Nowadays, therapeutic antibodies are the major and fastest growing class of biotherapeuticals. Since their invention, they are continuously developed to improve structural and functional... Show moreNowadays, therapeutic antibodies are the major and fastest growing class of biotherapeuticals. Since their invention, they are continuously developed to improve structural and functional characteristics. The high complexity of recently generated antibody derivatives, with various modifications induced during the manufacturing process itself leads to many proteoform variants of the desired product. These proteoforms can potentially exhibit altered activity. Therefore, an adequate characterization of the proteoforms, the assessment of their impact and careful monitoring of critical species is indispensable in order to guarantee effective and safe biopharmaceuticals. As the landscape of next-generation Ab formats continuously evolves, it is likewise of great importance to further develop appropriate analytical methods for their thorough attribute analysis. Hence, the focus of the research performed in this thesis is the development of multi-level approaches for the in-depth, primarily MS-based characterization of biopharmaceuticals to overcome the present restrictions and challenges arising e.g. by the implementation of complex Ab formats. Show less
Saint Lary, C.D. de; Kasbergen, L.M.R.; Bruijning-Verhagen, P.C.J.L.; Jeugd, H. van der; Chandler, F.; Hogema, B.M.; ... ; Visser, L.G. 2023
IntroductionIn 2020, the first Dutch West Nile virus (WNV) infected birds were detected through risk-targeted surveillance of songbirds. Retrospective testing of patients with unexplained... Show moreIntroductionIn 2020, the first Dutch West Nile virus (WNV) infected birds were detected through risk-targeted surveillance of songbirds. Retrospective testing of patients with unexplained neurological disease revealed human WNV infections in July and August 2020. Bird ringers are highly exposed to mosquito bites and possibly avian excrements during ringing activities. This study therefore investigates whether bird ringers are at higher risk of exposure to WNV and Usutu virus (USUV).MethodsDutch bird ringers were asked to provide a single serum sample (May – September 2021) and to fill out a survey. Sera were screened by protein microarray for presence of specific IgG against WNV and USUV non-structural protein 1 (NS1), followed by focus reduction virus neutralization tests (FRNT). Healthcare workers (2009–2010), the national immunity cohort (2016–2017) and blood donors (2021) were used as control groups without this occupational exposure.ResultsThe majority of the 157 participating bird ringers was male (132/157, 84%) and the median age was 62 years. Thirty-seven participants (37/157, 23.6%) showed WNV and USUV IgG microarray signals above background, compared to 6.4% (6/94) in the community cohort and 2.1% (2/96) in blood donors (p < 0.01). Two seroreactive bird ringers were confirmed WNV or USUV positive by FRNT. The majority of seroreactive bird ringers travelled to EU countries with reported WNV human cases (30/37, 81%) (p = 0.07). No difference was observed between bird ringers with and without previous yellow fever vaccination.DiscussionThe higher frequency of WNV and/or USUV IgG reactive bird ringers indicates increased flavivirus exposure compared to the general population, suggesting that individuals with high-exposure professions may be considered to complement existing surveillance systems. However, the complexity of serological interpretation in relation to location-specific exposure (including travel), and antibody cross-reactivity, remain a challenge when performing surveillance of emerging flaviviruses in low-prevalence settings. Show less
This thesis describes studies of individuals with systemic lupus erythematosus (SLE) presenting with neuropsychiatric (NP) symptoms at the Leiden NPSLE clinic. A diverse range of studies, including... Show moreThis thesis describes studies of individuals with systemic lupus erythematosus (SLE) presenting with neuropsychiatric (NP) symptoms at the Leiden NPSLE clinic. A diverse range of studies, including laboratory, radiological, clinical and patient´s reported outcomes are presented.The Leiden NPSLE clinic is a tertiary referral center for patients with SLE and neuropsychiatric (NP) symptoms. In the NPSLE clinic, patients are assessed by a multidisciplinary team. Thereafter, clinical, radiological and laboratory measures are weighed in a consensus meeting to correctly attribute the NP symptoms: related to lupus activity (NPSLE) or not. This extensive and standardized assessment of NPSLE, a rare and heterogenous disease lacking a gold standard, is unique and creates the opportunity to explore many aspects of NPSLE in well-defined phenotypes.In the first part of this thesis, we evaluate both classification and treatment of patients withSLE and NP symptoms. The second part of this thesis focuses on a diverse range of clinicaloutcomes of NPSLE, including both morbidity and mortality. The last part of this thesisassesses potential biomarkers for (specific manifestations of) NPSLE. Show less
Immunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19.... Show moreImmunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19. Importantly, effector functions and resulting inflammatory responses are impacted by the structure of N-glycans linked to the Fc-tail of IgG. Studying antibody glycosylation in emerging infectious diseases such as SARS-CoV-2 allows to gain insight into specific glycan signatures at the early stages of infection, and to investigate whether these reflect how the disease would progress. For example, low fucosylation is a common glyco-phenotypic signature of IgG1 produced against the spike (S) protein of severely ill SARS-CoV-2 infected patients early on in their disease course, but has likewise been described in other disease settings, where the antigen is presented in the context of host-cell membranes (Chapter 2). In this thesis, antibody glycomics signatures of SARS-CoV-2 infection and vaccination have been explored using an established liquid chromatography – mass spectrometry-based method relying on affinity-isolation and proteolytic digestion of both total and anti-S IgG. In Chapter 3, the glycosylation of SARS-CoV-2 anti-S IgG antibodies were found to be vastly skewed relative to total IgG and to change in a highly dynamic fashion. Moreover, IgG glycosylation was shown to be an early severity marker and showed patient stratification potential, with predicting power for intensive care admission within a hospitalized patient population. Early detection of a pro-inflammatory glycosylation pattern may provide a broader intervention window and decrease the number of ICU-admissions. Furthermore, anti-S IgG1 glycosylation levels obtained with LC-MS show promise to supplement clinical parameters and biomarkers of inflammation, that have together been used for the severity score calculation of hospitalized COVID-19 patients. Similarly to SARS-CoV-2 infection, antibodies generated against the spike protein upon BNT162b2 mRNA vaccination also induced a transient afucosylated anti-S IgG1 response in antigen naïve individuals, albeit to a lower extent than in severely ill patients, exemplifying the influence of the type of immunization on antibody glycosylation (Chapter 4). Upon vaccination, the observed initial, mild afucosylated response was additionally accompanied by low fucosyltransferase (FUT8) expression in antigen-specific plasma cells. Furthermore, the observed initial anti-S IgG afucosylation signature may aided mounting a stronger immune response, as indicated by its correlation with antibody amounts following the second vaccination dose. Given the impact of glycosylation on antibody function, deciphering theunderlying regulatory mechanisms influencing IgG glycosylation will be of great importance to better understand the inflammatory potential, vaccine efficacy and protective capacity of vaccine- or pathogen-induced IgG in both body fluids and tissues in the future.In Chapter 5 and 6, the reaction steps of a previously developed linkage-specific sialic acid derivatization workflow were studied in more detail. Key players in such reactions are catalyst, of which novel types with different physico-chemical properties were introduced in Chapter 5. In Chapter 6, prior lactone formation was found to be a prerequisite for subsequent amidation of α2,3-linked sialic acids, which proceeds via direct aminolysis of the C2 lactone. Together, these new insights will be beneficial for the rational optimization of high-throughput (MALDI-)MS-based glycomics and glycoproteomics workflows relying on linkage-specific sialic acid derivatization. Show less
Lim, W.H.; Adams, B.; Alexander, S.; Bouts, A.H.M.; Claas, F.; Collins, M.; ... ; Wong, G. 2021
Background: Parental donor kidney transplantation is the most common treatment option for children and adolescents with kidney failure. Emerging data from observational studies have reported... Show moreBackground: Parental donor kidney transplantation is the most common treatment option for children and adolescents with kidney failure. Emerging data from observational studies have reported improved short- and medium-term allograft outcomes in recipients of paternal compared to maternal donors. The INCEPTION study aims to identify potential differences in immunological compatibility between maternal and paternal donor kidneys and ascertain how this affects kidney allograft outcomes in children and adolescents with kidney failure.Methods: This longitudinal observational study will recruit kidney transplant recipients aged <= 18years who have received a parental donor kidney transplant across 4 countries (Australia, New Zealand, United Kingdom and the