Red blood cell blood group A antigen level affects the ability of heparin and PfEMP1 antibodies to disrupt Plasmodium falciparum rosettes.

BACKGROUND: The histo-blood group ABO system has been associated with adverse outcomes in COVID-19, thromboembolic diseases and Plasmodium falciparum malaria. An integral part of the severe malaria pathogenesis is rosetting, the adherence of parasite infected red blood cells (RBCs) to uninfected RBCs. Rosetting is influenced by the host's ABO blood group (Bg) and rosettes formed in BgA have previously been shown to be more resilient to disruption by heparin and shield the parasite derived surface antigens from antibodies. However, data on rosetting in weak BgA subgroups is scarce and based on investigations of relatively few donors. METHODS: An improved high-throughput flow cytometric assay was employed to investigate rosetting characteristics in an extensive panel of RBC donor samples of all four major ABO Bgs, as well as low BgA expressing samples. RESULTS: All non-O Bgs shield the parasite surface antigens from strain-specific antibodies towards P. falciparum erythrocyte membrane protein 1 (PfEMP1). A positive correlation between A-antigen levels on RBCs and rosette tightness was observed, protecting the rosettes from heparin- and antibody-mediated disruption. CONCLUSIONS: These results provide new insights into how the ABO Bg system affects the disease outcome and cautions against interpreting the results from the heterogeneous BgA phenotype as a single group in epidemiological and experimental studies.

Direct contact between Plasmodium falciparum and human B-cells in a novel co-culture increases parasite growth and affects B-cell growth.

BACKGROUND: Plasmodium falciparum parasites cause malaria and co-exist in humans together with B-cells for long periods of time. Immunity is only achieved after repeated exposure. There has been a lack of methods to mimic the in vivo co-occurrence, where cells and parasites can be grown together for many days, and it has been difficult with long time in vitro studies. METHODS AND RESULTS: A new method for growing P. falciparum in 5% CO2 with a specially formulated culture medium is described. This knowledge was used to establish the co-culture of live P. falciparum together with human B-cells in vitro for 10 days. The presence of B-cells clearly enhanced parasite growth, but less so when Transwell inserts were used (not allowing passage of cells or merozoites), showing that direct contact is advantageous. B-cells also proliferated more in presence of parasites. Symbiotic parasitic growth was verified using CESS cell-line and it showed similar results, indicating that B-cells are indeed the cells responsible for the effect. In malaria endemic areas, people often have increased levels of atypical memory B-cells in the blood, and in this assay it was demonstrated that when parasites were present there was an increase in the proportion of CD19 + CD20 + CD27 - FCRL4 + B-cells, and a contraction of classical memory B-cells. This effect was most clearly seen when direct contact between B-cells and parasites was allowed. CONCLUSIONS: These results demonstrate that P. falciparum and B-cells undoubtedly can affect each other when allowed to multiply together, which is valuable information for future vaccine studies.

Heparinoid sevuparin inhibits Streptococcus-induced vascular leak through neutralizing neutrophil-derived proteins.

Acute lung injury (ALI) and respiratory distress can develop as a consequence of sepsis with pathogens such as group A Streptococcus (GAS). In the pathogenesis of sepsis-associated ALI, endothelial barrier disruption brought on by phagocyte activation is considered a causative factor. Here, we find that sevuparin, a heparinoid with low anticoagulant activity, prevents neutrophil-induced lung plasma leakage in a murine model of systemic inflammation evoked by heat-killed GAS (hkGAS). Furthermore, using human neutrophils and endothelial cell monolayers, we demonstrate that sevuparin inhibits hkGAS-induced endothelial barrier disruption by neutralizing the activity of neutrophil-derived proteins. By mass spectrometry of neutrophil secretion, we identify proteins, including serprocidins, S100 proteins, and histone H4, that interact with sevuparin and that are responsible for the disruptive effect on endothelial integrity. Collectively, our results demonstrate the critical role of neutrophil-derived proteins in vascular hyperpermeability caused by GAS and suggest sevuparin as a potential therapeutic in acute neutrophilic inflammation.-Rasmuson, J., Kenne, E., Wahlgren, M., Soehnlein, O., Lindbom, L. Heparinoid sevuparin inhibits Streptococcus-induced vascular leak through neutralizing neutrophil-derived proteins.

