Expression of the MSPDBL2 antigen in a discrete subset of Plasmodium falciparum schizonts is regulated by GDV1 but may not be linked to sexual commitment.

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of the gametocyte development protein GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines, the variable proportions of MSPDBL2 positivity in schizonts do not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous hemagglutinin-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different from those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection. IMPORTANCE: Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions.

Antibody effector functions in malaria and other parasitic diseases: a few needles and many haystacks.

Many parasitic infections stimulate antibody responses in their mammalian hosts. The ability of these antibodies to protect against disease varies markedly. Research has revealed that functional properties of antibodies determine their role in protection against parasites. Investigations of antibodies against Plasmodium spp. have demonstrated a variety of functional activities, ranging from invasion inhibition and parasite growth inhibition to antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity. These activities have been demonstrated with a large variety of parasite molecules at multiple life cycle stages, highlighting the importance of functional antibody responses in malaria. Other parasitic infections have not yet been investigated in similar detail, but these mechanisms are likely to operate in nonmalarial parasitic infections as well. In this report, we review data on the role of functional antibody responses in protection from parasitic infections, highlighting discoveries in malaria, a parasite for which our knowledge base is the most advanced.

The impact of early life exposure to Plasmodium falciparum on the development of naturally acquired immunity to malaria in young Malawian children.

BACKGROUND: Antibodies targeting malaria blood-stage antigens are important targets of naturally acquired immunity, and may act as valuable biomarkers of malaria exposure. METHODS: Six-hundred and one young Malawian children from a randomized trial of prenatal nutrient supplementation with iron and folic acid or pre- and postnatal multiple micronutrients or lipid-based nutrient supplements were followed up weekly at home and febrile episodes were investigated for malaria from birth to 18 months of age. Antibodies were measured for 601 children against merozoite surface proteins (MSP1 19kD, MSP2), erythrocyte binding antigen 175 (EBA175), reticulocyte binding protein homologue 2 (Rh2A9), schizont extract and variant surface antigens expressed by Plasmodium falciparum-infected erythrocytes (IE) at 18 months of age. The antibody measurement data was related to concurrent malaria infection and to documented episodes of clinical malaria. RESULTS: At 18 months of age, antibodies were significantly higher among parasitaemic than aparasitaemic children. Antibody levels against MSP1 19kD, MSP2, schizont extract, and IE variant surface antigens were significantly higher in children who had documented episodes of malaria than in children who did not. Antibody levels did not differ between children with single or multiple malaria episodes before 18 months, nor between children who had malaria before 6 months of age or between 6 and 18 months. CONCLUSIONS: Antibodies to merozoite and IE surface antigens increased following infection in early childhood, but neither age at first infection nor number of malaria episodes substantially affected antibody acquisition. These findings have implications for malaria surveillance during early childhood in the context of elimination. Trials registration Clinical Trials Registration: NCT01239693 (Date of registration: 11-10-2010). URL: http://www.ilins.org.

Determination of CD Markers Profile of the Cell Line Infected by S15 Vaccine Strain of Theileria annulata Schizont Using RT-PCR Analysis.

The aim of this study was to identify the cell surface cluster of differentiation (CD) markers of the cell lines infected by Theileria annulata schizont. The CD molecules are very useful for the characterization of cells and different subpopulations of leukocytes. They are usually recognized by specific antibodies using flow cytometry and immunohistochemistry. In the current study, we applied reverse transcriptase-polymerase chain reaction (RT-PCR) to define the profile of cell surface markers in a cell line infected by an attenuated S15 vaccine strain of T. annulata schizont and a new laboratory-established cell line infected by a non-attenuated form. In order to determine the specific markers that can be used for excluding the non-attenuated cell lines, the characterization of the surface proteins profile of the S15 vaccine cell line is important. The RT-PCR was carried out by specifically designed primers using a panel of seven bovine CD markers, as well as beta-actin as an internal control house-keeping gene. We showed that both of the examined cell lines had a consistent expression of CD4, CD5, CD11a, CD14, CD43, and CD45 markers. However, the specific finding in this study was the expression of B-cell markers CD79a and CD5 by the newly-transformed cell line. On the other hand, CD5 as a marker for B-cell subset was expressed by S15 vaccine strain. In conclusion, we consider CD79a surface protein as a new marker for the cell lines infected by non-attenuated T. annulata schizont, while the cell lines infected by the vaccine strain do not express this marker. In addition, the identification of CD marker expression based on the RT-PCR assay might be a suitable and appropriate alternative technique for flow cytometry.

