This thesis is focused on the targeting of two antigens often expressed on the surface of cancer cells: MICA and HLA-E. Both antigens are members of the MHC-I family of proteins. They both act as... Show moreThis thesis is focused on the targeting of two antigens often expressed on the surface of cancer cells: MICA and HLA-E. Both antigens are members of the MHC-I family of proteins. They both act as ligands for the NK receptors on NK cells and CD8+ T cells. We target MICA using nanobodies, or VHHs, the small variable regions of camelid-derived heavy-chain only antibodies. The nanobodies are used as nanobody-drug conjugate, fused to the Mertansine-derivative DM1, or as the targeting domain of chimeric antigen receptors to create CAR NK cells. These nanobody-based immunotherapies selectively target and kill MICA-positive cancer cells. We target HLA-E with an antibody directed against a 13-amino acid epitope unique to the cytoplasmic tail of HLA-E. The antibody is specific to HLA-E and can be used for detection of HLA-E positive cells in flow cytometry and immunofluorescence and detection of HLA-E positive tumors in immunohistochemistry. Together with the epitope, the antibody can be used as a novel epitope tag for detection and purification of unrelated proteins. Show less
Khidir, S.J.H.; Toes, R.E.M.; Mulligen, E. van; Helm-van Mil, A.H.M. van der 2024
BackgroundWe aimed to estimate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence and describe its determinants and associated symptoms among unvaccinated healthcare... Show moreBackgroundWe aimed to estimate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence and describe its determinants and associated symptoms among unvaccinated healthcare workers (HCWs) after the first wave of the pandemic.MethodsHCWs from 13 Dutch hospitals were screened for antibodies against the spike protein of SARS-CoV-2 in June-July 2020 and after three months. Participants completed a retrospective questionnaire on determinants for occupational and community exposure to SARS-CoV-2 and symptoms suggestive of COVID-19 experienced since January 2020. The seroprevalence was calculated per baseline characteristic and symptom at baseline and after follow-up. Adjusted odds ratios (aOR) for seropositivity were determined using logistic regression.ResultsAmong 2328 HCWs, 323 (13.9%) were seropositive at enrolment, 49 of whom (15%) reported no previous symptoms suggestive of COVID-19. During follow-up, only 1% of the tested participants seroconverted. Seroprevalence was higher in younger HCWs compared to the mid-age category (aOR 1.53, 95% CI 1.07–2.18). Nurses (aOR 2.21, 95% CI 1.34–3.64) and administrative staff (aOR 1.87, 95% CI 1.02–3.43) had a higher seroprevalence than physicians. The highest seroprevalence was observed in HCWs in the emergency department (ED) (aOR 1.79, 95% CI 1.10–2.91), the lowest in HCWs in the intensive, high, or medium care units (aOR 0.47, 95% CI 0.31–0.71). Chronic respiratory disease, smoking, and having a dog were independently associated with a lower seroprevalence, while HCWs with diabetes mellitus had a higher seroprevalence. In a multivariable model containing all self-reported symptoms since January 2020, altered smell and taste, fever, general malaise/fatigue, and muscle aches were positively associated with developing antibodies, while sore throat and chills were negatively associated.ConclusionsThe SARS-CoV-2 seroprevalence in unvaccinated HCWs of 13 Dutch hospitals was 14% in June-July 2020 and remained stable after three months. A higher seroprevalence was observed in the ED and among nurses, administrative and young staff, and those with diabetes mellitus, while a lower seroprevalence was found in HCWs in intensive, high, or medium care, and those with self-reported lung disease, smokers, and dog owners. A history of altered smell or taste, fever, muscle aches and fatigue were independently associated with the presence of SARS-CoV-2 antibodies in unvaccinated HCWs. Show less
