Increased pfmdr1 copy number in Plasmodium falciparum isolates from Suriname

Amplification of the pfmdr1 gene is associated with clinical failures and reduced in vivo and in vitro sensitivity to both mefloquine and artemether–lumefantrine in South‐East Asia. Several African countries have reported the absence or very low prevalence of increased copy number, whilst South American reports are limited to Peru without and Venezuela with increased pfmdr1 multiplication. The relative pfmdr1 copy numbers were assessed in 68 isolates from Suriname collected from different endemic villages (2005) and from mining areas (2009). 11% of the isolates harbour multiple copies of the pfmdr1 gene. Isolates originating from mining areas do not yet display a higher tendency for increased copy number and no significant differences could be registered within a time span of 4 years, but the mere presence of increased copy number warrants caution and should be considered as an early warning sign for emerging drug resistance in Suriname and South America.     

Immunization of mice with Plasmodium TCTP delays establishment of Plasmodium infection

Translationally controlled tumour protein (TCTP) may play an important role in the establishment or maintenance of parasitemia in a malarial infection. In this study, the potential of TCTP as a malaria vaccine was investigated in two trials. In the initial vaccine trial, Plasmodium falciparum TCTP (PfTCTP) was expressed in Saccharomyces cerevisiae and used to immunize BALB/c mice. Following challenge with Plasmodium yoelii YM, parasitemia was significantly reduced during the early stages of infection. In the second vaccine trial, the TCTP from P. yoelii and P. berghei was expressed in Escherichia coli and used in several mouse malaria models. A significant reduction in parasitemia in the early stages of infection was observed in BALB/c mice challenged with P. yoelii YM. A significantly reduced parasitemia at each day leading up to a delayed and reduced peak parasitemia was also observed in BALB/c mice challenged with the nonlethal Plasmodium chabaudi (P.c.) chabaudi AS. These results suggest that TCTP has an important role for parasite establishment and may be important for pathogenesis.     

Neutrophil alterations in pregnancy‐associated malaria and induction of neutrophil chemotaxis by Plasmodium falciparum

Pregnancy‐associated malaria (PAM) is a severe form of the disease caused by sequestration of Plasmodium falciparum‐infected red blood cells (iRBCs) in the developing placenta. Pathogenesis of PAM is partially based on immunopathology, with frequent monocyte infiltration into the placenta. Neutrophils are abundant blood cells that are essential for immune defence but may also cause inflammatory pathology. Their role in PAM remains unclear. We analysed neutrophil alterations in the context of PAM to better understand their contribution to disease development. Pregnant women exposed to Plasmodium falciparum had decreased numbers of circulating neutrophils. Placental‐like BeWo cells stimulated with malaria parasites produced the neutrophil chemoattractant IL‐8 and recruited neutrophils in a trans‐well assay. Finally, immunostaining of a PAM placenta confirmed neutrophil accumulation in the intervillous space. Our data indicate neutrophils may play a role in placental malaria and should be more closely examined as an etiological agent in the pathophysiology of disease.     

Melatonin activates FIS1, DYN1, and DYN2 Plasmodium falciparum related‐genes for mitochondria fission: Mitoemerald‐GFP as a tool to visualize mitochondria structure

Malaria causes millions of deaths worldwide and is considered a huge burden to underdeveloped countries. The number of cases with resistance to all antimalarials is continuously increasing, making the identification of novel drugs a very urgent necessity. A potentially very interesting target for novel therapeutic intervention is the parasite mitochondrion. In this work, we studied in Plasmodium falciparum 3 genes coding for proteins homologues of the mammalian FIS1 (Mitochondrial Fission Protein 1) and DRP1 (Dynamin Related Protein 1) involved in mitochondrial fission. We studied the expression of P. falciparum genes that show ample sequence and structural homologies with the mammalian counterparts, namely FIS1, DYN1, and DYN2. The encoded proteins are characterized by a distinct pattern of expression throughout the erythrocytic cycle of P. falciparum, and their mRNAs are modulated by treating the parasite with the host hormone melatonin. We have previously reported that the knockout of the Plasmodium gene that codes for protein kinase 7 is essential for melatonin sensing. We here show that PfPk7 knockout results in major alterations of mitochondrial fission genes expression when compared to wild‐type parasites, and no change in fission proteins expression upon treatment with the host hormone. Finally, we have compared the morphological characteristics (using MitoTracker Red CMX Ros) and oxygen consumption properties of P. falciparum mitochondria in wild‐type parasites and PfPk7 Knockout strains. A novel GFP construct targeted to the mitochondrial matrix to wild‐type parasites was also developed to visualize P. falciparum mitochondria. We here show that, the functional characteristics of P. falciparum are profoundly altered in cells lacking protein kinase 7, suggesting that this enzyme plays a major role in the control of mitochondrial morphogenesis and maturation during the intra‐erythrocyte cell cycle progression.     

