many alternative high-density approaches like mass spectrometry.

Analyzing Signaling Pathways Using Antibody Arrays.

Cell signaling is comprised of complex networks that regulate homeostasis and human diseases. The analyses of such pathways would improve our understanding of disease pathology and direct drug development. However, it remains a great challenge to study pathways using traditional methods. We developed a high-throughput sandwich-based antibody array technology for the simultaneous detection of multiple targets, capable of identifying the relative expression levels or phosphorylation levels of major signaling pathway proteins. This array-based system features a nitrocellulose membrane or glass slide solid support, spotted with antibodies targeting key proteins of major signaling pathways, including RTK, EGFR, MAPK, AKT, apoptosis, TGFb, JAK/STAT, NFkB, and insulin receptor pathways. We employed these antibody arrays to investigate how the anti-cancer drugs, camptothecin and phorbol 12-myristate 13-acetate (PMA), alter protein phosphorylation in Jurkat and HeLa cells, respectively. Our array data suggest that camptothecin treatment induced DNA double-strand breaks in Jurkat cells and activated the DNA damage pathways ATM and Chk2, which then further induced apoptosis through caspase 3 and PARP. PMA induced the MAPK pathway in HeLa cells through the activation of ERK, CREB, and RSK1. These array results are consistent with previous studies using traditional methods and were validated with Western blotting. Our studies demonstrate that pathway antibody arrays provide a rapid, efficient, and multiplexed approach for profiling phosphorylated proteins.