The host response against infection with Plasmodium commonly raises self-reactivity as a side effect, and antibody deposition in kidney has been cited as a possible cause of kidney injury during... Show moreThe host response against infection with Plasmodium commonly raises self-reactivity as a side effect, and antibody deposition in kidney has been cited as a possible cause of kidney injury during severe malaria. In contrast, animal models show that infection with the parasite confers long-term protection from lethal lupus nephritis initiated by autoantibody deposition in kidney. We have limited knowledge of the factors that make parasite infection more likely to induce kidney damage in humans, or the mechanisms underlying protection from autoimmune nephritis in animal models. Our experiments with the autoimmune-prone Fc & gamma;R2B[KO] mice have shown that a prior infection with P. yoelii 17XNL protects from end-stage nephritis for a year, even when overall autoreactivity and systemic inflammation are maintained at high levels. In this report we evaluate post-infection alterations, such as hemozoin accumulation and compensatory changes in immune cells, and their potential role in the kidney-specific protective effect by Plasmodium. We ruled out the role of pigment accumulation with the use of a hemozoin-restricted P. berghei ANKA parasite, which induced a self-resolved infection that protected from autoimmune nephritis with the same mechanism as parasitic infections that accumulated normal levels of hemozoin. In contrast, adoptive transfer experiments revealed that bone marrow cells were altered by the infection and could transmit the kidney protective effect to a new host. While changes in the frequency of bone marrow cell populations after infection were variable and unique to a particular parasite strain, we detected a sustained bias in cytokine/chemokine expression that suggested lower fibrotic potential and higher Th1 bias likely affecting multiple cell populations. Sustained changes in bone marrow cell activation profile could have repercussions in immune responses long after the infection was cleared. Show less
Background Microscopic examination of thick and thin blood films is the gold standard in current guidelines for the diagnosis of malaria, but guidelines do not uniformly agree on which combination... Show moreBackground Microscopic examination of thick and thin blood films is the gold standard in current guidelines for the diagnosis of malaria, but guidelines do not uniformly agree on which combination of other methods should be used and when. Methods Three questionnaires were sent between March 2018 and September 2019 to laboratories subscribing to the external quality assessment scheme for the diagnosis of blood and intestinal parasites of the Dutch Foundation for Quality Assessment in Medical Laboratories in order to investigate how much variation in the laboratory diagnosis of malaria between different clinical laboratories is present in the Netherlands. Results The questionnaires were partially or fully completed by 67 of 77 (87%) laboratories. Only 9 laboratories reported 10 or more malaria positive patients per year. Most laboratories use a different diagnostic strategy, within office versus outside office hours depending on the screening assay result. Within office hours, 62.5% (35/56) of the responding laboratories perform an immunochromatographic test (ICT) in combination with microscopic examination of thick and thin blood films without additional examinations, such as Quantitative Buffy Coat and/or rtPCR analysis. Outside office hours 85.7% (48/56) of laboratories use an ICT as single screening assay and positive results are immediately confirmed by thick and thin blood films without additional examinations (89.6%, 43/48). In case of a negative ICT result outside office hours, 70.8% (34/48) of the laboratories perform microscopic examination of the thick film the next morning and 22.9% (11/48) confirm the negative ICT result immediately. Furthermore, substantial differences were found in the microscopic examinations of thick and thin blood films; the staining, theoretical sensitivity of the thick film and determination of parasitaemia. Conclusions This study demonstrated a remarkably high variation between laboratories in both their diagnostic strategy as well as their methods for microscopic examination for the diagnosis of malaria in a clinical setting, despite existing national and international guidelines. While the impact of these variations on the accuracy of the diagnosis of malaria is yet unknown, these findings should stimulate clinical laboratories to critically review their own diagnostic strategy. Show less
Malaria parasites are transmitted by Anopheles mosquitoes. During its life cycle in the mosquito vector the Plasmodium ookinete escapes the proteolytic milieu of the post-blood meal midgut by... Show moreMalaria parasites are transmitted by Anopheles mosquitoes. During its life cycle in the mosquito vector the Plasmodium ookinete escapes the proteolytic milieu of the post-blood meal midgut by traversing the midgut wall. This process requires penetration of the chitin-containing peritrophic matrix lining the midgut epithelium, which depends in part on ookinete-secreted chitinases. Plasmodium falciparum ookinetes have one chitinase (PfCHT1), whereas ookinetes of the avian-infecting parasite, P. gallinaceum, have two, a long and a short form, PgCHT1 and PgCHT2, respectively. Published data indicates that PgCHT2 forms a high molecular weight (HMW) reduction-sensitive complex; and one binding partner is the ookinete-produced von Willebrand A-domain-containing protein, WARP. Size exclusion chromatography data reported here show that P. gallinaceum PgCHT2 and its ortholog, P. falciparum PfCHT1 are covalently-linked components of a HMW chitinase-containing complex (> 1,300 kDa). Mass spectrometry of ookinete-secreted proteins isolated using a new chitin bead pull-down method identified chitinase-associated proteins in P. falciparum and P. gallinaceum ookinete-conditioned culture media. Mass spectrometry of this complex showed the presence of several micronemal proteins including von Willebrand factor A domain-related protein (WARP), ookinete surface enolase, and secreted ookinete adhesive protein (SOAP). To test the hypothesis that ookinete-produced PfCHT1 can form a high molecular homo-multimer or, alternatively, interacts with P. berghei ookinete-produced proteins to produce an HMW hetero-multimer, we created chimeric P. berghei parasites expressing PfCHT1 to replace PbCHT1, enabling the production of large numbers of PfCHT1-expressing ookinetes. We show that chimeric P. berghei ookinetes express monomeric PfCHT1, but a HMW complex containing PfCHT1 is not present. A better understanding of the chitinase-containing HMW complex may enhance development of next-generation vaccines or drugs that target malaria transmission stages. Show less
