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.     

Relation between hepatitis B carrier status and antibody against synthetic Plasmodium falciparum erythrocyte surface (pf155 - RESA) antigen

A survey on Plasmodium infection was carried out in gold mine camps located in the Brazilian Amazon. Antibody against P. falciparum ring-infected erythrocyte surface antigen (RESA) was quantified by an enzyme-immunoassay in order to assess P. falciparum exposure. Hepatitis B, a common infection in this area, was also investigated by serologic markers. Among 520 sampled subjects, 517 (99.4%) admitted previous symptomatic malaria, 106 (20.4%) had positive thick smears for malaria, 82.9% had HBV markers, and 7.1% were HBsAg positive. Anti-RESA titers was significantly lower in HBV carriers than in people with resolved HBV infection suggesting that the anti-RESA immune response could be supressed by HBV carrier status. Moreover, immunedeficient responses to both infections may take place in some subjects causing concomitant lower anti-RESA response and incapacity to clear HBV.     

Plasmodium falciparum: analysis of the interaction of antigen Pf155/RESA with the erythrocyte membrane

The location of the Plasmodium falciparum vaccine candidate antigen Pf155/RESA in the membrane of infected erythrocytes was analzyed by means of selective surface radioiodination and immunofluorescence of surface-modified cells. The lack of radiolabel in Pf155/RESA as well as its localization by immunofluorescence similar to that of the N-terminal region of erythrocyte band 3 suggests that the antigen is associated with the cytoplasmic phase of the erythrocyte membrane. In concordance with this, Pf155/RESA was detected by immunofluorescence on the surface of inside out membrane vesicles from P. falciparum-infected erythrocytes. Pf155/RESA from spent culture medium also bound to inside out membrane vesicles of normal erythrocytes as well as to cytoskeletal shells of such vesicles, but failed to bind to sealed right-side out membrane vesicles. Depletion of spectrin from the vesicles abolished antigen binding, suggesting that Pf155/RESA association with the erythrocyte cytoskeleton is mediated by spectrin.     

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.     

Plasmodium chabaudi: polymorphic and nonpolymorphic epitopes of the antigen Pch105/RESA.

The localization in the erythrocyte membrane of Pch105/RESA, the ring stage-infected erythrocyte surface antigen of Plasmodium chabaudi, the proposed analog to the vaccine candidate Pf155/RESA in P. falciparum, is here confirmed by the use of the immunogold technique in electron microscopy. Furthermore, a number of monoclonal antibodies to other P. chabaudi erythrocyte membrane antigens in the same molecular weight range as Pch105 were compared in different test systems. Data from immunoblotting of native and recombinant antigen as well as an inhibition ELISA indicate that Pch105 is identical to Pc96 and two other described antigens of 105 and 110 kDa. Pch105 could also be shown to have polymorphic epitopes, varying between different strains of P. chabaudi, without impact on the molecular weight.     

Pf155/RESA antigen is localized in dense granules of Plasmodium falciparum merozoites

Immunoelectron microscopy demonstrated the presence of Pf155/RESA in dense granules of Plasmodium falciparum merozoites rather than in micronemes as previously suggested. Since the dense granules are released after the merozoite enters the parasitophorous vacuole, the role of Pf155/RESA in invasion and subsequent steps of parasite development may differ from that of a molecule located in the micronemes.     

Class II restriction in mice to the malaria candidate vaccine ring infected erythrocyte surface antigen (RESA) as synthetic peptides or as expressed in recombinant vaccinia

The immune response to three peptides corresponding to the repeat regions of the malaria candidate vaccine ring infected E surface Ag (RESA) were studied. Both antibody responses and lymphocyte stimulation in mice injected with these peptides without carrier were found to be restricted to certain MHC class II haplotypes. Mice bearing IAk were strong responders to all three peptides. Mice bearing IAd were strong responders only to the 3' repeat peptides, the octamer and tetramer. Mice bearing Is or Iq did not respond to any repeat peptides. Remarkably, the pattern of genetic restriction of the antibody response to the entire RESA as expressed in vaccinia indicated that there were no other epitopes besides the three repeats. Because only one class II haplotype (i.e., k) out of five tested responded strongly to this peptide and only two out of five (i.e., k and d) responded to the octamer or tetramer, it may be difficult to achieve a good immune response against RESA in most or all humans.     

The surface of the Plasmodium falciparum-infected erythrocyte

In order to navigate its complex lifecycle, the malaria parasites must interactwith a range of host cells. Examples of this are the invasion of hepatocytes by sporozoites and erythrocyte invasion by merozoites. This requirement for cell recognition brings with it the need to display cognate ligands on the parasite surface, and therefore the capacity of the host to develop defences against the infection. Even at a stage where the intracellular nature of erythrocyte development would appear to offer an opportunity for the parasite to be immunologically "silent", parasite-derived proteins are found on the surface of the infected erythrocyte. This review will discuss the proteins found on or associated with the surface of the infected erythrocyte and the resulting phenotypes.     

