Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry

BACKGROUND: Naturally induced antibodies binding to surface antigens of Plasmodium falciparum-infected erythrocytes can be detected by direct agglutination of infected erythrocytes or by indirect immunofluorescence on intact, unfixed, infected erythrocytes. Agglutinating antibodies have previously been shown to recognise Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This protein is inserted by the parasite into the host cell membrane and mediates the adhesion to the venular endothelium of the host organism in vivo. METHODS: Erythrocytes infected at high parasitaemias with ethidium-bromide-labelled mature forms of P. falciparum parasites were sequentially exposed to immune plasma, goat anti-human immunoglobulin (Ig) G, and fluorescein-isothiocyanate-conjugated rabbit anti-goat Ig. Plasma antibodies recognising antigens exposed on the surface of parasitised erythrocytes were subsequently detected by two-colour flow cytometry. RESULTS: Binding of human antibodies to the surface of erythrocytes infected with adhesive strains of Plasmodium falciparum can be measured by the two-colour flow cytometry (FCM) assay described. In addition, we demonstrate that the adhesive capacity of a parasite isolate correlates with the capacity of human immune plasmas to label the isolate as detected by FCM. We also show that the antigens recognised by the labelling antibodies are strain specific and that their molecular weights are in the range previously described for PfEMP1 antigens. CONCLUSIONS: Our FCM assay predominantly detects antibodies that recognise PfEMP1 and thus constitutes a convenient assay for the analysis of acquisition, maintenance, and diversity of anti-PfEMP1-specific antibodies and for the examination of class and subclass characteristics.     

Plasmodium fragile: detection of a ring-infected erythrocyte surface antigen (RESA)

A ring-infected erythrocyte surface antigen (RESA) has been detected by modified immunofluorescence assay in erythrocytes infected with the simian malaria parasite, Plasmodium fragile. This RESA, of Mr 95,000, shares many characteristics with the RESA initially found in the human malaria parasite P. falciparum. Both antigens are found in the membrane of erythrocytes infected with young asexual parasite stages, in merozoite-enriched preparations, and in parasite culture supernatant. Since the RESA of P. falciparum has been shown to confer protective immunity and since P. fragile infection of rhesus monkeys mimics P. falciparum infection in humans, the finding of a RESA in P. fragile underlines the importance of this species as an animal model for antimalarial vaccines.     

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 isolate-specific. 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.     

Studies of murine malaria antigens using monoclonal antibodies. Production, selection, and characterization of antibodies

A panel of ten monoclonal antibodies made against Plasmodium chabaudi and Plasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs to P. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted with P. chabaudi antigens. Of the MAbs to P. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.     

Proteases in malaria-infected red blood cells

The discrimination between erythrocyte and Plasmodium proteases is now made easier by using synthetic fluorogenic substrates, high-pressure liquid chromatography, reliable methods of cell preparation, as well as radiolabeled extracts from in vitro cultures of P. falciparum. The reinvasion process of an erythrocyte by a merozoite involves specific proteinases, which were recently identified using fluorogenic peptidyl-AEC substrates and by analysis of schizont and merozoite extracts with the gelatin-SDS-PAGE method. The biological targets of both host and parasite proteinases are not yet well characterized because Plasmodium-infected red blood cells contain at least four compartments with different pH values, which could modulate the proteinase activities according to their pH range activity. The processing of the precursor for the major merozoite surface antigens involves cleavage of very specific peptidic bonds by, so far unknown, proteinases. The depletion of the erythrocyte cytoskeleton could depend on a 37 kD proteinase, which cleaves spectrin and the 4.1 component, as shown in P. berghei and P. falciparum species. In contrast to leupeptin, which inhibits the merozoite release from schizont-infected erythrocytes, the structural inhibitor analogous to the Val-Leu-Gly-Lys (or Arg) P. falciparum neutral proteinase substrates appears to block the invasion step of erythrocytes by merozoites and may open new trends in chemotherapeutical strategies.     