Background Anti-NMDA-receptor GluN1 antibodies (NMDAR1-abs) are present in an autoimmune encephalitis with severe neuropsychiatric symptoms. We aimed to estimate the impact of serum NMDAR1-abs on... Show moreBackground Anti-NMDA-receptor GluN1 antibodies (NMDAR1-abs) are present in an autoimmune encephalitis with severe neuropsychiatric symptoms. We aimed to estimate the impact of serum NMDAR1-abs on depressive symptoms years after first-ever ischemic stroke (IS).Methods Data were used from the PROSpective Cohort with Incident Stroke-Berlin (PROSCIS-B; NCT01363856). Serum NMDAR1-abs (IgM/IgA/IgG) were measured within 7 days after IS using cell-based assays. We defined seropositivity as titers >= 1:10, thereof low titers as <= 1:100 and high titers as >1:100. We used the Center for Epidemiological Studies-Depression (CES-D) scale to measure depressive symptoms at year one, two and three following IS. We calculated crude and confounder adjusted weighted generalized linear models to quantify the impact of NMDAR1-abs on CES-D assessed at three annual time-points.Results NMDAR1-abs were measured in 583 PROSCIS-B IS patients (mean age = 67 [SD = 13]; 42%female; median NIHSS = 2 [IQR = 1-4]) of whom 76 (13%; IgM: n = 49/IgA: n = 43/IgG: n = 2) were seropositive, 55 (9%) with low and 21 (4%) with high titers. CES-D regarded over all follow-up time-points was higher in seropositive patients (beta(crude) = 2.56 [95%CI = -0.34 to 5.45]; beta(adjusted) = 2.26 [95%CI = -0.68 to 5.20]) and effects were highest in patients with high titer (low titers: beta(crude) = 1.42 [95%CI = -1.79 to 4.62], beta(adjusted) = 0.53 [95%CI = -2.47 to 3.54]; high titers: beta(crude) = 5.85 [95%CI = 0.20 to 11.50]; beta(adjusted) = 7.20 [95%CI = 0.98 to 13.43]).Conclusion Patients with serum NMDAR1-abs (predominantly IgM&IgA) suffer more severe depressive symptoms after mild-to-moderate IS compared to NMDAR1-abs seronegative patients. Show less
The conserved region (Fc) of IgG antibodies dictates the interactions with designated receptors thus defining the immunological effector functions of IgG. Amino acid sequence variations in the Fc,... Show moreThe conserved region (Fc) of IgG antibodies dictates the interactions with designated receptors thus defining the immunological effector functions of IgG. Amino acid sequence variations in the Fc, recognized as subclasses and allotypes, as well as post-translational modifications (PTMs) modulate these interactions. Yet, the high similarity of Fc sequences hinders allotype-specific PTM analysis by state-of-the-art bottom-up methods and current subunit approaches lack sensitivity and face co-elution of near-isobaric allotypes.To circumvent these shortcomings, we present a nanoscale reversed-phase (RP) HPLC-MS workflow of intact Fc subunits for comprehensive characterization of Fc proteoforms in an allotype- and subclass-specific manner. Polyclonal IgGs were purified from individuals followed by enzymatic digestion releasing single chain Fc subunits (Fc/2) that were directly subjected to analysis. Chromatographic conditions were optimized to separate Fc/2 subunits of near-isobaric allotypes and subclasses allowing allotype and proteoform identification and quantification across all four IgG subclasses. The workflow was complemented by a semi-automated data analysis pipeline based on the open-source software Skyline followed by post-processing in R. The approach revealed pronounced differences in Fc glycosylation between donors, besides inter-subclass and inter-allotype variability within donors. Notably, partial occupancy of the N-glycosylation site in the CH3 domain of IgG3 was observed that is generally neglected by established approaches. The described method was benchmarked across several hundred runs and showed good precision and robustness.This methodology represents a first mature Fc subunit profiling approach allowing truly subclass- and allotype-specific Fc proteoform characterization beyond established approaches. The comprehensive information obtained paired with the high sensitivity provided by the miniaturization of the approach guarantees applicability to a broad range of research questions including clinically relevant (auto)antibody characterization or pharmacokinetics assessment of therapeutic IgGs. Show less