Transcriptomics reveal potential vaccine antigens and a drastic increase of upregulated genes during Theileria parva development from arthropod to bovine infective stages.

Theileria parva is a protozoan parasite transmitted by the brown ear tick Rhipicephalus appendiculatus that causes East Coast fever (ECF) in cattle, resulting in substantial economic losses in the regions of southern, eastern and central Africa. The schizont form of the parasite transforms the bovine host lymphocytes into actively proliferating cancer-like cells. However, how T. parva causes bovine host cells to proliferate and maintain a cancerous phenotype following infection is still poorly understood. On the other hand, current efforts to develop improved vaccines have identified only a few candidate antigens. In the present paper, we report the first comparative transcriptomic analysis throughout the course of T. parva infection. We observed that the development of sporoblast into sporozoite and then the establishment in the host cells as schizont is accompanied by a drastic increase of upregulated genes in the schizont stage of the parasite. In contrast, the ten highest gene expression values occurred in the arthropod vector stages. A comparative analysis showed that 2845 genes were upregulated in both sporozoite and schizont stages compared to the sporoblast. In addition, 647 were upregulated only in the sporozoite whereas 310 were only upregulated in the schizont. We detected low p67 expression in the schizont stage, an unexpected finding considering that p67 has been reported as a sporozoite stage-specific gene. In contrast, we found that transcription of p67 was 20 times higher in the sporoblast than in the sporozoite. Using the expression profiles of recently identified candidate vaccine antigens as a benchmark for selection for novel potential vaccine candidates, we identified three genes with expression similar to p67 and several other genes similar to Tp1-Tp10 schizont vaccine antigens. We propose that the antigenicity or chemotherapeutic potential of this panel of new candidate antigens be further investigated. Structural comparisons of the transcripts generated here with the existing gene models for the respective loci revealed indels. Our findings can be used to improve the structural annotation of the T. parva genome, and the identification of alternatively spliced transcripts.

Isolation and purification of glycosylphosphatidylinositols (GPIs) in the schizont stage of Theileria annulata and determination of antibody response to GPI anchors in vaccinated and infected animals.

BACKGROUND: Tropical theileriosis is widely distributed from North Africa to East Asia. It is a tick-borne disease caused by Theileria annulata, an obligate two-host intracellular protozoan parasite of cattle. Theileria annulata use leukocytes and red blood cells for completion of the life-cycle in mammalian hosts. The stage of Theileria annulata in monocytes and B lymphocytes of cattle is an important step in pathogenicity and diagnosis of the disease. Glycosylphosphatidylinositols (GPIs) are a distinct class of glycolipid structures found in eukaryotic cells and are implicated in several biological functions. GPIs are particularly abundant in protozoan parasites, where they are found as free glycolipids or attached to proteins in the plasma membrane. RESULTS: In this study we first isolated and purified schizonts of Theileria annulata from infected leukocytes in Theileria annulata vaccine cell line (S15) by aerolysin-percoll technique. Then, the free GPIs of schizont stage and isolated GPI from cell membrane glycoproteins were purified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography-mass spectrometry (GC-MS). Furthermore, enzyme linked immunosorbent assay (ELISA) on the serum samples obtained from naturally infected, as well as Theileria annulata-vaccinated animals, confirmed a significant (P < 0.01) high level of anti-GPI antibody in their serum. CONCLUSIONS: The results presented in this study show, to our knowledge for the first time, the isolation of GPI from the schizont stage of Theileria annulata and demonstrate the presence of anti-GPI antibody in the serum of naturally infected as well as vaccinated animals. This finding is likely to be valuable in studies aimed at the evaluation of chemically structures of GPIs in the schizont stage of Theileria annulata and also for pathogenicity and immunogenicity studies with the aim to develop GPI-based therapies or vaccines.

