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.

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.

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.

Identification of eight-protein biosignature for diagnosis of tuberculosis.

BACKGROUND: Biomarker-based tests for diagnosing TB currently rely on detecting Mycobacterium tuberculosis (Mtb) antigen-specific cellular responses. While this approach can detect Mtb infection, it is not efficient in diagnosing TB, especially for patients who lack aetiological evidence of the disease. METHODS: We prospectively enrolled three cohorts for our study for a total of 630 subjects, including 160 individuals to screen protein biomarkers of TB, 368 individuals to establish and test the predictive model and 102 individuals for biomarker validation. Whole blood cultures were stimulated with pooled Mtb-peptides or mitogen, and 640 proteins within the culture supernatant were analysed simultaneously using an antibody-based array. Sixteen candidate biomarkers of TB identified during screening were then developed into a custom multiplexed antibody array for biomarker validation. RESULTS: A two-round screening strategy identified eight-protein biomarkers of TB: I-TAC, I-309, MIG, Granulysin, FAP, MEP1B, Furin and LYVE-1. The sensitivity and specificity of the eight-protein biosignature in diagnosing TB were determined for the training (n=276), test (n=92) and prediction (n=102) cohorts. The training cohort had a 100% specificity (95% CI 98% to 100%) and 100% sensitivity (95% CI 96% to 100%) using a random forest algorithm approach by cross-validation. In the test cohort, the specificity and sensitivity were 83% (95% CI 71% to 91%) and 76% (95% CI 56% to 90%), respectively. In the prediction cohort, the specificity was 84% (95% CI 74% to 92%) and the sensitivity was 75% (95% CI 57% to 89%). CONCLUSIONS: An eight-protein biosignature to diagnose TB in a high-burden TB clinical setting was identified.

Discovering endometriosis biomarkers with multiplex cytokine arrays.

BACKGROUND: Chronic pelvic pain is often overlooked during primary examinations because of the numerous causes of such "vague" symptoms. However, this pain can often mask endometriosis, a smoldering disease that is not easily identified as a cause of the problem. As such, endometriosis has been shown to be a potentially long-term and often undiagnosed disease due to its vague symptoms and lack of any non-invasive testing technique. Only after more severe symptoms arise (severe pelvic pain, excessive vaginal bleeding, or infertility) is the disease finally uncovered by the attending physician. Due to the nature and complexity of endometriosis, high throughput approaches for investigating changes in protein levels may be useful for elucidating novel biomarkers of the disease and to provide clues to help understand its development and progression. METHODS: A large multiplex cytokine array which detects the expression levels of 260 proteins including cytokines, chemokines, growth factors, adhesion molecules, angiogenesis factors and other was used to probe biomarkers in plasma samples from endometriosis patients with the intent of detecting and/or understanding the cause of this disease. The protein levels were then analyzed using K-nearest neighbor and split-point score analysis. RESULTS: This technique identified a 14-marker cytokine profile with the area under the curve of 0.874 under a confidence interval of 0.81-0.94. Our training set further validated the panel for significance, specificity, and sensitivity to the disease samples. CONCLUSIONS: These findings show the utility and reliability of multiplex arrays in deciphering new biomarker panels for disease detection and may offer clues for understanding this mysterious disease.

New Insights into the Tumor Microenvironment Utilizing Protein Array Technology.

The tumor microenvironment (TME) is a considerably heterogeneous niche, which is created by tumor cells, the surrounding tumor stroma, blood vessels, infiltrating immune cells, and a variety of associated stromal cells. Intercellular communication within this niche is driven by soluble proteins synthesized by local tumor and stromal cells and include chemokines, growth factors, interferons, interleukins, and angiogenic factors. The interaction of tumor cells with their microenvironment is essential for tumorigenesis, tumor progression, growth, and metastasis, and resistance to drug therapy. Protein arrays enable the parallel detection of hundreds of proteins in a small amount of biological sample. Recent data have demonstrated that the application of protein arrays may yield valuable information regarding the structure and functional mechanisms of the TME. In this review, we will discuss protein array technologies and their applications in TME analysis to discern pathways involved in promoting the tumorigenic phenotype.

Cytokines in cancer drug resistance: Cues to new therapeutic strategies.

The development of oncoprotein-targeted anticancer drugs is an invaluable weapon in the war against cancer. However, cancers do not give up without a fight. They may develop multiple mechanisms of drug resistance, including apoptosis inhibition, drug expulsion, and increased proliferation that reduce the effectiveness of the drug. The collective work of researchers has highlighted the role of cytokines in the mechanisms of cancer drug resistance, as well as in cancer cell progression. Furthermore, recent studies have described how specific cytokines secreted by cancer stromal cells confer resistance to chemotherapeutic treatments. In order to gain a better understanding of mechanism of cancer drug resistance and a prediction of treatment outcome, it is imperative that correlations are established between global cytokine profiles and cancer drug resistance. Here we discuss the recent discoveries in this field of research and discuss their implications for the future development of effective anti-cancer medicines.

Application of multiplex immunoassay technology to investigations of ocular disease.

