Autoantibodies inhibit Plasmodium falciparum growth and are associated with protection from clinical malaria.

Many infections, including malaria, are associated with an increase in autoantibodies (AAbs). Prior studies have reported an association between genetic markers of susceptibility to autoimmune disease and resistance to malaria, but the underlying mechanisms are unclear. Here, we performed a longitudinal study of children and adults (n = 602) in Mali and found that high levels of plasma AAbs before the malaria season independently predicted a reduced risk of clinical malaria in children during the ensuing malaria season. Baseline AAb seroprevalence increased with age and asymptomatic Plasmodium falciparum infection. We found that AAbs purified from the plasma of protected individuals inhibit the growth of blood-stage parasites and bind P. falciparum proteins that mediate parasite invasion. Protected individuals had higher plasma immunoglobulin G (IgG) reactivity against 33 of the 123 antigens assessed in an autoantigen microarray. This study provides evidence in support of the hypothesis that a propensity toward autoimmunity offers a survival advantage against malaria.

Naturally Acquired Transmission-Blocking Immunity Against Different Strains of Plasmodium vivax in a Malaria-Endemic Area in Thailand.

BACKGROUND: Human immunity triggered by natural malaria infections impedes parasite transmission from humans to mosquitoes, leading to interest in transmission-blocking vaccines. However, immunity characteristics, especially strain specificity, remain largely unexplored. We investigated naturally acquired transmission-blocking immunity (TBI) against Plasmodium vivax, a major malaria parasite. METHODS: Using the direct membrane-feeding assay, we assessed TBI in plasma samples and examined the role of antibodies by removing immunoglobulins through protein G/L adsorption before mosquito feeding. Strain specificity was evaluated by conducting a direct membrane-feeding assay with plasma exchange. RESULTS: Blood samples from 47 patients with P vivax were evaluated, with 37 plasma samples successfully infecting mosquitoes. Among these, 26 showed inhibition before immunoglobulin depletion. Despite substantial immunoglobulin removal, 4 samples still exhibited notable inhibition, while 22 had reduced blocking activity. Testing against heterologous strains revealed some plasma samples with broad TBI and others with strain-specific TBI. CONCLUSIONS: Our findings indicate that naturally acquired TBI is mainly mediated by antibodies, with possible contributions from other serum factors. The transmission-blocking activity of plasma samples varied by the tested parasite strain, suggesting single polymorphic or multiple targets for naturally acquired TBI. These observations improve understanding of immunity against P vivax and hold implications for transmission-blocking vaccine development.

A systems serology approach to identifying key antibody correlates of protection from cerebral malaria in Malawian children.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins are expressed on the surface of infected erythrocytes, mediating parasite sequestration in the vasculature. PfEMP1 is a major target of protective antibodies, but the features of the antibody response are poorly defined. METHODS: In Malawian children with cerebral or uncomplicated malaria, we characterized the antibody response to 39 recombinant PfEMP1 Duffy binding like (DBL) domains or cysteine-rich interdomain regions (CIDRs) in detail, including measures of antibody classes, subclasses, and engagement with Fcγ receptors and complement. Using elastic net regularized logistic regression, we identified a combination of seven antibody targets and Fc features that best distinguished between children with cerebral and uncomplicated malaria. To confirm the role of the selected targets and Fc features, we measured antibody-dependent neutrophil and THP-1 cell phagocytosis of intercellular adhesion molecule-1 (ICAM-1) and endothelial protein C (EPCR) co-binding infected erythrocytes. RESULTS: The selected features distinguished between children with cerebral and uncomplicated malaria with 87% accuracy (median, 80-96% interquartile range) and included antibody to well-characterized DBLß3 domains and a less well-characterized CIDRγ12 domain. The abilities of antibodies to engage C1q and FcγRIIIb, rather than levels of IgG, correlated with protection. In line with a role of FcγRIIIb binding antibodies to DBLß3 domains, antibody-dependent neutrophil phagocytosis of ICAM-1 and EPCR co-binding IE was higher in uncomplicated malaria (15% median, 8-38% interquartile range) compared to cerebral malaria (7%, 30-15%, p < 0.001). CONCLUSIONS: Antibodies associated with protection from cerebral malaria target a subset of PfEMP1 domains. The Fc features of protective antibody response include engagement of FcγRIIIb and C1q, and ability to induce antibody-dependent neutrophil phagocytosis of infected erythrocytes. Identifying the targets and Fc features of protective immunity could facilitate the development of PfEMP1-based therapeutics for cerebral malaria.