The protozoan parasitePlasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by... Show moreThe protozoan parasitePlasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke thePlasmodium-associated autophagy-related (PAAR) response. This is characterised by a long-lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3-phosphate (PI3P)-labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P-labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3-labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminatePlasmodiumparasites. Show less
The genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the... Show moreThe genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the production of haploid parasites in the Plasmodium life cycle: the oocyst stage in mosquitoes and the liver and blood stages in mammalian hosts. The Plasmodium oocyst stage plays an important role in the production of the first generation of haploid parasites. Nuclear division is the most important event that occurs during the proliferation of all eukaryotes. However, obtaining the details of nuclear division at the oocyst stage is challenging owing to difficulties in preparation. In this study, we used focused-ion-beam-milling combined with scanning-electron-microscopy to report the 3D architecture during nuclear segregations in oocyst stage. This advanced technology allowed us to analyse the 3D details of organelle segregation inside the oocyst during sporogony formation. It was revealed that multiple nuclei were involved with several centrosomes in one germ nucleus during sporozoite budding (endopolygeny). Our high-resolution 3D analysis uncovered the endopolygeny-like nuclear architecture of Plasmodium in the definitive host. This nuclear segregation was different from that in the blood stage, and its similarity to other apicomplexan parasite nuclear divisions such as Sarcocystis is discussed. Show less
Nijhuis, R.H.T.; Lieshout, L. van; Verweij, J.J.; Claas, E.C.J.; Wessels, E. 2018
In this thesis we describe a set of studies, performed using rodent models of malaria, aimed to identify methods to improve vaccines consisting of live attenuated sporozoites, in particular... Show moreIn this thesis we describe a set of studies, performed using rodent models of malaria, aimed to identify methods to improve vaccines consisting of live attenuated sporozoites, in particular genetically attenuated parasites (GAP) vaccines. Studies in rodents and humans have shown that immunization with live-attenuated sporozoites can generate protective immunity, however induction of sterile protection in humans has required immunization with multiple vaccine doses and each dose consisting of relatively high numbers of sporozoites. Increasing the immunogenicity of whole sporozoite (wsp) vaccines can both reduce the number of sporozoites per dose and the number of vaccine doses. In the studies described in this thesis we attempted to increase GAP immunogenicity by: (i) adding adjuvants during GAP immunization; (ii) introducing genes encoding putative immunomodulatory proteins in the GAP genome to create ‘self-adjuvanting’ parasites; (iii) generating GAPs that arrest late into liver-stage development (LA-GAP) to increase antigen load and diversity during immunization; and (iv) exploring possibilities to genetically modify parasite to express vaccine antigens from different life cycle stages, in order to test the ability of parasites to induce immune responses against multiple life cycle stages and to inform the creation of a ‘multi-stage’ GAP vaccine. Show less
Geographical differences in the prevalence of allergic and chronic inflammatory diseases as well as in the efficacy of vaccines have been described. To improve our understanding of... Show moreGeographical differences in the prevalence of allergic and chronic inflammatory diseases as well as in the efficacy of vaccines have been described. To improve our understanding of immunological differences between various human populations, the innate and adaptive immune system and their responses to stimulation have been compared between Europeans and Africans. Furthermore, IgG glycosylation profiles have been compared between several populations and in response to vaccination. In addition, the immune response to controlled human malaria infection has been determined in Europeans and Africans by mass cytometry as well as the role of γδ T cells and CD4+ T cells in immune responses during natural malaria infection in Indonesian children. The results indicated striking differences that need to be taken into account. It might be necessary to develop population-specific vaccines and treatments against infectious, allergic, and chronic inflammatory diseases. Show less
Despite intense efforts over the past 50 years to develop a vaccine, there is currently no licensed malaria vaccine available. The limited success in inducing sufficient protection against malaria... Show moreDespite intense efforts over the past 50 years to develop a vaccine, there is currently no licensed malaria vaccine available. The limited success in inducing sufficient protection against malaria with subunit-vaccines has renewed an interest in whole-parasite vaccination strategies. While live-vaccines are hard to formulate and administer, they have been shown to confer long-lasting sterile immunity in humans. The aim of the work described in this thesis was to genetically engineer and characterize growth- and virulence-attenuated blood stage parasites (GAPBS) in the rodent malaria model, P. berghei. Specifically, the identification of GAPBS that produce only short-lived, low-level infections that can provoke strong and long-lasting protective immunity. The thesis describes improved methods to produce and screen potential GAPBS, specifically transfection methods to generate GAPBS and methods to analyze their blood stage growth-characteristics. In addition, we report the generation and characterization of a number of novel GAPBS that are virulence-attenuated and produce self-resolving infections in mice. These GAPBS are useful tools to better understand the induction of protective immunity against Plasmodium blood stages and may help to create an effective and broad acting antimalarial vaccine. Show less