Depleted circulatory complement-lysis inhibitor (CLI) in childhood cerebral malaria returns to normal with convalescence.

BACKGROUND: Cerebral malaria (CM), is a life-threatening childhood malaria syndrome with high mortality. CM is associated with impaired consciousness and neurological damage. It is not fully understood, as yet, why some children develop CM. Presented here is an observation from longitudinal studies on CM in a paediatric cohort of children from a large, densely-populated and malaria holoendemic, sub-Saharan, West African metropolis. METHODS: Plasma samples were collected from a cohort of children with CM, severe malarial anaemia (SMA), uncomplicated malaria (UM), non-malaria positive healthy community controls (CC), and coma and anemic patients without malaria, as disease controls (DC). Proteomic two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry were used in a discovery cohort to identify plasma proteins that might be discriminatory among these clinical groups. The circulatory levels of identified proteins of interest were quantified by ELISA in a prospective validation cohort. RESULTS: The proteome analysis revealed differential abundance of circulatory complement-lysis inhibitor (CLI), also known as Clusterin (CLU). CLI circulatory level was low at hospital admission in all children presenting with CM and recovered to normal level during convalescence (p < 0.0001). At acute onset, circulatory level of CLI in the CM group significantly discriminates CM from the UM, SMA, DC and CC groups. CONCLUSIONS: The CLI circulatory level is low in all patients in the CM group at admission, but recovers through convalescence. The level of CLI at acute onset may be a specific discriminatory marker of CM. This work suggests that CLI may play a role in the pathophysiology of CM and may be useful in the diagnosis and follow-up of children presenting with CM.

Effect of ABO blood group on asymptomatic, uncomplicated and placental Plasmodium falciparum infection: systematic review and meta-analysis.

BACKGROUND: Malaria clinical outcomes vary by erythrocyte characteristics, including ABO blood group, but the effect of ABO blood group on asymptomatic, uncomplicated and placental Plasmodium falciparum (P. falciparum) infection remains unclear. We explored effects of ABO blood group on asymptomatic, uncomplicated and placental falciparum infection in the published literature. METHODS: A systematic review and meta-analysis was performed using the preferred reporting items for systematic reviews and meta-analyses guidelines. Articles in Pubmed, Embase, Web of Science, CINAHL and Cochrane Library published before February 04, 2017 were searched without restriction. Studies were included if they reported P. falciparum infection incidence or prevalence, stratified by ABO blood group. RESULTS: Of 1923 articles obtained from the five databases (Embase = 728, PubMed = 620, Web of Science = 549, CINAHL = 14, Cochrane Library = 12), 42 met criteria for systematic review and 37 for meta-analysis. Most studies (n = 30) were cross-sectional, seven were prospective cohort, and five were case-control studies. Meta-analysis showed similar odds of uncomplicated P. falciparum infection among individuals with blood group A (summary odds ratio [OR] 0.96, 15 studies), B (OR 0.89, 15 studies), AB (OR 0.85, 10 studies) and non-O (OR 0.95, 17 studies) as compared to those with blood group O. Meta-analysis of four cohort studies also showed similar risk of uncomplicated P. falciparum infection among individuals with blood group non-O and those with blood group O (summary relative risk [RR] 1.03). Meta-analysis of six studies showed similar odds of asymptomatic P. falciparum infection among individuals with blood group A (OR 1.05), B (OR 1.03), AB (OR 1.23), and non-O (OR 1.07) when compared to those with blood group O. However, odds of active placental P. falciparum infection was significantly lower in primiparous women with non-O blood groups (OR 0.46, 95% confidence interval [CI] 0.23 - 0.69, I2 0.0%, three studies), particularly in those with blood group A (OR 0.41, 95% CI 0.003 - 0.82, I2 1.4%, four studies) than those with blood group O. CONCLUSIONS: This study suggests that ABO blood group may not affect susceptibility to asymptomatic and/or uncomplicated P. falciparum infection. However, blood group O primiparous women appear to be more susceptible to active placental P. falciparum infection.