Measuring Plasmodium falciparum Erythrocyte Invasion Phenotypes Using Flow Cytometry.

Having the ability to rapidly, accurately, and robustly measure Plasmodium falciparum merozoite invasion is a critical component in effective assessment of a blood stage vaccine's mechanism of action. Being able to measure invasion of erythrocytes accurately, objectively and in a high throughput fashion is of critical importance. Here, we describe a simple and robust flow cytometry method that allows for the measurement of the key invasion parameters of parasite multiplication rate and erythrocyte selectivity-both important determinants of disease severity-from the schizont to the ring stage of the parasite's life-cycle, thus separating invasion from growth of the parasite. Importantly, this method is able to accurately detect low levels of parasitemia and heterogeneity within the population that can be missed by enzymatic methods. Lastly, this method has been successfully adapted and employed in field based research settings for parasitemia measurements in vivo, ex vivo, and in vitro and to measure invasion inhibition by antibodies and the use of alternative pathways for invasion.

The value of local malaria strains for serological studies: local strains versus Palo Alto reference strain.

BACKGROUND: The standardization of the type of crude Plasmodium falciparum extracts for assays to evaluate the overall anti-blood-stage immune response in humans may be beneficial to malaria pre-elimination programmes. However, there is no consensus on which strain is appropriate for routine analyses. This study aimed to compare the responses of malaria IgG antibodies in serum collections from Dielmo and Ndiop to crude extracts of merozoites and schizonts of local and reference strains of P. falciparum. METHODS: Malaria antibodies were evaluated using serological tests for exposure to three local strains (0703, F15 and F16) and the P. falciparum reference Palo Alto strain (PA). A total of 218 sera collected in 2000 from inhabitants of the villages of Dielmo and Ndiop were used: 142 from Dielmo and 76 from Ndiop. The biological collection was used to evaluate by ELISA the prevalence of IgG antibodies against crude merozoite and schizont extracts. The genetics of the local and reference strains were compared. RESULTS: There was genetic divergence between strains 0703, F15, F16 and PA. IgG responses against local and reference strains correlated well (0.6 to 0.8; p<0.01). Ig G responses were highest to schizont and merozoite extracts from the field strain of P. falciparum 0703 adapted to in vitro culture. Extracts of P. falciparum strain 0703 isolated from a subject in Dielmo was the most widely recognized [91.3% (199/218) and 81.2% (177/218) for schizonts and merozoites, respectively], although the responses were high for merozoites from PA [85.3% (186/218)] the reference strain, and the two strains isolated from subjects living in Dakar: F15 [90.4% (197/218)] and F16 [72.5% (158/218)]. CONCLUSIONS: For serological studies, the local strain provided the most complete picture of exposure to transmission and malaria prevalence in the local context. However, for the standardization of this method by different laboratories, the reference strain appeared to perform sufficiently well to be used for the evaluation of malaria control programmes.

The impact of lipid-based nutrient supplementation on anti-malarial antibodies in pregnant women in a randomized controlled trial.

BACKGROUND: Malaria and undernutrition frequently coexist, especially in pregnant women and young children. Nutrient supplementation of these vulnerable groups might reduce their susceptibility to malaria by improving immunity. METHODS: Antibody immunity to antigens expressed by a placental-binding parasite isolate, a non-placental binding parasite isolate, merozoites and schizonts at enrolment (before 20 gestation weeks) and at 36 gestation weeks were measured in 1,009 Malawian pregnant women receiving a daily lipid-based nutrient supplement, multiple micronutrients or iron and folic acid, who were participants in a randomized clinical trial assessing the effects of nutrient supplementation on pregnancy outcomes and child development (registration ID: NCT01239693). RESULTS: Antibodies to placental-binding isolates significantly increased while antibodies to most merozoite antigens declined over pregnancy. Overall, after adjustment for covariates, the type of supplementation did not influence antibody levels at 36 gestation weeks or the rate of change in antibody levels from enrolment to 36 weeks. A negative association between maternal body mass index and opsonizing antibodies to placental-binding antigens (coefficient (95% CI) -1.04 (-1.84, -0.24), was observed. Similarly, women with higher socioeconomic status had significantly lower IgG and opsonizing antibodies to placental-binding antigens. Neither of these associations was significantly influenced by the supplementation type. CONCLUSIONS: In the current cohort nutrient supplementation did not affect anti-malarial antibody responses, but poor and undernourished mothers should be a priority group in future trials.