Background: Patients with immune-mediated inflammatory diseases (IMIDs) on immunosuppressants (ISPs) may have impaired long-term humoral immune responses and increased disease activity after SARS... Show moreBackground: Patients with immune-mediated inflammatory diseases (IMIDs) on immunosuppressants (ISPs) may have impaired long-term humoral immune responses and increased disease activity after SARS-CoV-2 infection. We aimed to investigate long-term humoral immune responses against SARS-CoV-2 and increased disease activity after a primary SARS-CoV-2 infection in unvaccinated IMID patients on ISPs. Methods: IMID patients on active treatment with ISPs and controls (i.e. IMID patients not on ISP and healthy controls) with a confirmed SARS-CoV-2 infection before first vaccination were included from an ongoing prospective cohort study (T2B! study). Clinical data on infections and increased disease activity were registered using electronic surveys and health records. A serum sample was collected before first vaccination to measure SARS-CoV-2 anti-receptor-binding domain (RBD) antibodies. Results: In total, 193 IMID patients on ISP and 113 controls were included. Serum samples from 185 participants were available, with a median time of 173 days between infection and sample collection. The rate of seropositive IMID patients on ISPs was 78% compared to 100% in controls (p < 0.001). Seropositivity rates were lowest in patients on anti-CD20 (40.0%) and anti-tumor necrosis factor (TNF) agents (60.5%), as compared to other ISPs (p < 0.001 and p < 0.001, respectively). Increased disease activity after infection was reported by 68 of 260 patients (26.2%; 95% CI 21.2-31.8%), leading to ISP intensification in 6 out of these 68 patients (8.8%). Conclusion: IMID patients using ISPs showed reduced long-term humoral immune responses after primary SARS-CoV-2 infection, which was mainly attributed to treatment with anti-CD20 and anti-TNF agents. Increased disease activity after SARS-CoV-2 infection was reported commonly, but was mostly mild. Show less
BackgroundPatients with immune-mediated inflammatory diseases (IMIDs) on immunosuppressants (ISPs) may have impaired long-term humoral immune responses and increased disease activity after SARS-CoV... Show moreBackgroundPatients with immune-mediated inflammatory diseases (IMIDs) on immunosuppressants (ISPs) may have impaired long-term humoral immune responses and increased disease activity after SARS-CoV-2 infection. We aimed to investigate long-term humoral immune responses against SARS-CoV-2 and increased disease activity after a primary SARS-CoV-2 infection in unvaccinated IMID patients on ISPs.MethodsIMID patients on active treatment with ISPs and controls (i.e. IMID patients not on ISP and healthy controls) with a confirmed SARS-CoV-2 infection before first vaccination were included from an ongoing prospective cohort study (T2B! study). Clinical data on infections and increased disease activity were registered using electronic surveys and health records. A serum sample was collected before first vaccination to measure SARS-CoV-2 anti-receptor-binding domain (RBD) antibodies.ResultsIn total, 193 IMID patients on ISP and 113 controls were included. Serum samples from 185 participants were available, with a median time of 173 days between infection and sample collection. The rate of seropositive IMID patients on ISPs was 78% compared to 100% in controls (p < 0.001). Seropositivity rates were lowest in patients on anti-CD20 (40.0%) and anti-tumor necrosis factor (TNF) agents (60.5%), as compared to other ISPs (p < 0.001 and p < 0.001, respectively). Increased disease activity after infection was reported by 68 of 260 patients (26.2%; 95% CI 21.2–31.8%), leading to ISP intensification in 6 out of these 68 patients (8.8%).ConclusionIMID patients using ISPs showed reduced long-term humoral immune responses after primary SARS-CoV-2 infection, which was mainly attributed to treatment with anti-CD20 and anti-TNF agents. Increased disease activity after SARS-CoV-2 infection was reported commonly, but was mostly mild. Show less