Exploring parasite heterogeneity using single-cell RNA-seq reveals a gene signature among sexual stage Plasmodium falciparum parasites.

The malaria parasite has a complex lifecycle, including several events of differentiation and stage progression, while actively evading immunity in both its mosquito and human hosts. Important parasite gene expression and regulation during these events remain hidden in rare populations of cells. Here, we combine a capillary-based platform for cell isolation with single-cell RNA-sequencing to transcriptionally profile 165 single infected red blood cells (iRBCs) during the intra-erythrocytic developmental cycle (IDC). Unbiased analyses of single-cell data grouped the cells into eight transcriptional states during IDC. Interestingly, we uncovered a gene signature from the single iRBC analyses that can successfully discriminate between developing asexual and sexual stage parasites at cellular resolution, and we verify five, previously undefined, gametocyte stage specific genes. Moreover, we show the capacity of detecting expressed genes from the variable gene families in single parasites, despite the sparse nature of data. In total, the single parasite transcriptomics holds promise for molecular dissection of rare parasite phenotypes throughout the malaria lifecycle.

A Thioredoxin Homologous Protein of Plasmodium falciparum Participates in Erythrocyte Invasion.

Invasion of erythrocytes by merozoites is required in the life cycle of malarial parasites. Proteins derived from the invasive merozoites are essential ligands for erythrocyte recognition and penetration. In this study, we report a novel protein that possesses a Trx domain-like structure of the thioredoxin family and is expressed on the surface of merozoites of the malaria parasite Plasmodium falciparum This protein, namely, PfTrx-mero protein, displayed a mutated sequence character at the Trx domain, but with a specific binding activity to human erythrocytes. Specific antibodies to the protein inhibited merozoite invasion into human erythrocytes. Immunization with a homologous protein of Plasmodium berghei strain ANKA also showed significant protection against lethal infection in mice. These results suggested that the novel PfTrx-like-mero protein expressed on the surface of merozoites is an important ligand participating in erythrocyte invasion and a potential vaccine candidate.

Levels of human proteins in plasma associated with acute paediatric malaria.

BACKGROUND: The intimate interaction between the pathophysiology of the human host and the biology of the Plasmodium falciparum parasite results in a wide spectrum of disease outcomes in malaria. Development of severe disease is associated with a progressively augmented imbalance in pro- and anti-inflammatory responses to high parasite loads and sequestration of parasitized erythrocytes. Although these phenomena collectively constitute common denominators for the wide variety of discrete severe malaria manifestations, the mechanistic rationales behind discrepancies in outcome are poorly understood. Exploration of the human pathophysiological response by variations in protein profiles in plasma presents an excellent opportunity to increase the understanding. This is ultimately required for better prediction, prevention and treatment of malaria, which is essential for ongoing elimination and eradication efforts. RESULTS: An affinity proteomics approach was used to analyse 541 paediatric plasma samples collected from community controls and patients with mild or severe malaria in Rwanda. Protein profiles were generated with an antibody-based suspension bead array containing 255 antibodies targetting 115 human proteins. Here, 57 proteins were identified with significantly altered levels (adjusted p-values < 0.001) in patients with malaria compared to controls. From these, the 27 most significant proteins (adjusted p-values < 10-14) were selected for a stringent analysis approach. Here, 24 proteins showed elevated levels in malaria patients and included proteins involved in acute inflammatory response as well as cell adhesion. The remaining three proteins, also implicated in immune regulation and cellular adhesivity, displayed lower abundance in malaria patients. In addition, 37 proteins (adjusted p-values < 0.05) were identified with increased levels in patients with severe compared to mild malaria. This set includes, proteins involved in tissue remodelling and erythrocyte membrane proteins. Collectively, this approach has been successfully used to identify proteins both with known and unknown association with different stages of malaria. CONCLUSION: In this study, a high-throughput affinity proteomics approach was used to find protein profiles in plasma linked to P. falciparum infection and malaria disease progression. The proteins presented herein are mainly involved in inflammatory response, cellular adhesion and as constituents of erythrocyte membrane. These findings have a great potential to provide increased conceptual understanding of host-parasite interaction and malaria pathogenesis.

