The importance of using WHO International Standards to harmonise SARS-CoV-2 serological assays.

The COVID-19 pandemic led to the rapid development of tests to diagnose SARS-CoV-2 infection and ascertain the prevalence of infection, along with the formulation of various treatments and vaccines. Globally, over 220 anti-SARS-CoV-2 serological assays have been developed for laboratory use, and many of these assays are currently used to assess immune responses against SARS-CoV-2. However, because these assays were independently developed by different manufacturers with different target antigens, immunoglobulin detection, technologies, and data reporting approaches, the results are not directly comparable, making it challenging to draw conclusions regarding immune responses at the population level. With deficiencies in assay validation, standardisation, and harmonisation, the inability to use and compare large datasets is becoming a major issue as serological data continue to increase. To help in addressing this issue, WHO established the first International Standard for the anti-SARS-CoV-2 immunoglobulin in late 2020. In this Personal View, we define the WHO International Standard for the anti-SARS-CoV-2 immunoglobulin, summarise the uses of primary versus secondary serology standards, recommend the use of such standards for data harmonisation, and list guidance and resources for using serology standards to improve data comparability.

Clinical Assessment of SARS-CoV-2 Antibodies in Oral Fluids Following Infection and Vaccination.

BACKGROUND: SARS-CoV-2 variants continue to circulate globally, even within highly vaccinated populations. The first-generation SARS-CoV-2 vaccines elicit neutralizing immunoglobin G (IgG) antibodies that prevent severe COVID-19 but induce only weak antibody responses in mucosal tissues. There is increasing recognition that secretory immunoglobin A (SIgA) antibodies in the upper respiratory tract and oral cavity are critical in interrupting virus shedding, transmission, and progression of disease. To fully understand the immune-related factors that influence SARS-CoV-2 dynamics at the population level, it will be necessary to monitor virus-specific IgG and SIgA in systemic and mucosal compartments. CONTENT: Oral fluids and saliva, with appropriate standardized collection methods, constitute a readily accessible biospecimen type from which both systemic and mucosal antibodies can be measured. Serum-derived IgG and immunoglobin A (IgA) are found in gingival crevicular fluids and saliva as the result of transudation, while SIgA, which is produced in response to mucosal infection and vaccination, is actively transported across salivary gland epithelia and present in saliva and passive drool. In this mini-review, we summarize the need for the implementation of standards, highly qualified reagents, and best practices to ensure that clinical science is both rigorous and comparable across laboratories and institutions. We discuss the need for a better understanding of sample stability, collection methods, and other factors that affect measurement outcomes and interlaboratory variability. SUMMARY: The establishment of best practices and clinical laboratory standards for the assessment of SARS-CoV-2 serum and mucosal antibodies in oral fluids is integral to understanding immune-related factors that influence COVID-19 transmission and persistence within populations.

The SeroNet Clinical and Translational Serology Task Force (CTTF) SARS-CoV-2 mucosal immunity methodological considerations and best practices workshop.

SARS-CoV-2 persists in certain populations, even with vaccination and boosters. Emerging evidence suggests that reductions in virus transmission and infection will likely require involvement of the mucosal immune system, especially secretory antibodies in the upper respiratory tract. The Clinical and Translational Serology Task Force (CTTF) within The National Cancer Institute (NCI)'s Serological Sciences Network for COVID-19 (SeroNet) hosted a workshop to review the status of development and standardization of mucosal sample collection methods and assays, identify challenges, and develop action plans to bridge gaps. Speakers presented data underscoring a role for secretory IgA in protection, mucosal markers as correlates of protection, methods for tracking and assessing mucosal antibodies, and lessons learned from other infectious agents. Perspectives from regulators and industry were put forward to guide mucosal vaccine development. Methodological considerations for optimizing collection protocols and assays and harmonizing data were highlighted. Rigorous studies, standardized protocols, controls, standards, and assay validation were identified as necessary to gain momentum in expanding SARS-CoV-2 vaccines to the mucosa.

Assay Harmonization Study To Measure Immune Response to SARS-CoV-2 Infection and Vaccines: a Serology Methods Study.