Muscle-specific kinase (MuSK) myasthenia gravis (MG) is a neuromuscular autoimmune disorder caused by predominantly IgG4 antibodies targeting the MuSK protein. IgG4 has the unique ability to... Show moreMuscle-specific kinase (MuSK) myasthenia gravis (MG) is a neuromuscular autoimmune disorder caused by predominantly IgG4 antibodies targeting the MuSK protein. IgG4 has the unique ability to exchange half-molecules with other IgG4s, resulting in monovalent binding to their antigen. To investigate if MuSK-antibody valency influences its pathogenicity, recombinant bivalent and functionally monovalent MuSK antibodies were generated from B-cell receptor sequences isolated from MuSK MG patients. Passive transfer studies revealed that monovalency amplifies MuSK antibody pathogenicity in vivo. This may be because monovalent MuSK antibodies inhibit MuSK signaling (antagonist), while bivalent MuSK antibodies activate MuSK signaling (agonist) in vitro. The binding epitope on MuSK further influences the consequences and pathogenicity of MuSK antibodies. Collectively, these results suggest that the pathophysiology in individual patients depends on their unique antibody composition and that class-switching to IgG4 is a critical step in developing MuSK MG. Furthermore, the IgG4 response against MuSK does not appear to result from a global increase in IgG4 responses, as MuSK MG patients only had mildly elevated serum IgG4. Instead, it is thought to be driven by the antigen itself. Importantly, MUSK and other MG associated genes are also expressed outside skeletal muscle. These locations are at risk for non-motor symptoms caused by autoantibodies or mutations, or for side-effects of targeted therapeutic strategies. Show less
Gharbharan, A.; Jordans, C.; Zwaginga, L.; Papageorgiou, G.; Geloven, N. van; Wijngaarden, P. van; ... ; CoV-Early study grp 2023
Objectives: The potential benefit of convalescent plasma (CP) therapy for coronavirus disease 2019 (COVID-19) is highest when administered early after symptom onset. Our objective was to determine... Show moreObjectives: The potential benefit of convalescent plasma (CP) therapy for coronavirus disease 2019 (COVID-19) is highest when administered early after symptom onset. Our objective was to determine the effectiveness of CP therapy in improving the disease course of COVID-19 among high-risk outpatients. Methods: A multicentre, double-blind randomized trial was conducted comparing 300 mL of CP with non-CP. Patients were >= 50 years, were symptomatic for <8 days, had confirmed RT-PCR or antigen test result for COVID-19 and had at least one risk factor for severe COVID-19. The primary endpoint was the highest score on a 5-point ordinal scale ranging from fully recovered (score = 1) or not (score = 2) on day 7, over hospital admission (score = 3), intensive care unit admission (score = 4) and death (score = 5) in the 28 days following randomization. Secondary endpoints were hospital admission, symptom duration and viral RNA excretion. Results: After the enrolment of 421 patients and the transfusion in 416 patients, recruitment was dis-continued when the countrywide vaccination uptake in those aged >50 years was 80%. Patients had a median age of 60 years, symptoms for 5 days, and 207 of 416 patients received CP therapy. During the 28 day follow-up, 28 patients were hospitalized and two died. The OR for an improved disease severity score with CP was 0.86 (95% credible interval, 0.59-1.22). The OR was 0.58 (95% CI, 0.33-1.02) for patients with <5 days of symptoms. The hazard ratio for hospital admission was 0.61 (95% CI, 0.28-1.34). No difference was found in viral RNA excretion or in the duration of symptoms. Conclusions: In patients with early COVID-19, CP therapy did not improve the 5-point disease severity score. Arvind Gharbharan, Clin Microbiol Infect 2023;29:208 (c) 2022 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). Show less
Coillie, J. van; Pongracz, T.; Rahmoller, J.; Chen, H.J.; Geyer, C.E.; Vught, L.A. van; ... ; UMC COVID-19 S HCW Study Grp 2022
Background: Afucosylated IgG1 responses have only been found against membrane-embedded epitopes, including anti-S in SARS-CoV-2 infections. These responses, intrinsically protective through... Show moreBackground: Afucosylated IgG1 responses have only been found against membrane-embedded epitopes, including anti-S in SARS-CoV-2 infections. These responses, intrinsically protective through enhanced Fc gamma RIIIa binding, can also trigger exacerbated pro-inflammatory responses in severe COVID-19. We investigated if the BNT162b2 SARS-CoV-2 mRNA also induced afucosylated IgG responses. Methods: Blood from vaccinees during the first vaccination wave was collected. Liquid chromatography-Mass spectrometry (LC-MS) was used to study anti-S IgG1 Fc glycoprofiles. Responsiveness of alveolar-like macrophages to produce proinflammatory cytokines in presence of sera and antigen was tested. Antigen-specific