Characterization of the Catalytic Subunits of the RNA Exosome-like Complex in Plasmodium falciparum.

The eukaryotic ribonucleic acid (RNA) exosome is a versatile multiribonuclease complex that mediates the processing, surveillance, and degradation of virtually all classes of RNA in both the nucleus and cytoplasm. The complex, composed of 10 to 11 subunits, has been widely described in many organisms. Bioinformatic analyses revealed that there may be also an exosome-like complex in Plasmodium falciparum, a parasite of great importance in public health, with eight predicted subunits having high sequence similarity to their counterparts in yeast and human. In this work, the putative RNA catalytic components, designated as PfRrp4, PfRrp41, PfDis3, and PfRrp6, were identified and systematically analyzed. Quantitative polymerase chain reaction (QPCR) analyses suggested that all of them were transcribed steadily throughout the asexual stage. The expression of these proteins was determined by Western blot, and their localization narrowed to the cytoplasm of the parasite by indirect immunofluorescence. The recombinant proteins of PfRrp41, PfDis3, and PfRrp6 exhibited catalytic activity for single-stranded RNA (ssRNA), whereas PfRrp4 showed no processing activity of both ssRNA and dsRNA. The identification of these putative components of the RNA exosome complex opens up new perspectives for a deep understanding of RNA metabolism in the malarial parasite P. falciparum.

Development of Plasmodium falciparum specific naïve, atypical, memory and plasma B cells during infancy and in adults in an endemic area.

BACKGROUND: B-cells are essential in immunity against malaria, but which sub-sets of B-cells specifically recognize Plasmodium falciparum and when they appear is still largely unknown. RESULTS: Using the flow cytometry technique for detection of P. falciparum specific (Pf+) B-cells, this study for the first time measured the development of Pf+ B cell (CD19+) phenotypes in Ugandan babies from birth up to nine months, and in their mothers. The babies showed increases in Pf+ IgG memory B-cells (MBCs), atypical MBCs, and plasma cells/blasts over time, but the proportion of these cells were still lower than in the mothers who displayed stable levels (5, 18, and 3%, respectively). Pf+ non-IgG+ MBCs and naïve B-cells binding to P. falciparum antigens were higher in the babies compared to the mothers (12 and 50%). In ELISA there was an increase in IgG and IgM antibodies over time in babies, and stable levels in mothers. At baby delivery, multigravidae mothers had a higher proportion of Pf+ IgG MBCs and less Pf+ naïve B-cells than primigravidae mothers. CONCLUSIONS: In newborns, naïve B-cells are a major player in recognizing P. falciparum. In adults, the high proportion of Pf+ atypical MBCs suggests a major role for these cells. Both in infants and adults, non-IgG+ MBCs were higher than IgG MBCs, indicating that these cells deserve more focus in future.

Affinity proteomics reveals elevated muscle proteins in plasma of children with cerebral malaria.