Differential T‐cell responses to a chimeric Plasmodium falciparum antigen; UB05‐09, correlates with acquired immunity to malaria

The development of a sterilizing and cost‐effective vaccine against malaria remains a major problem despite recent advances. In this study, it is demonstrated that two antigens of P. falciparum UB05, UB09 and their chimera UB05‐09 can serve as protective immunity markers by eliciting higher T‐cell responses in malaria semi‐immune subjects (SIS) than in frequently sick subjects (FSS) and could be used to distinguish these two groups. UB05, UB09 and UB05‐09 were cloned, expressed in E. coli, purified and used to stimulate PBMCs isolated from 63 subjects in a malaria endemic area, for IFN‐γ production, which was measured by the ELISpot assay. The polymorphism of UB09 gene in the malaria infected population was also studied by PCR/sequencing of the gene in P. falciparum field isolates. All three antigens were preferentially recognized by PBMCs from SIS. IFN‐γ production induced by these antigens correlated with the absence of fever and parasitaemia. UB09 was shown to be relatively well‐conserved in nature. It is concluded that UB05, UB09 and the chimera UB05‐09 posses T‐cell epitopes that are associated with protection against malaria and could thus be used to distinguish SIS from FSS eventhough acute infection with malaria has been shown to reduce cytokine production in some studies. Further investigations of these antigens as potential diagnostic and/or vaccine candidates for malaria are indicated.     

Th1 immune response to Plasmodium falciparum recombinant thrombospondin‐related adhesive protein (TRAP) antigen is enhanced by TLR3‐specific adjuvant,poly(I:C) in BALB/c mice

Sporozoite‐based malaria vaccines have provided a gold standard for malaria vaccine development, and thrombospondin‐related adhesive protein (TRAP) serves as the main vaccine candidate antigen on sporozoites. As recombinant malaria vaccine candidate antigens are poorly immunogenic, additional appropriate immunostimulants, such as an efficient adjuvant, are highly essential to modulate Th1‐cell predominance and also to induce a protective and long‐lived immune response. In this study, polyinosinic:polycytidylic acid [poly(I:C)], the ligand of TLR3, was considered as the potential adjuvant for vaccines targeting stronger Th1‐based immune responses. For this purpose, BALB/c mice were immunized with rPfTRAP delivered in putative poly(I:C) adjuvant, and humoural and cellular immune responses were determined in different immunized mouse groups. Delivery of rPfTRAP with poly(I:C) induced high levels and titres of persisted and also high‐avidity anti‐rPfTRAP IgG antibodies comparable to complete Freund's adjuvant (CFA)/incomplete Freund’s adjuvant (IFA) adjuvant after the second boost. In addition, rPfTRAP formulated with poly(I:C) elicited a higher ratio of IFN‐γ/IL‐5, IgG2a/IgG1, and IgG2b/IgG1 than with CFA/IFA, indicating that poly(I:C) supports the induction of a stronger Th1‐based immune response. This is a first time study which reveals the potential of rPfTRAP delivery in poly(I:C) to increase the level, avidity and durability of both anti‐PfTRAP cytophilic antibodies and Th1 cytokines.     

Antibody responses to surface antigens of Plasmodium falciparum gametocyte‐infected erythrocytes and their relation to gametocytaemia

An essential element for continuing transmission of Plasmodium falciparum is the availability of mature gametocytes in human peripheral circulation for uptake by mosquitoes. Natural immune responses to circulating gametocytes may play a role in reducing transmission from humans to mosquitoes. Here, antibody recognition of the surface of mature intra‐erythrocytic gametocytes produced either by a laboratory‐adapted parasite, 3D7, or by a recent clinical isolate of Kenyan origin (HL1204), was evaluated longitudinally in a cohort of Ghanaian school children by flow cytometry. This showed that a proportion of children exhibited antibody responses that recognized gametocyte surface antigens on one or both parasite lines. A subset of the children maintained detectable anti‐gametocyte surface antigen (GSA) antibody levels during the 5 week study period. There was indicative evidence that children with anti‐GSA antibodies present at enrolment were less likely to have patent gametocytaemia at subsequent visits (odds ratio = 0·29, 95% CI 0·06–1·05; P = 0·034). Our data support the existence of antigens on the surface of gametocyte‐infected erythrocytes, but further studies are needed to confirm whether antibodies against them reduce gametocyte carriage. The identification of GSA would allow their evaluation as potential anti‐gametocyte vaccine candidates and/or biomarkers for gametocyte carriage.     

