Population diversity and antibody selective pressure to Plasmodium falciparum MSP1 block2 locus in an African malaria-endemic setting.

BACKGROUND: Genetic evidence for diversifying selection identified the Merozoite Surface Protein1 block2 (PfMSP1 block2) as a putative target of protective immunity against Plasmodium falciparum. The locus displays three family types and one recombinant type, each with multiple allelic forms differing by single nucleotide polymorphism as well as sequence, copy number and arrangement variation of three amino acid repeats. The family-specific antibody responses observed in endemic settings support immune selection operating at the family level. However, the factors contributing to the large intra-family allelic diversity remain unclear. To address this question, population allelic polymorphism and sequence variant-specific antibody responses were studied in a single Senegalese rural community where malaria transmission is intense and perennial. RESULTS: Family distribution showed no significant temporal fluctuation over the 10 y period surveyed. Sequencing of 358 PCR fragments identified 126 distinct alleles, including numerous novel alleles in each family and multiple novel alleles of recombinant types. The parasite population consisted in a large number of low frequency alleles, alongside one high-frequency and three intermediate frequency alleles. Population diversity tests supported positive selection at the family level, but showed no significant departure from neutrality when considering intra-family allelic sequence diversity and all families combined. Seroprevalence, analysed using biotinylated peptides displaying numerous sequence variants, was moderate and increased with age. Reactivity profiles were individual-specific, mapped to the family-specific flanking regions and to repeat sequences shared by numerous allelic forms within a family type. Seroreactivity to K1-, Mad20- and R033 families correlated with the relative family genotype distribution within the village. Antibody specificity remained unchanged with cumulated exposure to an increasingly large number of alleles. CONCLUSION: The Pfmsp1 block2 locus presents a very large population sequence diversity. The lack of stable acquisition of novel antibody specificities despite exposure to novel allelic forms is reminiscent of clonal imprinting. The locus appears under antibody-mediated diversifying selection in a variable environment that maintains a balance between the various family types without selecting for sequence variant allelic forms. There is no evidence of positive selection for intra-family sequence diversity, consistent with the observed characteristics of the antibody response.

Antibodies elicited in adults by a primary Plasmodium falciparum blood-stage infection recognize different epitopes compared with immune individuals.

BACKGROUND: Asexual stage antibody responses following initial Plasmodium falciparum infections in previously healthy adults may inform vaccine development, yet these have not been as intensively studied as they have in populations from malaria-endemic areas. METHODS: Serum samples were collected over a six-month period from twenty travellers having returned with falciparum malaria. Fourteen of these were malaria-naïve and six had a past history of one to two episodes of malaria. Antibodies to seven asexual stage P. falciparum antigens were measured by ELISA. Invasion inhibitory antibody responses to the 19 kDa fragment of merozoite surface protein 1 (MSP119) were determined. RESULTS: Short-lived antibody responses were found in the majority of the subjects. While MSP119 antibodies were most common, MSP1 block 2 antibodies were significantly less frequent and recognized conserved domains. Antibodies to MSP2 cross-reacted to the dimorphic allelic families and anti-MSP2 isotypes were not IgG3 skewed as shown previously. MSP119 invasion inhibiting antibodies were present in 9/20 patients. A past history of malaria did not influence the frequency of these short-lived, functional antibodies (p = 0.2, 2-tailed Fisher's exact test). CONCLUSION: Adults infected with P. falciparum for the first time, develop relatively short-lived immune responses that, in the case of MSP119, are functional. Antibodies to the polymorphic antigens studied were particularly directed to allelic family specific, non-repetitive and conserved determinants and were not IgG subclass skewed. These responses are substantially different to those found in malaria immune individuals.

Regulation of protein phosphatase type 1 and cell cycle progression by PfLRR1, a novel leucine-rich repeat protein of the human malaria parasite Plasmodium falciparum.

The protein called 'suppressor of the dis2 mutant (sds22+)' is an essential regulator of cell division in fission and budding yeasts, where its deletion causes mitotic arrest. Its role in cell cycle control appears to be mediated through the activation of protein phosphatase type 1 (PP1) in Schizosaccharomyces pombe. We have identified the Plasmodium falciparum Sds22 orthologue, which we designated PfLRR1 as it belongs to the leucine-rich repeat protein family. We showed by glutathione-S-transferase pull-down assay that the PfLRR1 gene product interacts with PfPP1, that the PfLRR1-PfPP1 complex is present in parasite extracts and that PfLRR1 inhibits PfPP1 activity. Functional studies in Xenopus oocytes revealed that PfLRR1 interacted with endogenous PP1 and overcame the G2/M cell cycle checkpoint by promoting progression to germinal vesicle breakdown (GVBD). Confirmatory results showing the appearance of GVBD were observed when oocytes were treated with anti-PP1 antibodies or okadaic acid. Taken together, these observations suggest that PfLRR1 can regulate the cell cycle by binding to PP1 and regulating its activity.