Netherlands) between 1990 and 2020. High resolution human leukocyte antigen (HLA) typing of both recipients and corresponding parental donors will be undertaken, to provide an in-depth assessment of immunological compatibility. The primary outcome is a composite of de novo donor-specific anti-HLA antibody (DSA), biopsy-proven acute rejection or allograft loss up to 60-months post-transplantation. Secondary outcomes are de novo DSA, biopsyproven acute rejection, acute or chronic antibody mediated rejection or Chronic Allograft Damage Index (CADI) score of > 1 on allograft biopsy post-transplant, allograft function, proteinuria and allograft loss. Using principal component analysis and Cox proportional hazards regression modelling, we will determine the associations between defined sets of immunological and clinical parameters that may identify risk stratification for the primary and secondary outcome measures among young people accepting a parental donor kidney for transplantation. This study design will allow us to specifically investigate the relative importance of accepting a maternal compared to paternal donor, for families deciding on the best option for donation.Discussion: The INCEPTION study findings will explore potentially differential immunological risks of maternal and paternal donor kidneys for transplantation among children and adolescents. Our study will provide the evidence base underpinning the selection of parental donor in order to achieve the best projected long-term kidney transplant and overall health outcomes for children and adolescents, a recognized vulnerable population. Show less
In this thesis, mathematical modeling and simulation was applied as a tool to inform quantitative decision making in oncology drug discovery and development. Modeling based approaches were shown to... Show moreIn this thesis, mathematical modeling and simulation was applied as a tool to inform quantitative decision making in oncology drug discovery and development. Modeling based approaches were shown to be useful to understand the mechanism of action and deconvolve the complexities of novel biotherapeutic modalities being used to treat cancer, including monospecific and bispecific monoclonal antibodies and antibody drug conjugates. Several key observations and learnings were made. For example, modeling was shown to be a useful method to reduce animal experimentation, by enabling in vitro to in vivo correlations or use of simulation to replace experimental methodologies. Mechanism based modeling and simulation was found to be a useful means to translate from preclinical studies to the clinic to ensure progression of the best drug to clinical trials. These models could then be used to optimize design of clinical studies from selection of starting doses to recommended efficacious doses for pivotal trials. Modeling was shown to be beneficial to understand variability in the clinic and to identify factors impacting drug response in individual patients, paving the way for precision medicine strategies, informing clinical diagnostics, biomarkers, and doses for different oncology indications. Show less
Using a variety of relevant preclinical mouse models, this thesis not only showed several ways to improve antibody-based cancer immunotherapy but also demonstrates the importance of the choice of... Show moreUsing a variety of relevant preclinical mouse models, this thesis not only showed several ways to improve antibody-based cancer immunotherapy but also demonstrates the importance of the choice of the model for the translational relevance of the preclinical observations. Show less
Introduction: The aim of this narrative review is to provide an overview of the literature on the possible immunologic pathophysiology of psychiatric manifestations of neuropsychiatric systemic... Show moreIntroduction: The aim of this narrative review is to provide an overview of the literature on the possible immunologic pathophysiology of psychiatric manifestations of neuropsychiatric systemic lupus erythematosus (NPSLE).Methods: A systematic search on PubMed was conducted. English studies with full text availability that investigated the correlation between blood-brain barrier (BBB) dysfunction, intrathecal synthesis of antibodies, antibodies, cytokines, chemokines, metalloproteinases, complement and psychiatric NPSLE manifestations in adults were included.Results: Both transient BBB-dysfunction with consequent access of antibodies to the cerebrospinal fluid (CSF) and intrathecal synthesis of antibodies could occur in psychiatric NPSLE. Anti-phospholipid antibodies, anti-NMDA antibodies and anti-ribosomal protein p antibodies seem to mediate concentration dependent neuronal dysfunction. Interferon-a may induce microglial engulfment of neurons, direct neuronal damage and production of cytokines and chemokines in psychiatric NPSLE. Several cytokines, chemokines and matrix metalloproteinase-9 may contribute to the pathophysiology of psychiatric NPSLE by attracting and activating Th1-cells and B-cells.Discussion: This potential pathophysiology may help understand NPSLE and may have implications for the diagnostic management and therapy of psychiatric NPSLE. However, the presented pathophysiological model is based on correlations between potential immunologic etiologies and psychiatric NPSLE that remain questionable. More research on this topic is necessary to further elucidate the pathophysiology of NPSLE. Show less