Immunization against Plasmodium falciparum with recombinant polypeptides produced in Escherichia coli

Two proteins produced in recombinant Escherichia coli and containing amino acid sequences from the Plasmodium falciparum precursor to major merozoite surface antigens (PMMSA) have been partially purified. These proteins, together with a preparation of merozoites, have been used to immunize animals. The antibody response and the degree of protection were compared. Animals immunized with merozoites produced antibodies reacting with many P. falciparum proteins, whereas a response specific for PMMSA was detected in those receiving the recombinant material. Incomplete protection was conferred to both groups and there was no apparent correlation between antibody levels and protection.     

Severity of anaemia is associated with bone marrow haemozoin in children exposed to Plasmodium falciparum

There are no large‐scale ex vivo studies addressing the contribution of Plasmodium falciparum in the bone marrow to anaemia. The presence of malaria parasites and haemozoin were studied in bone marrows from 290 anaemic children attending a rural hospital in Mozambique. Peripheral blood infections were determined by microscopy and polymerase chain reactions. Bone marrow parasitaemia, haemozoin and dyserythropoiesis were microscopically assessed. Forty‐two percent (123/290) of children had parasites in the bone marrow and 49% (111/226) had haemozoin, overlapping with parasitaemia in 83% (92/111) of cases. Sexual and mature asexual parasites were highly prevalent (62% gametocytes, 71% trophozoites, 23% schizonts) suggesting their sequestration in this tissue. Sixteen percent (19/120) of children without peripheral infection had haemozoin in the bone marrow. Haemozoin in the bone marrow was independently associated with decreased Hb concentration (P = 0·005) and was more common in dyserythropoietic bone marrows (P = 0·010). The results of this ex vivo study suggest that haemozoin in the bone marrow has a role in the pathogenesis of malarial‐anaemia through ineffective erythropoiesis. This finding may have clinical implications for the development of drugs targeted to prevent and treat malarial‐anaemia.     

Plasmodium falciparum infection transmitted by transfusion: A cause of hemophagocytic syndrome after bone marrow tranplantation in a non‐endemic country

A 27‐year‐old man with severe aplastic anemia underwent bone marrow transplantation from his HLA identical brother in July 2016. Conditioning included ATGAM 30 mg/kg for 3 days and Cyclophosphamide 50 mg/kg for 4 days. The patient received several platelet and red blood cell transfusions before and after the conditioning. The patient received broad spectrum antibiotics and caspofungin because persistant febrile neutropenia without bacteriological or mycological documentation. Hemophagocytic syndrome was diagnosed on day +12. Steroids at 1 mg/kg were started on day +12. Fever resolved the same day but resumed 3 days later associated to intravascular hemolysis with no schizocytes on blood smears and negative DAT. Thick blood film smears performed on day +26 revealed Plasmodium falciparum parasites (parasitemia = 20%). Except the level of parasitemia, there were no signs of gravity. Quinine was started on day 26 at a loading dose of 15 mg/kg followed by 8 mg/kg three times a day for 20 doses. Fever vanished after 2 days. Parasitemia cleared in 3 days and remained negative thereafter. Investigations revealed that the patient was transfused by a red cell unit harvested in a voluntary donor native of a malaria endemic country. PCR for P. falciparum performed in this donor in the frame of investigations was positive. The patient is alive with a normal blood count 1 year after BMT.     

Human antibodies to the 19kDa C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 inhibit parasite growth in vitro

The 19kDa, C-terminal fragment of the major surface protein of Plasmodium falciparum (PfMSP1(19)) is a candidate for inclusion in a subunit malaria vaccine. In this study, we show that PfMSP1(19)-specific antibodies, affinity purified from malaria-immune human serum, can: (i) compete with invasion-inhibitory monoclonal antibodies for binding to PfMSP1(19) and (ii) mediate inhibition of parasite growth in vitro, in the absence of complement and mononuclear cells, at physiological antibody concentrations (100 micrograms/ml). Parasites expressing either the Kl or 3D7 allele of PfMSP1(19) were equally susceptible to inhibition of merozoite invasion, indicating that the target epitopes of inhibitory antibodies are conserved or cross-reactive. These studies suggest that vaccines designed to induce antibodies to PfMSP1(19) may protect against the high levels of malaria parasitaemia which are associated with clinical disease.     

Human erythrocyte remodelling during Plasmodium falciparum malaria parasite growth and egress

The intra-erythrocyte growth and survival of the malarial parasite Plasmodium falciparum is responsible for both uncomplicated and severe malaria cases and depends on the parasite's ability to remodel its host cell. Host cell remodelling has several functions for the parasite, such as acquiring nutrients from the extracellular milieu because of the loss of membrane transporters upon erythrocyte differentiation, avoiding splenic clearance by conferring cytoadhesive properties to the infected erythrocyte, escaping the host immune response by exporting antigenically variant proteins at the red blood cell surface. In addition, parasite-induced changes at the red blood cell membrane and sub-membrane skeleton are also necessary for the efficient release of the parasite progeny from the host cell. Here we review these cellular and molecular changes, which might not only sustain parasite growth but also prepare, at a very early stage, the last step of egress from the host cell.     

