Islet Autoantibody Standardization Program 2018 Workshop:Interlaboratory Comparison of Glutamic Acid Decarboxylase Autoantibody Assay Performance

BACKGROUND: The Islet Autoantibody Standardization Program (IASP) aims to improve the performance of immunoassays measuring type 1 diabetes (T1D)-associated autoantibodies and the concordance of results among laboratories. IASP organizes international interlaboratory assay comparison studies in which blinded serum samples are distributed to participating laboratories, followed by centralized collection and analysis of results, providing participants with an unbiased comparative assessment. In this report, we describe the results of glutamic acid decarboxylase autoantibody (GADA) assays presented in the IASP 2018 workshop. METHODS: In May 2018, IASP distributed to participants uniquely coded sera from 43 new-onset T1D patients, 7 multiple autoantibody-positive nondiabetic individuals, and 90 blood donors. Results were analyzed for the following metrics: sensitivity, specificity, accuracy, area under the ROC curve (ROC-AUC), partial ROC-AUC at 95% specificity (pAUC95), and concordance of qualitative and quantitative results. RESULTS: Thirty-seven laboratories submitted results from a total of 48 different GADA assays adopting 9 different formats. The median ROC-AUC and pAUC95 of all assays were 0.87 [interquartile range (IQR), 0.83-0.89] and 0.036 (IQR, 0.032-0.039), respectively. Large differences in pAUC95 (range, 0.001-0.0411) were observed across assays. Of formats widely adopted, bridge ELISAs showed the best median pAUC95 (0.039; range, 0.036-0.041). CONCLUSIONS: Several novel assay formats submitted to this study showed heterogeneous performance. In 2018, the majority of the best performing GADA immunoassays consisted of novel or established nonradioactive tests that proved on a par or superior to the radiobinding assay, the previous gold standard assay format for GADA measurement

Two Distinctly HLA-Associated Contiguous Linear Epitopes Uniquely Expressed Within the Islet Antigen 2 Molecule Are Major Autoantibody Epitopes of the Diabetes-Specific Tyrosine Phosphatase-Like Protein Autoantigens

AbstractThe related tyrosine phosphatase-like proteins islet Ag (IA)-2 and IA-2β are autoantigens of type 1 diabetes in humans. Autoantibodies are predominantly against IA-2, and IA-2-specific epitopes are major autoantibody targets. We used the close homology of IA-2 and IA-2β to design chimeras and mutants to identify humoral IA-2-specific epitopes. Two major IA-2 epitopes that are absent from the related autoantigens IA-2β and IA-2Δ 13 splice variant ICA512.bdc were found contiguous to each other within IA-2 juxtamembrane amino acids 611–620 (epitope JM1) and 621–630 (epitope JM2). JM1 and JM2 are recognized by sera from 67% of patients with IA-2 Abs, and relatives of patients with type 1 diabetes having Abs to either JM epitope had a >50% risk for developing type 1 diabetes within 6 years, even in the absence of diabetes-associated HLA genotypes. Remarkably, the presence of Abs to one of these two epitopes was mutually exclusive of the other; JM2 Abs and not JM1 Abs were found in relatives with HLA DR3/4, DR4/13, or DR1/4 genotypes; and the binding of autoantibodies to the JM2 epitope, but not the JM1 epitope, markedly affected proteolysis of IA-2. This is a unique demonstration of HLA-associated B cell responses to epitopes within a single autoantigen in humans and is consistent with modification of Ag processing by specific Ab-influencing peptide presentation by HLA molecules

Autoantibodies to truncated GAD(96-585) antigen stratify risk of early insulin requirement in adult-onset diabetes

We investigated whether characterisation of full-length (f-)GADA responses could identify early insulin requirement in adult-onset diabetes. In 179 f-GADA positive participants diagnosed with type 2 diabetes, we assessed associations of truncated (t-)GADA positivity, f-GADA IgG subclasses, and f-GADA affinity with early insulin requirement (<5 years), type 1 diabetes genetic risk score (T1D GRS), and C-peptide. t-GADA positivity was lower in f-GADA positive without early insulin in comparison to f-GADA positive type 2 diabetes requiring insulin within 5 years, and type 1 diabetes (75% vs. 91% and 95% respectively, p<0.0001). t-GADA positivity (in those f-GADA positive) identified a group with a higher type 1 diabetes genetic susceptibility (mean T1D GRS 0.248 vs. 0.225, p=0.003), lower C-peptide (1156 pmol/L vs. 4289 pmol/L, p=1x10-7), and increased IA-2A positivity (23% vs. 6%, p=0.03). In survival analysis, t-GADA positivity was associated with early insulin requirement compared with those only positive for f-GADA, independently from age of diagnosis, f-GADA titre and duration of diabetes [adjusted HR 5.7 (95% CI 1.4, 23.5), p=0.017]. The testing of t-GADA in f-GADA positive individuals with type 2 diabetes identifies those who have genetic and clinical characteristics comparable to type 1 diabetes and stratifies those at higher risk of early insulin requirement