The aim of this thesis was to develop tools to detect and quantify HLA-specific memory B cells in peripheral blood of HLA-immunized individuals and to assess the applicability of the newly... Show moreThe aim of this thesis was to develop tools to detect and quantify HLA-specific memory B cells in peripheral blood of HLA-immunized individuals and to assess the applicability of the newly developed assays in the setting of clinical transplantation. Transplant patients who have already made a defense response to the foreign HLA on donor organ have an increased risk of developing antibody-mediated rejection, which may adversely affect the survival of the graft. In diagnostic HLA laboratories, serum of the patients is tested for the presence of HLA-specific antibodies before and after transplantation. Serum HLA antibodies are produced by plasma cells located in the bone marrow however, memory B cells can also play a role in antibody production against the donor organ. So far, the role of these cells has been neglected in the diagnostics. The research conducted in my PhD thesis enabled us to develop methods to detect HLA-specific memory B cells. By this means, patients who may potentially harbor HLA-specific memory B cells such as repeat transplant candidates, women receiving transplants from their partner or child and patients undergoing desensitization treatments may benefit from the assays that are described in this PhD thesis. Show less
Therapeutic proteins have become very successful in the treatment of various chronic and life-threatening diseases. However, besides their benefits, therapeutic proteins seem to have a common... Show moreTherapeutic proteins have become very successful in the treatment of various chronic and life-threatening diseases. However, besides their benefits, therapeutic proteins seem to have a common problem - the response of a patient’s immune system against the protein. This means that the immune system of the patient actively removes the drug from the body, thereby potentially decreasing or reversing the effect of the therapy. By now there is strong consensus that damaged and aggregated proteins are important risk factors. Protein aggregates are, due to their heterogeneity and often low quantity, challanging to characterize. Further, there is a large academic interest in understanding the mechanisms of aggregation and the role of non-proteinaceous particles in the process of protein aggregation and unwanted immunogenicity in order to design more effective and safe protein-based medicines. This PhD thesis supported that research effort by developing and improving analytical methodologies to detect the size, quantity and other properties of protein aggregates and particles, especially in the relevant nano- and micrometer size range. These techniques were then applied to study a so far unknown nanoparticulate impurity in pharmaceutical-grade sugars. Further, the results shown in this thesis revealed that these nanoparticulate impurities pose a threat to protein stability. Show less
The work presented in this thesis aimed at increasing our understanding of the effect of helminths on Plasmodium spp. immune response in co-infected individuals living in endemic countries. It... Show moreThe work presented in this thesis aimed at increasing our understanding of the effect of helminths on Plasmodium spp. immune response in co-infected individuals living in endemic countries. It presents data from studies conducted in rural and semi urban areas of Lambaréné (Gabon) where the burden of malaria and helminths is particularly important. Although scarce previous studies have indicated an effect of helminths on malaria outcomes and immune response to Plasmodium spp. parasite in co-infected subjects. However it is still debated how consistent is this effect across study sites and teams and what its immunological basis is. Show less
