Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2.

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterized samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries.

Screening for Type 1 Diabetes in the General Population: A Status Report and Perspective.

Most screening programs to identify individuals at risk for type 1 diabetes have targeted relatives of people living with the disease to improve yield and feasibility. However, ∼90% of those who develop type 1 diabetes do not have a family history. Recent successes in disease-modifying therapies to impact the course of early-stage disease have ignited the consideration of the need for and feasibility of population screening to identify those at increased risk. Existing population screening programs rely on genetic or autoantibody screening, and these have yielded significant information about disease progression and approaches for timing for screening in clinical practice. At the March 2021 Type 1 Diabetes TrialNet Steering Committee meeting, a session was held in which ongoing efforts for screening in the general population were discussed. This report reviews the background of these efforts and the details of those programs. Additionally, we present hurdles that need to be addressed for successful implementation of population screening and provide initial recommendations for individuals with positive screens so that standardized guidelines for monitoring and follow-up can be established.

Activated but functionally impaired memory Tregs are expanded in slow progressors to type 1 diabetes.

AIMS/HYPOTHESIS: Slow progressors to type 1 diabetes are individuals positive for multiple pancreatic islet autoantibodies who have remained diabetes-free for at least 10 years; regulation of the autoimmune response is understudied in this group. Here, we profile CD4+ regulatory T cells (Tregs) in a small but well-characterised cohort of extreme slow progressors with a median age 43 (range 31-72 years), followed up for 18-32 years. METHODS: Peripheral blood samples were obtained from slow progressors (n = 8), age- and sex-matched to healthy donors. One participant in this study was identified with a raised HbA1c at the time of assessment and subsequently diagnosed with diabetes; this donor was individually evaluated in the analysis of the data. Peripheral blood mononuclear cells (PBMCs) were isolated, and to assess frequency, phenotype and function of Tregs in donors, multi-parameter flow cytometry and T cell suppression assays were performed. Unsupervised clustering analysis, using FlowSOM and CITRUS (cluster identification, characterization, and regression), was used to evaluate Treg phenotypes. RESULTS: Unsupervised clustering on memory CD4+ T cells from slow progressors showed an increased frequency of activated memory CD4+ Tregs, associated with increased expression of glucocorticoid-induced TNFR-related protein (GITR), compared with matched healthy donors. One participant with a raised HbA1c at the time of assessment had a different Treg profile compared with both slow progressors and matched controls. Functional assays demonstrated that Treg-mediated suppression of CD4+ effector T cells from slow progressors was significantly impaired, compared with healthy donors. However, effector CD4+ T cells from slow progressors were more responsive to Treg suppression compared with healthy donors, demonstrated by increased suppression of CD25 and CD134 expression on effector CD4+ T cells. CONCLUSIONS/INTERPRETATIONS: We conclude that activated memory CD4+ Tregs from slow progressors are expanded and enriched for GITR expression, highlighting the need for further study of Treg heterogeneity in individuals at risk of developing type 1 diabetes.

Slow progressors to type 1 diabetes lose islet autoantibodies over time, have few islet antigen-specific CD8+ T cells and exhibit a distinct CD95hi B cell phenotype.

AIMS/HYPOTHESIS: The aim of this study was to characterise islet autoantibody profiles and immune cell phenotypes in slow progressors to type 1 diabetes. METHODS: Immunological variables were compared across peripheral blood samples obtained from slow progressors to type 1 diabetes, individuals with newly diagnosed or long-standing type 1 diabetes, and healthy individuals. Polychromatic flow cytometry was used to characterise the phenotypic attributes of B and T cells. Islet autoantigen-specific B cells were quantified using an enzyme-linked immunospot (ELISpot) assay and islet autoantigen-specific CD8+ T cells were quantified using peptide-HLA class I tetramers. Radioimmunoassays were used to detect islet autoantibodies. Sera were assayed for various chemokines, cytokines and soluble receptors via ELISAs. RESULTS: Islet autoantibodies were lost over time in slow progressors. Various B cell subsets expressed higher levels of CD95 in slow progressors, especially after polyclonal stimulation, compared with the corresponding B cell subsets in healthy donors (p < 0.05). The phenotypic characteristics of CD4+ and CD8+ T cells were similar in slow progressors and healthy donors. Lower frequencies of CD4+ T cells with a central memory phenotype (CD27int, CD127+, CD95int) were observed in slow progressors compared with healthy donors (mean percentage of total CD4+ T cells was 3.00% in slow progressors vs 4.67% in healthy donors, p < 0.05). Autoreactive B cell responses to proinsulin were detected at higher frequencies in slow progressors compared with healthy donors (median no. of spots was 0 in healthy donors vs 24.34 in slow progressors, p < 0.05) in an ELISpot assay. Islet autoantigen-specific CD8+ T cell responses were largely absent in slow progressors and healthy donors. Serum levels of DcR3, the decoy receptor for CD95L, were elevated in slow progressors compared with healthy donors (median was 1087 pg/ml in slow progressors vs 651 pg/ml in healthy donors, p = 0.06). CONCLUSIONS/INTERPRETATION: In this study, we found that slow progression to type 1 diabetes was associated with a loss of islet autoantibodies and a distinct B cell phenotype, consistent with enhanced apoptotic regulation of peripheral autoreactivity via CD95. These phenotypic changes warrant further studies in larger cohorts to determine their functional implications.

A novel and rapid method to quantify Treg mediated suppression of CD4 T cells.

Measuring regulatory T cell suppression provides important insight into T cell dysfunction in autoimmune disease. However, to date, suppression assays are limited by the requirement for freshly isolated cells, and significant cell numbers. Here, we present a novel and rapid in vitro assay using effector T cell surface expression of both CD25 and CD134 as a surrogate marker of regulatory T cell-mediated suppression. This surface marker-based suppression assay works for frozen samples and for samples with limited cell numbers. It is also shorter taking two days to complete compared to the four days required for proliferation-based assays. Furthermore, this assay works with both in vitro expanded and natural Tregs, as well as anti-CD3/anti-CD28 bead-based and APC stimulation conditions. In conclusion, we have developed and validated a new suppression assay for cryopreserved samples with limited cell numbers that may be helpful to investigate T cell regulation in the context of infection or autoimmune diseases.