Assessment of the neutrophilic antibody-dependent respiratory burst (ADRB) response to Plasmodium falciparum.

Semi-immunity against Pf malaria is based on a combination of cellular and humoral immune responses. PMNs and IgGs are considered important components of this process, but the underlying mechanisms are unclear. We investigated the neutrophilic ADRB by analyzing the production of ROS in response to Pf antigen-specific IgGs bound to solid-phase immobilized antigens (sADRB) or whole merozoites (mADRB). We found that the PMN stimulations in each assay were based on different underlying mechanisms, demonstrating the importance of the assay set-up for the evaluation of antibody-triggered PMN responses. In the sADRB assay, ROS were produced externally, and by specific blocking of CD32(a)/FcγRII(a), the immediate neutrophilic response was abolished, whereas the removal of CD16(b)/FcγRIII(b) had no substantial effect. The key role of CD32(a) was confirmed using CD16(b)-deficient PMNs, in which similar changes of neutrophilic ADRB profiles were recorded after treatment. In the mADRB assay, ROS were produced almost exclusively within the cell, suggesting that the underlying mechanism was phagocytosis. This was confirmed using an additional phagocytosis assay, in which PMNs specifically ingested merozoites opsonized with Ghanaian plasma IgGs, seven times more often than merozoites opsonized with European plasma IgGs (P<0.001). Our data show that assay set-ups used to evaluate the responses of PMNs and perhaps other effector cells must be chosen carefully to evaluate the appropriate cellular responses. Our robust, stable, and well-characterized methods could therefore be useful in malaria vaccine studies to analyze the antimalarial effector function of antibodies.

Dramatic declines in seropositivity as determined with crude extracts of Plasmodium falciparum schizonts between 2000 and 2010 in Dielmo and Ndiop, Senegal.

BACKGROUND: Programmes of pre-elimination of malaria have been implemented in Senegal since 2010, and the burden of malaria has decreased substantially. These changes in the epidemiology should be monitored with effective tools that allow changes in patterns of transmission to be estimated. In Dielmo and Ndiop, two villages of Senegal with different malaria endemicity, infections have been followed longitudinally for 20 years, during which time there have been several control interventions leading to substantial decreases of transmission. This study aimed to compare malaria antibody responses of the inhabitants of these two villages, between 2000 and 2010, using schizont crude extracts of a local strain of P. falciparum (Pf Sch07/03). METHODS: Sera collected from inhabitants of the two villages (141 from Dielmo and 79 from Ndiop in 2000; 143 from Dielmo and 79 from Ndiop in 2010) were used to assess the prevalence of antibodies against crude schizont extracts of Pf Sch07/03. Three ages groups were defined: [5-9] yrs, [10-14] yrs and [15-19] yrs. Statistical comparisons were performed. Seroprevalence and the magnitude of antibody responses were compared between age groups, villages and periods. RESULTS: Overall seroprevalence to P.fSch07/03 decreased between 2000 and 2010 in both villages: from 94.4% to 44.4% in Dielmo and from 74.4% to 34.6% in Ndiop. The difference between Dielmo and Ndiop was highly significant in 2000 (p<0.001) but not in 2010 (p >0.20). The decrease in seroprevalence was larger in younger (more than 40%) than older (less than 19%) inhabitants. Longitudinal monitoring of the younger group showed that seroprevalence decreased between 2000 and 2010 in Dielmo from 98.7 to 79.3, but not in Ndiop from 67.6 to 66.7. The magnitude of antibody responses in seropositive individuals was significantly higher in 2000 than 2010 for both villages. CONCLUSIONS: Crude extracts of P. falciparum are appropriate tools for evaluating malaria prevalence at different periods, and in both low and high endemic area. Using crude extracts from local strains to assess transmission may allow efficient evaluation of the consequences of control programs on malaria transmission.

Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.

Synthesis and evaluation of monoclonal antibody against Plasmodium falciparum merozoite surface antigen 2.

OBJECTIVE: To assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins. METHODS: Mice were immunized with a schizont extract to stimulate the immune system to make antibodies against different antigens of the late stage parasite including production of antibodies against different domains of Plasmodium falciparum (P. falciparum) MSP-2. B lymphocytes of immunized mice were extracted from the spleen and the fusion was performed using NS-1 myeloma cells and the hybridoma cells were assayed by ELISA either with a schizont extract or different domains of MSP-2 and/or by IFAT with whole schizont preparation. Fusion of NS-1 and spleen cells was performed. The positive hybrids were cloned and ELISA was applied against different dilutions. The positive clones were transferred to a small tissue culture flask and after developing they were assayed against schizont extract and the different MSP-2 domains. The positive clones were expanded to large (75 cm(2)) flask and cultured under the same conditions, checking them using both ELISA and IFAT and the positive cells were frozen as soon as possible. RESULTS: A total number of 7 fusions including 26 plates (2 496 wells) were performed, of which 1 336 hybrids were produced and the overall efficiency (1 336/2496 × 100) was about 53%. ELISA was performed to detect the positive hybrids against crude schizont extract by which the highest frequency to crude schizont extract was found for the supernatant of the hybrids produced in fusion number 3 (66 out of 315 hybrids). The supernatant of both B5 and F1 hybridoma cells were more positive against domain 2 of the MSP-2 recombinant protein in Western blotting test. Western blotting results also showed that different domains of the MSP-2 recombinant protein and also the MSP-2 of the P. falciparum parasite were recognized by some of the positive clones and also immune sera. CONCLUSIONS: Bringing together all the results of this study it has been confirmed that some clones have recognized both schizont extract and different domains of the MSP-2 recombinant protein and therefore confirming the quality of the MSP-2 domains.

Structural and biochemical characterization of Plasmodium falciparum 12 (Pf12) reveals a unique interdomain organization and the potential for an antiparallel arrangement with Pf41.

Plasmodium falciparum is the most devastating agent of human malaria. A major contributor to its virulence is a complex lifecycle with multiple parasite forms, each presenting a different repertoire of surface antigens. Importantly, members of the 6-Cys s48/45 family of proteins are found on the surface of P. falciparum in every stage, and several of these antigens have been investigated as vaccine targets. Pf12 is the archetypal member of the 6-Cys protein family, containing just two s48/45 domains, whereas other members have up to 14 of these domains. Pf12 is strongly recognized by immune sera from naturally infected patients. Here we show that Pf12 is highly conserved and under purifying selection. Immunofluorescence data reveals a punctate staining pattern with an apical organization in late schizonts. Together, these data are consistent with an important functional role for Pf12 in parasite-host cell attachment or invasion. To infer the structural and functional diversity between Pf12 and the other 11 6-Cys domain proteins, we solved the 1.90 Å resolution crystal structure of the Pf12 ectodomain. Structural analysis reveals a unique organization between the membrane proximal and membrane distal domains and clear homology with the SRS-domain containing proteins of Toxoplasma gondii. Cross-linking and mass spectrometry confirm the previously identified Pf12-Pf41 heterodimeric complex, and analysis of individual cross-links supports an unexpected antiparallel organization. Collectively, the localization and structure of Pf12 and details of its interaction with Pf41 reveal important insight into the structural and functional properties of this archetypal member of the 6-Cys protein family.

Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin.

BACKGROUND AND PURPOSE: Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria. EXPERIMENTAL APPROACH: Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed. KEY RESULTS: Borrelidin protected against lethal malaria at 0.25 mg·kg⁻¹·day⁻¹. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts. CONCLUSIONS AND IMPLICATIONS: Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.