Haemoglobin C and S role in acquired immunity against Plasmodium falciparum malaria

A recently proposed mechanism of protection for haemoglobin C (HbC; beta6Glu-->Lys) links an abnormal display of PfEMP1, an antigen involved in malaria pathogenesis, on the surface of HbC infected erythrocytes together with the observation of reduced cytoadhesion of parasitized erythrocytes and impaired rosetting in vitro. We investigated the impact of this hypothesis on the development of acquired immunity against Plasmodium falciparum variant surface antigens (VSA) encoding PfEMP1 in HbC in comparison with HbA and HbS carriers of Burkina Faso. We measured: i) total IgG against a single VSA, A4U, and against a panel of VSA from severe malaria cases in human sera from urban and rural areas of Burkina Faso of different haemoglobin genotypes (CC, AC, AS, SC, SS); ii) total IgG against recombinant proteins of P. falciparum asexual sporozoite, blood stage antigens, and parasite schizont extract; iii) total IgG against tetanus toxoid. Results showed that the reported abnormal cell-surface display of PfEMP1 on HbC infected erythrocytes observed in vitro is not associated to lower anti- PfEMP1 response in vivo. Higher immune response against the VSA panel and malaria antigens were observed in all adaptive genotypes containing at least one allelic variant HbC or HbS in the low transmission urban area whereas no differences were detected in the high transmission rural area. In both contexts the response against tetanus toxoid was not influenced by the beta-globin genotype. These findings suggest that both HbC and HbS affect the early development of naturally acquired immunity against malaria. The enhanced immune reactivity in both HbC and HbS carriers supports the hypothesis that the protection against malaria of these adaptive genotypes might be at least partially mediated by acquired immunity against malaria.     

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.     

Plasmodium vivax: malarial proteins associated with the membrane-bound caveola-vesicle complexes and cytoplasmic cleft structures of infected erythrocytes

The identification of antigens of parasite origin associated with the altered membrane of Plasmodium vivax-infected erythrocytes was undertaken in this study. The 125I-lactoperoxidase catalyzed surface radiolabeling of trophozoite-infected erythrocytes revealed new bands of 95 and 70 kDa not labeled in normal erythrocytes. Erythrocyte membrane-enriched preparations from [35S]methionine biosynthetically labeled-infected erythrocytes also indicated that in addition to bands at 95 and 70 kDa, several other parasite proteins were possibly membrane associated. Five monoclonal antibodies (Mabs) reactive with P. vivax produced an immunofluorescent pattern of numerous small dots scattered over the entire infected erythrocyte. This pattern mimics that of Schuffner's stippling; small red dots seen in Giemsa-stained P. vivax-infected erythrocytes, which represent accumulations of dye in caveola-vesicle complexes (CVC). Four of the monoclonal antibodies immunoprecipitated a Triton X-100 detergent-insoluble 95-kDa parasite protein which was localized by immunofluorescent assay and immunoelectron microscopy exclusively to the CVC. Two of these Mabs were immunofluorescence reactive with the surface of intact infected erythrocytes in suspension. The fifth Mab, which also localized exclusively to the CVC structures, immunoprecipitated a Triton X-100 extractable protein of 70 kDa. Two other monoclonal antibodies reacted exclusively with the numerous membranous cleft structures found in the cytoplasm of infected erythrocytes. This cleft-associated parasite antigen was 28 kDa in size. Some of these Mabs recognize epitopes and produce similar IFA patterns on erythrocytes infected with P. cynomolgi, P. knowlesi, and P. ovale parasites, but not with P. falciparum- or P. brasilianum-infected erythrocytes.     

The Infection of Duck and Goose Erythrocytes by the Mammalian Malaria Parasite, Plasmodium berghei

The infection of mice and baby rats by both Plasmodium lophurae , an avian parasite, and Plasmodium berghei , a mammalian malaria parasite, prompted investigation of the likelihood of P. berghei infecting avian erythrocytes. Though erythrocytes of chick embryos were not infected, those of the goose and duck embryos were. In both these cells the morphology of the parasite was markedly different from that seen in mammalian erythrocytes. Infections were transitory and it was impossible to find parasites after 4 days. Examination of the hosts of both species of parasites showed a rather wide range and examination of the susceptibility of the duck erythrocyte indicated that this cell was peculiarly receptive to infection by a variety of plasmodia.     

Plasmodium berghei: immunosuppression of the cell-mediated immune response induced by nonviable antigenic preparations

In this work, plasmodial antigens were examined for their ability to suppress the cellular immune response during lethal Plasmodium berghei infection. Splenic enlargement and the number and function of white spleen cells were assessed after injection of normal mice with irradiated parasitized erythrocytes (IPE) or with parasitized erythrocytes (PE) membranes. Both IPE and PE membranes caused splenomegaly and an increase in the number of splenic white cells with concurrent alteration of the relative proportions of T cells and macrophages. The percentage of T lymphocytes was fractionally diminished, but there was a marked increase in Lyt 2.2 positive (suppressor and cytotoxic) T subsets and in the number of splenic macrophage precursors. The pathological enlargement of the spleen was induced by various plasma membrane-derived antigens containing both proteins and carbohydrates. Splenocytes of mice injected with liposomes containing deoxycholate-treated PE or PE fractions showed both diminished interleukin 2 production and a decreased response to mitogen. It appears that some of the changes in the cellular immune response during P. berghei infection are a consequence of the massive provision of a wide spectrum of antigens, capable of suppressing the immune response. Thus, it may be appropriate to evaluate the possible negative effect of parasite epitopes that are candidates for vaccine.     

