Structure and activity of glycosylphosphatidylinositol anchors of Plasmodium falciparum

The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum are thought to be etiologic agents of malaria based on their ability to induce proinflammatory cytokine production by macrophages and cause symptoms that resemble severe malaria illness in animals. This review summarizes the published information on the structures of P. falciparum GPIs, structure-activity relationship, and anti-GPI antibodies in the host.     

Cerebral malaria is associated with IgG2 and IgG4 antibody responses to recombinant Plasmodium falciparum RIFIN antigen

RIFIN proteins belong to the largest Plasmodium falciparum multicopy family of variant surface antigens (VSA) expressed by infected erythrocytes. VSA antibodies have been shown to be associated with protection against malaria. Here, antibody subclass responses to a recombinant RIFIN protein (RIF-29) in 116 Ghanaian children were determined by ELISA to investigate the relationship between severe malaria and anti-RIF-29 antibodies. The study group was composed of 23 children diagnosed exclusively for cerebral malaria and 35 children who had non-cerebral severe malaria. The remaining 58 individuals were age-, gender- and area-matched asymptomatic controls. The finding that IgG1 and IgG3 responses predominated in severe malaria patients compared to matched controls suggests that these antibodies are not protective, but are most probably induced by a current infection, an observation substantiated by the equally high reactivity to both recombinant RIF-29 protein and to P. falciparum crude lysate proteins. The exclusive detection of IgG2 and IgG4 antibodies to RIF-29 protein only in cerebral malaria children brings to mind the possibility that these antibodies are pathogenic. This is a new finding that may go some way towards explaining why these children are at risk of developing the life-threatening form of cerebral malaria.     

Lack of Association of the HbE Variant with Protection from Cerebral Malaria in Thailand

Hemoglobin E (HbE; beta26Glu --> Lys) is the most common variant of the beta-globin gene in Southeast Asia; it has been suggested that it confers resistance against Plasmodium falciparum malaria. In this study 306 adult patients with P. falciparum malaria (198 mild and 108 cerebral malaria patients) living in northwest Thailand were investigated to examine whether the HbE variant is associated with protection from cerebral malaria. Our results revealed that the sample allele frequency of HbE was not significantly different between mild (7.3%) and cerebral malaria (7.4%) patients. Thus, the HbA/HbE polymorphism would not be a major genetic factor influencing the onset of cerebral malaria in Thailand.     

Distribution of IgG subclass antibodies specific for Plasmodium falciparum glutamate-rich-protein molecule in sickle cell trait children with asymptomatic infections

Polymorphism in the beta-globin gene (hemoglobin S) has been associated with protection against severe forms of malaria. In a cross-sectional study, 180 young Gabonese children with and without sickle cell trait and harboring asymptomatic Plasmodium falciparum infections, were assessed for the responses to recombinant protein containing the conserved region of glutamate-rich protein (GLURP). We reported increased age-dependence of antibody prevalence and levels of total IgG (p<0.0001), IgG1 (p=0.009), and IgG3 (p<0.03) antibodies to GLURP with a cut-off at 5 years of age. Whatever the hemoglobin type, cytophilic antibodies (IgG1 and IgG3) were prevalent, but GLURP-specific IgG4 antibodies were detected at significantly (p<0.05) lower levels in HbAS children. We showed that the distribution of non-cytophilic IgG antibodies differs according to the hemoglobin type and to the malaria antigens tested. This may have possible implication for the clearance of malaria parasites and for protection against severe malaria.     

Plasmodium falciparum glycosylphosphatidylinositol toxin interacts with the membrane of non-parasitized red blood cells: a putative mechanism contributing to malaria anemia

Following exposure to synthetic Plasmodium falciparum glycosylphosphatidylinositol (P.f.-GPI), red blood cells (RBCs) reacted with antibodies in the serum of a patient with severe acute P. falciparum malaria. Carbohydrate microarray analysis of the patient's serum confirmed the presence of both, IgM and IgG antibodies against P.f.-GPI. The antibodies failed to bind to RBCs when P.f.-GPI lacking the lipid portion was applied. Addition of the detergent Triton X-100 during preincubation with P.f.-GPI resulted in increased recognition. Recognition of P.f.-GPI was dependent on the concentrations of synthetic P.f.-GPI, the serum and the numbers of RBCs. IgM antibodies dominated P.f.-GPI-sensitized RBCs recognition. Recognition by IgM antibodies proved highest during the 1stweek of acute malaria and decreased during the following 2weeks as assessed by flow cytometry and carbohydrate microarray analysis. These results strongly support the notion that released P.f.-GPI can insert into non-parasitized RBC membranes and results in recognition by circulating anti-GPI antibodies and possibly subsequent elimination. This process may contribute to malaria-associated anemia.     