Corrigendum: Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors.

This corrects the article DOI: 10.1038/nature24994.

Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors.

Malaria is among the most serious infectious diseases affecting humans, accounting for approximately half a million deaths each year. Plasmodium falciparum causes most life-threatening cases of malaria. Acquired immunity to malaria is inefficient, even after repeated exposure to P. falciparum, but the immune regulatory mechanisms used by P. falciparum remain largely unknown. Here we show that P. falciparum uses immune inhibitory receptors to achieve immune evasion. RIFIN proteins are products of a polymorphic multigene family comprising approximately 150-200 genes per parasite genome that are expressed on the surface of infected erythrocytes. We found that a subset of RIFINs binds to either leucocyte immunoglobulin-like receptor B1 (LILRB1) or leucocyte-associated immunoglobulin-like receptor 1 (LAIR1). LILRB1-binding RIFINs inhibit activation of LILRB1-expressing B cells and natural killer (NK) cells. Furthermore, P. falciparum-infected erythrocytes isolated from patients with severe malaria were more likely to interact with LILRB1 than erythrocytes from patients with non-severe malaria, although an extended study with larger sample sizes is required to confirm this finding. Our results suggest that P. falciparum has acquired multiple RIFINs to evade the host immune system by targeting immune inhibitory receptors.

Comparison of Antibody Responses and Parasite Clearance in Artemisinin Therapeutic Efficacy Studies in the Democratic Republic of Congo and Asia.

BACKGROUND: Understanding the effect of immunity on Plasmodium falciparum clearance is essential for interpreting therapeutic efficacy studies designed to monitor emergence of artemisinin drug resistance. In low-transmission areas of Southeast Asia, where resistance has emerged, P. falciparum antibodies confound parasite clearance measures. However, variation in naturally acquired antibodies across Asian and sub-Saharan African epidemiological contexts and their impact on parasite clearance re yet to be quantified. METHODS: In an artemisinin therapeutic efficacy study, antibodies to 12 pre-erythrocytic and erythrocytic P. falciparum antigens were measured in 118 children with uncomplicated P. falciparum malaria in the Democratic Republic of Congo (DRC) and compared with responses in patients from Asian sites, described elsewhere. RESULTS: Parasite clearance half-life was shorter in DRC patients (median, 2 hours) compared with most Asian sites (median, 2-7 hours), but P. falciparum antibody levels and seroprevalences were similar. There was no evidence for an association between antibody seropositivity and parasite clearance half-life (mean difference between seronegative and seropositive, -0.14 to +0.40 hour) in DRC patients. CONCLUSIONS: In DRC, where artemisinin remains highly effective, the substantially shorter parasite clearance time compared with Asia was not explained by differences in the P. falciparum antibody responses studied.

