Autoantibodies to Ezrin are an early sign of pancreatic cancer in humans and in genetically engineered mouse models

BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy with only a 5% 5-year survival rate. Reliable biomarkers for early detection are still lacking. The goals of this study were (a) to identify early humoral responses in genetically engineered mice (GEM) spontaneously developing PDAC; and (b) to test their diagnostic/predictive value in newly diagnosed PDAC patients and in prediagnostic sera. METHODS AND RESULTS: The serum reactivity of GEM from inception to invasive cancer, and in resectable or advanced human PDAC was tested by two-dimensional electrophoresis Western blot against proteins from murine and human PDAC cell lines, respectively. A common mouse-to-human autoantibody signature, directed against six antigens identified by MALDI-TOF mass spectrometry, was determined. Of the six antigens, Ezrin displayed the highest frequency of autoantibodies in GEM with early disease and in PDAC patients with resectable disease. The diagnostic value of Ezrin-autoantibodies to discriminate PDAC from controls was further shown by ELISA and ROC analyses (P < 0.0001). This observation was confirmed in prediagnostic sera from the EPIC prospective study in patients who eventually developed PDAC (with a mean time lag of 61.2 months between blood drawing and PDAC diagnosis). A combination of Ezrin-autoantibodies with CA19.9 serum levels and phosphorylated α-Enolase autoantibodies showed an overall diagnostic accuracy of 0.96 ± 0.02. CONCLUSIONS: Autoantibodies against Ezrin are induced early in PDAC and their combination with other serological markers may provide a predictive and diagnostic signature

Immunoassay for quantification of antigen-specific IgG fucosylation

BACKGROUND: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (FcγR). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell FcγRs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. METHODS: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives FcγRIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (FcγR-IgG ELISA). FINDINGS: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R2=0.93, p < 0.0001). The FEASI method was then used to quantify IgG levels and fucosylation in COVID-19 convalescent plasma which was independently validated by LC-MS. INTERPRETATION: FEASI can be reliably implemented to measure relative and absolute IgG Fc fucosylation and quantify binding of antigen-specific IgG to FcγR in a high-throughput manner accessible to all diagnostic and research laboratories. FUNDING: This work was funded by the Stichting Sanquin Bloedvoorziening (PPOC 19-08 and SQI00041) and ZonMW 10430 01 201 0021

Divergent SARS-CoV-2-specific T and B cell responses in severe but not mild COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2 specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T cell compartment, and indicative of vascular leakage. The observed disparate T and B cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients. This article is protected by copyright. All rights reserved

Divergent SARS-CoV-2-specific T- and B-cell responses in severe but not mild COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2-specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T-cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T-cell compartment, and indicative of vascular leakage. The observed disparate T- and B-cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients

Saliva SARS-CoV-2 antibody prevalence in children

COVID-19 patients produce circulating and mucosal antibodies. In adults, specific saliva antibodies have been detected. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We therefore assessed SARS-CoV-2-specific antibody prevalence in serum and saliva in children in the Netherlands. We assessed SARS-CoV-2 antibody prevalence in serum and saliva of 517 children attending medical services in the Netherlands (irrespective of COVID-19 exposure) from April to October 2020. The prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N)-specific IgG and IgA were evaluated with an exploratory Luminex assay in serum and saliva and with the Wantai SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay in serum. Using the Wantai assay, the RBD-specific antibody prevalence in serum was 3.3% (95% confidence interval [CI]. 1.9 to 5.3%). With the Luminex assay, we detected heterogeneity between antibodies for S, RBD, and N antigens, as IgG and IgA prevalence ranged between 3.6 and 4.6% in serum and between 0 and 4.4% in saliva. The Luminex assay also revealed differences between serum and saliva, with SARS-CoV-2-specific IgG present in saliva but not in serum for 1.5 to 2.7% of all children. Using multiple antigen assays, the IgG prevalence for at least two out of three antigens (S, RBD, or N) in serum or saliva can be calculated as 3.8% (95% CI, 2.3 to 5.6%). Our study displays the heterogeneity of the SARS-CoV-2 antibody response in children and emphasizes the additional value of saliva antibody detection and the combined use of different antigens. IMPORTANCE Comprehending humoral immunity to SARS-CoV-2, including in children, is crucial for future public health and vaccine strategies. Others have suggested that mucosal antibody measurement could be an important and more convenient tool to evaluate humoral immunity compared to circulating antibodies. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We show the heterogeneity of SARS-CoV-2 antibodies, in terms of both antigen specificity and differences between circulating and mucosal antibodies, emphasizing the additional value of saliva antibody detection next to detection of antibodies in serum