B cells were characterized and glycosyltransferase levels were investigated by Fluorescence-Activated Cell Sorting (FACS). Findings: Initial transient afucosylated anti-S IgG1 responses were found in naive vaccinees, but not in antigen -experienced ones. All vaccinees had increased galactosylated and sialylated anti-S IgG1. Both naive and antigen -experienced vaccinees showed relatively low macrophage activation potential, as expected, due to the low antibody levels for naive individuals with afucosylated IgG1, and low afucosylation levels for antigen-experienced individuals with high levels of anti-S. Afucosylation levels correlated with FUT8 expression in antigen-specific plasma cells in naive individuals. Interestingly, low fucosylation of anti-S IgG1 upon seroconversion correlated with high anti-S IgG levels after the second dose. Interpretation: Here, we show that BNT162b2 mRNA vaccination induces transient afucosylated anti-S IgG1 responses in naive individuals. This observation warrants further studies to elucidate the clinical context in which potent afucosylated responses would be preferred. Copyright (c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Show less
Purpose: Five patients who underwent uncomplicated retinal pigment epithelium (RPE)-choroid transplantation for neovascular age-related macular degeneration developed a destructive inflammatory... Show morePurpose: Five patients who underwent uncomplicated retinal pigment epithelium (RPE)-choroid transplantation for neovascular age-related macular degeneration developed a destructive inflammatory reaction causing subretinal fluid accumulation and extensive RPE atrophy in the graft. We hypothesized that this inflammation could be caused by an auto-immune response against the graft, resulting in circulating auto-antibodies. The aim of our study was to examine a potential autoimmune origin, which would allow a more targeted therapy approach. Methods: Five above-mentioned patients and four control groups of five patients each were included: 1) after uncomplicated RPE-choroid transplantation, 2) after full macular translocation, 3) treated with anti-vascular endothelial growth factor, and 4) healthy controls. Histopathology of rejected graft tissue was performed using standard procedures. Presence of RPE-choroid autoantibodies in serum was examined by indirect immunofluorescence and Western blot, and human leukocyte antigen (HLA) typing was performed. Results: Histopathological examination of an explanted graft showed infiltration of T-lymphocytes and macrophages in the choroid and RPE, and an increased number of B-cell lymphocytes were found in the choroid. Indirect immunofluorescence showed weak RPE-choroid autoantibody immunoreactivity in three patients of different groups. Western blot did not show specific RPE-choroid autoantibody immunoreactivity and no difference of HLA genotypes between the groups was found. Conclusions: Although local mononuclear inflammatory cell infiltration and a high number of B-lymphocytes were observed in an explanted graft, we did not detect serological evidence of an autoimmune origin of the postoperative inflammation using direct immunofluorescence and Western Blot. Alternatively, the graft failure may have been caused by local innate inflammation, triggered by breakdown of tolerance. Based on our current findings of this small study group, we have no rationale to pursue therapies targeted towards autoreactive graft failure. More research is needed to confirm our findings. Show less
Objective We aimed to investigate whether serum anti-N-methyl-D-aspartate-receptor GluN1 (previously NR1) antibody (NMDAR1-abs) seropositivity impacts cognitive function (CF) in the long term... Show moreObjective We aimed to investigate whether serum anti-N-methyl-D-aspartate-receptor GluN1 (previously NR1) antibody (NMDAR1-abs) seropositivity impacts cognitive function (CF) in the long term following ischemic stroke. Methods Data were used from the PROSpective Cohort with Incident Stroke-Berlin. NMDAR1-abs (IgM/IgA/IgG) were measured with cell-based assays from serum obtained within 7 days after the first-ever stroke. Seropositivity was defined as titers >= 1:10, low titers as <= 1:100 and high titers as > 1:100. We assessed CF at 1, 2 and 3 years after stroke with the Telephone Interview