Identifying Targets of Protective Antibodies against Severe Malaria in Papua, Indonesia, Using Locally Expressed Domains of Plasmodium falciparum Erythrocyte Membrane Protein 1.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a diverse family of multidomain proteins expressed on the surface of malaria-infected erythrocytes, is an important target of protective immunity against malaria. Our group recently studied transcription of the var genes encoding PfEMP1 in individuals from Papua, Indonesia, with severe or uncomplicated malaria. We cloned and expressed domains from 32 PfEMP1s, including 22 that were upregulated in severe malaria and 10 that were upregulated in uncomplicated malaria, using a wheat germ cell-free expression system. We used Luminex technology to measure IgG antibodies to these 32 domains and control proteins in 63 individuals (11 children). At presentation to hospital, levels of antibodies to PfEMP1 domains were either higher in uncomplicated malaria or were not significantly different between groups. Using principal component analysis, antibodies to 3 of 32 domains were highly discriminatory between groups. These included two domains upregulated in severe malaria, a DBLß13 domain and a CIDRα1.6 domain (which has been previously implicated in severe malaria pathogenesis), and a DBLδ domain that was upregulated in uncomplicated malaria. Antibody to control non-PfEMP1 antigens did not differ with disease severity. Antibodies to PfEMP1 domains differ with malaria severity. Lack of antibodies to locally expressed PfEMP1 types, including both domains previously associated with severe malaria and newly identified targets, may in part explain malaria severity in Papuan adults.

Naturally acquired antibody kinetics against Plasmodium vivax antigens in people from a low malaria transmission region in western Thailand.

BACKGROUND: Plasmodium vivax (P. vivax) is the dominant Plasmodium spp. causing the disease malaria in low-transmission regions outside of Africa. These regions often feature high proportions of asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. However, previous work has failed to address the presence and maintenance of such antibody responses to P. vivax particularly in low-transmission regions. METHODS: We followed 34 patients in western Thailand after symptomatic P. vivax infections to monitor antibody kinetics over 9 months, during which no recurrent infections occurred. We assessed total IgG, IgG subclass and IgM levels to up to 52 P. vivax proteins every 2-4 weeks using a multiplexed Luminex® assay and identified protein-specific variation in antibody longevity. Mathematical modelling was used to generate the estimated half-life of antibodies, long-, and short-lived antibody-secreting cells. RESULTS: Generally, an increase in antibody level was observed within 1-week post symptomatic infection, followed by an exponential decay of different rates. We observed mostly IgG1 dominance and IgG3 sub-dominance in this population. IgM responses followed similar kinetic patterns to IgG, with some proteins unexpectedly inducing long-lived IgM responses. We also monitored antibody responses against 27 IgG-immunogenic antigens in 30 asymptomatic individuals from a similar region. Our results demonstrate that most antigens induced robust and long-lived total IgG responses following asymptomatic infections in the absence of (detected) boosting infections. CONCLUSIONS: Our work provides new insights into the development and maintenance of naturally acquired immunity to P. vivax and will guide the potential use of serology to indicate immune status and/or identify populations at risk.

Comparison of total immunoglobulin G antibody responses to different protein fragments of Plasmodium vivax Reticulocyte binding protein 2b.

BACKGROUND: Plasmodium vivax is emerging as the dominant and prevalent species causing malaria in near-elimination settings outside of Africa. Hypnozoites, the dormant liver stage parasite of P. vivax, are undetectable to any currently available diagnostic test, yet are a major reservoir for transmission. Advances have been made to harness the naturally acquired immune response to identify recent exposure to P. vivax blood-stage parasites and, therefore, infer the presence of hypnozoites. This in-development diagnostic is currently able to detect infections within the last 9-months with 80% sensitivity and 80% specificity. Further work is required to optimize protein expression and protein constructs used for antibody detection. METHODS: The antibody response against the top performing predictor of recent infection, P. vivax reticulocyte binding protein 2b (PvRBP2b), was tested against multiple fragments of different sizes and from different expression systems. The IgG induced against the recombinant PvRBP2b fragments in P. vivax infected individuals was measured at the time of infection and in a year-long observational cohort; both conducted in Thailand. RESULTS: The antibody responses to some but not all different sized fragments of PvRBP2b protein are highly correlated with each other, significantly higher 1-week post-P. vivax infection, and show potential for use as predictors of recent P. vivax infection. CONCLUSIONS: To achieve P. vivax elimination goals, novel diagnostics are required to aid in detection of hidden parasite reservoirs. PvRBP2b was previously shown to be the top candidate for single-antigen classification of recent P. vivax exposure and here, it is concluded that several alternative recombinant PvRBP2b fragments can achieve equal sensitivity and specificity at predicting recent P. vivax exposure.

Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytes.

Dynamin 1 is a neuronal endocytic protein that participates in vesicle formation by scission of invaginated membranes. Dynamin 1 is also expressed in the kidney; however, its physiological significance to this organ remains unknown. Here, we show that dynamin 1 is crucial for microtubule organization and stabilization in glomerular podocytes. By immunofluorescence and immunoelectron microscopy, dynamin 1 was concentrated at microtubules at primary processes in rat podocytes. By immunofluorescence of differentiated mouse podocytes (MPCs), dynamin 1 was often colocalized with microtubule bundles, which radially arranged toward periphery of expanded podocyte. In dynamin 1-depleted MPCs by RNAi, α-tubulin showed a dispersed linear filament-like localization, and microtubule bundles were rarely observed. Furthermore, dynamin 1 depletion resulted in the formation of discontinuous, short acetylated α-tubulin fragments, and the decrease of microtubule-rich protrusions. Dynamins 1 and 2 double-knockout podocytes showed dispersed acetylated α-tubulin and rare protrusions. In vitro, dynamin 1 polymerized around microtubules and cross-linked them into bundles, and increased their resistance to the disassembly-inducing reagents Ca2+ and podophyllotoxin. In addition, overexpression and depletion of dynamin 1 in MPCs increased and decreased the nocodazole resistance of microtubules, respectively. These results suggest that dynamin 1 supports the microtubule bundle formation and participates in the stabilization of microtubules.

The malaria parasite Plasmodium falciparum in red blood cells selectively takes up serum proteins that affect host pathogenicity.

BACKGROUND: The malaria parasite Plasmodium falciparum is a protozoan that develops in red blood cells (RBCs) and requires various host factors. For its development in RBCs, nutrients not only from the RBC cytosol but also from the extracellular milieu must be acquired. Although the utilization of host nutrients by P. falciparum has been extensively analysed, only a few studies have reported its utilization of host serum proteins. Hence, the aim of the current study was to comprehensively identify host serum proteins taken up by P. falciparum parasites and to elucidate their role in pathogenesis. METHODS: Plasmodium falciparum was cultured with human serum in vitro. Uptake of serum proteins by parasites was comprehensively determined via shotgun liquid chromatography-mass spectrometry/mass spectrometry and western blotting. The calcium ion concentration in serum was also evaluated, and coagulation activity of the parasite lysate was assessed. RESULTS: Three proteins, vitamin K-dependent protein S, prothrombin, and vitronectin, were selectively internalized under sufficient Ca2+ levels in the culture medium. The uptake of these proteins was initiated before DNA replication, and increased during the trophozoite and schizont stages, irrespective of the assembly/disassembly of actin filaments. Coagulation assay revealed that prothrombin was activated and thereby induced blood coagulation. CONCLUSIONS: Serum proteins were taken up by parasites under culture conditions with sufficient Ca2+ levels. This uptake phenomenon was associated with their pathogenicity.

Host immunity to Plasmodium falciparum and the assessment of emerging artemisinin resistance in a multinational cohort.

