Haemoglobin C and S in natural selection against Plasmodium falciparum malaria: a plethora or a single shared adaptive mechanism?

Conclusive evidence exists on the protective role against clinical Plasmodium falciparum malaria of Haemoglobin S (beta 6Glu-->Val) and HbC (HbC; beta 6Glu-->Lys), both occurring in sub-Saharan Africa. However, the mechanism/s of the protection exerted remain/s debated for both haemoglobin variants, HbC and HbS. Recently, an abnormal display of PfEMP1, an antigen involved in malaria pathogenesis, was reported on HbAC and HbCC infected erythrocytes that showed reduced cytoadhesion and impaired rosetting in vitro. On this basis it has been proposed that HbC protection might be attributed to the reduced PfEMP1-mediated adherence of parasitized erythrocytes in the microvasculature. Furthermore, impaired cytoadherence was observed in HbS carriers suggesting for the first time a convergence in the protection mechanism of these two haemoglobin variants. We investigated the impact of this hypothesis on the development of acquired immunity against P. falciparum variant surface antigens (VSA) encoding PfEMP1 in HbC and HbS carriers in comparison with HbA of Burkina Faso. Higher immune response against a VSA panel and several malaria antigens were observed in all adaptive genotypes containing at least one allelic variant HbC or HbS in the low transmission urban area whereas no differences were detected in the high transmission rural area. In both contexts the response against tetanus toxoid was not influenced by the beta-globin genotype. Thus, these findings suggest that both HbC and HbS affect the early development of naturally acquired immunity against malaria. We reviewed the hypothesized mechanisms so far proposed in light of these recent results.     

Human pregnancy-associated malaria-specific B cells target polymorphic, conformational epitopes in VAR2CSA

Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes (IEs) that bind to chondroitin sulphate A (CSA) in the placenta by PAM-associated clonally variant surface antigens (VSA). Pregnancy-specific VSA (VSA(PAM)), which include the PfEMP1 variant VAR2CSA, are targets of IgG-mediated protective immunity to PAM. Here, we report an investigation of the specificity of naturally acquired immunity to PAM, using eight human monoclonal IgG1 antibodies that react exclusively with intact CSA-adhering IEs expressing VSA(PAM). Four reacted in Western blotting with high-molecular-weight (> 200 kDa) proteins, while seven reacted with either the DBL3-X or the DBL5-epsilon domains of VAR2CSA expressed either as Baculovirus constructs or on the surface of transfected Jurkat cells. We used a panel of recombinant antigens representing DBL3-X domains from P. falciparum field isolates to evaluate B-cell epitope diversity among parasite isolates, and identified the binding site of one monoclonal antibody using a chimeric DBL3-X construct. Our findings show that there is a high-frequency memory response to VSA(PAM), indicating that VAR2CSA is a primary target of naturally acquired PAM-specific protective immunity, and demonstrate the value of human monoclonal antibodies and conformationally intact recombinant antigens in VSA characterization.     

Antibodies against PfEMP1, RIFIN, MSP3 and GLURP are acquired during controlled Plasmodium falciparum malaria infections in naïve volunteers

Antibodies to polymorphic antigens expressed during the parasites erythrocytic stages are important mediators of protective immunity against P. falciparum malaria. Therefore, polymorphic blood stage antigens like MSP3, EBA-175 and GLURP and variant surface antigens PfEMP1 and RIFIN are considered vaccine candidates. However, to what extent these antibodies to blood stage antigens are acquired during naive individuals' first infections has not been studied in depth. Using plasma samples collected from controlled experimental P. falciparum infections we show that antibodies against variant surface antigens, PfEMP1 and RIFIN as well as MSP3 and GLURP, are acquired during a single short low density P. falciparum infection in non-immune individuals including strain transcendent PfEMP1 immune responses. These data indicate that the immunogenicity of the variant surface antigens is similar to the less diverse merozoite antigens. The acquisition of a broad and strain transcendent repertoire of PfEMP1 antibodies may reflect a parasite strategy of expressing most or all PfEMP1 variants at liver release optimizing the likelihood of survival and establishment of chronic infections in the new host.     

