Plasmodium falciparum variant surface antigen expression patterns during malaria

The variant surface antigens expressed on Plasmodium falciparum-infected erythrocytes are potentially important targets of immunity to malaria and are encoded, at least in part, by a family of var genes, about 60 of which are present within every parasite genome. Here we use semi-conserved regions within short var gene sequence "tags" to make direct comparisons of var gene expression in 12 clinical parasite isolates from Kenyan children. A total of 1,746 var clones were sequenced from genomic and cDNA and assigned to one of six sequence groups using specific sequence features. The results show the following. (1) The relative numbers of genomic clones falling in each of the sequence groups was similar between parasite isolates and corresponded well with the numbers of genes found in the genome of a single, fully sequenced parasite isolate. In contrast, the relative numbers of cDNA clones falling in each group varied considerably between isolates. (2) Expression of sequences belonging to a relatively conserved group was negatively associated with the repertoire of variant surface antigen antibodies carried by the infected child at the time of disease, whereas expression of sequences belonging to another group was associated with the parasite "rosetting" phenotype, a well established virulence determinant. Our results suggest that information on the state of the host-parasite relationship in vivo can be provided by measurements of the differential expression of different var groups, and need only be defined by short stretches of sequence data.     

Haemoglobin C and S role in acquired immunity against Plasmodium falciparum malaria

A recently proposed mechanism of protection for haemoglobin C (HbC; beta6Glu-->Lys) links an abnormal display of PfEMP1, an antigen involved in malaria pathogenesis, on the surface of HbC infected erythrocytes together with the observation of reduced cytoadhesion of parasitized erythrocytes and impaired rosetting in vitro. We investigated the impact of this hypothesis on the development of acquired immunity against Plasmodium falciparum variant surface antigens (VSA) encoding PfEMP1 in HbC in comparison with HbA and HbS carriers of Burkina Faso. We measured: i) total IgG against a single VSA, A4U, and against a panel of VSA from severe malaria cases in human sera from urban and rural areas of Burkina Faso of different haemoglobin genotypes (CC, AC, AS, SC, SS); ii) total IgG against recombinant proteins of P. falciparum asexual sporozoite, blood stage antigens, and parasite schizont extract; iii) total IgG against tetanus toxoid. Results showed that the reported abnormal cell-surface display of PfEMP1 on HbC infected erythrocytes observed in vitro is not associated to lower anti- PfEMP1 response in vivo. Higher immune response against the VSA panel and 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. These findings suggest that both HbC and HbS affect the early development of naturally acquired immunity against malaria. The enhanced immune reactivity in both HbC and HbS carriers supports the hypothesis that the protection against malaria of these adaptive genotypes might be at least partially mediated by acquired immunity against malaria.     

A distinct 5' flanking var gene region regulates Plasmodium falciparum variant erythrocyte surface antigen expression in placental malaria

The Plasmodium falciparum multigene var family codes for approximately 50 variant adhesive proteins expressed in a mutually exclusive manner at the surface of infected red blood cells (iRBCs). Switching expression of var genes can lead to fundamental changes in the adhesive and antigenic properties of iRBCs. For example, a specific phenotypic switch in adhesion from CD36 to chondroitin sulphate A (CSA) is associated with malaria pathogenesis in pregnant women. The factors and DNA elements that control the expression of a particular member of the var gene family during gestational malaria remains enigmatic. Here, we report that the subtelomeric FCR3 varCSA is expressed under the control of a unique DNA element of 1.8 kb, whereas the other members of the var multigene family are flanked by common regulatory elements. The 5' varCSA-type element is conserved as a single copy in laboratory strains and clinical isolates from Brazil and West Africa and contains two distinct repetitive elements of 150 bp and 60 bp respectively. The 5' varCSA-type sequence tags a var gene in the 3D7 genome that is homologous to the FCR3 varCSA gene. A recombinant DBL gamma domain of this var gene showed specific binding to CSA. This subtelomeric varCSA gene is transcribed in the opposite sense when compared with the usual orientation of telomere-adjacent var genes. This unique arrangement might explain why the varCSA gene is relatively conserved in genetically distinct parasites despite being located in a highly recombinogenic chromosome compartment. The 5' untranslated region (UTR) of the varCSA-type sequence is also transcribed in placental isolates that bind to CSA, illustrating an important role for the unique 5' varCSA-type sequence in the regulation of var genes involved in malaria pathogenesis in pregnant women. However, this promoter is not always found to be transcribing var genes selected for expression of products that bind to CSA in vitro. Our work identifies a sequence tag for the identification of varCSA genes in placental isolates for the first time.     