Artemisinin-resistant falciparum malaria, defined by a slow-clearance phenotype and the presence of kelch13 mutants, has emerged in the Greater Mekong Subregion. Naturally acquired immunity to malaria clears parasites independent of antimalarial drugs. We hypothesized that between- and within-population variations in host immunity influence parasite clearance after artemisinin treatment and the interpretation of emerging artemisinin resistance. Antibodies specific to 12 Plasmodium falciparum sporozoite and blood-stage antigens were determined in 959 patients (from 11 sites in Southeast Asia) participating in a multinational cohort study assessing parasite clearance half-life (PCt1/2) after artesunate treatment and kelch13 mutations. Linear mixed-effects modeling of pooled individual patient data assessed the association between antibody responses and PCt1/2.P. falciparum antibodies were lowest in areas where the prevalence of kelch13 mutations and slow PCt1/2 were highest [Spearman ρ = -0.90 (95% confidence interval, -0.97, -0.65), and Spearman ρ = -0.94 (95% confidence interval, -0.98, -0.77), respectively]. P. falciparum antibodies were associated with faster PCt1/2 (mean difference in PCt1/2 according to seropositivity, -0.16 to -0.65 h, depending on antigen); antibodies have a greater effect on the clearance of kelch13 mutant compared with wild-type parasites (mean difference in PCt1/2 according to seropositivity, -0.22 to -0.61 h faster in kelch13 mutants compared with wild-type parasites). Naturally acquired immunity accelerates the clearance of artemisinin-resistant parasites in patients with falciparum malaria and may confound the current working definition of artemisinin resistance. Immunity may also play an important role in the emergence and transmission potential of artemisinin-resistant parasites.

PV1 Protein from Plasmodium falciparum Exhibits Chaperone-Like Functions and Cooperates with Hsp100s.

Plasmodium falciparum parasitophorous vacuolar protein 1 (PfPV1), a protein unique to malaria parasites, is localized in the parasitophorous vacuolar (PV) and is essential for parasite growth. Previous studies suggested that PfPV1 cooperates with the Plasmodium translocon of exported proteins (PTEX) complex to export various proteins from the PV. However, the structure and function of PfPV1 have not been determined in detail. In this study, we undertook the expression, purification, and characterization of PfPV1. The tetramer appears to be the structural unit of PfPV1. The activity of PfPV1 appears to be similar to that of molecular chaperones, and it may interact with various proteins. PfPV1 could substitute CtHsp40 in the CtHsp104, CtHsp70, and CtHsp40 protein disaggregation systems. Based on these results, we propose a model in which PfPV1 captures various PV proteins and delivers them to PTEX through a specific interaction with HSP101.

A male gametocyte osmiophilic body and microgamete surface protein of the rodent malaria parasite Plasmodium yoelii (PyMiGS) plays a critical role in male osmiophilic body formation and exflagellation.

Anopheles mosquitoes transmit Plasmodium parasites of mammals, including the species that cause malaria in humans. Malaria pathology is caused by rapid multiplication of parasites in asexual intraerythrocytic cycles. Sexual stage parasites are also produced during the intraerythrocytic cycle and are ingested by the mosquito, initiating gametogenesis and subsequent sporogonic stage development. Here, we present a Plasmodium protein, termed microgamete surface protein (MiGS), which has an important role in male gametocyte osmiophilic body (MOB) formation and microgamete function. MiGS is expressed exclusively in male gametocytes and microgametes, in which MiGS localises to the MOB and microgamete surface. Targeted gene disruption of MiGS in a rodent malaria parasite Plasmodium yoelii 17XNL generated knockout parasites (ΔPyMiGS) that proliferate normally in erythrocytes and form male and female gametocytes. The number of MOB in male gametocyte cytoplasm is markedly reduced and the exflagellation of microgametes is impaired in ΔPyMiGS. In addition, anti-PyMiGS antibody severely blocked the parasite development in the Anopheles stephensi mosquito. MiGS might thus be a potential novel transmission-blocking vaccine target candidate.

The malaria parasite RhopH protein complex interacts with erythrocyte calmyrin identified from a comprehensive erythrocyte protein library.

