Inhibit Merozoite Invasion Research Articles

Antibodies to a conserved-motif peptide sequence of the Plasmodium falciparum thrombospondin-related anonymous protein and circumsporozoite protein recognize a 78-kilodalton protein in the asexual blood stages of the parasite and inhibit merozoite invasion in vitro.

Athrombospondin-related anonymous protein (TRAP) of the human malaria parasite Plasmodium falciparum shares highly conserved amino acid sequence motifs with the circumsporozoite protein of all plasmodia sequenced so far, as well as with unrelated proteins like thrombospondin and properdin. Although it was first described as an asexual blood stages protein, there has been some controversy about its expression in these stages. Pursuant to our interest in the conserved sequences within the malaria antigens, we synthesized an 18-residue peptide (18-mer) representing a conserved motif of TRAP and raised polyclonal antibodies against it. In an immunoblot assay in which we probed proteins from the asexual blood stages of the parasite, we found that this antibody recognized predominantly a 78-kDa protein in the whole parasite lysate. Furthermore, in another immunoblot, the recombinant TRAP constructs containing the conserved-motif sequence were distinctly recognized by the antipeptide antibodies, whereas a construct lacking the motif sequence was not, suggesting that the antibodies specifically cross-reacted with a protein which might be a TRAP-like protein present in the asexual blood stages of the parasite. Also, in an immunofluorescence assay, this antibody brightly stained the acetone-fixed trophozoites of the parasite. Most significantly, anti-18-mer immunoglobulin G, as well as antipeptide antibody against a smaller (nonamer) construct representing the most conserved motif within the 18-mer, inhibited the merozoite invasion of erythrocytes in a dose-dependent manner. These results provide evidence of the expression of TRAP or a TRAP-like protein in the asexual blood stages of the parasite and of a possible role of the conserved motifs in the parasite-host cell interaction during the process of invasion.

Plasmodium falciparum:Differential Parasite Growth Inhibition Mediated by Antibodies to the Antigens Pf332 and Pf155/RESA

Plasmodium falciparum-reactive antibodies can inhibit growth of parasite blood stagesin vitroby interfering at different stages of parasite development. Antibodies reactive with repeat sequences in Pf155/ring-infected surface antigen (Pf155/RESA) or in antigen Pf332 inhibit parasite growth as determined by a reduction of newly infected erythrocytes. Antibodies to Pf155/RESA have been implicated in merozoite invasion inhibition. Since it was considered unlikely that antibodies to the late stage antigen Pf332 act on merozoite invasion, we investigated at what developmental stage this growth inhibition takes place. Parasites were cultured in the presence of antibodies to repeat sequences of either Pf332 or Pf155/RESA and were examined with regard to the distribution of different stages and their morphology. As expected, antibodies to both antigens decreased the number of ring stages after reinfection. They did not affect the intraerythrocytic development of ring stages or trophozoites. However, antibodies to Pf332 induced high levels of schizonts displaying an abnormal morphology while antibodies to Pf155/RESA induced considerably lower levels of degenerated schizonts. Thus, Pf332-reactive antibodies seem to interfere with the schizont development by blocking the rupture of mature schizonts or, alternatively, by interfering intraerythrocytically with late stage parasites.

The effects of bistratene A on the development of Plasmodium falciparum in culture

The effects of the marine ascidian compound bistratene A on in vitro cultures of Plasmodium falciparum were assessed. Concentrations from 0 to 1.5 μg ml −1 of the compound were tested. The parasitaemia in asynchronous cultures treated with bistratene A increased normally over the first 40 h, then decreased leaving only gametocytes. When synchronized cultures were treated with a constant dose of 50 ng ml −1, gametocytes developed more rapidly than they did in control cultures. In addition, gametocytes developed in drug-treated cultures of P. falciparum that normally do not produce gametocytes. Bistratene A appears to inhibit merozoite invasion as well as to induce gametocytogenesis.

Immunogenicity in rabbits and monkeys of influenza ISCOMs conjugated with repeated sequences of the Plasmodium falciparum antigen Pf155/RESA.

Rabbits and monkeys were immunized with two fusion proteins, ZZ-M3 and ZZ-M5, coupled to pre-formed influenza virus membrane glycoprotein ISCOMs. The fusion proteins comprise two IgG-binding domains from staphylococcal protein A (ZZ) and repeated amino acid sequences from the C-terminal (M3) or central (M5) repeat regions of the Plasmodium falciparum antigen Pf155/RESA. The induced antibody responses were of long duration, could be efficiently boosted and were comparable to those obtained with Freund's Adjuvant. The produced antibodies reacted with M3, M5, protein A and the influenza glycoprotein, recognized Pf155/RESA and inhibited merozoite invasion in vitro. These results suggest that coupling of immunogens to pre-formed ISCOMs may be a basis for construction of multivalent subunit vaccines.