The Coronavirus disease 2019 (COVID-19) pandemic presented the scientific community with an immediate need for accurate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology assays, resulting in an expansion of assay development, some without following a rigorous quality control and validation, and with a wide range of performance characteristics. Vast amounts of data have been gathered on SARS-CoV-2 antibody response; however, performance and ability to compare the results have been challenging. This study seeks to analyze the reliability, sensitivity, specificity, and reproducibility of a set of widely used commercial, in-house, and neutralization serology assays, as well as provide evidence for the feasibility of using the World Health Organization (WHO) International Standard (IS) as a harmonization tool. This study also seeks to demonstrate that binding immunoassays may serve as a practical alternative for the serological study of large sample sets in lieu of expensive, complex, and less reproducible neutralization assays. In this study, commercial assays demonstrated the highest specificity, while in-house assays excelled in antibody sensitivity. As expected, neutralization assays demonstrated high levels of variability but overall good correlations with binding immunoassays, suggesting that binding may be reasonably accurate as well as practical for the study of SARS-CoV-2 serology. All three assay types performed well after WHO IS standardization. The results of this study demonstrate there are high performing serology assays available to the scientific community to rigorously dissect antibody responses to infection and vaccination. IMPORTANCE Previous studies have shown significant variability in SARS-CoV-2 antibody serology assays, highlighting the need for evaluation and comparison of these assays using the same set of samples covering a wide range of antibody responses induced by infection or vaccination. This study demonstrated that there are high performing assays that can be used reliably to evaluate immune responses to SARS-CoV-2 in the context of infection and vaccination. This study also demonstrated the feasibility of harmonizing these assays against the International Standard and provided evidence that the binding immunoassays may have high enough correlation with the neutralization assays to serve as a practical proxy. These results represent an important step in standardizing and harmonizing the many different serological assays used to evaluate COVID-19 immune responses in the population.

Simultaneous inhibition of CXCR1/2, TGF-ß, and PD-L1 remodels the tumor and its microenvironment to drive antitumor immunity.

BACKGROUND: Despite the success of immune checkpoint blockade therapy in the treatment of certain cancer types, only a small percentage of patients with solid malignancies achieve a durable response. Consequently, there is a need to develop novel approaches that could overcome mechanisms of tumor resistance to checkpoint inhibition. Emerging evidence has implicated the phenomenon of cancer plasticity or acquisition of mesenchymal features by epithelial tumor cells, as an immune resistance mechanism. METHODS: Two soluble factors that mediate tumor cell plasticity in the context of epithelial-mesenchymal transition are interleukin 8 (IL-8) and transforming growth factor beta (TGF-ß). In an attempt to overcome escape mechanisms mediated by these cytokines, here we investigated the use of a small molecule inhibitor of the IL-8 receptors CXCR1/2, and a bifunctional agent that simultaneously blocks programmed death ligand 1 (PD-L1) and traps soluble TGF-ß. RESULTS: We demonstrate that simultaneous inhibition of CXCR1/2, TGF-ß, and PD-L1 signaling synergizes to reduce mesenchymal tumor features in murine models of breast and lung cancer, and to markedly increase expression of tumor epithelial E-cadherin while reducing infiltration with suppressive granulocytic myeloid-derived suppressor cells, significantly enhancing T-cell infiltration and activation in tumors, and leading to improved antitumor activity. CONCLUSIONS: This study highlights the potential benefit of combined blockade of CXCR1/2 and TGF-ß signaling for modulation of tumor plasticity and potential enhancement of tumor responses to PD-L1 blockade. The data provide rationale for the evaluation of this novel approach in the clinic.

Low Intratumoral Mast Cells Are Associated With a Higher Risk of Prostate Cancer Recurrence.

BACKGROUND: Mast cells are of interest in prostate cancer because they possess both pro- and anti-tumorigenic properties and are present in the tumor microenvironment. We studied the association of mast cell count and densities with prostate cancer recurrence using tissue microarrays (TMAs) for 462 men who recurred (cases) and 462 controls that were matched to the cases nested in a cohort of radical prostatectomy patients. METHODS: Dual-immunostaining for mast cell tryptase and epithelial cytokeratin-8 and whole slide image analysis were used to assess total mast cell number, mast cell density (mast cell number/tissue area), and mast cell number per epithelial or stromal area in TMA spots containing tumor (up to 4 per man). We used conditional logistic regression to estimate the odds ratio (OR) and 95% confidence interval of recurrence for the mean, minimum, and maximum mast cell parameters in tumor tissue among each man's TMA spots. RESULTS: After taking into account matching factors of age, race, Gleason sum, and pathologic stage, higher minimum mast cell density in the tumor (comparing highest to lowest quartiles: OR = 0.58, 95% CI 0.40-0.86; P-trend = 0.004) was associated with a lower risk of recurrence. Patterns for mast cell number and ratio of mast cell number to epithelial or stromal area were similar to those for mast cell density. CONCLUSIONS: Our results suggest that intratumoral mast cells may be protective against prostate cancer recurrence and could potentially serve as a prognostic biomarker after prostatectomy. Prostate 77: 412-424, 2017. © 2016 Wiley Periodicals, Inc.