Abnormal PfEMP1/knob display on Plasmodium falciparum-infected erythrocytes containing hemoglobin variants: fresh insights into malaria pathogenesis and protection

Hemoglobin (Hb) variants are associated with reduced risk of life-threatening Plasmodium falciparum malaria syndromes, including cerebral malaria and severe malarial anemia. Despite decades of research, the mechanisms by which common Hb variants - sickle HbS, HbC, α-thalassemia, fetal HbF - protect African children against severe and fatal malaria have not been fully elucidated. In vitro experimental and epidemiological data have long suggested that Hb variants do not confer malaria protection by restricting the growth of parasites in red blood cells (RBCs). Recently, four Hb variants were found to impair cytoadherence, the binding of P. falciparum-infected RBCs (PfRBCs) to microvascular endothelial cells (MVECs), a centrally important event in both parasite survival and malaria pathogenesis in humans. Impaired cytoadherence is associated with abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite's major cytoadherence ligand and virulence factor, on the surface of host RBCs. We propose a model in which Hb variants allow parasites to display relatively low levels of PfEMP1, sufficient for sequestering PfRBCs in microvessels and avoiding their clearance from the bloodstream by the spleen. By preventing the display of high levels of PfEMP1, Hb variants may weaken the binding of PfRBCs to MVECs, compromising their ability to activate endothelium and initiate the downstream microvascular events that drive the pathogenesis of malaria.     

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.     

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.     

Production of human monoclonal antibodies against various antigens of erythrocyte stages of Plasmodium falciparum

Peripheral blood lymphocytes from 20 individuals living in a malaria endemic area (Burkina Faso) were transformed with Epstein-Barr virus. No antigen specific selection, nor stimulation of B-cells were performed prior to transformation. 20 cell lines were established, 14 secreted polyclonal antibodies directed against erythrocytic stages of Plasmodium falciparum. 4 lines were cloned and the supernatant analysed and characterised against Plasmodium falciparum antigens.     

3D7-Derived Plasmodium falciparum erythrocyte membrane protein 1 is a frequent target of naturally acquired antibodies recognizing protein domains in a particular pattern independent of malaria transmission intensity

Protection against Plasmodium falciparum malaria is largely mediated by IgG against surface Ags such as the erythrocyte membrane protein 1 family (PfEMP1) responsible for antigenic variation and sequestration of infected erythrocytes. PfEMP1 molecules can be divided into groups A, B/A, B, C, and B/C. We have previously suggested that expression of groups A and B/A PfEMP1 is associated with severe disease and that Abs to these molecules are acquired earlier in life than Abs to PfEMP1 belonging to groups B, B/C, and C PfEMP1. In this study, we compared the acquisition of IgG to 20 rPfEMP1 domains derived from 3D7 in individuals living under markedly different malaria transmission intensity and were unable to find differences in the Ab acquisition rate to PfEMP1 of different groupings (A, B, or C) or domain type (alpha, beta, gamma, delta, epsilon, or x). Abs were acquired early in life in individuals living in the high transmission village and by the age of 2-4 years most individuals had Abs against most constructs. This level of reactivity was found at the age of 10-20 years in the medium transmission village and was never reached by individuals living under low transmission. Nevertheless, the sequence by which individuals acquired Abs to particular constructs was largely the same in the three villages. This indicates that the pattern of PfEMP1 expression by parasites transmitted at the different sites was similar, suggesting that PfEMP1 expression is nonrandom and shaped by host-parasite relationship factors operating at all transmission intensities.     

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.     

The flip-side of Cytoadherence: immune selection, antigenic variation and the var Genes of Plasmodium falciparum

In areas where Plasmodium falciparum is endemic, the natural immunity acquired by people exposed to frequent malaria infection is likely to have a differential selective impact upon different parasite genotypes. It has been suggested that the immune response directed against the variant antigen PfEMP1, which is expressed on the infected erythrocyte surface, is a crucial determinant of parasite population structure and favours the existence of distinct strains, or Varotypes. Here, Colin Sutherland summarizes current knowledge of the var multigene family, which encodes the PfEMP1 variants, and suggests that this information may allow certain predictions of the strain hypothesis to be tested directly.     