Autoantibodies toward ATP4A and ATP4B subunits of gastric proton pump H+,K+-ATPase are reliable serological pre-endoscopic markers of corpus atrophic gastritis

INTRODUCTION: Noninvasive assessment of corpus atrophic gastritis (CAG), a condition at increased risk of gastric cancer, is based on the measurement of pepsinogens, gastrin, and Helicobacter pylori antibodies. Parietal cell autoantibodies (PCAs) against the gastric proton pump (ATP4) are potential serological biomarkers of CAG. The purpose of this study was to compare the diagnostic performance of PCA and pepsinogen I tests in patients with clinical suspicion of CAG with the histopathological evaluation of gastric biopsies as reference standard. METHODS: A prospective case-finding study was performed on 218 naive adult patients (131 women, median age 65 years) who underwent gastric biopsies to confirm/exclude CAG. Patients with histopathological CAG were defined as cases, conversely as controls. Autoantibodies against the individual alpha (ATP4A) and beta (ATP4B) subunits of ATP4 were measured by luciferase immunoprecipitation, and global PCA and pepsinogen I by enzyme-linked immunosorbent assay. RESULTS: Histopathology classified 107 subjects (49%) as cases (CAG+, autoimmune 81.2%, and multifocal extensive 18.8%) and 111 subjects (51%) as controls (CAG−). In cases, ATP4A, ATP4B, and PCA titers were increased compared with controls, whereas pepsinogen I was reduced (P < 0.0001 for all). ATP4B, ATP4A, and pepsinogen I tests showed sensitivities of 77%, 75%, and 73% and specificities of 88%, 88%, and 80%, respectively. The receiver operating characteristic (ROC) area under the ROC curve (AUC) of these serological biomarkers confirmed their ability to discriminate cases from controls (ATP4B = 0.838, ATP4A = 0.826, pepsinogen I = 0.775, and PCA = 0.805), whereas the partial ROC-pAUC90 analysis showed that the ATP4B test had the best diagnostic performance (P = 0.008 vs ATP4; P = 0.0002 vs pepsinogen I). The presence of autoimmune or extensive gastritis was not significantly different between ATP4B positive or negative cases (P = 0.217). DISCUSSION: PCAs are promising serological biomarkers for the identification of CAG in high-risk individuals, particularly in an autoimmune pattern but also in an extensive-multifocal atrophy pattern

Detection of antibodies directed to the N-terminal region of GAD is dependent on assay format and contributes to differences in the specificity of GAD autoantibody assays for type 1 diabetes

Autoantibodies to glutamate decarboxylase (GADA) are sensitive markers of islet autoimmunity and type 1 diabetes. They form the basis of robust prediction models and are widely used for recruitment of subjects at high risk of type 1 diabetes to prevention trials. However GADA are also found in many individuals at low risk of diabetes progression. To identify the sources of diabetes irrelevant GADA reactivity therefore, we analyzed data from the 2009 and 2010 Diabetes Autoantibody Standardization Program GADA workshop and found that binding of healthy control sera varied according to assay type. Characterization of control sera found positive by radiobinding assay, but negative by ELISA showed that many of these sera reacted to epitopes in the N-terminal region of the molecule. This finding prompted development of an N-terminally truncated GAD65 radiolabel, (35)S-GAD65(96-585), which improved the performance of most GADA radiobinding assays (RBAs) participating in an Islet Autoantibody Standardization Program GADA substudy. These detailed workshop comparisons have identified a source of disease-irrelevant signals in GADA RBAs and suggest that N-terminally truncated GAD labels will enable more specific measurement of GADA in type 1 diabetes

The first 142 amino acids of glutamate decarboxylase do not contribute to epitopes recognized by autoantibodies associated with Type 1 diabetes

Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOy lated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4-and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention

Post hoc analysis of a randomized, double-blind, prospective trial evaluating a CXCR1/2 inhibitor in new-onset type 1 diabetes: endo-metabolic features at baseline identify a subgroup of responders