Fcgamma receptor IIA and IIIB polymorphisms are associated with susceptibility to cerebral malaria

Human FcgammaRIIA and FcgammaRIIIB exhibit genetic polymorphisms, FcgammaRIIA-131H/R and FcgammaRIIIB-NA1/NA2, coding for different capacities for IgG binding and phagocytosis. Recently, FcgammaRIIA-131R was reported to be associated with protection against high-density Plasmodium falciparum infection in Kenya. Furthermore, FcgammaRIIIB-NA1/NA2 polymorphism was shown to influence FcgammaRIIA function in an allele-specific manner. In this study, we examined a possible association of FcgammaRIIA-131H/R and FcgammaRIIIB-NA1/NA2 polymorphisms with malaria severity in 107 cerebral malaria patients, 157 non-cerebral severe malaria patients, and 202 mild malaria controls living in northwest Thailand. This study reveals that, with the FcgammaRIIIB-NA2 allele, the FcgammaRIIA-131H/H genotype is associated with susceptibility to cerebral malaria (OR 1.85, 95% CI 1.14-3.01; P=0.012), although these polymorphisms are not individually involved in the disease severity. Our results suggest that FcgammaRIIA-131H/R and FcgammaRIIIB-NA1/NA2 polymorphisms have an interactive effect on host defense against malaria infection.     

Low antibodies against Plasmodium falciparum and imbalanced pro-inflammatory cytokines are associated with severe malaria in Mozambican children: a case-control study

ABSTRACT: BACKGROUND: The factors involved in the progression from Plasmodium falciparum infection to severe malaria (SM) are still incompletely understood. Altered antibody and cellular immunity against P. falciparum might contribute to increase the risk of developing SM. METHODS: To identify immune responses associated with SM, a sex- and age-matched case-control study was carried out in 134 Mozambican children with SM (cerebral malaria, severe anaemia, acidosis and/or respiratory distress, prostration, hypoglycaemia, multiple seizures) or uncomplicated malaria (UM). IgG and IgM against P. falciparum lysate, merozoite antigens (MSP-119, AMA-1 and EBA-175), a Duffy binding like (DBL)-alpha rosetting domain and antigens on the surface of infected erythrocytes were measured by ELISA or flow cytometry. Plasma concentrations of IL-12p70, IL-2, IFN-gamma, IL-4, IL-5, IL-10, IL-8, IL-6, IL- 1beta, TNF, TNF-beta and TGF-beta1 were measured using fluorescent bead immunoassays. Data was analysed using McNemar's and Signtest. RESULTS: Compared to UM, matched children with SM had reduced levels of IgG against DBLalpha (P < 0.001), IgM against MSP-119 (P = 0.050) and AMA-1 (P = 0.047), TGF-beta1 (P <0.001) and IL-12 (P = 0.039). In addition, levels of IgG against P. falciparum lysate and IL-6 concentrations were increased (P = 0.004 and P = 0.047, respectively). Anti-DBLalpha IgG was the only antibody response associated to reduced parasite densities in a multivariate regression model (P = 0.026). CONCLUSIONS: The lower levels of antibodies found in children with SM compared to children with UM were not attributable to lower exposure to P. falciparum in the SM group. IgM against P. falciparum and specific IgG against a rosetting PfEMP1 domain may play a role in the control of SM, whereas an imbalanced pro-inflammatory cytokine response may exacerbate the severity of infection. A high overlap in symptoms together with a limited sample size of different SM clinical groups reduced the power to identify immunological correlates for particular forms of SM.     

Immunogenicity of C-terminus of Plasmodium falciparum merozoite surface protein 1 expressed as a non-glycosylated polypeptide in yeast

The C-terminal region of the merozoite surface protein 1 (MSP1_(19)) is one of the mostpromising vaccine candidates against the erythrocytic forms of malaria.In the present study,a gene encodingPlasmodium falciparum MSP1_(19) was expressed in yeast Pichia pastoris.A non-glycosylated form of therecombinant protein MSP1_(19) was purified from culture medium.This recombinant protein maintains itsantigenicity.Significant immune responses were seen in C57BL/6 mice after the second immunization.Moreover,the specific antibodies recognized the native antigens of P.falciparum,The prevailing isotypesof immunoglobulin (Ig)G associated with immunization were IgG1,IgG2a and IgG2b.The antibodiesisolated from mouse sera immunized with MSP1_(19) can inhibit parasite growth in vitro.Based on theseimmunological studies,we concluded that MSP1_(19) deserves further evaluation in pre-clinical immunizationsagainst P.falciparum.     