Monoclonal antibodies against a specific surface determinant on malarial (Plasmodium knowlesi) merozoites block erythrocyte invasion

Twelve hybridoma cell lines secreting monoclonal antibodies against Plasmodium knowlesi merozoites have been produced. Antibodies from 3 of the 12 lines agglutinated merozoites. The 2 monoclonal antibodies (13C11 and 16F8) that markedly agglutinated merozoites blocked merozoite invasion of erythrocytes. Of these 2 lines, the one that induced the most agglutination also blocked invasion most effectively. The third monoclonal antibody (53B3) caused minimal agglutination of merozoites and did not block invasion, nor did the other 9 nonagglutinating antibodies. The 2 blocking monoclonal antibodies bound to antigens around the entire surface of merozoites, as demonstrated by immunoelectron microscopy, and precipitated a single biosynthetically labeled protein of apparent m.w. of 250,000. None of the nonagglutinating lines precipitated this protein. Monoclonal antibodies 13C11 and 16F8 reacted with a common antigenic determinant on a Malaysian and a Philippine strain of P. knowlesi in that they blocked invasion and precipitated a 250,000 m.w. protein from both. Sera from immune monkeys also precipitated this 250,000 m.w. protein.     

Plasmodium vivax: cloning and expression of a major blood-stage surface antigen

Plasmodium vivax is a highly prevalent malaria pathogen of man; the following report is the first to describe the cloning and expression of a major asexual erythrocytic stage antigen of this species. The screening of a genomic DNA expression library with a monoclonal antibody directed against a 200-kDa surface component (Pv200) of the more mature schizonts of P. vivax led to the selection of a recombinant bacterial clone which produced a fusion protein. Mouse and rabbit immune sera raised against the purified fusion protein recognized the 200-kDa parasite antigen on Western blots and reacted with the surface of segmenters by immunofluorescence. Sequencing of the 1.9-kb P. vivax DNA insert coding for this fusion protein revealed a 45-47% homology at the nucleotide level with the P. falciparum gene of a parasite surface antigen, Pf195, which has been shown to be a promising candidate for a malaria vaccine in primates and in man.     

Plasmodium falciparum: differential parasite reactivity of rabbit antibodies to repeated sequences in the antigen Pf155/RESA

For selection of immunogens capable of inducing high levels of antibodies reactive with the Plasmodium falciparum antigen Pf155/RESA, rabbits were immunized with synthetic peptides corresponding to sequences based on the repeat subunits EENVEHDA and (EENV)2 from the C-terminus of this antigen. The antibodies obtained were analyzed with regard to binding to synthetic peptides in ELISA and to reactivity with parasite antigens by immunofluorescence or immunoblotting. All antisera reacted with both the peptides EENVEHDA and (EENV)2 as well as with Pf155/RESA. Antibody fractions specific for each of the two peptides were prepared by affinity chromatography on insolubilized peptides. Strong reactivity with antigens in the membrane of erythrocytes infected with early stages of the parasite as well as reactivity with Pf155/RESA in immunoblotting correlated with reactivity of antibody with (EENV)2. Antibody preparations reactive with EENVEHDA and depleted of (EENV)2 reactivity showed only a weak reactivity with Pf155/RESA but reacted also with P. falciparum polypeptides of 250, 210, and 88 kDa. In immunofluorescence, these antibodies stained mainly the intraerythrocytic parasite. Both EENVEHDA- and (EENV)2-specific antibodies inhibited merozoite reinvasion in P. falciparum in vitro cultures, the latter antibodies being the most efficient. This study defines the specificity and cross-reactivity with other P. falciparum antigens of antibodies to the C-terminal repeats of Pf155/RESA.     

Antigens on the Plasmodium falciparum infected erythrocyte surface are parasite derived: a reply

In this article Chris Newbold and Kevin Marsh describe the evidence for the co-existence of both modified host proteins and of parasite determinants at the infected erythrocyte surface. The stable characteristics of infected cells may in part stem from parasite-induced changes in band 3 molecules, thus explaining some of the cytoadherence properties of uninfected, but abnormal cells (as in sickle-cell disease and diabetes). However, Newbold and Marsh suggest that it is difficult to explain the astonishing diversity of antigens that have been observed at the surface of infected red cells unless such molecules have been synthesized by the parasite.     

Plasmodium falciparum-infected erythrocyte receptor(s) for CD36 and thrombospondin are restricted to knobs on the erythrocyte surface.