for Cognitive Status-modified (TICS-m) and used crude and propensity score adjusted inverse probability weighted generalized linear models to estimate the impact of NMDAR1-abs serostatus on TICS-m. Results Data on NMDAR1-abs (median day of sampling = 4[IQR = 2-5]) were available in 583/621 PROSCIS-B patients (39% female; median NIHSS = 2[IQR = 1-4]; median MMSE = 28[IQR:26-30]), of whom 76(13%) were seropositive (IgM: n = 48/IgA: n = 43/IgG: n = 2). Any NMDAR1-abs seropositivity had no impact on TICS-m compared to seronegative patients (beta crude = 0.69[95%CI = - 0.84 to 2.23]; beta adjusted = 0.65[95%CI = - 1.00 to 2.30]). Patients with low titers scored better on TICS-m compared to seronegative patients (beta crude = 2.33[95%CI = 0.76 to 3.91]; beta adjusted = 2.47[95%CI = 0.75 to 4.19]); in contrast, patients with high titers scored lower on TICS-m (beta crude = -2.82[95%CI = - 4.90 to - 0.74], beta adjusted = - 2.96[95%CI = - 5.13 to - 0.80]), compared to seronegative patients. Conclusion In our study, NMDAR1-abs seropositivity did not affect CF over 3 years after a first mild to moderate ischemic stroke. CF differed according to NMDAR1-abs serum titer, with patients with high NMDAR1-abs titers having a less favorable cognitive outcome compared to seronegative patients. Show less
Background: Antibodies against mycobacterial proteins are highly specific, but lack sensitivity, whereas cytokines have been shown to be sensitive but not very specific in the diagnosis of... Show moreBackground: Antibodies against mycobacterial proteins are highly specific, but lack sensitivity, whereas cytokines have been shown to be sensitive but not very specific in the diagnosis of tuberculosis (TB). We assessed combinations between antibodies and cytokines for diagnosing TB. Methods: Immuoglubulin (Ig) A and IgM antibody titres against selected mycobacterial antigens including Apa, NarL, Rv3019c, PstS1, LAM, "Kit 1" (MTP64 and Tpx)", and "Kit 2" (MPT64, Tpx and 19 kDa) were evaluated by ELISA in plasma samples obtained from individuals under clinical suspicion for TB. Combinations between the antibody titres and previously published cytokine responses in the same participants were assessed for diagnosing active TB. Results: Antibody responses were more promising when used in combination (AUC of 0.80), when all seven antibodies were combined. When anti-"Kit 1"-IgA levels were combined with five host cytokine biomarkers, the AUC increased to 97% (92-100%) with a sensitivity of 95% (95% CI, 73-100%), and specificity of 88.5% (95% CI, 68.7-97%) achieved after leave-one-out cross validation. Conclusion: When used in combination, IgA titres measured with ELISA against multiple Mycobacterium tuberculosis antigens may be useful in the diagnosis of TB. However, diagnostic accuracy may be improved if the antibodies are used in combination with cytokines. Show less
Antibody-based drugs are the largest class of biopharmaceuticals produced. They are currently employed to treat diverse diseases such as cancer, cardiovascular or neuronalogical diseases. Whereas... Show moreAntibody-based drugs are the largest class of biopharmaceuticals produced. They are currently employed to treat diverse diseases such as cancer, cardiovascular or neuronalogical diseases. Whereas some years ago, efforts were focused on developing classical monoclonal antibodies (mAbs), nowadays there is a trend towards engineered antibodies with enhanced functions, such as bispecific antibodies (BsAbs). These molecules increase on structural complexity and bring new challenges for their analytical characterization. Furthermore, the continuous development of new antibody treatments and molecules also increases the pressure in the analytical labs longing for more automation and high-throughput mass spectrometric analysis. This thesis addresses these aspects and provide solutions for improved and efficient characterization of novel antibody therapeutics focusing on mass spectrometric approaches. These approaches are based on multidimensional liquid chromatography or capillary electrophoresis hyphenated to mass spectrometry or on the application of in-source decay fragmentation. Next to structural characterization, in this thesis MS-based approaches have been developed for their functional characterization permitting to study the relationships between antibody structure and function. Show less