Cloning and expression in Escherichia coli of a surface antigen of Plasmodium falciparum merozoites.

A complementary DNA library was constructed from mRNA purified from asexual blood forms of Plasmodium falciparum. Among the members of this library we have identified a plasmid (pMC31-1) coding for a polypeptide exposed at the surface of merozoites, the invasive stage of the asexual cycle. This plasmid was identified by direct expression using both polyclonal and monoclonal antibodies specific for a schizont polypeptide of 200 kd which has been shown to be processed to an 83-kd polypeptide expressed at the surface of merozoites. The cDNA portion of the pMC31-1 plasmid hybridizes with DNA from three isolates of P. falciparum. Antisera raised against extracts of Escherichia coli harbouring pMC31-1 react with surface and internal structures of schizonts and with the surface of merozoites from all the isolates of P. falciparum examined. These results suggest that plasmid pMC31-1 encodes an antigen of value for the development of a vaccine against malaria.     

Primary structure of a Plasmodium falciparum malaria antigen located at the merozoite surface and within the parasitophorous vacuole

DNA encoding an antigen of 101,000 apparent molecular weight from the human malaria parasite Plasmodium falciparum was cloned and sequenced. Genomic DNA from the Camp strain covering the complete coding region along with cDNA from the FCR3 strain covering 81% of the coding region were obtained. The cloned DNA specified a full-length protein of 743 amino acids which included two tandemly repeated regions, one near the amino terminus containing eight hexapeptide repeats of sequence TVNDEDED, and the second near the carboxyl terminus containing primarily KE and KEE repeats. The latter repeated region is encoded by a 174-base stretch of mRNA containing only a single pyrimidine. Except for a putative leader sequence located at the amino terminus of the protein, the protein is hydrophilic and highly charged with a calculated isoelectric point of 5.6. Sequences from the Camp and FCR3 strains are very close and are also nearly identical to the partial cDNA sequence of the acidic basic repeated antigen (ABRA) protein from the FC27 strain (Stahl, H.D., Bianco, A.E., Crewther, R.F., Anders, R.F., Kyne, A.P., Coppel, R. L., Mitchell, G.F., Kemp, D.J., and Brown, G.V. (1986) Mol. Biol. Med. 3, 351-368). ABRA was previously shown to be located at the merozoite surface and in the parasitophorous vacuole. Because of its location and because it becomes complexed to merozoites when schizonts rupture in the presence of immune serum, ABRA is a candidate component of a malaria vaccine.     

Testosterone response of hepatic gene expression in female mice having acquired testosterone-unresponsive immunity to Plasmodium chabaudi malaria

Blood-stage malaria of Plasmodium chabaudi is characterized by its responsiveness to testosterone (T): T suppresses development of protective immunity, whereas once acquired immunity is T-unresponsive. Here, we have analyzed the liver, a T target and lymphoid organ with anti-malaria activity, for its T-responsiveness of gene expression in immune mice. Using Affymetrix microarray technology, in combination with quantitative RT-PCR, we have identified (i) T-unresponsive expression of newly acquired mRNAs encoding diverse sequences of IgG- and IgM-antibodies, (ii) 24 genes whose expression has become T-unresponsive including those encoding the T(H)2 response promoting EHMT2 and the erythrocyte membrane protein band 7.2 STOM, (iii) T-unresponsive expression of mRNAs for the cytokines IL-1β, IL-6, TNFα, and IFNγ, as well as iNOS, which are even not inducible by infection, and (iv) 35 genes retaining their T-responsiveness, which include those encoding the infection-inducible acute phase proteins SAA1, SAA2, and ORM2 as well as those of liver metabolism which encode the T-downregulated female-prevalent enzymes CYP2B9, CYP2B13, CYP3A41, CYP7A1, and SULT2A2 and the T-upregulated male-prevalent enzymes CYP2D9, CYP7B1, UGT2B1, HSD3B2, HSD3B5, respectively. The mRNA of the latter T-metabolizing enzyme is even 5-fold increased by T, suggesting a decrease in the effective T concentrations in the liver of immune mice. Collectively, our data suggest that the liver, which has acquired a selective T-unresponsiveness of gene expression, contributes to the acquired T-unresponsive, antibody-mediated protective immunity to blood-stage malaria of P. chabaudi.     