Hemozoin induces lung inflammation and correlates with malaria-associated acute respiratory distress syndrome.

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial growth factor. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand 2, and keratinocyte-derived chemokine/CXCL1), cytokines (IL-1ß, IL-6, IL-10, TNF, and transforming growth factor-ß), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-2, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.

Differential acquisition of human antibody responses to Plasmodium falciparum according to intensity of exposure to Anopheles bites.

Malaria immunity is modulated by many environmental and epidemiological factors. This study evaluates the influence of a hitherto unstudied environmental-epidemiological factor, namely the impact of human exposure to Anopheles bites on the isotype profile of acquired antibody responses to Plasmodium falciparum. In two Senegalese villages where the intensity of exposure to Anopheles bites was markedly different (high and low exposure), specific IgG1 and IgG3 responses to P. falciparum whole schizont extract (WSE) and circumsporozoite protein (CSP) were evaluated at the peak of Anopheles exposure (September) and later (December) in a cohort of 120 children aged 3-8 years. Multivariate analysis showed a significantly lower IgG1 response against P. falciparum WSE and CSP in children highly exposed to Anopheles bites (Gankette) compared to those who were weakly exposed (Mboula). In contrast, in both villages, parasitemia and increasing age were strongly associated with higher IgG1 and IgG3 levels. We hypothesize that high exposure to Anopheles bites could inhibit IgG1-dependent responsiveness to P. falciparum known to induce protective immune responses against malaria. The impact of mosquito saliva on the regulation of specific protective immunity may need to be taken into account in epidemiological studies and trials for malaria vaccines.

Control of Plasmodium falciparum erythrocytic cycle: γδ T cells target the red blood cell-invasive merozoites.

The control of Plasmodium falciparum erythrocytic parasite density is essential for protection against malaria, because it prevents pathogenesis and progression toward severe disease. P falciparum blood-stage parasite cultures are inhibited by human Vγ9Vδ2 γδ T cells, but the underlying mechanism remains poorly understood. Here, we show that both intraerythrocytic parasites and the extracellular red blood cell-invasive merozoites specifically activate Vγ9Vδ2 T cells in a γδ T cell receptor-dependent manner and trigger their degranulation. In contrast, the γδ T cell-mediated antiparasitic activity only targets the extracellular merozoites. Using perforin-deficient and granulysin-silenced T-cell lines, we demonstrate that granulysin is essential for the in vitro antiplasmodial process, whereas perforin is dispensable. Patients infected with P falciparum exhibited elevated granulysin plasma levels associated with high levels of granulysin-expressing Vδ2(+) T cells endowed with parasite-specific degranulation capacity. This indicates in vivo activation of Vγ9Vδ2 T cells along with granulysin triggering and discharge during primary acute falciparum malaria. Altogether, this work identifies Vγ9Vδ2 T cells as unconventional immune effectors targeting the red blood cell-invasive extracellular P falciparum merozoites and opens novel perspectives for immune interventions harnessing the antiparasitic activity of Vγ9Vδ2 T cells to control parasite density in malaria patients.

Blood-stage Plasmodium berghei infection generates a potent, specific CD8+ T-cell response despite residence largely in cells lacking MHC I processing machinery.

Murine cerebral malaria is a complex disease caused by Plasmodium berghei ANKA infection. Several cell types, including CD8(+) T cells, are essential effectors of disease. Although the use of transgenic parasites expressing model antigens has revealed the induction of cytotoxic T lymphocytes (CTL) specific for these model antigens, there is no direct evidence for a response to authentic blood-stage parasite antigens, nor any knowledge of its magnitude. Our studies show that there is a dramatic primary parasite-specific CTL response, akin to viral immunity, reaching approximately 30% of splenic CD8(+) T cells, with many producing interferon-γ and tumor necrosis factor-α. These cells express granzyme B and other markers of specific responders, are cytolytic, and respond to a broad array of major histocompatibility complex (MHC) I-restricted epitopes, 5 of which are identified here. Our studies indicate that vigorous CTL responses can be induced to pathogens even when they largely reside in red blood cells, which lack MHC I processing machinery.