Evidence that invasion-inhibitory antibodies specific for the 19-kDa fragment of merozoite surface protein-1 (MSP-1 19) can play a protective role against blood-stage Plasmodium falciparum infection in individuals in a malaria endemic area of Africa

The C-terminal 19-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP-1(19)) is a target of protective Abs against blood-stage infection and a leading candidate for inclusion in a human malaria vaccine. However, the precise role, relative importance, and mechanism of action of Abs that target this protein remain unclear. To examine the potential protective role of Abs to MSP-1(19) in individuals naturally exposed to malaria, we conducted a treatment time to infection study over a 10-wk period in 76 residents of a highland area of western Kenya during a malaria epidemic. These semi-immune individuals were not all equally susceptible to reinfection with P. falciparum following drug cure. Using a new neutralization assay based on transgenic P. falciparum expressing the P. chabaudi MSP-1(19) orthologue, individuals with high-level MSP-1(19)-specific invasion-inhibitory Abs (>75th percentile) had a 66% reduction in the risk of blood-stage infection relative to others in the population (95% confidence interval, 3-88%). In contrast, high levels of MSP-1(19) IgG or IgG subclass Abs measured by enzyme immunoassay with six different recombinant MSP-1(19) Ags did not correlate with protection from infection. IgG Abs measured by serology and functional invasion-inhibitory activity did not correlate with each other. These findings implicate an important protective role for MSP-1(19)-specific invasion inhibitory Abs in immunity to blood-stage P. falciparum infection, and suggest that the measurement of MSP-1(19) specific inhibitory Abs may serve as an accurate correlate of protection in clinical trials of MSP-1-based vaccines.     

Synthetic glycosylphosphatidylinositol microarray reveals differential antibody levels and fine specificities in children with mild and severe malaria

Glycosylphosphatidylinositol (GPI) glycolipids abound on the cell surface at the merozoite stage of Plasmodium falciparum life cycle are a central toxin in malaria. The contribution of GPI specific humoral immune responses to protection against malaria pathology is not clear, since studies on the correlation between anti-GPI antibody titers and disease severity have yielded contradictory results. Here, we present the application of a carbohydrate microarray based on synthetic PfGPI glycans to assess levels and fine specificities of anti-GPI antibody responses in healthy and malaria diseased individuals. Furthermore, the age dependent development of humoral immune responses against GPI in malaria-exposed children was investigated. Anti-GPI antibodies were only rarely found in children under the age of 18 months. Sera from subjects with severe malaria and healthy children contained antibodies that recognized predominantly synthetic Man₃-GPI and Man₄-GPIs. In contrast, antibodies in sera of children with mild malaria also showed substantial reactivity with truncated glycans comprising glucosamine–inositol moieties without mannose or with only one or two mannose residues.     

The importance of human FcgammaRI in mediating protection to malaria

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcgammaRI. This important finding documents the capacity of FcgammaRI to mediate potent antimalaria immunity and supports the development of FcgammaRI-directed therapy for human malaria.     

Elevated anti-malarial IgE in asymptomatic individuals is associated with reduced risk for subsequent clinical malaria

Immunological characteristics were assessed for prospective risk of clinical malaria in a longitudinally followed population in a holoendemic area of Tanzania. Baseline characteristics including crude Plasmodium falciparum extract-specific IgE and IgG; total IgE; and parasitological indices, e.g. number of P. falciparum clones, were investigated among 700 asymptomatic individuals. Cox regression analysis estimated the risk of succumbing to a new clinical episode during a 40 weeks follow up. High anti-P. falciparum IgE levels were associated with reduced risk of acute malaria in all age groups independently of total IgE levels. Statistically significant reduced odds ratio of 0.26 (95% CI, 0.09-0.72, P=0.010) and 0.44 (95% CI, 0.19-0.99, P=0.047) for the two highest fifths, respectively was observed after adjustment for age, sex, total IgE, numbers of parasite clones per infection and HIV-1 seropositivity. In contrast, high levels of malaria specific IgG or total IgE were not associated with reduced risk to succumb to a new clinical episode. A protective effect of asymptomatic multiclonal P. falciparum infections was also confirmed. For the first time, anti-malarial IgE levels in asymptomatic individuals in endemic area are found to be associated with reduced risk for subsequent malaria disease. Specific IgE antibodies may play role in maintaining anti-malarial immunity, or indicate other aspects of immune function relevant for protection against malaria.     

