This thesis presents the development of quantitative imaging tools to study parasite migration. Since migration is crucial for malaria parasites to continue their life cycle, factors influencing... Show moreThis thesis presents the development of quantitative imaging tools to study parasite migration. Since migration is crucial for malaria parasites to continue their life cycle, factors influencing their migration capability may also impact the efficacy of malaria vaccine candidates. Here, imaging of parasite migration was used to gain insights that can support the development of antiparasitic vaccines. SMOOT (Sporozoite Motility Orienting and Organizing Tool) was developed and established as a quantitative software analysis tool for tracking the migration of malaria sporozoites in vitro and in human skin explant. This tool provides a readout with high kinematic detail, enabling the quantitative characterization of novel factors influencing the migration capability of malaria sporozoites. Subsequently, the study of sporozoite migration was expanded beyond in vitro and ex vivo models. A hybrid tracer labeling approach for malaria sporozoites was developed and used to reveal the in vivo dissemination of malaria sporozoites in a murine model. This multimodal imaging approach was also applied to investigate human skin invasion by helminth larvae. This thesis concludes with a review of the broader potential for imaging technology to advance the development of new diagnostic methods, therapeutic interventions and vaccines for combating parasitic infections. Show less
Korne, C.M. de; Schuijlenburg, R. van; Sijtsma, J.C.; Bes, H.M. de; Baalbergen, E.; Azargoshasb, S.; ... ; Roestenberg, M. 2022
Antibodies can prevent malaria by neutralizing the infectious Plasmodium falciparum sporozoites (SPZ) before they establish an infection in the liver. Circumsporozoite protein (CSP), the most... Show moreAntibodies can prevent malaria by neutralizing the infectious Plasmodium falciparum sporozoites (SPZ) before they establish an infection in the liver. Circumsporozoite protein (CSP), the most abundant surface protein of SPZ is the leading candidate for passive (and subunit) immunization approaches against malaria. Comprehensive assessment of the parasite-inhibitory capacity of anti-CSP monoclonal antibodies (mAbs) is an important step in advancing CSP-based immunization strategies. In this study, we employed a quantitative imaging-based motility assay to quantify the effect of anti-CSP mAbs on SPZ motility, both in vitro and in human skin. Our assay provided a quantitative measure of mAb parasite-inhibitory capacity through measurement of the half-maximal motility inhibitory concentration (IC50M) value for anti-CSP mAbs (IC50M 2A10: 24 nM, IC50M 3SP2: 71 nM). We found a sevenfold discrepancy between the IC50M and the binding saturation concentration measured by ELISA, possibly related to the observed shedding of CSP-mAb complexes during SPZ movement. In a subset of SPZ (5%), in vitro motility was unaffected by the presence of 2A10 while 3SP2 was able to completely block movement. In our ex vivo skin explant model, SPZ proved less susceptible to anti-CSP mAbs compared to SPZ in an in vitro environment. By quantitatively assessing motility, we created a valuable tool that can be used for comprehensive assessment of anti-CSP mAb potency. Insight that will help deepen our understanding of anti-CSP mAb potency and guide selection of the most promising anti-CSP mAbs for downstream clinical development. Show less
Zawistowska-Deniziak, A.; Lambooij, J.M.; Kalinowska, A.; Patente, T.A.; Lapinski, M.; Zande, H.J.P. van der; ... ; Guigas, B. 2022
Background:The parasitic trematode Fasciola hepatica evades host immune defenses through secretion of various immunomodulatory molecules. Fatty Acid Binding Proteins (fhFABPs) are among the main... Show moreBackground:The parasitic trematode Fasciola hepatica evades host immune defenses through secretion of various immunomodulatory molecules. Fatty Acid Binding Proteins (fhFABPs) are among the main excreted/secreted proteins and have been shown to display anti-inflammatory properties. However, little is currently known regarding their impact on dendritic cells (DCs) and their subsequent capacity to prime specific CD4(+) T cell subsets. Methodology/Principal Findings:The immunomodulatory effects of both native F. hepatica extracts and recombinant fhFABPs were assessed on monocyte-derived human DCs (moDCs) and the underlying mechanism was next investigated using various approaches, including DC-allogenic T cell co-culture and DC phenotyping through transcriptomic, proteomic and FACS analyses. We mainly showed that fhFABP1 induced a tolerogenic-like phenotype in LPS-stimulated moDCs characterized by a dose-dependent increase in the cell-surface tolerogenic marker CD103 and IL-10 secretion, while DC co-stimulatory markers were not affected. A significant decrease in secretion of the pro-inflammatory cytokines IL-12p70 and IL-6 was also observed. In addition, these effects were associated with an increase in both Th2-on-Th1 ratio and IL-10 secretion by CD4(+) T cells following DC-T cell co-culture. RNA sequencing and targeted proteomic analyses identified thrombospondin-1 (TSP-1) as a non-canonical factor highly expressed and secreted by fhFABP1-primed moDCs. The effect of fhFABP1 on T cell skewing was abolished when using a TSP-1 blocking antibody during DC-T cell co-culture. Immunomodulation by helminth molecules has been linked to improved metabolic homeostasis during obesity. Although fhFABP1 injection in high-fat diet-fed obese mice induced a potent Th2 immune response in adipose tissue, it did not improved insulin sensitivity or glucose homeostasis. Conclusions/Significance: We show that fhFABP1 modulates T cell polarization, notably by promoting DC TSP-1 secretion in vitro, without affecting metabolic homeostasis in a mouse model of type 2 diabetes. Show less