Plasmodium berghei and Plasmodium chabaudi: A neutral endopeptidase in parasite extracts and plasma of infected animals

By using a sensitive fluorometric method with Val-Leu-Gly-Arg-3-amino-9-ethylcarbazole (VLGR-AEC) as a substrate, two endopeptidase activities were identified in two fractions of Sephacryl S-200 gel filtration from soluble P. berghei and P. chabaudi extracts. Controls with normal mouse erythrocytes, with leukocytes, and with reticulocyte enriched blood and different washing procedures during the preparation of soluble P. berghei extracts showed that the MW >200 kDa fraction was a contaminant from erythrocytes and exhibited an optimal pH activity of 8.2. In contrast, the fraction 130 kDa was related to P. berghei and P. chabaudi and exhibited an optimal pH activity of 7.4. The two enzyme activities were compared with eight different substrates. The parasite endopeptidase showed a strong activity with Val-Leu-Gly-Lys-AEC (VLGK-AEC) and Ser-Gly-Lys-AEC (SGK-AEC) as substrates; in contrast, the mouse host endopeptidase poorly cleaved the VLGK-AEC and did not cleave SGK-AEC. Presence of the hydrophobic benzyl group on serine reduced the hydrolizing properties of P. berghei endopeptidase: the reverse was observed with host endopeptidase. The hydrolysis of the N-polyhydroxyalcanoyl-VLGK-AEC substrate by the parasite neutral endopeptidase strongly increased with the schizogonic stage, as shown with synchronized P. chabaudi in mice. By its physiological pH and specificity the release of this enzyme in mouse plasma during the infection could be of interest in a peptidyl-drug Strategy.     

Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions

Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium-infected erythrocytes, the contribution of lipid-mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol-rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium-infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.     

Pantothenic Acid Metabolism During Avian Malaria Infection: Pantothenate Kinase Activity in Duck Erythrocytes and in Plasmodium lophurae*

SYNOPSIS. The initial enzymatic reaction in the pathway of coenzyme A genesis from pantothenic acid, i.e., the conversion of pantothenic acid to phosphopantothenic acid catalyzed by pantothenic acid kinase, was found in extracts of avian erythrocytes but not in extracts of Plasmodium lophurae or intact parasites. The activity of the kinase was significantly higher in extracts prepared from normal duck erythrocytes than those infected with P. lophurae. The apparent absence of pantothenic acid kinase in P. lophurae is corroborative support for nutritional studies(7,9) which suggest that the avian malarial parasite is dependent for its supply of coenzyme A on the host cell.     

Histo-blood group p: biosynthesis of globoseries glycolipids in EBV-transformed B cell lines

The genetic and biosynthetic basis of the histo-blood group P-system is not fully understood. Individuals with the rare p phenotype do not express the three glycolipid antigens (P^k, P and P₁) of this system, probably because of deficiencies in glycosyltransferases involved in their biosynthesis. Iiukaet al. [Iiuka S, Chen SH, Yoshida A (1986)Biochem Biophys Res Commun 137: 1187–95], however, previously reported that detergent extracts from an EBV-transformed B cell line derived from a p individual did express the glycosyltransferase activity (P^k transferase) assumed to be missing in this blood group status. Here, we have reinvestigated the antigen expression and glycosyltransferase activities in two p individuals by analysing EBV-transformed cell lines as well as erythrocytes to confirm the blood group P status. The thin layer chromatography glycolipid profile of extracts from erythrocytes and EBV-transformed B cell lines showed characteristic accumulation of lactosylceramide and absence of P^k and P antigens. Glycosyltransferase activities of the B cell lines were analysed using glycolipid substrates and both extracts were found to contain lactosylceramide synthetase and P transferase activities but to be completely devoid of P^k transferase activity. The presented data indicate that p individuals, in contrast to previous reports, do not express a functional P^k glycosyltransferase.Dedicated to Professor S. Hakomori in the occasion of his 65th birthday from two of his past posdoc's.     