The generation and evaluation of two panels of epitope-matched mouse IgG1, IgG2a, IgG2b and IgG3 antibodies specific for Plasmodium falciparum and Plasmodium yoelii merozoite surface protein 1-19 (MSP1(19))

Murine immunoglobulin G (IgG) plays an important role in mediating protective immune responses to malaria. We still know relatively little about which IgG subclasses protect against this disease in mouse models, although IgG2a and IgG2b are considered to be the most potent and dominate in successful passive transfer experiments in rodent malarias. To explore the mechanism(s) by which the different mouse IgG subclasses may mediate a protective effect, we generated mouse IgG1, IgG2a, IgG2b and IgG3 specific for the C-terminal 19-kDa region of Plasmodium falciparum merozoite surface protein 1 (PfMSP1(19)), and to the homologous antigen from Plasmodium yoelii (P. yoelii), both major targets of protective immune responses. This panel of eight IgGs bound antigen with an affinity comparable to that seen for their epitope-matched parental monoclonal antibodies (mAbs) from which they were derived, although for reasons of yield, we were only able to explore the function of mouse IgG1 recognizing PfMSP1(19) in detail, both in vitro and in vivo. Murine IgG1 was as effective as the parental human IgG from which it was derived at inducing NADPH-mediated oxidative bursts and degranulation from neutrophils. Despite showing efficacy in in vitro functional assays with neutrophils, the mouse IgG1 failed to protect against parasite challenge in vivo. The lack of protection afforded by MSP1(19)-specific IgG1 against parasite challenge in wild type mice suggests that this Ab class does not play a major role in the control of infection with mouse malaria in the Plasmodium berghei transgenic model.     

Anti-malaria humoral responses in children exposed to Plasmodium falciparum and Schistosoma haematobium

Antibody responses directed against the Plasmodium falciparum antigens, total extract, anti-merozoite surface protein-3 (MSP3b) and glutamate-rich protein (Glurp-R0) were studied in 42 children exposed to both Schistosoma haematobium and P. falciparum infections. The association between levels of the anti-malaria IgG subclasses and IgM with host age, sex, schistosome infection intensity and schistosome specific antibodies was studied before chemotherapeutic treatment of schistosome infections. This showed a significant negative association between schistosome infection intensity and levels of IgG1, IgG3, and IgG4 directed against malaria total extract antigen, and a positive association between levels of anti-schistosome soluble egg antigen IgG2, IgG3, and IgG4 and levels of the same subclasses directed against malaria total extract antigens. The effect of treating schistosome infections with praziquantel on malaria specific responses was also studied. This treatment resulted in increases in significant IgG4 levels against MSP3b and IgM against Glurp R0. Treatment also resulted in a significant decrease in IgG4 levels against Glurp R0. Host age, sex or pre-treatment infection intensity was not associated with the magnitude of change in the two IgG4 responses while males showed a significantly higher increase in levels of IgM. The results suggest cross reactivity between schistosome and malaria antigens in this population.     

Acquired immunity and postnatal clinical protection in childhood cerebral malaria

By analysing data on the age distribution of cerebral malaria among sites of different transmission intensities, we conclude that the most plausible explanation for the epidemiological patterns seen is that (i) cerebral malaria is caused by a distinct set of Plasmodium falciparum antigenic types; (ii) these antigenic types or 'CM strains' are very common and induce strong strain-specific immunity; and (iii) the postnatal period of protection against cerebral malaria is much longer than the period of protection against other forms of severe disease. The alternative hypothesis that cerebral malaria may be caused by any 'strain' of P. falciparum is compatible with the data only if a single exposure is sufficient to protect against further episodes. This is not consistent with observations on the history of exposure of patients with cerebral malaria. Finally, it is clear that although the delayed peak in incidence of cerebral malaria (with age) can be generated by assuming that subsequent exposures carry a higher risk of disease, such an explanation is not compatible with the observation that severe disease rates are low among infants and young children in areas of high transmissibility.     

Plasmodium falciparum: chondroitin sulfate A is the major receptor for adhesion of parasitized erythrocytes in the placenta

Plasmodium falciparum parasites that sequester in the placenta bind to the molecule chondroitin sulfate A (CSA). Women become resistant to malaria during pregnancy as they acquire antibodies that inhibit parasite adhesion to CSA, suggesting that a vaccine against placental malaria is feasible. Hyaluronic acid (HA) and non-immune IgG have also been proposed as receptors for P. falciparum adhesion in the placenta, but evidence for their roles is inconclusive. In this study, CSA, HA, and IgG were simultaneously assessed for their relative contributions to placental adhesion. Placental parasites collected in Tanzania uniformly adhered to the molecule CSA, and soluble CSA completely inhibited adhesion of most samples to placental cryosections. Three of 46 placental parasite samples also adhered to immobilized HA, but HA failed to inhibit adhesion of any placental parasites to placental cryosections. Similarly, non-immune IgG and protein A failed to inhibit adhesion of parasite samples to placental cryosection. P. falciparum adhesion in the placenta appears to be a non-redundant process that requires CSA as a receptor. Vaccines that elicit functional antibodies against CSA-binding parasites may confer resistance to pregnancy malaria.     

