A 22 kDa protein associated with the Plasmodium falciparum merozoite surface protein-1 complex.

The Plasmodium falciparum merozoite surface protein-1 (MSP-1) is synthesized as a precursor of approximately 195 kDa and is processed to form a complex of polypeptides on the surface of free merozoites. As a result of a second processing event, the entire MSP-1 complex is shed from the surface, apart from a C-terminal fragment that remains anchored to the merozoite membrane. We have identified a 22 kDa protein (p22) on the surface of merozoites by cell surface radioiodination and indirect immunofluorescence assay on unfixed free merozoites. p22 is also a component of the shed MSP-1 complex where it is present in part as a 19 kDa form (p22(19)) as shown by immunochemical and peptide mapping analyses. The soluble complex contains MSP-1-derived polypeptides and p22 in approximately stoichiometrically equal amounts. N-terminal amino acid sequence analyses of p22/p22(19) showed that the protein is not derived from the MSP-1 precursor.

Plasmodium falciparum proteinases and red blood cell invasion.

Malarial proteinases of the erythrocytic life-cycle are used to design new inhibitors capable of blocking the parasite's development. The Merozoite Proteinase for Erythrocytic Invasion (MPEI) of Plasmodium falciparum, a neutral proteinase, and the acidic Pf37 proteinase acting on spectrin as substrate, are good candidates for this kind of strategy.

Lymphocyte response in vitro to Plasmodium falciparum merozoite antigens in donors from a holoendemic area.

Crude merozoite antigens from P. falciparum were used to analyse the proliferative response of peripheral blood mononuclear cells from 114 inhabitants of the village of Dielmo (Senegal, West Africa), who are exposed continuously to malaria transmission. The high or low responses to merozoite antigens obtained in lymphocyte stimulation assays were correlated to the presence or absence of parasites, to the IFN-gamma production and to the HLA-phenotype. High responders produced high levels of IFN-gamma while low responders did not secrete IFN-gamma (23/27). The two HLA phenotypes HLA-B51 and HLA-DR1 were significantly associated with high response (p < 0.05).

[Analysis of the antibody response to merozoite antigens in a malaria holoendemic area]. / Analyse de la réponse anticorps aux antigènes de mérozoïtes dans une zone d'holoendémicité palustre.

In the aim to determine the possible role of HLA-antigens in malaria infection, sera from 50 HLA-typed donors from Dielmo (Senegal) were tested in immunoblotting (using crude merozoites as antigen) and immunoprecipitation (using detergent-extracts from surface-iodinated merozoite as antigen). The donors were previously tested on lymphocyte proliferation in vitro and gamma-interferon production and grouped into two classes: high responders and low responders. In immunoblotting and immunoprecipitation experiments, no specific differences were found in the antibody reactivity with native merozoite antigen in individuals with high (HR) or low (LR) in vitro proliferative T cell responses. In other words, both groups of responders, high and low, showed antibodies in their sera against a wide range of different parasite antigens; although between individual donors striking differences were found. Individual donors had developed different levels of antibodies, or no antibodies at all, against individual natural antigens. These differences, however, could not be correlated with HR or LR. The band patterns obtained were compared with HLA-antigens of donors phenotypes. Results showed that there was no correlation found between the different merozoite antigens recognized by sera of the different donors or groups of donors (HR and LR) and the donors' HLA-phenotypes. The fact that donors with HLA-B51 all recognized (MSP1(42) and donors with DR1 recognized MSP1(19), was not a convincing correlation.

Lymphocyte response in vitro to Plasmodium falciparum merozoite antigens in donors from a holoendemic area.

Crude merozoite antigens from Plasmodium falciparum were used to evaluate the proliferative response of peripheral mononuclear cells (PBMCs) from 114 inhabitants of the village of Dielmo (Senegal, West Africa) exposed continuously to malaria transmission. The high or low responses to merozoite antigens obtained in lymphocyte stimulation assays were correlated with the presence or absence of parasites, IFN-gamma production and HLA phenotype. The high responders produced high levels of IFN-gamma, in contrast to the low responders, most of whom did not secrete IFN-gamma (23/27). Among others, the two HLA phenotypes HLA-B51 and HLA-DR1 were significantly associated with a high response (P < 0.05).

Inhibitory monoclonal antibodies recognise epitopes adjacent to a proteolytic cleavage site on the RAP-1 protein of Plasmodium falciparum.