Malaria merozoite apical organelles; microneme and rhoptry secreted proteins play functional roles during and following invasion of host erythrocytes. Among numerous proteins, the rhoptries discharge high molecular weight proteins known as RhopH complex. Recent reports suggest that the RhopH complex is essential for growth and survival of the malaria parasite within erythrocytes. However, an in-depth understanding of the host-parasite molecular interactions is indispensable. Here we utilized a comprehensive mouse erythrocyte protein library consisting of 443 proteins produced by a wheat germ cell-free system, combined with AlphaScreen technology to identify mouse erythrocyte calmyrin as an interacting molecule of the rodent malaria parasite Plasmodium yoelii RhopH complex (PyRhopH). The PyRhopH interaction was dependent on the calmyrin N-terminus and divalent cation capacity. The finding unveils a recommendable and invaluable usefulness of our comprehensive mouse erythrocyte protein library together with the AlphaScreen technology in investigating a wide-range of host-parasite molecular interactions.

Correction: An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors.

[This corrects the article DOI: 10.1371/journal.ppat.1005306.].

An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors.

Nucleotide variation patterns across species are shaped by the processes of natural selection, including exposure to environmental pathogens. We examined patterns of genetic variation in two sister species, Anopheles gambiae and Anopheles coluzzii, both efficient natural vectors of human malaria in West Africa. We used the differentiation signature displayed by a known coordinate selective sweep of immune genes APL1 and TEP1 in A. coluzzii to design a population genetic screen trained on the sweep, classified a panel of 26 potential immune genes for concordance with the signature, and functionally tested their immune phenotypes. The screen results were strongly predictive for genes with protective immune phenotypes: genes meeting the screen criteria were significantly more likely to display a functional phenotype against malaria infection than genes not meeting the criteria (p = 0.0005). Thus, an evolution-based screen can efficiently prioritize candidate genes for labor-intensive downstream functional testing, and safely allow the elimination of genes not meeting the screen criteria. The suite of immune genes with characteristics similar to the APL1-TEP1 selective sweep appears to be more widespread in the A. coluzzii genome than previously recognized. The immune gene differentiation may be a consequence of adaptation of A. coluzzii to new pathogens encountered in its niche expansion during the separation from A. gambiae, although the role, if any of natural selection by Plasmodium is unknown. Application of the screen allowed identification of new functional immune factors, and assignment of new functions to known factors. We describe biochemical binding interactions between immune proteins that underlie functional activity for malaria infection, which highlights the interplay between pathogen specificity and the structure of immune complexes. We also find that most malaria-protective immune factors display phenotypes for either human or rodent malaria, with broad specificity a rarity.

Identification of a novel merozoite surface antigen of Plasmodium vivax, PvMSA180.

BACKGROUND: Although a number of Plasmodium vivax proteins have been identified, few have been investigated as potential vaccine candidates. This study characterized the Plasmodium vivax merozoite surface antigen 180 (PvMSA180, PVX_094920), a novel P. vivax antigenic protein. METHODS: The target gene was amplified as four overlapping domains (D1, D2, D3 and D4) to enable expression of the recombinant protein using cell-free and bacterial expression systems. The recombinant PvMSA180 proteins were used in protein microarrays to evaluate the humoral immune response of 72 vivax-infected patients and 24 vivax-naïve individuals. Antibodies produced in mice against the PvMSA180-D1 and -D4 domains were used to assess the subcellular localization of schizont-stage parasites with immunofluorescence assays. A total of 51 pvmsa180 sequences from 12 countries (41 sequences from PlasmoDB and 6 generated in this study) were used to determine the genetic diversity and genealogical relationships with DNAsp and NETWORK software packages, respectively. RESULTS: PvMSA180 consists of 1603 amino acids with a predicted molecular mass of 182 kDa, and has a signal peptide at the amino-terminus. A total of 70.8% of patients (51/72) showed a specific antibody response to at least one of the PvMSA180 domains, and 20.8% (15/72) exhibited a robust antibody response to at least three of the domains. These findings suggest that PvMSA180 is targeted by the humoral immune response during natural infection with P. vivax. Immunofluorescence analysis demonstrated that PvMSA180 is localized on the merozoite surface of schizont-stage parasites, and pvmsa180 sequences originating from various geographic regions worldwide showed low genetic diversity. Twenty-two haplotypes were found, and haplotype 6 (Hap_6, 77%) of pvmsa180 was detected in isolates from six countries. CONCLUSIONS: A novel P. vivax surface protein, PvMSA180, was characterized in this study. Most of P. vivax-infected patients had specific antibodies against particular antigenic domains, indicating that this protein is immunogenic in naturally exposed populations. Genetic analysis of worldwide isolates showed that pvmsa180 is less polymorphic than other well-known candidates and that some haplotypes are common to several countries. However, additional studies with a larger sample size are necessary to evaluate the antibody responses in geographically separated populations, and to identify the function of PvMSA180 during parasite invasion.

