Jennifer K. Thompson

Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of P. falciparum

Resistance of Plasmodium falciparum to chloroquine shares features with the multidrug resistance (MDR) phenotype of mammalian tumor cells. We report here the sequence of pfmdr, the P. falciparum homolog of mdr. We show that pfmdr is amplified in some chloroquine-resistant parasites but not in any of the sensitive isolates examined and that pfmdr transcript levels are increased. The gene is located on chromosome 5, and in one chloroquine-resistant line with an amplified pfmdr gene, chromosome 5 is greatly enlarged. The chromosome heterogeneity is due to varying copy numbers of different-sized pfmdr-containing amplicons. The existence of an mdr gene in P. falciparum and its amplification in some chloroquine-resistant lines greatly adds to the circumstantial evidence that pfmdr mediates chloroquine resistance in these lines.

Heterochromatin silencing and locus repositioning linked to regulation of virulence genes in Plasmodium falciparum.

The malaria parasite Plasmodium falciparum undergoes antigenic variation to evade host immune responses through switching expression of variant surface proteins encoded by the var gene family. We demonstrate that both a subtelomeric transgene and var genes are subject to reversible gene silencing. Var gene silencing involves the SIR complex as gene disruption of PfSIR2 results in activation of this gene family. We also demonstrate that perinuclear gene activation involves chromatin alterations and repositioning into a location that may be permissive for transcription. Together, this implies that locus repositioning and heterochromatic silencing play important roles in the epigenetic regulation of virulence genes in P. falciparum.

A var gene promoter controls allelic exclusion of virulence genes in Plasmodium falciparum malaria

Mono-allelic expression of gene families is used by many organisms to mediate phenotypic variation of surface proteins. In the apicomplexan parasite Plasmodium falciparum, responsible for the severe form of malaria in humans, this is exemplified by antigenic variation of the highly polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1, encoded by the 60-member var gene family, represents a major virulence factor due to its central role in immune evasion and intravascular parasite sequestration. Mutually exclusive expression of PfEMP1 is controlled by epigenetic mechanisms involving chromatin modification and perinuclear var locus repositioning. Here we show that a var promoter mediates the nucleation and spreading of stably inherited silenced chromatin. Transcriptional activation of this promoter occurs at the nuclear periphery in association with chromosome-end clusters. Additionally, the var promoter sequence is sufficient to infiltrate a transgene into the allelic exclusion programme of var gene expression, as transcriptional activation of this transgene results in silencing of endogenous var gene transcription. These results show that a var promoter is sufficient for epigenetic silencing and mono-allelic transcription of this virulence gene family, and are fundamental for our understanding of antigenic variation in P. falciparum. Furthermore, the PfEMP1 knockdown parasites obtained in this study will be important tools to increase our understanding of P. falciparum-mediated virulence and immune evasion.

Homologous Recombination within Subtelomeric Repeat Sequences Generates Chromosome Size Polymorphisms in P. falciparum

We present restriction maps for chromosomes 1 and 2 of six cloned lines of P. falciparum. These delineate the locations of eight genetic markers, including genes for five antigens. In parasites from diverse areas, chromosome structure is conserved in central regions but is polymorphic both in length and sequence near the telomeres. The telomeres and adjacent sequences comprise a conserved structure at the ends of most P. falciparum chromosomes. However, the subtelomeric zones are polymorphic and coincide with the locations of a repetitive element (rep20). Deletions of rep20 generate clones of P. falciparum that lack rep20 on one or both ends of chromosomes 1 or 2, and larger deletions remove telomere-proximal genes as well. The chromosome length polymorphisms can therefore be largely explained by recombination within these blocks of repeats, a mechanism that is also important in the generation of diversity in genes for repetitive antigens of P. falciparum.

A novel ligand from Plasmodium falciparum that binds to a sialic acid‐containing receptor on the surface of human erythrocytes

Invasion of the merozoite form of Plasmodium falciparum into human erythrocytes involves multiple receptor–ligand interactions. The EBA175 protein of P. falciparum has been shown to be the ligand that binds to a sialic acid‐dependent site on glycophorin A. We have identified a novel P. falciparum ligand, termed erythrocyte‐binding antigen 140 (EBA140), that shares structural features and homology with EBA175. Subcellular localization of EBA140 suggests that it is located in the micronemes, the same localization as EBA175. EBA140 binds to a sialic acid‐dependent receptor on the surface of human erythrocytes. Binding of EBA140 to this erythrocyte receptor is sensitive to neuraminidase and resistant to trypsin, proteinase K and pronase. The protease‐resistant properties of the erythrocyte receptor suggests that it is not glycophorin A or C. Additionally, analysis of mutant erythrocytes from humans has shown that EBA140 does not bind glycophorin B. Interestingly, we have identified a parasite line that lacks the eba140 gene, suggesting that this protein is not essential for in vitro invasion. These results suggest that EBA140 may be involved in merozoite invasion using a sialic acid‐dependent receptor on human erythrocytes.