This thesis contains a variety of information about the natural and vaccine induced immunity against the human papillomavirus. The spontaneously induced HPV-specific humoral response after... Show moreThis thesis contains a variety of information about the natural and vaccine induced immunity against the human papillomavirus. The spontaneously induced HPV-specific humoral response after infection was assessed in population-based studies. The vaccine-induced changes in HPV-seroprevalence among the HPV unvaccinated Dutch population aged 0-89 years, where we compared the HPV-seroprevalence before the introduction of the HPV vaccine with data of approximately six years post-implementation of the national HPV vaccination program. Also, the HPV immune status of the Dutch Caribbean population just after introduction of HPV vaccination was determined. Moreover, the longitudinal relation between the hr-HPV antibody levels and the prevalence of HPV infections in three-dose vaccinated girls were studied. And more insight was gained into humoral and cellular immune responses after just a one-dose of the HPV vaccine. At last, the kinetics of innate and adaptive immune responses directly after vaccination different HPV vaccines were investigated. In the coming years some important changes are expected regarding HPV screening and vaccination. The effectiveness of the one-dose schedule will become clear as clinical trials end. In the Netherlands, a sex-neutral vaccination will be implemented soon. These changes will need to be monitored to provide scientific answers about the effectiveness and immunogenicity. Show less
This thesis describes two studies on the efficacy and safety of vaccinations in patients stable autoimmune myasthenia gravis, one with tetanus revaccination and one with influenza vaccination. Of... Show moreThis thesis describes two studies on the efficacy and safety of vaccinations in patients stable autoimmune myasthenia gravis, one with tetanus revaccination and one with influenza vaccination. Of both vaccinations, the humoral response and clinical parameters of the disease are described. For tetanus revaccination, also the cellular response is described. Furthermore, the validation of a disease specific quality of life questionnaire is described. Show less
Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well... Show moreChanges in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson's r correlation coefficient of 0.8208 between the two methods. Show less
Background: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of... Show moreBackground: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of inflammatory (auto)immune disorders and have also been linked to protection against viral infection and discussed in the context of vaccine-induced protection. In contrast, sialylated IgG Abs have shown immunosuppressive effects.Objective: We sought to investigate IgG glycosylation programming during the germinal center (GC) reaction following immunization of mice with a foreign protein antigen and different adjuvants.Methods: Mice were analyzed for GC T-cell, B-cell, and plasma cell responses, as well as for antigen-specific serum IgG subclass titers and Fc glycosylation patterns.Results: Different adjuvants induce distinct IgG(+) GC B-cell responses with specific transcriptomes and expression levels of the alpha 2,6-sialyltransferase responsible for IgG sialylation that correspond to distinct serum IgG Fc glycosylation patterns. Low IgG Fc sialylation programming in GC B cells was overall highly dependent on the Foxp3(-) follicular helper T (TFH) cell-inducing cytokine IL-6, here in particular induced by water-inoil adjuvants and Mycobacterium tuberculosis. Furthermore, low IgG Fc sialylation programming was dependent on adjuvants that induced IL-27 receptor-dependent IFN-gamma(+) TFH1 cells, IL-6/IL-23-dependent IL-17A(+) T-FH17 cells, and high ratios of TFH cells to Foxp31 follicular regulatory T cells. Here, the 2 latter were dependent on M tuberculosis and its cord factor.Conclusion: This study's findings regarding adjuvant-dependent GC responses and IgG glycosylation programming may aid in the development of novel vaccination strategies to induce IgG Abs with both high affinity and defined Fc glycosylation patterns in the GC. Show less