Wit, E.M.K.; Beurden, F. van; Kleinjan, G.H.; Grivas, N.; Korne, C.M. de; Buckle, T.; ... ; Poel, H.G. van der 2021
Introduction Previous studies indicated that location and amount of detected sentinel lymph nodes (SLNs) in prostate cancer (PCa) are influenced where SLN-tracer is deposited within the prostate.... Show moreIntroduction Previous studies indicated that location and amount of detected sentinel lymph nodes (SLNs) in prostate cancer (PCa) are influenced where SLN-tracer is deposited within the prostate. To validate whether intratumoral (IT) tracer injection helps to increase identification of tumor-positive lymph nodes (LNs) better than intraprostatic (IP) tracer injection, a prospective randomized phase II trial was performed.Methods PCa patients with a > 5% risk of lymphatic involvement were randomized between ultrasound-guided transrectal injection of indocyanine green-[Tc-99m]Tc-nanocolloid in 2 depots of 1 mL in the tumor (n = 55, IT-group) or in 4 depots of 0.5 mL in the peripheral zone of the prostate (n = 58, IP-group). Preoperative lymphoscintigraphy and SPECT/CT were used to define the location of the SLNs. SLNs were dissected using combination of radio- and fluorescence-guidance, followed by extended pelvic LN dissection and robot-assisted radical prostatectomy. Outcome measurements were number of tumor-bearing SNs, tumor-bearing LNs, removed nodes, number of patients with nodal metastases, and metastasis-free survival (MFS) of 4-7-year follow-up data.Results IT-injection did not result in significant difference of removed SLNs (5.0 vs 6.0, p = 0.317) and histologically positive SLNs (28 vs 22, p = 0.571). However, in IT-group, the SLN-positive nodes were 73.7% of total positive nodes compared to 37.3% in IP-group (p = 0.015). Moreover, significantly more node-positive patients were found in IT-group (42% vs 24%, p = 0.045), which did not result in worse MFS. In two patients (3.6%) from whom the IT-tracer injection only partly covered intraprostatic tumor spread, nodal metastases in ePLND without tumor-positive SNs were yielded.Conclusions The percentage-positive SLNs found after IT-injection were significantly higher compared to IP-injection. Significantly more node-positive patients were found using IT-injection, which did not affect MFS. IT-injection failed to detect nodal metastases from non-index satellite lesions. Therefore, we suggest to combine IT- and IP-tracer injections in men with visible tumor on imaging. Show less
Malaria vaccine candidates based on live, attenuated sporozoites have led to high levels of protection. However, their efficacy critically depends on the sporozoites' ability to reach and infect... Show moreMalaria vaccine candidates based on live, attenuated sporozoites have led to high levels of protection. However, their efficacy critically depends on the sporozoites' ability to reach and infect the host liver. Administration via mosquito inoculation is by far the most potent method for inducing immunity but highly impractical. Here, we observed that intradermal syringe-injected Plasmodium berghei sporozoites (syrSPZ) were 3-fold less efficient in migrating to and infecting mouse liver than mosquito-inoculated sporozoites (msqSPZ). This was related to a clustered dermal distribution (2-fold decreased median distance between syrSPZ and msqSPZ) and, more importantly, a 1.4 fold (significantly)-slower and more erratic movement pattern. These erratic movement patterns were likely caused by alteration of dermal tissue morphology (.15 -mm intercellular gaps) due to injection of fluid and may critically decrease sporozoite infectivity. These results suggest that novel microvolume-based administration technologies hold promise for replicating the success of mosquito-inoculated live, attenuated sporozoite vaccines.IMPORTANCE Malaria still causes a major burden on global health and the economy. The efficacy of live, attenuated malaria sporozoites as vaccine candidates critically depends on their ability to migrate to and infect the host liver. This work sheds light on the effect of different administration routes on sporozoite migration. We show that the delivery of sporozoites via mosquito inoculation is more efficient than syringe injection; however, this route of administration is highly impractical for vaccine purposes. Using confocal microscopy and automated imaging software, we demonstrate that syringe-injected sporozoites do cluster, move more slowly, and display more erratic movement due to alterations in tissue morphology. These findings indicate that microneedle-based engineering solutions hold promise for replicating the success of mosquito-inoculated live, attenuated sporozoite vaccines. Show less
Winkel, B.M.F.; Pelgrom, L.R.; Schuijlenburg, R. van; Baalbergen, E.; Ganesh, M.S.; Gerritsma, H.; ... ; Roestenberg, M. 2020