AimIn a recent randomized, multicenter trial (NCT02814838) a short-term anti-inflammatory treatment with ladarixin (LDX; an inhibitor of the CXCR1/2 chemokine receptors) did not show benefit on preserving residual beta cell function in new-onset type 1 diabetes. We present a post hoc analysis of trial patients in the predefined subgroup analysis developed according to baseline daily insulin requirement (DIR) tertiles.MethodA double-blind, randomized (2:1), placebo-controlled study was conducted in 45 men and 31 women (aged 18–46 years) within 100 days of the first insulin administration. Patients received LDX (400 mg twice daily) for three cycles of 14 days on/14 days off, or placebo. The primary endpoint was the area under the curve for C-peptide [AUC (0–120 min)] in response to a 2-h mixed meal tolerance test (MMTT) at week 13 ± 1. Seventy-five patients completed the week 13 MMTT and were divided into three groups according to the DIR tertiles: lower, ≤ 0.23U/kg/die (n = 25); middle, 0.24–0.40 U/kg/die (n = 24); upper, ≥ 0.41 U/kg/die (n = 26).ResultsWhen considering the patients in the upper tertile (HIGH-DIR), C-peptide AUC (0–120 min) at 13 weeks was higher in the LDX group (n = 16) than in the placebo (n = 10) group [difference: 0.72 nmol/L (95% CI 0.9–1.34), p = 0.027]. This difference reduced over time (0.71 nmol/L at 26 weeks, p = 0.04; 0.42 nmol/L at 52 weeks, p = 0.29), while it has never been significant at any time in patients in the lower and/or middle tertile (LOW-DIR). We characterized at baseline the HIGH-DIR and found that endo-metabolic (HOMA-B, adiponectin, and glucagon-to-C-peptide ratio) and immunologic (chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemoattractant protein 1 (MCP1) and Vascular Endothelial Growth Factor (VEGF)) features distinguished this group from LOW-DIR.ConclusionWhile LDX did not prevent the progressive loss of beta-cell function in the majority of treated subjects, the post hoc analysis suggests that it could work in subjects with HIGH-DIR at baseline. As we found differences in endo-metabolic and immunologic parameters within this subgroup, this generates the hypothesis that the interactions between host factors and drug action can contribute to its efficacy. Further research is needed to evaluate this hypothesis

Young infants exhibit robust functional antibody responses and restrained IFN-γ production to SARS-CoV-2

Severe COVID-19 appears rare in children. This is unexpected, especially in young infants, who are vulnerable to severe disease caused by other respiratory viruses. We evaluate convalescent immune responses in four infants under 3 months old with confirmed COVID-19 who presented with mild febrile illness, alongside their parents, and adult controls recovered from confirmed COVID-19. Although not statistically significant, compared to seropositive adults, infants have high serum levels of IgG and IgA to SARS-CoV-2 spike protein with corresponding functional ability to block SARS-CoV-2 cellular entry. Infants also exhibit robust saliva anti-spike IgG and IgA responses. Spike-specific IFN-γ production by infant peripheral blood mononuclear cells appears restrained, but the frequency of spike-specific IFN-γ and/or TNF-ɑ producing T cells is comparable between infants and adults. On principal component analysis, infant immune responses appear distinct from their parents. Robust functional antibody responses alongside restrained IFN-γ production may help protect infants from severe COVID-19

A novel LIPS assay for insulin autoantibodies

Insulin autoantibodies (IAA) are often the first marker of autoimmunity detected in children in the preclinical phase of type 1 diabetes (T1D). Currently, the vast majority of laboratories adopt the radiobinding micro-assay (RBA) for measuring IAA. Our aim was to replace RBA with a novel non-radioactive IAA Luciferase Immuno Precipitation System (LIPS) assay with improved performance. We developed (pro)insulin antigens with alternative placements of a NanoLuc (TM) luciferase reporter (NLuc). Performance in LIPS was evaluated by testing sera from new onset T1D (n = 80), blood donors (n = 123), schoolchildren (n = 186), first-degree relatives (FDRs) from the Bart's Oxford family study (n = 53) and from the Belgian Diabetes Registry (n = 136), coded sera from the Islet Autoantibody Standardization Program (IASP) (T1D n = 50, blood donors n = 90). IAA LIPS based on B chain-NLuc proinsulin or B chain-NLuc insulin, in which NLuc was fused at the C-terminus of the insulin B chain, required only 2 mu L of serum and a short incubation time, showed high concordance with RBA (Spearman r = 0.866 and 0.833, respectively), high assay performance (B chain-NLuc proinsulin ROC-AUC = 0.894 and B chain-NLuc insulin ROC-AUC = 0.916), and an adjusted sensitivity at 95% specificity ranking on par with the best assays submitted to the two most recent IASP workshops. In FDRs, the IAA LIPS showed improved discrimination of progressors to T1D compared to RBA. We established a novel high-performance non-radioactive IAA LIPS that might replace the current gold standard RBA and find wide application in the study of the IAA response in T1D

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