Assessment of Plasmodium falciparum resistance to ferroquine (SSR97193) in field isolates and in W2 strain under pressure.

BACKGROUND: Ferroquine (FQ), or SSR97193, is a novel antimalarial drug currently in phase I clinical trials. FQ is a unique organometallic compound designed to overcome the chloroquine (CQ) resistance problem. FQ revealed to be equally active on CQ-sensitive and CQ-resistant Plasmodium falciparum laboratory strains and field isolates. FQ is also curative on rodent malaria parasites. As FQ will be tested in patients, the potential for resistance to this drug was evaluated. METHODS: The relationship between CQ-resistant transporter gene genotype and susceptibility to FQ were studied in 33 Cambodian P. falciparum field isolates previously studied for their in vitro response to CQ. In parallel, the ability of the CQ-resistant strain W2, to become resistant to FQ under drug pressure was assessed. RESULTS: The IC50 values for FQ in field isolates were found to be unrelated to mutations occurring in the P. falciparum chloroquine resistance transporter (PfCRT) or to the level of expression of the corresponding mRNA. In vitro, under a drug pressure of 100 nM of FQ, transient survival was observed in only one of two experiments. CONCLUSION: Field isolates studies and experimental drug pressure experiments showed that FQ overcomes CQ resistance, which reinforces the potential of this compound as a new antimalarial drug.

Blood typing in Saimiri sciureus monkeys: influence of anti-red blood cell alloantibodies on Plasmodium falciparum parasitaemia in vivo.

The Saimiri sciureus monkey is a well-established host for experimental studies with human malaria parasites. During the course of iterative inoculations with Plasmodium falciparum parasitised red blood cells (RBC), anti-RBC alloantibodies were detected in the sera of two of eight Saimiri monkeys. These anti-RBC antibodies were further used to investigate RBC phenotypes in 35 colony-reared Saimiri monkeys by flow cytometry. Three RBC phenotypes (named I-III) were observed. Their distribution was I (86%), II (11%) and III (3%). Using the Palo Alto FUP-2 strain, a variant P. falciparum line insensitive to hyperimmune serum and the passive transfer of anti-RBC alloantibodies, a dramatic drop in parasite growth was documented in an incompatible monkey.

Human antibodies to the polymorphic block 2 domain of the Plasmodium falciparum merozoite surface protein 1 (MSP-1) exhibit a highly skewed, peptide-specific light chain distribution.

Antibodies to polymorphic block 2 of the Plasmodium falciparum merozoite surface protein 1 (MSP-1) present a paradoxical association with acquired protection against clinical malaria, while showing restricted and fixed specificity, reminiscent of antigenic sin. We report here that these antibodies present a highly imbalanced, peptide-specific light chain distribution. This was not observed with several other parasite-derived peptides or antigens. These data point to a skewed immune response to MSP-1 block 2 that is constrained both in specificity and chain usage. This is the first report of a biased response to polymorphic epitopes of a surface antigen in malaria parasites.

Identification of a mitochondrial superoxide dismutase with an unusual targeting sequence in Plasmodium falciparum.

The intraerythrocytic stages of Plasmodium falciparum are exposed to oxidative stress and require functional anti-oxidant systems to survive. In addition to the parasite's known iron-dependent superoxide dismutase PfSOD1, a second SOD gene (PfSOD2) interrupted by 8 introns was identified on chromosome 6. Molecular modelling shows that the structure of PfSOD2 is similar to other iron-dependent SODs and phylogenetic analysis suggests PfSOD1 and PfSOD2 are the result of an ancestral gene duplication. The deduced amino acid sequence of PfSOD2 is similar to PfSOD1 but has a long N-terminal extension. Immunofluorescence studies show that PfSOD1 is cytosolic, whereas the N-terminal extension of PfSOD2 targets a green fluorescent protein fusion into the parasite's mitochondrion. Both SOD genes are transcribed during the erythrocytic cycle with PfSOD1 mRNA levels up to 35-fold higher than those of PfSOD2. Northern blots demonstrated that the mRNA levels of both SOD genes are up-regulated upon exposure to oxidative stress.

Double staining of Plasmodium falciparum nucleic acids with hydroethidine and thiazole orange for cell cycle stage analysis by flow cytometry.