Background and objectives The histopathologic classification for ANCA-associated GN distinguishes four classes on the basis of patterns of injury. In the original validation study, these classes... Show moreBackground and objectives The histopathologic classification for ANCA-associated GN distinguishes four classes on the basis of patterns of injury. In the original validation study, these classes were ordered by severity of kidney function loss as follows: focal, crescentic, mixed, and sclerotic. Subsequent validation studies disagreed on outcomes in the crescentic and mixed classes. This study, driven by the original investigators, provides several analyses in order to determine the current position of the histopathologic classification of ANCA-associated GN.Design, setting, participants, & measurements Avalidation study was performed with newly collected data from 145 patients from ten centers worldwide, including an analysis of interobserver agreement on the histopathologic evaluation of the kidney biopsies. This study also included a meta-analysis on previous validation studies and a validation of the recently proposed ANCA kidney risk score.Results The validation study showed that kidney failure at 10-year follow-up was significantly different between the histopathologic classes (P < 0.001). Kidney failure at 10-year follow-up was 14% in the crescentic class versus 20% in the mixed class (P=0.98). In themeta-analysis, no significant difference in kidney failure was also observed when crescentic class was compared with mixed class (relative risk, 1.15; 95% confidence interval, 0.94 to 1.41). When we applied the ANCA kidney risk score to our cohort, kidney survival at 3 years was 100%, 96%, and 77% in the low-, medium-, andhigh-risk groups, respectively (P<0.001). These survival percentages are higher compared with the percentages in the original study.Conclusions The crescentic and mixed classes seem to have a similar prognosis, also after adjusting for differences in patient populations, treatment, and interobserver agreement. However, at this stage, we are not inclined to merge the crescentic and mixed classes because the reported confidence intervals do not exclude important differences in prognosis and because an important histopathologic distinction would be lost. Show less
Muscle-specific kinase (MuSK) plays a critical role in establishing and maintaining neuromuscular synapses. Antibodies derived from immunizing animals with MuSK were important tools to help detect... Show moreMuscle-specific kinase (MuSK) plays a critical role in establishing and maintaining neuromuscular synapses. Antibodies derived from immunizing animals with MuSK were important tools to help detect MuSK and its activity. The role of antibodies in MuSK-related research got an extra dimension when autoantibodies to MuSK were found to cause myasthenia gravis (MG) in 2001. Active immunization with MuSK or passive transfer of polyclonal purified IgG(4) fractions from patients reproduced myasthenic muscle weakness in a range of animal models. Polyclonal patient-purified autoantibodies were furthermore found to block agrin-Lrp4-MuSK signaling, explaining the synaptic disassembly, failure of neuromuscular transmission and ultimately muscle fatigue observed in vivo. MuSK autoantibodies are predominantly of the IgG4 subclass. Low levels of other subclass MuSK antibodies coexist, but their role in the pathogenesis is unclear. Patient-derived monoclonal antibodies revealed that MuSK antibody subclass and valency alters their functional effects and possibly their pathogenicity. Interestingly, recombinant functional bivalent MuSK antibodies might even have therapeutic potential for a variety of neuromuscular disorders, due to their agonistic nature on the MuSK signaling cascade. Thus, MuSK antibodies have proven to be helpful tools to study neuromuscular junction physiology, contributed to our understanding of the pathophysiology of MuSK MG and might be used to treat neuromuscular disorders. The source of MuSK antibodies and consequently their (mixed) polyclonal or monoclonal nature were important confounding factors in these experiments. Here we review the variety of MuSK antibodies described thus far, the insights they have given us and their potential for the future. Show less