Effect of different fractions of heparin on Plasmodium falciparum merozoite invasion of red blood cells in vitro.

Heparin and various heparin fractions were separated according to differences in molecular weight or affinity for antithrombin III and used for the inhibition of Plasmodium falciparum merozoite invasion of red blood cells in vitro. No variation in sensitivity to heparin was found among the four strains of P. falciparum tested; all required approximately 5 micrograms/ml (0.5 U/ml) of heparin for 50% inhibition of invasion. The most efficient fraction of heparin was the one with low affinity for antithrombin III. Its 50% inhibition concentration was 1 microgram/ml, indicating that it was more efficient than unfractionated heparin and other heparin fractions. The effect of heparin was reversible, since washing of heparin-treated cultures containing mainly schizonts showed no inhibition of merozoite invasion. The results suggest that a heparin fraction with no anticoagulant effect might be useful in the treatment of patients with falciparum malaria.

Involvement of Pf155/RESA and cross-reactive antigens in Plasmodium falciparum merozoite invasion in vitro

Lines of Plasmodium falciparum FCR3 either expressing or not expressing the blood-stage antigen Pf155/RESA were used to analyze the possible involvement of this antigen in the merozoite invasion process in vitro. Antibodies from human sera, affinity purified on synthetic peptides corresponding to C-terminal repeated sequences in Pf155/RESA, were shown to inhibit merozoite invasion of both types of parasites with similar efficiency. Reversal of the invasion inhibition by fusion proteins containing repeated sequences of Pf155/RESA but not of the cross-reactive antigens Ag332 and Pf11.1 indicated that the inhibitory antibodies had similar target antigens in both Pf155/RESA+ and Pf155/RESA- parasites that involved cross-reacting epitopes present in Pf155/RESA. Rabbit antibodies specific for Pf155/RESA repeats inhibited merozoite invasion of Pf155/RESA expressing parasites efficiently but had no or very small effect on the invasion of Pf155/RESA-deficient parasites. In contrast, rabbit antibodies specific for Ag332 repeats as well as human antibodies affinity purified on synthetic Ag332 peptides inhibited merozoite invasion of both types of parasites with high efficiency. A similar inhibition pattern was seen with the human monoclonal antibody 33G2, which has specificity for Ag332 but also cross-reacts with Pf155/RESA and Pf11.1. Taken together, our data suggest that Pf155/RESA and related cross-reactive antigens as well as Ag332 are involved in the merozoite invasion process and may constitute targets for invasion inhibitory antibodies.

Plasmodium falciparum: An invasion inhibitory human monoclonal antibody is directed against a malarial glycolipid antigen

A Plasmodium falciparum malaria blood stage antigen was detected using a human monoclonal antibody (MAb A52A6) obtained from a clinically immune donor. Immunofluorescence analysis showed that the MAb reacted with the intracellular parasite throughout the asexual blood stage cycle as well as with gametocytes. The MAb also reacted with the surface of erythrocytes containing late stage P. falciparum parasites. The antigen seen by the MAb was species- but not strain- or isolatespecific. At rupture of the infected erythrocytes, antigenic material was deposited on the membrane of uninfected cells surrounding the parasite. At merozoite invasion MAb reactive material was present on the invaginating erythrocyte membrane, indicating an involvement of the antigen in the invasion process. This was also indicated by the high capacity of the MAb to inhibit merozoite invasion in vitro. The antigen appears to be a phosphoglycolipid, sensitive to phospholipase and present in lipid extracts of P. falciparum-infected erythrocytes.

Lack of correlation between red cell invasion by merozoites and anti-heat shock protein-70 antibody levels in malaria patients' sera

A total of 172 sera samples were collected from individuals who were living in Piyawli-Jaitwarpur village in Ghaziabad district (U.P.), India. They had suffered from falciparum malaria attack, and were cured with antimalarial drugs 1–2 weeks prior to sample collection. These samples were divided into nine groups according to their age. The pooled sera from each group were tested for the presence of anti-schizont and anti-heat shock protein (hsp) −70 antibodies, as well as for parasite growth inhibition in vitro. All sera samples showed significant levels of antibodies against schizont antigens and these levels increased with age. The sera also contained anti-hsp-70 antibodies but at lower levels and did not follow the same age-related pattern as seen with schizont antibodies. The sera from each group significantly inhibited merozoite invasion in vitro. However the same was not true for other blood stage parasites; the 2–15 years age group sera did not show significant growth inhibition of rings, trophozoites and schizonts. No correlation was observed between anti-hsp-70 antibody levels and inhibition of merozoite invasion. It is therefore concluded that the antibodies preventing the merozoite invasion could be other than anti-hsp-70 antibodies. The candidature of hsp-70 for P. falciparum malaria vaccine thus needs to be re-evaluated.