Author summary Malaria continues to be the deadliest parasitic disease worldwide, and an effective vaccine yielding sterile immunity does not yet exist. Attenuated parasites can induce sterile... Show moreAuthor summary Malaria continues to be the deadliest parasitic disease worldwide, and an effective vaccine yielding sterile immunity does not yet exist. Attenuated parasites can induce sterile protection in both human and rodent models for malaria, but these vaccines need to be administered directly into the bloodstream in order to convey protection; administration via the skin results in a much-reduced efficacy. We hypothesized this is caused by an early immune regulation initiated at the first site of contact with the immune system: the skin. However, the human skin stage of malaria has not been investigated to date. We used human antigen presenting cells as well as whole human skin explants to investigate (dermal) immune responses and found thatPlasmodiumsporozoites are able to suppress immune responses by inducing regulatory macrophages. Our study provides new insights in the mechanism of early immune regulation exploited byPlasmodiumparasites and can help to explain why intradermal vaccination using whole attenuated sporozoites results in reduced protection.Professional antigen-presenting cells (APCs), like macrophages (M phi s) and dendritic cells (DCs), are central players in the induction of natural and vaccine-induced immunity to malaria, yet very little is known about the interaction of SPZ with human APCs. Intradermal delivery of whole-sporozoite vaccines reduces their effectivity, possibly due to dermal immunoregulatory effects. Therefore, understanding these interactions could prove pivotal to malaria vaccination. We investigated human APC responses to recombinant circumsporozoite protein (recCSP), SPZ and anti-CSP opsonized SPZ both in monocyte derived MoDCs and MoM phi s. Both MoDCs and MoM phi s readily took up recCSP but did not change phenotype or function upon doing so. SPZ are preferentially phagocytosed by MoM phi s instead of DCs and phagocytosis greatly increased after opsonization. Subsequently MoM phi s show increased surface marker expression of activation markers as well as tolerogenic markers such as Programmed Death-Ligand 1 (PD-L1). Additionally they show reduced motility, produce interleukin 10 and suppressed interferon gamma (IFN gamma) production by antigen specific CD8(+)T cells. Importantly, we investigated phenotypic responses to SPZ in primary dermal APCs isolated from human skin explants, which respond similarly to their monocyte-derived counterparts. These findings are a first step in enhancing our understanding of pre-erythrocytic natural immunity and the pitfalls of intradermal vaccination-induced immunity. Show less
Duszenko, N.; Willigen, D.M. van; Welling, M.M.; Korne, C.M. de; Schuijlenburg, R. van; Winkel, B.M.F.; ... ; Roestenberg, M. 2020
In an era of antimicrobial resistance, a better understanding of the interaction between bacteria and the sentinel immune system is needed to discover new therapeutic targets for combating... Show moreIn an era of antimicrobial resistance, a better understanding of the interaction between bacteria and the sentinel immune system is needed to discover new therapeutic targets for combating bacterial infectious disease. Sentinel immune cells such as macrophages phagocytose intact bacteria and thereby initiate ensuing immune responses. The bacterial surface composition is a key element that determines the macrophage signaling. To study the role of the bacterial cell surface composition in immune recognition, we developed a platform technology for altering bacterial surfaces in a controlled manner with versatile chemical scaffolds. We show that these scaffolds are efficiently loaded onto both Gram-positive and -negative bacteria and that their presence does not impair the capacity of monocyte-derived macrophages to phagocytose bacteria and subsequently signal to other components of the immune system. We believe this technology thus presents a useful tool to study the role of bacterial cell surface composition in disease etiology and potentially in novel interventions utilizing intact bacteria for vaccination. Show less
Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet... Show moreHelminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte-derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC-SIGN (CD209). Blocking DC-SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N-glycans with terminal Galβ1-4(Fucα1-3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid-linked glycans, including LewisX, a known ligand for DC-SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD-L1. Furthermore, schistosomula EVs increased expression of IL-12 and IL-10 by moDCs, which was partly dependent on the interaction with DC-SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC-SIGN and EV-associated glycoconjugates in parasite-induced immune modulation. Show less
Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet... Show moreHelminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte-derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC-SIGN (CD209). Blocking DC-SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N-glycans with terminal Gal beta 1-4(Fuc alpha 1-3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid-linked glycans, including LewisX, a known ligand for DC-SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD-L1. Furthermore, schistosomula EVs increased expression of IL-12 and IL-10 by moDCs, which was partly dependent on the interaction with DC-SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC-SIGN and EV-associated glycoconjugates in parasite-induced immune modulation. Show less