Plasmodium falciparum variant surface antigen expression varies between isolates causing severe and nonsevere malaria and is modified by acquired immunity

In areas of endemic parasite transmission, protective immunity to Plasmodium falciparum malaria is acquired over several years with numerous disease episodes. Acquisition of Abs to parasite-encoded variant surface Ags (VSA) on the infected erythrocyte membrane is important in the development of immunity, as disease-causing parasites appear to be those not controlled by preexisting VSA-specific Abs. In this work we report that VSA expressed by parasites from young Ghanaian children with P. falciparum malaria were commonly and strongly recognized by plasma Abs from healthy children in the same area, whereas recognition of VSA expressed by parasites from older children was weaker and less frequent. Independent of this, parasites isolated from children with severe malaria (cerebral malaria and severe anemia) were better recognized by VSA-specific plasma Abs than parasites obtained from children with nonsevere disease. This was not due to a higher infection multiplicity in younger patients or in patients with severe disease. Our data suggest that acquisition of VSA-specific Ab responses gradually restricts the VSA repertoire that is compatible with parasite survival in the semi-immune host. This appears to limit the risk of severe disease by discriminating against the expression of VSA likely to cause life-threatening complications, such as cerebral malaria and severe anemia. Such VSA seem to be preferred by parasites infecting a nonimmune host, suggesting that VSA expression and switching are not random, and that the VSA expression pattern is modulated by immunity. This opens the possibility of developing morbidity-reducing vaccines targeting a limited subset of common and particularly virulent VSA.     

Mass spectrometric analysis of Plasmodium falciparum erythrocyte membrane protein-1 variants expressed by placental malaria parasites

Surface proteins from Plasmodium falciparum are important malaria vaccine targets. However, the surface proteins previously identified are highly variant and difficult to study. We used tandem mass spectrometry to characterize the variant antigens (Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)) expressed on the surface of malaria-infected erythrocytes that bind to chondroitin sulfate A (CSA) in the placenta. Whereas PfEMP1 variants previously implicated as CSA ligands were detected, in unselected parasites four novel variants were detected in CSA-binding or placental parasites but not in unselected parasites. These novel PfEMP1 variants require further study to confirm whether they play a role in placental malaria.     

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.     

Induction of Strain-Transcending Antibodies Against Group A PfEMP1 Surface Antigens from Virulent Malaria Parasites

Sequence diversity in pathogen antigens is an obstacle to the development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. Here we identified the PfEMP1 variants transcribed by P. falciparum strains selected for a virulence-associated adhesion phenotype (IgM-positive rosetting). The parasites transcribed a subset of Group A PfEMP1 variants characterised by an unusual PfEMP1 architecture and a distinct N-terminal domain (either DBLα1.5 or DBLα1.8 type). Antibodies raised in rabbits against the N-terminal domains showed functional activity (surface reactivity with live infected erythrocytes (IEs), rosette inhibition and induction of phagocytosis of IEs) down to low concentrations (<10 µg/ml of total IgG) against homologous parasites. Furthermore, the antibodies showed broad cross-reactivity against heterologous parasite strains with the same rosetting phenotype, including clinical isolates from four sub-Saharan African countries that showed surface reactivity with either DBLα1.5 antibodies (variant HB3var6) or DBLα1.8 antibodies (variant TM284var1). These data show that parasites with a virulence-associated adhesion phenotype share IE surface epitopes that can be targeted by strain-transcending antibodies to PfEMP1. The existence of shared surface epitopes amongst functionally similar disease-associated P. falciparum parasite isolates suggests that development of therapeutic interventions to prevent severe malaria is a realistic goal.     

Identification of residues in the Cmu4 domain of polymeric IgM essential for interaction with Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)

The binding of nonspecific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Herein we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4beta domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped Abs, IgM mutants, and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM H chain and requires the IgM Cmu4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4beta domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cmu4 domain are equivalent to those regions of IgG and IgA recognized by Fc-binding proteins from bacteria, suggesting that this region of Ig molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and that it shows functional similarities with Fc-binding proteins from pathogenic bacteria.     