Features and prognosis of severe malaria caused by Plasmodium falciparum, Plasmodium vivax and mixed Plasmodium species in Papua New Guinean children

Background

Mortality from severe pediatric falciparum malaria appears low in Oceania but Plasmodium vivax is increasingly recognized as a cause of complications and death. The features and prognosis of mixed Plasmodium species infections are poorly characterized. Detailed prospective studies that include accurate malaria diagnosis and detection of co-morbidities are lacking.

Methods and Findings

We followed 340 Papua New Guinean (PNG) children with PCR-confirmed severe malaria (77.1% P. falciparum, 7.9% P. vivax, 14.7% P. falciparum/vivax) hospitalized over a 3-year period. Bacterial cultures were performed to identify co-incident sepsis. Clinical management was under national guidelines. Of 262 children with severe falciparum malaria, 30.9%, 24.8% and 23.2% had impaired consciousness, severe anemia, and metabolic acidosis/hyperlactatemia, respectively. Two (0.8%) presented with hypoglycemia, seven (2.7%) were discharged with neurologic impairment, and one child died (0.4%). The 27 severe vivax malaria cases presented with similar phenotypic features to the falciparum malaria cases but respiratory distress was five times more common (P = 0.001); one child died (3.7%). The 50 children with P. falciparum/vivax infections shared phenotypic features of mono-species infections, but were more likely to present in deep coma and had the highest mortality (8.0%; P = 0.003 vs falciparum malaria). Overall, bacterial cultures were positive in only two non-fatal cases. 83.6% of the children had alpha-thalassemia trait and seven with coma/impaired consciousness had South Asian ovalocytosis (SAO).

Conclusions

The low mortality from severe falciparum malaria in PNG children may reflect protective genetic factors other than alpha-thalassemia trait/SAO, good nutrition, and/or infrequent co-incident sepsis. Severe vivax malaria had similar features but severe P. falciparum/vivax infections were associated with the most severe phenotype and worst prognosis.     

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.     

Evidence that the erythrocyte invasion ligand PfRh2 is a target of protective immunity against Plasmodium falciparum malaria

Abs targeting blood-stage Ags of Plasmodium falciparum are important in acquired immunity to malaria, but major targets remain unclear. The P. falciparum reticulocyte-binding homologs (PfRh) are key ligands used by merozoites during invasion of erythrocytes. PfRh2a and PfRh2b are functionally important members of this family and may be targets of protective immunity, but their potential role in human immunity has not been examined. We expressed eight recombinant proteins covering the entire PfRh2 common region, as well as PfRh2a- and PfRh2b-specific regions. Abs were measured among a cohort of 206 Papua New Guinean children who were followed prospectively for 6 mo for reinfection and malaria. At baseline, Abs were associated with increasing age and active infection. High levels of IgG to all PfRh2 protein constructs were strongly associated with protection from symptomatic malaria and high-density parasitemia. The predominant IgG subclasses were IgG1 and IgG3, with little IgG2 and IgG4 detected. To further understand the significance of PfRh2 as an immune target, we analyzed PfRh2 sequences and found that polymorphisms are concentrated in an N-terminal region of the protein and seem to be under diversifying selection, suggesting immune pressure. Cluster analysis arranged the sequences into two main groups, suggesting that many of the haplotypes identified may be antigenically similar. These findings provide evidence suggesting that PfRh2 is an important target of protective immunity in humans and that Abs act by controlling blood-stage parasitemia and support its potential for vaccine development.     