Eye-derived fluids, including tears, aqueous humour and vitreous humour often contain molecular signatures of ocular disease states. These signatures can be composed of cytokines, chemokines, growth factors, proteases and soluble receptors. However, the small quantities (<10 µl) of these fluids severely limit the detection of these proteins by traditional enzyme-linked immunosorbent assay or Western blot. To maximise the amount of information generated from the analysis of these specimens, many researchers have employed multiplex immunoassay technologies for profiling the expression or modification of multiple proteins from minute sample volumes.

Identification and characterization of monoclonal antibodies against GP73 for use as a potential biomarker in liver cancer screening and diagnosis.

Golgi membrane protein 1, or GP73, is recently being evaluated as a novel cancer biomarker against prostate cancer, lung adenocarcinoma, and hepatocellular carcinoma (HCC). In the microenvironment of HCC, GP73 expression levels are significantly elevated. It is this elevation that may prove more specific and sensitive for HCC detection than that of the traditional biomarker, alpha-fetoprotein (AFP). This may be especially true if it can be measured and identified earlier in the diagnostic process. We sought to develop a testing platform to measure GP73 levels for the purposes of earlier diagnostic screening of at risk patients. We expressed recombinant GP73 protein to use as an immunogen in order to develop several monoclonal anti-GP73 antibodies. Three clones, 1D7, 2B2, and 5B4, were identified with all three having a higher than 1:5,000,000 titer. These clones were then isotyped and validated to bind the immunogen protein. Different combinations of antibody pairs were then tested in order to create a functional sandwich antibody pair. Using this pair on liver disease patient serum samples, we found that GP73 was significantly elevated when compared to healthy control patient serum (P < 0.0001). Average GP73 levels in HCC patients was 284.0 ng/mL, slightly higher than liver disease patients (265.6 ng/mL), and significantly elevated over normal serum levels is (74.86 ng/mL). The area under the receiver-operating characteristic curve (ROC) for GP73 to detect liver cancer was 0.98 (95% CI, 0.95 to 1.00; P < 0.0001), and GP73 levels had a sensitivity of 97%, a specificity of 87% for detecting liver cancer. By contrast, the sensitivity and specificity of liver disease detection was 76% and 97%, respectively. We then tested detection of 74 serum samples (n control = 46, n liver disease = 7, n liver cancer = 21) by our ELISA testing methodology and commercial kit simultaneously. The results found that our kit and the commercial kit had a good linear correlation coefficient, r(2) = 0.932. Together these clones and our ELISA pair may prove extremely useful in the detection and monitoring of GP73 in HCC and other at risk patients.

Tumor-induced perturbations of cytokines and immune cell networks.

Until recently, the intrinsically high level of cross-talk between immune cells, the complexity of immune cell development, and the pleiotropic nature of cytokine signaling have hampered progress in understanding the mechanisms of immunosuppression by which tumor cells circumvent native and adaptive immune responses. One technology that has helped to shed light on this complex signaling network is the cytokine antibody array, which facilitates simultaneous screening of dozens to hundreds of secreted signal proteins in complex biological samples. The combined applications of traditional methods of molecular and cell biology with the high-content, high-throughput screening capabilities of cytokine antibody arrays and other multiplexed immunoassays have revealed a complex mechanism that involves multiple cytokine signals contributed not just by tumor cells but by stromal cells and a wide spectrum of immune cell types. This review will summarize the interactions among cancerous and immune cell types, as well as the key cytokine signals that are required for tumors to survive immunoediting in a dormant state or to grow and spread by escaping it. Additionally, it will present examples of how probing secreted cell-cell signal networks in the tumor microenvironment (TME) with cytokine screens have contributed to our current understanding of these processes and discuss the implications of this understanding to antitumor therapies.

Enhanced chemosensitization in multidrug-resistant human breast cancer cells by inhibition of IL-6 and IL-8 production.

Drug resistance remains a major hurdle to successful cancer treatment. Many mechanisms such as overexpression of multidrug-resistance related proteins, increased drug metabolism, decreased apoptosis, and impairment of signal transduction pathway can contribute multidrug resistance (MDR). Recent studies strongly suggest a close link between cytokines and drug resistance. To identify new targets involved in drug resistance, we established a multidrug-resistant human breast cancer cell line MCF-7/R and examined the cytokine profile using cytokine antibody array technology. Among 120 cytokines/chemokines screened, IL-6, IL-8, and 13 other proteins were found to be markedly increased in drug-resistant MCF-7/R cell line as compared to sensitive MCF-7/S cell line, while 7 proteins were specifically reduced in drug-resistant MCF-7/R cells. Neutralizing antibodies against IL-6 and IL-8 partially reversed the drug resistance of MCF-7/R to paclitaxel and doxorubicin, while a neutralizing antibody against MCP-1 had no significant effect. Inhibition of endogenous IL-6 or IL-8 by siRNA technology significantly enhanced drug sensitivity of MCF-7/R cells. Furthermore, overexpression of IL-6 or IL-8 expression by transfection increased the ADM resistance in MCF-7/S cells. Our data suggest that increased expression levels of IL-6 and IL-8 may contribute to MDR in human breast cancer cells.

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.

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.

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.

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.

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.

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.