Identification and Prioritization of Merozoite Antigens as Targets of Protective Human Immunity to Plasmodium falciparum Malaria for Vaccine and Biomarker Development

The development of effective malaria vaccines and immune biomarkers of malaria is a high priority for malaria control and elimination. Ags expressed by merozoites of Plasmodium falciparum are likely to be important targets of human immunity and are promising vaccine candidates, but very few Ags have been studied. We developed an approach to assess Ab responses to a comprehensive repertoire of merozoite proteins and investigate whether they are targets of protective Abs. We expressed 91 recombinant proteins, located on the merozoite surface or within invasion organelles, and screened them for quality and reactivity to human Abs. Subsequently, Abs to 46 proteins were studied in a longitudinal cohort of 206 Papua New Guinean children to define Ab acquisition and associations with protective immunity. Ab responses were higher among older children and those with active parasitemia. High-level Ab responses to rhoptry and microneme proteins that function in erythrocyte invasion were identified as being most strongly associated with protective immunity compared with other Ags. Additionally, Abs to new or understudied Ags were more strongly associated with protection than were Abs to current vaccine candidates that have progressed to phase 1 or 2 vaccine trials. Combinations of Ab responses were identified that were more strongly associated with protective immunity than responses to their single-Ag components. This study identifies Ags that are likely to be key targets of protective human immunity and facilitates the prioritization of Ags for further evaluation as vaccine candidates and/or for use as biomarkers of immunity in malaria surveillance and control.

Association between naturally acquired antibodies to erythrocyte-binding antigens of Plasmodium falciparum and protection from malaria and high-density parasitemia.

BACKGROUND Antibodies targeting blood stage antigens are important in protection against malaria, but the principle targets remain unclear. Erythrocyte-binding antigens (EBAs) are important erythrocyte invasion ligands used by merozoites and may be targets of protective immunity, but there are limited data examining their potential importance. METHODS We examined antibodies among 206 Papua New Guinean children who were treated with antimalarials at enrollment and observed prospectively for 6 months for reinfection and malaria. Immunoglobulin (Ig) G, IgG subclasses, and IgM to different regions of EBA175, EBA140, and EBA181 expressed as recombinant proteins were assessed in comparison with several other merozoite antigens. RESULTS High levels of IgG to each of the EBAs were strongly associated with protection from symptomatic malaria and high density parasitemia, but not with risk of reinfection per se. The predominant IgG subclasses were either IgG1 or IgG3, depending on the antigen. The predominance of IgG1 versus IgG3 reflected structural features of specific regions of the proteins. IgG3 was most strongly associated with protection, even for those antigens that had an IgG1 predominant response. CONCLUSIONS The EBAs appear important targets of acquired protective immunity. These findings support their further development as vaccine candidates.

MOLECULAR VARIATION IN A NOVEL POLYMORPHIC ANTIGEN ASSOCIATED WITH PLASMODIUM FALCIPARUM MEROZOITES

A cDNA clone encoding part of a novel polymorphic merozoite antigen from Plasmodium falciparum was isolated by screening a cDNA library with human immune serum from Papua New Guinea. Immunofluorescence microscopy and immunoblotting with affinity-purified antibodies recognized a highly polymorphic antigen, Ag956, present in schizonts and merozoites. Biosynthetic labeling and immunoprecipitation experiments demonstrated that Ag956 is proteolytically cleaved during merozoite maturation. The complete genomic sequence of Ag956 from the D10 clone of P. falciparum isolate FC27 encodes a secreted protein of calculated molecular mass 43,243 that is very hydrophilic and contains a region of unusual heptad repeats of the general structure AXXAXXX. This antigen has been named the secreted polymorphic antigen associated with merozoites (SPAM). The sequence of a second SPAM allele from the 3D7 clone of isolate NF54 reveals that the alanine heptad repeats and the hydrophilic C-terminal half of the protein are conserved. Variation among SPAM alleles is the result of deletions and amino acid substitutions in non-repetitive sequences within and flanking the alanine heptad-repeat domain. Heptad repeats in which the a and d position contain hydrophobic residues generate amphipathic alpha-helices which give rise to helical bundles or coiled-coil structures in proteins. Thus, SPAM is the first example of a P. falciparum antigen in which a repetitive sequence has features characteristic of a well-defined structural element.

Reticulocyte and Erythrocyte Binding-Like Proteins Function Cooperatively in Invasion of Human Erythrocytes by Malaria Parasites

ABSTRACT Plasmodium falciparum causes the most severe form of malaria in humans and invades erythrocytes using multiple ligand-receptor interactions. Two important protein families involved in erythrocyte binding are the erythrocyte binding-like (EBL) and the reticulocyte binding-like (RBL or P. falciparum Rh [PfRh]) proteins. We constructed P. falciparum lines lacking expression of EBL proteins by creating single and double knockouts of the corresponding genes for eba-175, eba-181, and eba-140 and show that the EBL and PfRh proteins function cooperatively, consistent with them playing a similar role in merozoite invasion. We provide evidence that PfRh and EBL proteins functionally interact, as loss of function of EBA-181 ablates the ability of PfRh2a/b protein antibodies to inhibit merozoite invasion. Additionally, loss of function of some ebl genes results in selection for increased transcription of the PfRh family. This provides a rational basis for considering PfRh and EBL proteins for use as a combination vaccine against P. falciparum. We immunized rabbits with combinations of PfRh and EBL proteins to test the ability of antibodies to block merozoite invasion in growth inhibition assays. A combination of EBA-175, PfRh2a/b, and PfRh4 recombinant proteins induced antibodies that potently blocked merozoite invasion. This validates the use of a combination of these ligands as a potential vaccine that would have broad activity against P. falciparum.

The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking

The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein–protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.