Interactions between merozoite surface proteins 1, 6, and 7 of the malaria parasite Plasmodium falciparum

Merozoites of the malaria parasite Plasmodium falciparum expose at their surface a large multiprotein complex, composed of proteolytically processed, noncovalently associated products of at least three genes, msp-1, msp-6, and msp-7. During invasion of erythrocytes, this complex is shed from the surface except for a small glycosylphosphatidylinositol-anchored portion originating from MSP-1. The proteolytic cleavage separating the C-terminal portion of MSP-1 is required for successful invasion. Little is known about the structure and function of the abundant and essential multipartite complex. Using heterologously produced MSP-1, MSP-6, and MSP-7 in precursor and with the exception of MSP-7 in processed form, we have studied in vitro the complex formation between the different proteins to identify the interaction partners within the complex. Both MSP-6(36) and MSP-7 bind only to MSP-1 subunits that are shed, but although MSP-6(36) contacts just subunit p38, MSP-7 interacts with p83, p30, and p38. The intact C-terminal region of MSP-6 is required for the association with p38 as well as for its multimerization into tetramers. Furthermore, our data suggest that only the processed form and not the precursor form of MSP-1 interacts with MSP-6(36). MSP-6- as well as MSP-7-specific rabbit antibodies inhibit parasite multiplication in vitro as shown previously for antibodies directed against MSP-1. Our findings raise interesting questions with regard to proteolysis-mediated mechanisms of maturation of the MSP-1-MSP-6-MSP-7 complex and to the mode by which antibodies directed against this complex interfere with parasite multiplication.     

Gametocyte clearance in uncomplicated and severe Plasmodium falciparum malaria after artesunate-mefloquine treatment in Thailand

Artemisinin-based combination therapy (ACT) is currently promoted as a strategy for treating both uncomplicated and severe falciparum malaria, targeting asexual blood-stage Plasmodium falciparum parasites. However, the effect of ACT on sexual-stage parasites remains controversial. To determine the clearance of sexual-stage P. falciparum parasites from 342 uncomplicated, and 217 severe, adult malaria cases, we reviewed and followed peripheral blood sexual-stage parasites for 4 wk after starting ACT. All patients presented with both asexual and sexual stage parasites on admission, and were treated with artesunate-mefloquine as the standard regimen. The results showed that all patients were asymptomatic and negative for asexual forms before discharge from hospital. The percentages of uncomplicated malaria patients positive for gametocytes on days 3, 7, 14, 21, and 28 were 41.5, 13.1, 3.8, 2.0, and 2.0%, while the percentages of gametocyte positive severe malaria patients on days 3, 7, 14, 21, and 28 were 33.6, 8.2, 2.7, 0.9, and 0.9%, respectively. Although all patients were negative for asexual parasites by day 7 after completion of the artesunate-mefloquine course, gametocytemia persisted in some patients. Thus, a gametocytocidal drug, e.g., primaquine, may be useful in combination with an artesunate-mefloquine regimen to clear gametocytes, so blocking transmission more effectively than artesunate alone, in malaria transmission areas.     

Protection against Plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3

In humans, sterile immunity against malaria can be consistently induced through exposure to the bites of thousands of irradiated infected mosquitoes. The same level of protection has yet to be achieved using subunit vaccines. Recent studies have indicated an essential function for intrahepatic parasites, the stage after the mosquito bite, and thus for antigens expressed during this stage. We report here the identification of liver-stage antigen 3, which is expressed both in the mosquito and liver-stage parasites. This Plasmodium falciparum 200-kilodalton protein is highly conserved, and showed promising antigenic and immunogenic properties. In chimpanzees (Pan troglodytes), the primates most closely related to humans and that share a similar susceptibility to P. falciparum liver-stage infection, immunization with LSA-3 induced protection against successive heterologous challenges with large numbers of P. falciparum sporozoites.     