BACKGROUND: Microarray analyses of stage-specific gene expression of Plasmodium falciparum require purification of RNAs from highly synchronized cultures. To date, no reliable method to control the quality of synchronization of P. falciparum cultures is available. METHODS: A double-staining method using hydroethidine and thiazole orange for nucleic acid staining was carried out to compare by flow cytometric analysis the nucleic acid labeling of synchronized P. falciparum in cultures at different time points of the 48-h intraerythrocytic cycle. RESULTS: With this method, we determined the quality of culture synchronization in schizont and ring stages. Nucleic acid analysis, based on thiazole orange fluorescence, clearly showed that low levels of schizonts in ring cultures results in a high contamination of ring nucleic acids by schizonts. Conversely, nucleic acids from trophozoite or schizont cultures containing ring stages did not present a significant contamination by ring nucleic acids. CONCLUSION: The results demonstrated a very low nucleic acid content in the ring stage when compared with the high nucleic acid content of schizont-stage parasites. The rapid and reliable flow cytometric strategy using hydroethidine- and thiazole orange-stained parasite nucleic acids allows monitoring of the purity of the preparation, thus greatly improving the quality assessment of parasite cultures, a critical step to study gene expression patterns.

Regulation of antigen-specific immunoglobulin G subclasses in response to conserved and polymorphic Plasmodium falciparum antigens in an in vitro model.

Cytophilic antibodies (Abs) play a critical role in protection against Plasmodium falciparum blood stages, yet little is known about the parameters regulating production of these Abs. We used an in vitro culture system to study the subclass distribution of antigen (Ag)-specific immunoglobulin G (IgG) produced by peripheral blood mononuclear cells (PBMCs) from individuals exposed to P. falciparum or unexposed individuals. PBMCs, cultivated with or without cytokines and exogenous CD40/CD40L signals, were stimulated with a crude parasite extract, recombinant vaccine candidates derived from conserved Ags (19-kDa C terminus of merozoite surface protein 1 [MSP1(19)], R23, and PfEB200), or recombinant Ags derived from the polymorphic Ags MSP1 block 2 and MSP2. No P. falciparum-specific Ab production was detected in PBMCs from unexposed individuals. PBMCs from donors exposed frequently to P. falciparum infections produced multiple IgG subclasses when they were stimulated with the parasite extract but usually only one IgG subclass when they were stimulated with a recombinant Ag. Optimal Ab production required addition of interleukin-2 (IL-2) and IL-10 for all antigenic preparations. The IgG subclass distribution was both donor and Ag dependent and was only minimally influenced by the exogenous cytokine environment. In vitro IgG production and subclass distribution correlated with plasma Abs to some Ags (MSP1(19), R23, and MSP2) but not others (PfEB200 and the three MSP1 block 2-derived Ags). Data presented here suggest that intrinsic properties of the protein Ag itself play a major role in determining the subclass of the Ab response, which has important implications for rational design of vaccine delivery.

Plasmodium falciparum merozoite surface protein 1 (MSP1): genotyping and humoral responses to allele-specific variants.

The present study is the first to investigate Plasmodium falciparum merozoite surface protein 1 (MSP1) allele-specific humoral responses in residents of central Africa. In endemic areas, acquired immune responses to malaria are assumed to reflect the need to be infected with a large number of antigenically diverse parasite populations. In the work presented here, the relationship between antibody specificity and the infecting parasite genotype was investigated in asymptomatic subjects and patients with uncomplicated malaria in order to possibly clarify the relationship between anti-MSP1 block2 antibodies and clinical malaria. Overall isolates were typed by nested PCR using allele-specific primers of the P. falciparum MSP1 gene to identify the infecting parasite genotype. The K1 type was the predominant allelic family in both clinical groups. Polyinfection (number of isolates with more than one parasite genotype) and the complexity of infections (mean number of parasite genotype per infected subject) were higher in isolates from asymptomatic individuals. Total immunoglobulins G (IgG) responses to schizont crude extract antigens and to MSP1 variant-specific peptides were assessed by ELISA test. More than 90% of the sera reacted against schizont extract, whatever the clinical group and the K1 seroprevalence was the highest in both clinical groups. Our results showed an age-dependence in the number of different variants of MSP1 block2 recognised by serum. Indeed, isolates from older (>14 years) subjects showed lower multiplicity of infection and higher was the mean number of different MSP1 variants recognised by their serum. This corresponded to the age reported for the acquisition of anti-parasite immunity under high malaria endemicity. The contribution of variant-specific immunity in asymptomatic malaria infections is discussed.