Synthetic GPI array to study antitoxic malaria response

Parasite glycosylphosphatidylinositol (GPI) is an important toxin in malaria disease, and people living in malaria-endemic regions often produce high levels of anti-GPI antibodies. The natural anti-GPI antibody response needs to be understood to aid the design of an efficient carbohydrate-based antitoxin vaccine. We present a versatile approach based on a synthetic GPI glycan array to correlate anti-GPI antibody levels and protection from severe malaria.     

Widespread occurrence of antibodies against circumsporozoite protein and against blood forms of Plasmodium vivax, P. falciparum and P. malariae in Brazilian wild monkeys

BACKGROUND: A survey of malaria antibodies was carried out over 7 years and a total of 777 serum samples from wild monkeys were collected in three distinct ecological areas of Brazil where autochthonous malaria has been reported: the 'Cerrado' (similar to savanna), the Atlantic Forest and the Atlantic Semideciduous Forest. METHODS: We carried out enzyme-linked immunosorbent assay to investigate the presence of IgG antibodies against peptides of the circumsporozoite protein (CSP) repeat region of 'classic'Plasmodium vivax, P. vivax VK247, human P. vivax-like/P. simiovale, P. brasilianum/P. malariae and P. falciparum. We also carried out immunofluorescence assay with asexual forms of P. vivax, P. malariae and P. falciparum. RESULTS: The high prevalence of antibodies against CSP in all areas indicates that the monkeys had intense contact with sporozoites from infected anophelines. The immune response against asexual forms of Plasmodium in the monkeys from the Atlantic Forest indicates the development of the infection. CONCLUSIONS: We discuss the possibility of monkeys being malaria reservoirs in non-endemic areas.     

Combined effects of Gm or Km immunoglobulin allotypes and age on antibody responses to Plasmodium falciparum VarO rosetting variant in Benin

Clinical protection of Beninese children against Plasmodium falciparum malaria was shown to be influenced by immunoglobulin (IG) Gm and Km allotypes, and related to seroreactivity with the rosette-forming VarO-antigenic variant. IgG to the VarO-infected erythrocyte surface, IgG1 and IgG3 to PfEMP1-NTS-DBL1α(1)-VarO were higher in the under 4-year-old children carrying the Gm 5,6,13,14;1,17 phenotype. In contrast, surface-reactive IgG, total IgG, IgG1 and IgG3 to NTS-DBL1α(1)- and DBL2βC2-VarO domains were lower in the above 4-year-old children harbouring the Km1 allotype. These data outline an age-related association of antibodies against malaria antigens and IG allotype distribution.     

Malaria resistance genes are associated with the levels of IgG subclasses directed against Plasmodium falciparum blood-stage antigens in Burkina Faso

ABSTRACT: BACKGROUND: HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms have been associated with malaria resistance in humans, whereas cytophilic immunoglobulin G (IgG) antibodies are thought to play a critical role in immune protection against asexual blood stages of the parasite. Furthermore, HBB, IL4, TNF, and FCGR2A have been associated with both malaria resistance and IgG levels. This suggests that some malaria resistance genes influence the levels of IgG subclass antibodies. METHODS: In this study, the effect of HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms on the levels of IgG responses against Plasmodium falciparum blood-stage extract was investigated in 220 individuals living in Burkina Faso. The Pearson's correlation coefficient among IgG subclasses was determined. A family-based approach was used to assess the association of polymorphisms with anti-P. falciparum IgG, IgG1, IgG2, IgG3 and IgG4 levels. RESULTS: After applying a multiple test correction, several polymorphisms were associated with IgG subclass or IgG levels. There was an association of i) haemoglobin C with IgG levels; ii) the FcgammaRIIa H/R131 with IgG2 and IgG3 levels; iii) TNF-863 with IgG3 levels; iv) TNF-857 with IgG levels; and, v) TNF1304 with IgG3, IgG4, and IgG levels. CONCLUSION: Taken together, the results support the hypothesis that some polymorphisms affect malaria resistance through their effect on the acquired immune response, and pave the way towards further comprehension of genetic control of an individual's humoral response against malaria.