Relationships between clinical protection and antibodies to Plasmodium falciparum RESA (ring-infected erythrocyte surface antigen) peptides

A longitudinal study involving 76 individuals living in Dafinso and Vallée du Kou (near Bobo-Dioulasso, Burkina Faso, West Africa) was performed in June 1987 (beginning of the transmission period), August-September 1987 (during) and January 1988 (after). The serological antibody (Ab) responses against synthetic peptides representing repeat amino acid sequences of the P. falciparum Ring-Infected Erythrocyte Surface Antigen (RESA): (EENV)5, (EENVEHDA)4, (DDEHVEEPTVA)2 were evaluated by ELISA. The clinical longitudinal study during the transmission period allowed us to define three different groups in terms of age and occurrence of clinical malarial attack (greater than 5000 parasites mm-3 of blood and axillary fever greater than 37.7 degrees C). Levels (A620) of Ab to (EENVEHDA)4 and (DDEHVEEPTVA)2 were correlated with age. The adult group (III) had the highest prevalences of Ab to RESA peptides. No significant difference was found between groups of children with or without malaria attack. Nevertheless, at the beginning of the transmission period, children who had at least one malaria attack during the study presented the lowest level of antibodies to RESA peptides.     

Polymorphism and epitope sharing between the alleles of merozoite surface protein-1 of Plasmodium falciparum among Indian isolates

A comparison was made between local malaria transmission and malaria imported by travellers to identify the utility of national and regional annual parasite index (API) in predicting malaria risk and its value in generating recommendations on malaria prophylaxis for travellers. Regional malaria transmission data was correlated with malaria acquired in Latin America and imported into the USA and nine European countries. Between 2000 and 2004, most countries reported declining malaria transmission. Highest API's in 2003/4 were in Surinam (287.4) Guyana (209.2) and French Guiana (147.4). The major source of travel associated malaria was Honduras, French Guiana, Guatemala, Mexico and Ecuador. During 2004 there were 6.3 million visits from the ten study countries and in 2005, 209 cases of malaria of which 22 (11%) were Plasmodium falciparum. The risk of adverse events are high and the benefit of avoided benign vivax malaria is very low under current policy, which may be causing more harm than benefit.     

RESA-IFA assay in Plasmodium falciparum malaria, observations on relationship between serum antibody titers, immunity, and antigenic diversity

RESA-IFA assays were conducted using 63 adult sera from 7 different malarious areas against 7 different strains of Plasmodium falciparum, and 28 children's sera against 3 different parasite strains. Generally, where immunity to malaria was high, there was little or no antigenic diversity among the different strains examined. However, where sera were collected from semi-immunes, or from children, some variation in the RESA-IFA endpoint titers was discernible. Correlation between antibody titers determined by RESA-IFA and in vitro parasite invasion inhibition was not complete. Sera having high RESA-IFA titers were predictably inhibitory; however, many sera having low RESA-IFA titers were as inhibitory as sera having high titers, indicating that antibodies with specificities different from the RESA may be as important, or more important, to clinical immunity to blood-stage infections.     

Plasmodium falciparum: structural and functional domains of the mature-parasite-infected erythrocyte surface antigen

The mature parasite-infected erythrocyte surface antigen (MESA) is a protein exported to the membrane skeleton of the infected red cell, where it forms a strong noncovalent interaction with the host red cell protein, protein 4.1. The complete gene structure of MESA from the Ugandan isolate Palo Alto is described. Comparison to the previously reported MESA sequence from the Papua New Guinean cloned line D10 reveals strong conservation of the general gene structure of a short first exon and a long second exon. The exact exon/intron boundaries were determined by the generation and sequencing of a cDNA from this region. The MESA gene from both isolates consists of seven blocks of repeats that are identical in order. Repeat blocks are conserved to a high degree; however, differences are noted in most blocks in the form of scattered mutations or differences in repeat numbers. Previous work had shown that synthetic peptides spanning a 19-residue region could inhibit the binding of MESA to protein 4.1. Removal of this region from MESA almost completely abolished the binding of MESA to IOVs. Sequencing of this region from a number of laboratory and field isolates demonstrates complete conservation of the cytoskeletal binding domain and flanking sequences.