Passive immunization of Aotus monkeys with human antibodies to the Plasmodium falciparum antigen Pf155/RESA

In order to assess the protective effects of anti-Pf155/RESA antibodies of different specificities in vivo, passive immunizations of Aotus monkeys were performed. Antibodies reactive with the Pf155/RESA repeat sequences (EENV)2 and EENVEHDA were isolated from the immunoglobulin G (IgG) fraction of a pool of plasmas from Liberia by affinity chromatography on synthetic peptides. The two fractions of antibodies differed in specificity but displayed similar capacities to inhibit merozoite invasion in Plasmodium falciparum in vitro cultures. Four groups of monkeys (named groups I to IV) were injected with (i) 160 mg of total control IgG, (ii) 2 mg of IgG affinity purified on (EENV)2, (iii) 2 mg of IgG affinity purified on EENVEHDA, and (iv) 160 mg of total immune IgG, respectively. The monkeys were then challenged with P. falciparum-infected erythrocytes, and the levels of parasitemia and hematocrits as well as other serological parameters were determined daily. Although all groups developed parasitemia, groups II and IV tended to show lower mean daily levels. Three monkeys of group II and two monkeys (each) of groups III and IV self cured the infections, but so did one monkey from the group treated with control IgG (group I). The serum levels of transfused antibodies were low at the peak of parasitemia, suggesting that clearance of parasites was mediated by immune responses mounted by the monkeys. The results indicate that antibodies to epitopes formed by repeats of Pf155/RESA may depress P. falciparum parasitemias and thus that immunogens based on such repeats should be suitable components in a subunit vaccine against asexual stages of P. falciparum.

Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion.

The Plasmodium falciparum gene encoding erythrocyte binding antigen-175 (EBA-175), a putative receptor for red cell invasion (Camus, D., and T. J. Hadley. 1985. Science (Wash. DC). 230:553-556.), has been isolated and characterized. DNA sequencing demonstrated a single open reading frame encoding a translation product of 1,435 amino acid residues. Peptides corresponding to regions on the deduced amino acid sequence predicted to be B cell epitopes were assessed for immunogenicity. Immunization of mice and rabbits with EBA-peptide 4, a synthetic peptide encompassing amino acid residues 1,062-1,103, produced antibodies that recognized P. falciparum merozoites in an indirect fluorescent antibody assay. When compared to sera from rabbits immunized with the same adjuvant and carrier protein, sera from rabbits immunized with EBA-peptide 4 inhibited merozoite invasion of erythrocytes in vitro by 80% at a 1:5 dilution. Furthermore, these sera inhibited the binding of purified, authentic EBA-175 to erythrocytes, suggesting that their activity in inhibiting merozoite invasion of erythrocytes is mediated by blocking the binding of EBA-175 to erythrocytes. Since the nucleotide sequence of EBA-peptide 4 is conserved among seven strains of P. falciparum from throughout the world (Sim, B. K. L. 1990. Mol. Biochem. Parasitol. 41:293-296.), these data identify a region of the protein that should be a focus of vaccine development efforts.

In Vitro Growth Inhibition of Plasmodium falciparum by Sera from Different Regions of the Philippines

Sera from different malaria endemic regions of the Republic of the Philippines were compared for their ability to inhibit growth of Plasmodium falciparum in vitro. Dialyzed serum was added to synchronous cultures containing schizonts for either the total 48 hr test period or only the last 24 hr in order to analyze the effects on erythrocytic invasion and intraerythrocytic growth, respectively. Reduction in 3H-hypoxanthine uptake was used to determine the percent of inhibition compared to nonimmune serum. One hundred seventy sera from Mindanao and Palawan in the South, the centrally located island of Mindoro, and Luzon in the North, were tested against 4 P. falciparum strains from the Philippines and 1 from Africa. Indirect fluorescent antibody titers were not predictive of inhibition. Inhibition of merozoite invasion rather than intraerythrocytic parasite growth is suggested by this study. Generally, sera were more inhibitory to parasite strains from the same geographical area than to those from more remote areas.