The low-molecular-weight rhoptry-associated protein (RAP) complex of Plasmodium falciparum consists of at least two gene products, RAP-1 and RAP-2, and has the ability to immunise Saimiri monkeys against experimental P. falciparum infection. Several monoclonal antibodies specifically recognise this complex and in this study we show that purified immunoglobulin derived from these monoclonals is capable of inhibiting parasite growth in vitro. It has previously been shown that RAP-1 initially appears as an 80-kDa protein (p80) in early schizogony and is processed to a 65-kDa protein (p65) in late schizogony. Several of the inhibitory monoclonals recognise both the 80- and 65-kDa proteins by Western blot analysis suggesting that they recognise linear epitopes on RAP-1. We have mapped these epitopes by testing the reactivity of the monoclonals against fragments of the rap-1 gene expressed as beta-galactosidase fusion proteins and subsequently against synthetic peptides. All of the epitopes map to a region 10-20 amino acids C-terminal to the proteolytic cleavage site for the processing of p80 to p65 at amino acid 190. We also show that the 65-kDa protein is not present in purified merozoites, suggesting that its generation is associated with merozoite release rather than erythrocyte invasion. These results are discussed with respect to possible inhibitory mechanisms for the monoclonals.

[In vitro T cell response to merozoite antigens in the area of continuous malaria transmission]. / Réponse cellulaire T in vitro aux antigènes de mérozoïtes en zone de transmission continue du paludisme.

T CELL response in vitro to merozoite extracts of P falciparum was tested in holoendémic area (Dielmo, Region of Fatick). 58 individuals (29 adults and 29 children) were sampled. Mononuclear cells from peripheral blood were cultured with the presence of merozoites antigens during 7 days. Cells were pulsed at day 6 and 3H-thymidine incorporation was measured 16h later. T Cell response high or low was evaluated by Stimulation Index (SI). This is defined as mean cpm expérimental cells/mean cpm control cells. Two groups of Responders have been identified: High Résponders: SI > or = 5 48% of subjects Low Responders: SI < 5 52% of subjects. A microparasitaemia have been found in 19 donnors without illness. 14 of these are Low Responders (89%). It was found that SI increased with the number year of exposure.

A single fragment of a malaria merozoite surface protein remains on the parasite during red cell invasion and is the target of invasion-inhibiting antibodies.

A complex of polypeptides derived from a precursor is present on the surface of the malaria merozoite. During erythrocyte invasion only a small fragment from this complex is retained on the parasite surface and carried into the newly infected red cell. Antibodies to this fragment will interrupt invasion.

The N-terminal amino acid sequences of the Plasmodium falciparum (FCB1) merozoite surface antigens of 42 and 36 kilodalton, both derived from the 185-195-kilodalton precursor.

The N-terminal amino acid sequences of the 42- and 36-kDa Plasmodium falciparum (strain FCB1) merozoite surface polypeptides, both processing fragments from the 185-195-kDa polymorphic glycoprotein, have been obtained. The N-terminus of the 42-kDa fragment is located in the amino acid sequence of the precursor molecule at amino acid residue 1255 (numbering according to Mackay, M., Goman, M., Bone, N., Hyde, J.E., Scaife, J., Certa, U., Stunnenberg, H. and Bujard, H. (1985) EMBO J. 4, 3823-3829). The peptide bond cleaved during processing is Glu-Ala. This fragment is derived from the C-terminal end of the precursor molecule. The N-terminus of the 36-kDa fragment is located in the precursor molecule at amino acid residue 902 (numbering as above), and the bond cleaved is Asn-Asp.

46-53 kilodalton glycoprotein from the surface of Plasmodium falciparum merozoites.

A 46-53 kDa glycoprotein antigen of Plasmodium falciparum merozoites has been identified using a murine monoclonal antibody that inhibits infection of human erythrocytes in vitro. Immunofluorescence screening with the antibody of greater than 250 isolates of the parasite finds the inhibitory epitope expressed by only 18% of strains. The glycoprotein is metabolically labelled with methionine, cysteine, histidine and glucosamine but incorporates little lysine or leucine. It is synthesized early in schizogony and remains, without any apparent processing, on the surface of released merozoites where it is demonstrated by immuno-electronmicroscopy and also by vectorial radio-iodination.

Purification and identification of a neutral endopeptidase in Plasmodium falciparum schizonts and merozoites.

An endopeptidase specific to the Plasmodium falciparum erythrocytic schizont stage and to free merozoites was detected using the fluorogenic GlcA-Val-Leu-Gly-Lys(or Arg)-AEC substrate. The enzyme was purified by high performance liquid chromatography (HPLC); its optimal activity was around pH 7.5 and its isoelectric point was 4.4. The molecular weight of the enzyme was about 68,000, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The endopeptidase was strongly inhibited by thiol proteinase inhibitors, leupeptin, and antipain. The possible involvement of this neutral endopeptidase in the reinvasion process is discussed.