Systemic inflammation and sequestration of parasitized erythrocytes are central processes in the pathophysiology of severe Plasmodium falciparum childhood malaria. However, it is still not understood why some children are more at risks to develop malaria complications than others. To identify human proteins in plasma related to childhood malaria syndromes, multiplex antibody suspension bead arrays were employed. Out of the 1,015 proteins analyzed in plasma from more than 700 children, 41 differed between malaria infected children and community controls, whereas 13 discriminated uncomplicated malaria from severe malaria syndromes. Markers of oxidative stress were found related to severe malaria anemia while markers of endothelial activation, platelet adhesion and muscular damage were identified in relation to children with cerebral malaria. These findings suggest the presence of generalized vascular inflammation, vascular wall modulations, activation of endothelium and unbalanced glucose metabolism in severe malaria. The increased levels of specific muscle proteins in plasma implicate potential muscle damage and microvasculature lesions during the course of cerebral malaria.

Phagocytosis-inducing antibodies to Plasmodium falciparum upon immunization with a recombinant PfEMP1 NTS-DBL1α domain.

BACKGROUND: Individuals living in endemic areas gradually acquire natural immunity to clinical malaria, largely dependent on antibodies against parasite antigens. There are many studies indicating that the variant antigen PfEMP1 at the surface of the parasitized red blood cell (pRBC) is one of the major targets of the immune response. It is believed that antibodies against PfEMP1 confer protection by blocking sequestration (rosetting and cytoadherence), inducing antibody-dependent cellular-inhibitory effect and opsonizing pRBCs for phagocytosis. METHODS: A recombinant NTS-DBL1α domain from a rosette-mediating PfEMP1 was expressed in Escherichia coli. The resulting protein was purified and used for immunization to generate polyclonal (goat) and monoclonal (mouse) antibodies. The antibodies' ability to opsonize and induce phagocytosis in vitro was tested and contrasted with the presence of opsonizing antibodies naturally acquired during Plasmodium falciparum infection. RESULTS: All antibodies recognized the recombinant antigen and the surface of live pRBCs, however, their capacity to opsonize the pRBCs for phagocytosis varied. The monoclonal antibodies isotyped as IgG2b did not induce phagocytosis, while those isotyped as IgG2a were in general very effective, inducing phagocytosis with similar levels as those naturally acquired during P. falciparum infection. These monoclonal antibodies displayed different patterns, some of them showing a concentration-dependent activity while others showed a prozone-like effect. The goat polyclonal antibodies were not able to induce phagocytosis. CONCLUSION: Immunization with an NTS-DBL1-α domain of PfEMP1 generates antibodies that not only have a biological role in rosette disruption but also effectively induce opsonization for phagocytosis of pRBCs with similar activity to naturally acquired antibodies from immune individuals living in a malaria endemic area. Some of the antibodies with high opsonizing activity were not able to disrupt rosettes, indicating that epitopes of the NTS-DBL1-α other than those involved in rosetting are exposed on the pRBC surface and are able to induce functional antibodies. The ability to induce phagocytosis largely depended on the antibody isotype and on the ability to recognize the surface of the pRBC regardless of the rosette-disrupting capacity.

ARAM: an automated image analysis software to determine rosetting parameters and parasitaemia in Plasmodium samples.