Antibodies against PfEMP1, RIFIN,MSP3 and GLURP Are Acquired during Controlled Plasmodium falciparum Malaria Infections in Na?ve Volunteers

Antibodies to polymorphic antigens expressed during the parasites erythrocytic stages are important mediators of protective immunity against P. falciparum malaria. Therefore, polymorphic blood stage antigens like MSP3, EBA-175 and GLURP and variant surface antigens PfEMP1 and RIFIN are considered vaccine candidates. However, to what extent these antibodies to blood stage antigens are acquired during naive individuals'' first infections has not been studied in depth. Using plasma samples collected from controlled experimental P. falciparum infections we show that antibodies against variant surface antigens, PfEMP1 and RIFIN as well as MSP3 and GLURP, are acquired during a single short low density P. falciparum infection in non-immune individuals including strain transcendent PfEMP1 immune responses. These data indicate that the immunogenicity of the variant surface antigens is similar to the less diverse merozoite antigens. The acquisition of a broad and strain transcendent repertoire of PfEMP1 antibodies may reflect a parasite strategy of expressing most or all PfEMP1 variants at liver release optimizing the likelihood of survival and establishment of chronic infections in the new host.     

Different response to Plasmodium falciparum in west African sympatric ethnic groups: possible implications for malaria control strategies

The comparison of malaria indicators among populations with different genetic backgrounds and uniformly exposed to the same parasite strains, is one of the approaches to the study of human heterogeneities in the response to the infection. The results of our comparative studies conducted in Burkina Faso, West Africa, showed consistent interethnic differences in Plasmodium falciparum infection rates, malaria morbidity, prevalence and levels of antibodies to various P. falciparum antigens, and genetic background. The differences in the immune response were not explained by the entomological observations which indicated substantially uniform exposure to infective bites. The presence in the same epidemiological context of individuals characterized by different immune reactivity to malaria represents an ideal opportunity to study the possible relationships between the baseline level of anti-malaria immunity of a population and the protective efficacy of control measures based on the reduction of transmission. In spite of similar reduction of entomological inoculation rates obtained by permethrin-impregnated curtains, ethnic- and age-dependent efficacy was observed. These studies demonstrate the existence of marked interethnic differences in the susceptibility to P. falciparum malaria, probably involving the genetic regulation of humoral immune responses. These differences should be considered in the development of anti-malaria vaccines and in the evaluation and application of malaria control strategies.     

Recovery of adhesion to chondroitin-4-sulphate in Plasmodium falciparum varCSA disruption mutants by antigenically similar PfEMP1 variants

Protection against maternal malaria has been associated with the acquisition of a specific antibody response that prevents adhesion of Plasmodium falciparum-infected erythrocytes to the glycosaminoglycan chondroitin-4-sulphate (CSA), which is present in the placental intervillous space. These antibodies are directed against variant forms of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) that mediate binding to CSA. We have generated insertional disruption mutants of the gene encoding the CSA-binding phenotype in the P. falciparum clone FCR3 (varCSA) to test the hypothesis that strategies targeting the parasite's determinant for this adhesive phenotype may prevent sequestration of infected erythrocytes in the placenta and hence the development of maternal malaria. The varCSA-disruption mutants were initially unable to adhere to CSA; however, they could recover the phenotype after repeated selection over CSA. We show that recovery of CSA binding is varCSA independent and mediated by the activation of a novel var variant. Importantly, the corresponding PfEMP1 protein reacts with a monoclonal antibody recognizing the DBL3 gamma domain of the varCSA gene product, indicating that the DBL3 gamma CSA-binding domains are conserved between these PfEMP1-binding variants. Our data support strategies exploring these conserved epitopes as vaccine candidates against maternal malaria.     

Malarone treatment failure not associated with previously described mutations in the cytochrome b gene

The efficacy of pyrimethamine-sulfadoxine in the treatment of uncomplicated falciparum malaria in young children of a malaria holoendemic area in rural Burkina Faso is reported. Of 28 children treated with a standard single dose of pyrimethamine-sulfadoxine and followed-up over 14 days, only one Late Treatment Failure and four Late Parasitological Failures were observed, all with low-grade parasitaemia. In this area of very restricted use of pyrimethamine-sulfadoxine, the drug appears to be still sufficiently effective in the treatment of malaria. These findings provide further evidence for the justification of continued use of pyrimethamine-sulfadoxine as a second-line treatment for malaria in Burkina Faso.