Adherence of Plasmodium falciparum-infected RBCs (PRBC) to endothelial cells causes PRBC sequestration in cerebral microvessels and is considered to be a major contributor to the pathogenesis of cerebral malaria. Both CD36 and thrombospondin (TSP) are glycoproteins that mediate PRBC adherence to endothelial cells in vitro. Because they are both expressed on the surface of endothelial cells, they probably contribute to PRBC sequestration and vascular occlusion in vivo. By applying affinity labeling of receptor binding sites with purified ligands, we showed for the first time that both CD36 and TSP can bind independently to the PRBC surface and that the PRBC receptor(s) for CD36 and TSP are localized specifically to the electron-dense knob protrusions of the PRBC surface. These findings may help in efforts to develop a malaria vaccine to prevent cerebral malaria.     

Structure of the RESA gene of Plasmodium falciparum.

We have determined the nucleotide sequence of the gene encoding the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum, an antigen that has been shown to confer protective immunity on monkeys. The sequence has enabled us to predict the structure of the RESA gene and the amino acid sequence of its protein product. The gene consists of two exons with a short intron located near the 5' end of the coding region. A hydrophobic amino acid segment predicted for the 3' end of exon 1 is consistent with the possibility that exon 1 encodes trafficking signal sequences. We show that restriction fragment length polymorphisms can be used to define two different alleles of RESA, represented by isolates FC27 and NF7, and compare the FC27 sequence with that of a long cDNA clone from NF7 described previously.     

Erratum: Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates

[This corrects the article on p. 225 in vol. 7, PMID: 2452082.].     

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) destabilizes erythrocyte membrane skeleton

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) is a parasite-derived protein that appears on the cytoplasmic surface of the host cell membrane in the later stages of the parasite's development where it associates with membrane skeleton. We have recently demonstrated that a 60-residue fragment (FIa1, residues 38-97) of PfEMP3 bound to spectrin. Here we show that this polypeptide binds specifically to a site near the C terminus of alpha-spectrin at the point that spectrin attaches to actin and protein 4.1R in forming the junctions of the membrane skeletal network. We further show that this polypeptide disrupts formation of the ternary spectrin-actin-4.1R complex in solution. Importantly, when incorporated into the cell, the PfEMP3 fragment causes extensive reduction in shear resistance of the cell. We conjecture that the loss of mechanical cohesion of the membrane may facilitate the exit of the mature merozoites from the cell.     

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene.     

Plasmodium falciparum antigens on the surface of the gametocyte-infected erythrocyte

Background

The asexual blood stages of the human malaria parasite Plasmodium falciparum produce highly immunogenic polymorphic antigens that are expressed on the surface of the host cell. In contrast, few studies have examined the surface of the gametocyte-infected erythrocyte.

Methodology/Principal Findings

We used flow cytometry to detect antibodies recognising the surface of live cultured erythrocytes infected with gametocytes of P. falciparum strain 3D7 in the plasma of 200 Gambian children. The majority of children had been identified as carrying gametocytes after treatment for malaria, and each donated blood for mosquito-feeding experiments. None of the plasma recognised the surface of erythrocytes infected with developmental stages of gametocytes (I–IV), but 66 of 194 (34.0%) plasma contained IgG that recognised the surface of erythrocytes infected with mature (stage V) gametocytes. Thirty-four (17.0%) of 200 plasma tested recognised erythrocytes infected with trophozoites and schizonts, but there was no association with recognition of the surface of gametocyte-infected erythrocytes (odds ratio 1.08, 95% C.I. 0.434–2.57; P = 0.851). Plasma antibodies with the ability to recognise gametocyte surface antigens (GSA) were associated with the presence of antibodies that recognise the gamete antigen Pfs 230, but not Pfs48/45. Antibodies recognising GSA were associated with donors having lower gametocyte densities 4 weeks after antimalarial treatment.

Conclusions/Significance

We provide evidence that GSA are distinct from antigens detected on the surface of asexual 3D7 parasites. Our findings suggest a novel strategy for the development of transmission-blocking vaccines.     

Apical membrane antigen 1 plays a central role in erythrocyte invasion by Plasmodium species

Apical membrane antigen 1 (AMA1) is an asexual blood-stage protein expressed in the invasive merozoite form of Plasmodia species, which are the causative agent of malaria. We have complemented the function of Plasmodium falciparum AMA1 (PfAMA1) with a divergent AMA1 transgene from Plasmodium chabaudi (PcAMA1). It was not possible to disrupt the PfAMA1 gene using 'knock-out' plasmids, although we demonstrate that the PfAMA1 gene can be targeted by homologous recombination. These experiments suggest that PfAMA1 is critical, perhaps essential, for blood-stage growth. Importantly, we showed that PcAMA1 expression in P. falciparum provides trans-species complementation to at least 35% of the function of endogenous PfAMA1 in human red cells. Furthermore, expression of this transgene in P. falciparum leads to more efficient invasion of murine erythrocytes. These results indicate an important role for AMA1 in the invasion of red blood cells (RBCs) across divergent Plasmodium species.