Epitope specificity and capacity to inhibit parasite growth in vitro of human antibodies to repeat sequences of the Plasmodium falciparum antigen Ag332

It has earlier been shown that the Plasmodium falciparum-reactive human monoclonal antibody 33G2 inhibits parasite growth in vitro as well as cytoadherence of infected red blood cells to melanoma cells in vitro. MoAb 33G2 recognizes an epitope of the P. falciparum antigen Ag332 and cross-reactive determinants in Pf 155/RESA and Pf 11.1 located in repetitive regions containing sequences of regularly spaced pairs of glutamic acid. To study whether antibodies of this specificity frequently occur in human immune sera and if they could be of importance for protective immunity, antibodies were affinity purified on MoAb 33G2 reactive Ag332 peptides. The epitope specificity of the affinity purified antibodies, determined by the Pepscan method, resembled that of MoAb 33G2, but showed differences in fine specificity. The antibodies cross-reacted to some extent with Pf 11.1 and Pf 155/RESA repeat peptides as detected by peptide ELISA and Pepscan. In indirect immunofluorescence all purified antibodies displayed a dotted pattern of staining of late stage infected red blood cells of two lines of the P. falciparum strain FCR3, including a Pf 155/RESA deficient line. The in vitro growth of these two lines was efficiently inhibited by the affinity purified antibodies, indicating that their inhibitory effect was mainly due to reactivity with antigens other than Pf 155/RESA. This, and the fact that Pf 11.1 has been shown not to be expressed by the asexual stages suggests that Ag332 may be an important target for potentially protective antibodies in vivo and that Ag332 based immunogens are of interest for development of malaria subunit vaccines.     

A longitudinal study of naturally acquired cellular and humoral immune responses to a merozoite surface protein (MSP1) of Plasmodium falciparum in an area of seasonal malaria transmission

A longitudinal study of cellular and serological responses to the major merozoite surface protein of Plasmodium falciparum (PfMSP1) has been conducted in a malaria immune population living in The Gambia, where malaria transmission is seasonally endemic. Recombinant or native proteins representing the sequence of PfMSP1 from the Wellcome strain of P. falciparum were used in in vitro lymphocyte proliferation, cytokine and antibody assays. Cellular responses of individual donors fluctuated over time, independent of seasonal changes in malaria transmission whereas anti-PfMSP1 antibody levels were remarkably stable. At a population level, IFNγ responses were both more prevalent and of greater magnitude at the end of the rainy (malaria transmission) season than during the dry season. Responses of individuals lving in a rural village were compared with those of individuals living in an urban area with much lower levels of malaria transmission. Malaria infections were more likely to be symptomatic in urban dwellers than in inhabitants of rural villages but no significant differences in the level or prevalence of cellular or serological responses were seen between the two groups. However, urban dwellers with current symptomatic malaria infections had somewhat lower anti-PfMSP1 antibody levels than their healthy, non-parasitaemic neighbours.     

Leishmania donovani infection drives the priming of human monocyte‐derived dendritic cells during Plasmodium falciparum co‐infections

Functional impairment of dendritic cells (DCs) is part of a survival strategy evolved by Leishmania and Plasmodium parasites to evade host immune responses. Here, the effects of co‐exposing human monocyte‐derived DCs to Leishmania donovani promastigotes and Plasmodium falciparum‐infected erythrocytes were investigated. Co‐stimulation resulted in a dual, dose‐dependent effect on DC differentiation which ranged from semi‐mature cells, secreting low interleukin(‐12p70 levels to a complete lack of phenotypic maturation in the presence of high parasite amounts. The effect was mainly triggered by the Leishmania parasites, as illustrated by their ability to induce semi‐mature, interleukin‐10‐producing DCs, that poorly responded to lipopolysaccharide stimulation. Conversely, P. falciparum blood‐stage forms failed to activate DCs and only slightly interfered with lipopolysaccharide effects. Stimulation with high L. donovani concentrations triggered phosphatidylserine translocation, whose onset presented after initiating the maturation impairment process. When added in combination, the two parasites could co‐localize in the same DCs, confirming that the leading effects of Leishmania over Plasmodium may not be due to mutual exclusion. Altogether, these results suggest that in the presence of visceral leishmaniasis–malaria co‐infections, Leishmania‐driven effects may overrule the more silent response elicited by P. falciparum, shaping host immunity towards a regulatory pattern and possibly delaying disease resolution.