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors

Objectives: Characterisation of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT-PCR-positive SARS-CoV-2 convalescent adults during the first 250 days after onset of symptoms. Methods: We measured antibody responses to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT-PCR-positive SARS-CoV-2 convalescent adults. With a median of 5 (range 2-18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed-effects modelling. Results: All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels declined with median half-lives of 62 and 59 days, respectively, 2-5 months after symptom onset, and several-fold variation in half-lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow-up, which points towards long-term immunological memory. The magnitude of the anti-RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in-house developed competitive assay. Conclusion: The result of this study gives valuable insight into the long-term longitudinal response of antibodies to SARS-CoV-2

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors

Objectives: Characterisation of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT-PCR-positive SARS-CoV-2 convalescent adults during the first 250 days after onset of symptoms. Methods: We measured antibody responses to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT-PCR-positive SARS-CoV-2 convalescent adults. With a median of 5 (range 2-18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed-effects modelling. Results: All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels declined with median half-lives of 62 and 59 days, respectively, 2-5 months after symptom onset, and several-fold variation in half-lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow-up, which points towards long-term immunological memory. The magnitude of the anti-RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in-house developed competitive assay. Conclusion: The result of this study gives valuable insight into the long-term longitudinal response of antibodies to SARS-CoV-2

Immunoassay for quantification of antigen-specific IgG fucosylation

Background: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (FcγR). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell FcγRs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. Methods: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives FcγRIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (FcγR-IgG ELISA). Findings: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R2=0.93, p < 0.0001). The FEASI method was then used to quantify IgG levels and fucosylation in COVID-19 convalescent plasma which was independently validated by LC-MS. Interpretation: FEASI can be reliably implemented to measure relative and absolute IgG Fc fucosylation and quantify binding of antigen-specific IgG to FcγR in a high-throughput manner accessible to all diagnostic and research laboratories. Funding: This work was funded by the Stichting Sanquin Bloedvoorziening (PPOC 19-08 and SQI00041) and ZonMW 10430 01 201 0021

Development of a SARS-CoV-2 total antibody assay and the dynamics of antibody response over time in hospitalized and nonhospitalized patients with COVID-19

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infections often cause only mild disease that may evoke relatively low Ab titers compared with patients admitted to hospitals. Generally, total Ab bridging assays combine good sensitivity with high specificity. Therefore, we developed sensitive total Ab bridging assays for detection of SARS-CoV-2 Abs to the receptor-binding domain (RBD) and nucleocapsid protein in addition to conventional isotype-specific assays. Ab kinetics was assessed in PCRconfirmed, hospitalized coronavirus disease 2019 (COVID-19) patients (n = 41) and three populations of patients with COVID- 19 symptoms not requiring hospital admission: PCR-confirmed convalescent plasmapheresis donors (n = 182), PCR-confirmed hospital care workers (n = 47), and a group of longitudinally sampled symptomatic individuals highly suspect of COVID-19 (n = 14). In nonhospitalized patients, the Ab response to RBD is weaker but follows similar kinetics, as has been observed in hospitalized patients. Across populations, the RBD bridging assay identified most patients correctly as seropositive. In 11/14 of the COVID-19-suspect cases, seroconversion in the RBD bridging assay could be demonstrated before day 12; nucleocapsid protein Abs emerged less consistently. Furthermore, we demonstrated the feasibility of finger-prick sampling for Ab detection against SARS-CoV-2 using these assays. In conclusion, the developed bridging assays reliably detect SARS-CoV-2 Abs in hospitalized and nonhospitalized patients and are therefore well suited to conduct seroprevalence studies