Sixty‐six kilodalton‐related antigens of Plasmodium knowlesi are merozoite surface antigens associated with the apical prominence

We have previously reported that Fab fragments of a monoclonal antibody to the 66 kD-related antigens of Plasmodium knowlesi inhibit merozoite invasion of erythrocytes, strongly suggesting a role for these antigens as merozoite receptors for red cells. In this paper we have examined the distribution of these antigens on the surface of free merozoites. Rapid immunofluorescence microscopy on free, unfixed cells demonstrated a preferential association of antigen with a polar region of merozoites. This was confirmed, and localized essentially to the apical region, by immunoelectron microscopy under a variety of fixation regimes. Metabolic inhibitors did not affect the distribution, suggesting that apical localization is not due to capping induced by cross-linking antibodies. These observations suggest that 66 kD-related antigens may play a role in the orientation of the apical prominence of merozoites towards the red cell, and/or in the junction formation that occurs subsequent to orientation.

Vaccination trials in rhesus monkeys with a minor, invariant, Plasmodium knowlesi 66 kD merozoite antigen.

A minor Plasmodium knowlesi 66 kD antigen, which plays an essential role in merozoite invasion, has been shown to be stable in distinct variants and strains of the parasite, and in the face of a specific immune response from the host. Parasites were unable to produce novel molecule(s) to replace it functionally, even in the presence of specific immune pressure. Rhesus monkeys immunized with the purified 66 kD antigen, with saponin as adjuvant, produced antibody which inhibited merozoite invasion of red cells in vitro. Four out of six immunized rhesus monkeys demonstrated clinically effective immunity when challenged at a time of known or presumed high inhibitory antibody titre. When immunization failed to protect, it was ascribed to insufficient levels of specific antibody attributable either to a suboptimal dose of antigen or the use of an inadequate adjuvant.

Characterization of Cross-Reactive Blood-Stage Antigens of the Plasmodium cynomolgi Complex Using Anti-Plasmodium vivax Monoclonal Antibodies

Five out of 18 monoclonal antibodies (moAB's) produced against blood stages of a Brazilian (Belem) strain of Plasmodium vivax were shown to cross-react with all of the 11 strains of the P. cynomolgi complex that were assayed. The 5 moAB's produced 3 different patterns of immunofluorescence, identical for both P. vivax and P. cynomolgi. Three of these moAB's appeared to react with antigens associated with the cytoplasm or membranes of infected erythrocytes. By Western blot analysis, 2 of these 3 moAB's identified an antigen with an apparent molecular weight of 31 kDa in extracts of parasitized erythrocytes of both species; the third of these moAB's reacted with an antigen with an apparent molecular weight of 95 kDa. By immunofluorescence, the 2 other moAB's reacted only with parasites at all developmental stages. The target antigen of these 2 moAB's was not identified. Immunoradiometric assays indicated that the moAB's are directed against 3 or possibly 4 distinct nonrepetitive epitopes. None of the moAB's inhibited merozoite invasion or growth of the parasites in an in vitro culture system of the Berok strain of P. cynomolgi.

Affinity-purified antibodies to ring-infected erythrocyte surface antigen do not correlate with merozoite invasion inhibition in Plasmodium falciparum.

We affinity purified, from malaria-immune serum, antibody to the ring-infected erythrocyte surface antigen (RESA), using petri dishes containing a monolayer of Plasmodium falciparum ring-infected erythrocytes. Except for one out of eight samples, the purified antibody positive by RESA-immunofluorescent assay was not inhibitory to the in vitro invasion of merozoites into erythrocytes in three geographically distinct strains of P. falciparum. However, the initial high level of merozoite-inhibiting antibodies of the intact serum samples remained in the immunoglobulin G fraction from which the RESA antibodies had been removed by affinity chromatography. These results suggest that, although in some cases RESA-immunofluorescent assay-positive antibodies may be inhibitory to merozoite invasion, there are more important antibodies capable of merozoite invasion inhibition.

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.

Rabbit and human antibodies to a repeated amino acid sequence of a Plasmodium falciparum antigen, Pf 155, react with the native protein and inhibit merozoite invasion.