Korne, C.M. de; Wit, E.M.; Jong, J. de; Olmos, R.A.V.; Buckle, T.; Leeuwen, F.W.B. van; Poel, H.G. van der 2019
Given the number of global malaria cases and deaths, the need for a vaccine against Plasmodium falciparum (Pf) remains pressing. Administration of live, radiation-attenuated Pf sporozoites can... Show moreGiven the number of global malaria cases and deaths, the need for a vaccine against Plasmodium falciparum (Pf) remains pressing. Administration of live, radiation-attenuated Pf sporozoites can fully protect malaria-naive individuals. Despite the fact that motility of these attenuated parasites is key to their infectivity and ultimately protective efficacy, sporozoite motility in human tissue (e.g. skin) remains wholly uncharacterized to date. We show that the ability to quantitatively address the complexity of sporozoite motility in human tissue provides an additional tool in the development of attenuated sporozoite vaccines. We imaged Pf movement in the skin of its natural host and compared wild-type and radiation-attenuated GFP-expressing Pf sporozoites. Using custom image analysis software and human skin explants we were able to quantitatively study their key motility features. This head-to-head comparison revealed that radiation attenuation impaired the capacity of sporozoites to vary their movement angle, velocity and direction, promoting less refined movement patterns. Understanding and overcoming these changes in motility will contribute to the development of an efficacious attenuated parasite malaria vaccine. Show less
BackgroundThe protective efficacy of the most promising malaria whole-parasite based vaccine candidates critically depends on the parasite's potential to migrate in the human host. Key components... Show moreBackgroundThe protective efficacy of the most promising malaria whole-parasite based vaccine candidates critically depends on the parasite's potential to migrate in the human host. Key components of the parasite motility machinery (e.g. adhesive proteins, actin/myosin-based motor, geometrical properties) have been identified, however the regulation of this machinery is an unknown process.MethodsIn vitro microscopic live imaging of parasites in different formulations was performed and analysed, with the quantitative analysis software SMOOTIn vitro, their motility; their adherence capacity, movement pattern and velocity during forward locomotion.ResultsSMOOT(In vitro) enabled the detailed analysis of the regulation of the motility machinery of Plasmodium berghei in response to specific (macro)molecules in the formulation. Albumin acted as an essential supplement to induce parasite attachment and movement. Glucose, salts and other whole serum components further increased the attachment rate and regulated the velocity of the movement.ConclusionsBased on the findings can be concluded that a complex interplay of albumin, glucose and certain salts and amino acids regulates parasite motility. Insights in parasite motility regulation by supplements in solution potentially provide a way to optimize the whole-parasite malaria vaccine formulation. Show less
Grivas, N.; Roest, R.C. van der; Korne, C.M. de; KleinJan, G.H.; Sikorska, K.; Schoots, I.G.; ... ; Poel, H.G. van der 2019
Introduction: The skin stage of malaria is a vital and vulnerable migratory life stage of the parasite. It has been characterised in rodent models, but remains wholly uninvestigated for human... Show moreIntroduction: The skin stage of malaria is a vital and vulnerable migratory life stage of the parasite. It has been characterised in rodent models, but remains wholly uninvestigated for human malaria parasites. To enable in depth analysis of not genetically modified (non-GMO) Plasmodium falciparum (Pf) sporozoite behaviour in human skin, we devised a labelling technology (Cy5M(2), targeting the sporozoite mitochondrion) that supports tracking of individual non-GMO sporozoites in human skin.Methods: Sporozoite labelling with Cy5M(2) was performed in vitro as well as via the feed of infected Anopheles mosquitos. Labelling was validated using confocal microscopy and flow cytometry and the fitness of labelled sporozoites was determined by analysis of infectivity to human hepatocytes in vitro, and in vivo in a rodent infection model. Using confocal video microscopy and custom software, single-sporozoite tracking studies in human skin-explants were performed.Results: Both in vitro and in mosquito labelling strategies yielded brightly fluorescent sporozoites of three different Plasmodium species. Cy5M(2) uptake colocalized with MitoTracker (R) green and could be blocked using the known Translocator protein (TSPO)-inhibitor PK11195. This method supported the visualization and subsequent quantitative analysis of the migration patterns of individual non-GMO Pf sporozoites in human skin and did not affect the fitness of sporozoites.Conclusions: The ability to label and image non-GMO Plasmodium sporozoites provides the basis for detailed studies on the human skin stage of malaria with potential for in vivo translation. As such, it is an important tool for development of vaccines based on attenuated sporozoites and their route of administration. Show less