In this research heavy chain antibody fragments (VHHs) are developed as potential tools for non-invasive in vivo imaging of Alzheimer__s disease (AD) and Cerebral Amyloid Angiopathy (CAA). First... Show moreIn this research heavy chain antibody fragments (VHHs) are developed as potential tools for non-invasive in vivo imaging of Alzheimer__s disease (AD) and Cerebral Amyloid Angiopathy (CAA). First the generation of antibody fragments, directed against A_ is described. VHHs that were selected from different libraries showed differential affinity for different A_ epitopes when used for immunohistochemistry. These observations indicate that the VHHs are the first immunologic probes with the capacity to differentiate between parenchymal and vascular beta amyloid aggregates. Next, in chapter three the VHH are assessed on their ability to cross the blood-brain barrier using an established in vitro blood brain barrier co-culture system. VHH ni3A showed the highest transmigration efficiency of all tested VHHs. This transport is, in part, facilitated by a 3 amino acid substitution in its N-terminal domain. Additional studies indicated that the mechanism of transcellular passage of ni3A is by active transport. The data described in chapter four provides preliminary proof that these VHHs have the capacity to target A_ depositions in vivo. Consequently, the VHHs are promising tools for further development as imaging agents for the differential diagnosis of A_-related neurodegenerative diseases like CAA and AD. Show less
Major advancements in the understanding of the immune system have provided us with the opportunity for rational design of therapeutic immunological interventions. The notion that dendritic cells ... Show moreMajor advancements in the understanding of the immune system have provided us with the opportunity for rational design of therapeutic immunological interventions. The notion that dendritic cells (DC) play a crucial role in the activation of T lymphocytes has made DC biology of central importance for vaccine development. Accordingly, efficient delivery of antigen to DCs is one of main objectives in vaccine development. In this thesis, antibody-mediated antigen targeting is evaluated as a potential antigen delivery strategy for therapeutic vaccination. Complexes of protein antigen and antigen-specific antibodies are natural formulations that bind to Fc__ receptors. Fc__R ligation on DCs leads to efficient uptake, DC maturation and presentation of the antigen to T lymphocytes. Interaction of Ag-Ab complexes with Fc__Rs on DCs provides a link between the humoral and cellular arms of the immune response. This thesis contains an extensive evaluation of Fc__R-mediated antigen delivery to dendritic cells in the context of T lymphocyte-mediated immunotherapy. In addition, it contains a detailed analysis of Fc__R function on DCs and addresses the kinetics of cross-presentation of antigen after Fc__R-mediated uptake. Show less
Worldwide, more than a billion people are infected with helminths. These worm infections are chronic in nature and can lead to considerable morbidity. Immunologically these infections are... Show moreWorldwide, more than a billion people are infected with helminths. These worm infections are chronic in nature and can lead to considerable morbidity. Immunologically these infections are interesting; chronic helminth infections are characterized by skewing towards a T helper 2 type response as well as regulatory responses. The latter is thought to prevent strong immune responses against parasitic worms, allowing their long-term survival and restricting pathology. This regulatory network is thought to also temper responses to non-helminth antigens, like allergens or self antigens, possibly leading to lower prevalence of allergies and autoimmune diseases in subjects that are chronically infected with helminths. Thus, helminths may bear molecules that have potential therapeutic action against allergies and possibly other inflammatory diseases. However, on the other side of the coin, this would predict that helminth infected subjects might not respond strongly to third party antigens like vaccines. Helminth derived molecules have been identified that induce T helper 2 and regulatory responses via modulation of dendritic cells and some appear to do so via TLR signaling. New insights into these pathways could be useful to antagonize suppression and hence boost vaccine efficacy or to optimize suppression induced by helminth derived molecules and control inflammatory diseases. Show less
Huidig beleid schrijft voor dat voor de bloedtransfusie plaatsvindt, het serum van de patient getest wordt op irreguliere anti-stoffen van rode bloedcellen. In dit boek onderzoekt Henk Schonewille... Show moreHuidig beleid schrijft voor dat voor de bloedtransfusie plaatsvindt, het serum van de patient getest wordt op irreguliere anti-stoffen van rode bloedcellen. In dit boek onderzoekt Henk Schonewille of dit beleid moet veranderen om bloedtransfusie veiliger te maken. Current pre-transfusion policy requires the patient's serum to be tested for the presence of irregular red blood cell antibodies. In case of an alloantibody, red blood cells lacking the corresponding antigen are transfused after an antoglobulin crossmatch. The aim of the study in this thesis is primarily to investigate whether this policy should change to improve transfusion safety Show less