BACKGROUND: Rosetting is associated with severe malaria and a primary cause of death in Plasmodium falciparum infections. Detailed understanding of this adhesive phenomenon may enable the development of new therapies interfering with rosette formation. For this, it is crucial to determine parameters such as rosetting and parasitaemia of laboratory strains or patient isolates, a bottleneck in malaria research due to the time consuming and error prone manual analysis of specimens. Here, the automated, free, stand-alone analysis software automated rosetting analyzer for micrographs (ARAM) to determine rosetting rate, rosette size distribution as well as parasitaemia with a convenient graphical user interface is presented. METHODS: Automated rosetting analyzer for micrographs is an executable with two operation modes for automated identification of objects on images. The default mode detects red blood cells and fluorescently labelled parasitized red blood cells by combining an intensity-gradient with a threshold filter. The second mode determines object location and size distribution from a single contrast method. The obtained results are compared with standardized manual analysis. Automated rosetting analyzer for micrographs calculates statistical confidence probabilities for rosetting rate and parasitaemia. RESULTS: Automated rosetting analyzer for micrographs analyses 25 cell objects per second reliably delivering identical results compared to manual analysis. For the first time rosette size distribution is determined in a precise and quantitative manner employing ARAM in combination with established inhibition tests. Additionally ARAM measures the essential observables parasitaemia, rosetting rate and size as well as location of all detected objects and provides confidence intervals for the determined observables. No other existing software solution offers this range of function. The second, non-malaria specific, analysis mode of ARAM offers the functionality to detect arbitrary objects. CONCLUSIONS: Automated rosetting analyzer for micrographs has the capability to push malaria research to a more quantitative and statistically significant level with increased reliability due to operator independence. As an installation file for Windows © 7, 8.1 and 10 is available for free, ARAM offers a novel open and easy-to-use platform for the malaria community to elucidate resetting.

Differences in affinity of monoclonal and naturally acquired polyclonal antibodies against Plasmodium falciparum merozoite antigens.

BACKGROUND: Malaria is a major global cause of deaths and a vaccine is urgently needed. RESULTS: We have employed the P. falciparum merozoite antigens MSP2-3D7/FC27 and AMA1, used them in ELISA, and coupled them in different ways using surface plasmon resonance (SPR) and estimated affinity (measured as kd) of monoclonal as well as naturally-acquired polyclonal antibodies in human plasma. There were major differences in kd depending on how the antigens were immobilized and where the His-tag was placed. For AMA1 we could see correlations with invasion inhibition. Using different immobilizations of proteins in SPR, we could see only moderate correlations with levels of antibodies in ELISA, indicating that in ELISA the proteins were not uniformly bound and that antibodies with many specificities exist in natural immunisation. The correlations between ELISA and SPR were enhanced when only parasite positive samples were included, which may indicate that high affinity antibodies are difficult to maintain over long periods of time. We found higher kd values for MSP2 (indicating lower affinity) compared to AMA1, which might be partly explained by MSP2 being an intrinsically disordered protein, while AMA1 is globular. CONCLUSIONS: For future vaccine studies and for understanding immunity, it is important to consider how to present proteins to the immune system to achieve highest antibody affinities.

Novel flow cytometry technique for detection of Plasmodium falciparum specific B-cells in humans: increased levels of specific B-cells in ongoing infection.

BACKGROUND: Malaria caused by Plasmodium falciparum is still a major health threat in endemic areas especially for children below 5 years of age. While it is recognized that antibody immunity plays an important role in controlling the disease, knowledge of the mechanisms of sustenance and natural boosting of immunity is very limited. Before, it has not been possible to investigate malaria specific B-cells directly in flow cytometry, making it difficult to know how much of a B cell response is due to malaria, or how much is due to other immunological stimulators. METHODS: This study developed a technique using quantum dots and schizont extract made from ghosts of infected erythrocytes, to be able to investigate P. falciparum specific B-cells, something that has never been done before. RESULTS: Major differences in P. falciparum specific B-cells were found between samples from immune (22.3 %) and non-immune (1.7 %) individuals. Samples from parasite positive individuals had the highest proportions of specific B-cells (27.9 %). CONCLUSION: The study showed increased levels of P. falciparum-specific B-cells in immune individuals, with the highest levels in active malaria infections, using a new technique that opens up new possibilities to study how these cells are sustained in vivo after natural infections. It will also be useful in vaccine studies.

A comparative study on the heparin-binding proteomes of Toxoplasma gondii and Plasmodium falciparum.