Absence of in vivo selection for K13 mutations after artemether-lumefantrine treatment in Uganda.

BACKGROUND: Individual drug treatment may select resistant parasites in the human body, a process termed in vivo selection. Some single nucleotide polymorphisms in Plasmodium falciparum chloroquine-resistance transporter (pfcrt) and multidrug resistance gene 1 (pfmdr1) genes have been reportedly selected after artemether-lumefantrine treatment. However, there is a paucity of data regarding in vivo selection of P. falciparum Kelch propeller domain (pfkelch13) polymorphisms, responsible for artemisinin-resistance in Asia, and six putative background mutations for artemisinin resistance; D193Y in ferredoxin, T484I in multiple resistance protein 2, V127M in apicoplast ribosomal protein S10, I356T in pfcrt, V1157L in protein phosphatase and C1484F in phosphoinositide-binding protein. METHODS: Artemether-lumefantrine efficacy study with a follow-up period of 28 days was conducted in northern Uganda in 2014. The above-mentioned genotypes were comparatively analysed before drug administration and on days; 3, 7, and 28 days after treatment. RESULTS: In 61 individuals with successful follow-up, artemether-lumefantrine treatment regimen was very effective with PCR adjusted efficacy of 95.2%. Among 146 isolates obtained before treatment, wild-type alleles were observed in 98.6% of isolates in pfkelch13 and in all isolates in the six putative background genes except I356T in pfcrt, which had 2.4% of isolates as mixed infections. In vivo selection study revealed that all isolates detected in the follow-up period harboured wild type alleles in pfkelch13 and the six background genes. CONCLUSION: Mutations in pfkelch13 and the six background genes may not play an important role in the in vivo selection after artemether-lumefantrine treatment in Uganda. Different mechanisms might rather be associated with the existence of parasites after treatment.

Differences in PfEMP1s recognized by antibodies from patients with uncomplicated or severe malaria.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants are encoded by var genes and mediate pathogenic cytoadhesion and antigenic variation in malaria. PfEMP1s can be broadly divided into three principal groups (A, B and C) and they contain conserved arrangements of functional domains called domain cassettes. Despite their tremendous diversity there is compelling evidence that a restricted subset of PfEMP1s is expressed in severe disease. In this study antibodies from patients with severe and uncomplicated malaria were compared for differences in reactivity with a range of PfEMP1s to determine whether antibodies to particular PfEMP1 domains were associated with severe or uncomplicated malaria. METHODS: Parts of expressed var genes in a severe malaria patient were identified by RNAseq and several of these partial PfEMP1 domains were expressed together with others from laboratory isolates. Antibodies from Papuan patients to these parts of multiple PfEMP1 proteins were measured. RESULTS: Patients with uncomplicated malaria were more likely to have antibodies that recognized PfEMP1 of Group C type and recognized a broader repertoire of group A and B PfEMP1s than patients with severe malaria. CONCLUSION: These data suggest that exposure to a broad range of group A and B PfEMP1s is associated with protection from severe disease in Papua, Indonesia.