Bacterial Prostatitis Enhances 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine (PhIP)-Induced Cancer at Multiple Sites.

Dietary carcinogens, such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and chronic inflammation have each been implicated as etiologic agents in prostate cancer. We hypothesized that bacterial prostatitis would accelerate PhIP-induced preinvasive lesions in the rat prostate. Male Fischer 344 rats were assigned into 4 groups: Control (untreated), PhIP (200 ppm in the diet for 20 weeks), Escherichia coli (E. coli, prostatic inoculation in week 10), or PhIP + E. coli. Study animals were monitored for a total of 52 weeks and were euthanized as necessary based on strict criteria for health status and tumor burden. Animals treated with E. coli initially developed acute and chronic inflammation in all lobes of the prostate, whereas inflammation was observed predominantly in the ventral lobe at time of death. PhIP + E. coli-treated animals exhibited a marked decrease in survival compared with PhIP-alone-treated animals as a result of an increase in the number of invasive cancers that developed at multiple sites, including the skin, small intestine, and Zymbal's gland. Despite their earlier mortality, PhIP + E. coli-treated animals developed an increased average number of precancerous lesions within the prostate compared with PhIP-treated animals, with a significantly increased Ki-67 index. Multiplexed serum cytokine analysis indicated an increase in the level of circulating IL6 and IL12 in PhIP + E. coli-treated animals. Elevated serum IL6 levels correlated with the development of precancerous lesions within the prostate. These results suggest that bacterial infections and dietary carcinogens, two conceivably preventable cancer risk factors, may synergistically promote tumorigenesis.

The role of inflammation in prostate cancer.

In the United States and in "Westernized" countries, the prevalence of both prostate cancer and prostate inflammation is very high, indicating that the two pathologies could be causally related. Indeed, chronic inflammation is now regarded as an "enabling" characteristic of human cancer. Prostate cancer incidence is thought to be mediated in part by genetics, but also by environmental exposures, including the same exposures that may contribute to the development of prostatic inflammation. As our understanding of the role of inflammation in cancer deepens, it is increasingly apparent that "inflammation" as a whole is a complex entity that does not always play a negative role in cancer etiology. In fact, inflammation can play potentially dichotomous (both pro and antitumorigenic) roles depending on the nature and the cellular makeup of the immune response. This chapter will focus on reviewing the current state of knowledge on the role of innate and adaptive immune cells within the prostate tumor microenvironment and their seemingly complex role in prostate cancer in preventing versus promoting initiation and progression of the disease.

Infection of Xenotransplanted Human Cell Lines by Murine Retroviruses: A Lesson Brought Back to Light by XMRV.

Infection of xenotransplanted human cells by xenotropic retroviruses is a known phenomenon in the scientific literature, with examples cited since the early 1970s. However, arguably, until recently, the importance of this phenomenon had not been largely recognized. The emergence and subsequent debunking of Xenotropic Murine leukemia virus-Related Virus (XMRV) as a cell culture contaminant as opposed to a potential pathogen in several human diseases, notably prostate cancer and Chronic Fatigue Syndrome, highlighted a potential problem of murine endogenous gammaretroviruses infecting commonly used human cell lines. Subsequent to the discovery of XMRV, many additional cell lines that underwent xenotransplantation in mice have been shown to harbor murine gammaretroviruses. Such retroviral infection poses the threat of not only confounding experiments performed in these cell lines via virus-induced changes in cellular behavior but also the potential infection of other cell lines cultured in the same laboratory. Thus, the possibility of xenotropic retroviral infection of cell lines may warrant additional precautions, such as periodic testing for retroviral sequences in cell lines cultured in the laboratory.

Development of peptide nucleic acid probes for detection of the HER2 oncogene.

Peptide nucleic acids (PNAs) have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes.

Synthesis of novel cyclic NGR/RGD peptide analogs via on resin click chemistry.

Targeted drug deliveries as well as high resolution imaging of cancerous tissues and organs via specific cancer cell markers have become important in chemotherapeutic interventions of cancer treatment. Short peptides such as RGD and NGR are showing promising results for targeted drug delivery and in vivo imaging. We have applied on resin Huisgen's 1,3-dipolar cycloaddition to synthesize new cyclic RGD and NGR peptide analogs. Preliminary binding assays of these new analogs by fluorescence polarization indicates specific binding to purified CD13 (Aminopeptidase N) and cell lysates from MCF-7 and SKOV-3 cancer cell lines.