Toxoplasma gondii is an obligatory intracellular apicomplexan parasite which exploits host cell surface components in cell invasion and intracellular parasitization. Sulfated glycans such as heparin and heparan sulfate have been reported to inhibit cell invasion by T. gondii and other apicomplexan parasites such as Plasmodium falciparum. The aim of this study was to investigate the heparin-binding proteome of T. gondii. The parasite-derived components were affinity-purified on the heparin moiety followed by MS fingerprinting of the proteins. The heparin-binding proteins of T. gondii and P. falciparum were compared based on functionality and affinity to heparin. Among the proteins identified, the invasion-related parasite ligands derived from tachyzoite/merozoite surface and the secretory organelles were prominent. However, the profiles of the proteins were different in terms of affinity to heparin. In T. gondii, the proteins with highest affinity to heparin were the intracellular components with functions of parasite development contrasted to that of P. falciparum, of which the rhoptry-derived proteins were prominently identified. The profiling of the heparin-binding proteins of the two apicomplexan parasites not only explained the mechanism of heparin-mediated host cell invasion inhibition, but also, to a certain extent, revealed that the action of heparin on the parasite extended after endocytosis.

Label-free microfluidic enrichment of ring-stage Plasmodium falciparum-infected red blood cells using non-inertial hydrodynamic lift.

BACKGROUND: Understanding of malaria pathogenesis caused by Plasmodium falciparum has been greatly deepened since the introduction of in vitro culture system, but the lack of a method to enrich ring-stage parasites remains a technical challenge. Here, a novel way to enrich red blood cells containing parasites in the early ring stage is described and demonstrated. METHODS: A simple, straight polydimethylsiloxane microchannel connected to two syringe pumps for sample injection and two height reservoirs for sample collection is used to enrich red blood cells containing parasites in the early ring stage (8-10 h p.i.). The separation is based on the non-inertial hydrodynamic lift effect, a repulsive cell-wall interaction that enables continuous and label-free separation with deformability as intrinsic marker. RESULTS: The possibility to enrich red blood cells containing P. falciparum parasites at ring stage with a throughput of ~12,000 cells per hour and an average enrichment factor of 4.3 ± 0.5 is demonstrated. CONCLUSION: The method allows for the enrichment of red blood cells early after the invasion by P. falciparum parasites continuously and without any need to label the cells. The approach promises new possibilities to increase the sensitivity of downstream analyses like genomic- or diagnostic tests. The device can be produced as a cheap, disposable chip with mass production technologies and works without expensive peripheral equipment. This makes the approach interesting for the development of new devices for field use in resource poor settings and environments, e.g. with the aim to increase the sensitivity of microscope malaria diagnosis.

Subclass responses and their half-lives for antibodies against EBA175 and PfRh2 in naturally acquired immunity against Plasmodium falciparum malaria.

BACKGROUND: Plasmodium falciparum EBA175 and PfRh2 belong to two main families involved in parasite invasion, and both are potential vaccine candidates. Current knowledge is limited regarding which target antigens and subclasses of antibodies are actually important for protection, and how naturally acquired immunity is achieved. METHODS: Repeated blood samples were collected from individuals in Nigeria over a period of almost one year. ELISA was used to analyse subclasses of IgG responses. RESULTS: For both EBA175 (region III-V) and (a fragment of) PfRh2, the dominant antibody responses consisted of IgG1 and IgG3 followed by IgG2, while for PfRh2 there was also a relatively prominent response for IgG4. High levels of IgG1, IgG2 and IgG3 for EBA175 and total IgG for PfRh2 correlated significantly with a lower parasitaemia during the study period. Children with HbAS had higher levels of some subclasses compared to children with HbAA, while in adults the pattern was the opposite. The half-lives of IgG2 and IgG4 against EBA175 were clearly shorter than those for IgG1 and IgG3. CONCLUSION: EBA175 and PfRh2 are potential targets for protective antibodies since both correlated with lower parasitaemia. The shorter half-lives for IgG2 and IgG4 might explain why these subclasses are often considered less important in protection against malaria. Triggering the right subclass responses could be of critical importance in a successful vaccine. Further studies are needed to evaluate the role of haemoglobin polymorphisms and their malaria protective effects in this process.