Isolation and functional characterization of Plasmodium falciparum merozoite antigens.

Merozoite surface proteins are thought to play an important role during the invasion of red blood cells by merozoites. In this article the strategies for the chromatographic isolation and for the functional and molecular characterisation of isolated antigens from freshly harvested Plasmodium falciparum merozoites from cultures are described.

A 46,000 dalton Plasmodium falciparum merozoite surface glycoprotein not related to the 185,000-195,000 dalton schizont precursor molecule: isolation and characterization.

A 46,000 dalton glycoprotein was isolated by extraction of freshly harvested P. falciparum merozoites (FCB1 strain), followed by gel electrophoresis of the extract and electroelution. The antigen is present in the late ring, trophozoite, schizont, and segmenter stages and is localized on the merozoite surface at the end of schizogony. It is not related to the 185,000-195,000 dalton schizont antigen. An antiserum against the 46,000 dalton antigen inhibits invasion of erythrocytes by merozoites. The isolated antigen is identical to the antigen against which monoclonal antibody (mcab) 13.4 is directed.

Fragments of the polymorphic Mr 185,000 glycoprotein from the surface of isolated Plasmodium falciparum merozoites form an antigenic complex.

Merozoites of the human malaria parasite Plasmodium falciparum express on their surface several antigens derived from a polymorphic glycoprotein precursor of Mr 185,000 synthesised earlier on by trophozoites and schizonts. A panel of 18 monoclonal antibodies against a range of different specificities of the precursor was used to characterise its mature products in spontaneously released merozoites. Merozoites released by [35S]methionine or [14C]glucosamine-labelled schizonts, or surface 125I-labelled purified merozoites, were extracted in detergents, and the antigens were detected by immunoprecipitation or Western blotting. We show that a nonglycosylated peptide of Mr 80,000 and two glycosylated fragments of Mr 40,000 and Mr 16,000, all derived from the precursor, are exposed on the surface of the mature merozoite. Precipitations from extracts in different detergents indicate that the 80 and 40 kDa fragments can form a non-covalent complex with each other and two additional major surface antigens of 36 and 22 kDa. Several antibodies react strongly with the complex but not with its dissociated subunits, thus indicating presence of conformational epitopes. Other epitopes are positively mapped on different dissociated subunits by immunoprecipitation and Western blotting. The 80 and 40 kDa antigens each carry a different polymorphic marker epitope, and both of these markers are absent on the 16 kDa fragment. The 40 and 16 kDa glycoproteins share common epitopes, and the latter may be derived from the former fragments. Only epitopes present on the 16 kDa antigen, but not those specific for the larger fragments, are detectable by immunofluorescence in the ring-stage. This indicates that the whole or a part of the 16 kDa antigen remains on the parasite through the invasion process.

Isolation and characterization of the 80,000 dalton Plasmodium falciparum merozoite surface antigen.

The 80,000 dalton merozoite surface protein, derived from the 185,000-195,000 dalton schizont precursor, was isolated from detergent extracts of naturally-released merozoites using chromatographic procedures. A rabbit antiserum raised against this antigen was used for characterizing the 80,000 dalton fragment. The antiserum did not inhibit merozoite invasion or parasite growth in vitro, suggesting that this merozoite surface antigen is not directly involved in the invasion process of the merozoite into the host erythrocyte.

The relationship to knobs of the 92,000 D protein specific for knobby strains of Plasmodium falciparum.

A 92,000 D protein was identified associated with the membrane of host erythrocytes infected with the FCB1 Plasmodium falciparum strain from Colombia. The same protein was identified in the knob-forming Gambian (and the Malayan Camp) strain, but was not present in all the corresponding knobless strains. In the FCB1 strain as well as in the FCR3 strain the protein is synthesized during the ring-stage period. The cleavage products of the 92,000 D protein were investigated by peptide mapping following limited proteolytic digestion with Staphylococcus aureus V8 protease. The 92,000 D protein cleavage products from both the Colombian and the Gambian strains were identical. Moreover, both the proteins were sensitive to trypsin and chymotrypsin and also to treatment with neuraminidase. Enzymatic removal of the protein from the erythrocyte membrane by trypsin or chymotrypsin did not affect parasite maturation. The merozoites thus produced were fully invasive and the morphology of the knobs was unaltered. When the erythrocyte membrane was treated with trypsin before the time of synthesis of the 92,000 D protein, it was not possible to identify the protein in membranes of later stages of infected erythrocytes, indicating that the protein cannot be inserted into the membrane cytoskeleton compartment. Knobs, however, were formed more or less normally, suggesting that it is not the accumulation of this protein which products the knobs.