The Plasmodium falciparum-derived antigen of Mr 155,000 designated Pf 155, deposited in the membrane of infected erythrocytes, contains at least two blocks of tandemly repeated amino acid sequences. The peptide Glu-Glu-Asn-Val-Glu-His-Asp-Ala, which corresponds to a subunit of a C-terminally located repeat, was synthesized. Rabbits immunized with the octapeptide conjugated with either keyhole limpet hemocyanine or tetanus toxoid formed antibodies against the octapeptide. These antibodies reacted with Pf 155 as detected by immunoblotting or a modified immunofluorescence assay. Sera from humans exposed to P. falciparum also contained antibodies binding to the octapeptide in a dot-blot immunoassay. Their anti-octapeptide titers were correlated with their immunofluorescence titers in the assay detecting Pf 155 and other parasite antigens in the membrane of infected erythrocytes. Human octapeptide-reactive antibodies were isolated on an affinity column with the octapeptide conjugated to bovine serum albumin as ligand. These human antibodies reacted with Pf 155 in immunoblotting and strongly stained the surface of infected erythrocytes in the modified immunofluorescence assay. Approximately 20% of this immunofluorescence activity in a high-titered human serum could be recovered from the octapeptide column, indicating that a significant fraction of these anti-parasite antibodies react with epitopes associated with the octapeptide. Furthermore, the human octapeptide-reactive antibodies very efficiently inhibited merozoite reinvasion into erythrocytes in vitro. Similarly purified rabbit antibodies also significantly inhibited reinvasion. Our results suggest that the C-terminal segment of repeated peptides in Pf 155 is a major antigenic region of the molecule and may contain target sites for protective immunity in P. falciparum malaria.

A rhoptry antigen of Plasmodium falciparum contains conserved and variable epitopes recognized by inhibitory monoclonal antibodies

Four monoclonal antibodies produced against Plasmodium falciparum recognize an antigen in merozoites that is localized in rhoptries, as judged by a punctate, double dot fluorescence pattern. All four antibodies bound to the same affinity purified antigen in a two site immunoradiometric assay. Immunoprecipitation of antigen by monoclonal antibody followed by sodium dodecyl sulphate-polyacrlamide gel electrophoresis yielded protein bands of 80, 66 and 42 kDa. Western blotting gave bands of 80 and 66 kDa only with three of the antibodies: the fourth did not blot. Based on protease inhibitor data the 66 kDa band is considered to be a cleavage product of the 80 kDa band, but the 42 kDa band does not appear to derive from the latter and may be a coprecipitation product. This group of antigens labels with both [ 35S]methionine and [ 3H]histidine. Two of the monoclonal antibodies inhibited merozoite invasion of erythrocytes. One of these inhibitors recognizes a variable epitope, whereas the second recognizes a highly conserved epitope present in all 106 primary isolates of P. falciparum tested from Brazil, Thailand and Papua New Guinea.

Isolation and Cultivation of Plasmodium falciparum Using Adult Bovine Serum

RPMI 1640 medium supplemented with adult bovine serum and hypoxanthine was superior to human serum-supplemented medium for the isolation of new strains of Plasmodium falciparum in Sudan. Similar observations in Indonesia have since confirmed our results. The chloroquine sensitivity of new isolates was identical in either human or bovine serum. Once acclimated to culture conditions P. falciparum strains grew better when using human serum. Erythrocyte-specific antibody present in adult bovine serum slightly inhibited merozoite invasion of uninfected cells. Removal of this cross-reactive antibody from bovine serum increased parasite multiplication to the level obtained in human serum.

Characterization with monoclonal antibodies of a surface antigen of Plasmodium falciparum merozoites.

The merozoite, the extracellular form of the erythrocyte stage of the malarial parasite, invades the erythrocyte and develops intracellularly. Cloned hybridoma cell lines secreting monoclonal antibodies directed against the merozoite surface were selected by indirect immunofluorescent assay by using intact isolated merozoites. Monoclonal antibodies to a 200,000 m.w. merozoite surface antigen were selected and were used to characterize this protein and its role in erythrocyte invasion. Immunoelectron microscopy demonstrated that the antigen was located exclusively on the merozoite surface coat, distributed evenly over the entire surface. The 200,000 m.w. protein incorporated [3H]glucosamine, suggesting that it is a glycoprotein and could be purified to homogeneity by using immuno-affinity chromatography. Freshly isolated, invasive merozoites retained the 200,000 m.w. antigen, but the protein was rapidly cleaved to proteins of 90,000 and 50,000 m.w. when the merozoite was extracellular. The 50,000 m.w. fragment was retained the epitope binding to monoclonal antibody 5B1 and were labeled with [3H]glucosamine. Monoclonal antibodies against the 200,000 m.w. antigen partially inhibited merozoite invasion into erythrocytes.