Plasmodium lophurae: Antibody-induced movement and capping of surface membranes of erythrocyte-free malarial parasites

Surface antigens of the avian malarial parasite, Plasmodium lophurae, and its host cell, the duckling erythrocyte, were visualized at the ultrastructural level using rabbit antisera and ferritin-labeled goat anti-rabbit IgG. Rabbit antisera to P. lophurae caused an aggregation of parasite and parasitophorous vacuole surface membrane antigens, a phenomenon known as capping. Capping required living plasmodia and did not occur if parasites had been fixed with glutaraldehyde prior to exposure to antisera. Antisera against duckling erythrocytes did not cross-react with erythrocyte-free malarial parasites, and did not form caps on the surface of the red blood cell. Antiplasmodial sera did not react with normal or malaria-infected red cells. These results suggest that surface membrane proteins of the intracellular plasmodium are capable of lateral movement.     

Induction of protective immunity against Schistosoma mansoni by a non living vaccine. I. Partial characterization of antigens recognized by antibodies from mice immunized with soluble schistosome extracts

A single intradermal injection of frozen and thawed schistosomula in conjunction with the bacterial adjuvant Mycobacterium bovis strain Bacille Calmette Guerin, Phipps substrain (BCG) induced significant levels of resistance to challenge Schistosoma mansoni infection in C57BL/6 mice. Immunization with the aqueous fraction remaining after 100,000 X G centrifugation of the larval lysate was also protective under these conditions, suggesting that some immunogenic determinants may not be membrane associated. Frozen-thawed cercariae and soluble components of adult worms also protected against challenge infection in these experiments. These observations indicate that soluble immunogens are present in both early and late developmental stages of the parasite, and therefore may be good candidate antigens for an immunochemically defined vaccine against schistosomiasis. Induction of humoral reactivity against soluble or membrane antigens was examined in mice protected against cercarial challenge by prior exposure to frozen-thawed larvae, soluble larval, or soluble adult antigens plus BCG. Animals that were immunized with frozen-thawed larvae produced low but significant levels of antibodies against larval surface antigens when examined by indirect immunofluorescence or by immunoprecipitation of surface-labeled schistosomula. Mice immunized with soluble antigens, however, showed negligible antibody reactivity against surface membrane antigens. Because mice immunized with soluble antigens were resistant to challenge infection, these results strongly suggest that anti-surface membrane reactivity is not required in the mechanism of protective immunity in this model. Sera from mice immunized with either total freeze-thaw larval lysate or soluble schistosome extracts all showed strong reactivity against soluble antigens, as detected by ELISA. Western blot analysis showed these antisera to react with a restricted number of high m.w. antigens that were present both in schistosomula and in adult worms. These antigens are therefore likely to play a major role in the development of resistance in this model as immunogens and/or as targets of protective immune response.     

The Plasmodium falciparum heat shock protein 40, Pfj4, associates with heat shock protein 70 and shows similar heat induction and localisation patterns

Human cerebral malaria is caused by the protozoan parasite Plasmodium falciparum, which establishes itself within erythrocytes. The normal body temperature in the human host could constitute a possible source of heat stress to the parasite. Molecular chaperones belonging to the heat shock protein (Hsp) class are thought to be important for parasite subsistence in the host cell, as the expression of some members of this family has been reported to increase upon heat shock. In this paper we investigated the possible functions of the P. falciparum heat shock protein DnaJ homologue Pfj4, a type II Hsp40 protein. We analysed the ability of Pfj4 to functionally replace Escherichia coli Hsp40 proteins in a dnaJ cbpA mutant strain. Western analysis on cellular fractions of P. falciparum-infected erythrocytes revealed that Pfj4 expression increased upon heat shock. Localisation studies using immunofluorescence and immuno-electron microscopy suggested that Pfj4 and P. falciparum Hsp70, PfHsp70-1, were both localised to the parasites nucleus and cytoplasm. In some cases, Pfj4 was also detected in the erythrocyte cytoplasm of infected erythrocytes. Immunoprecipitation studies and size exclusion chromatography indicated that Pfj4 and PfHsp70-1 may directly or indirectly interact. Our results suggest a possible involvement of Pfj4 together with PfHsp70-1 in cytoprotection, and therefore, parasite survival inside the erythrocyte.     

Analysis of Cross-Reactive Antigens of Phytophthora fragariae and Strawberry and their Relation to Resistance and Susceptibility

The presence of cross-reactive antigens between five isolates of P, fragariae (Pf 1, Pf 2, Pf 3, Pf 10 and Pf 11) belonging to five physiological races of the fungus and five strawberry cultivars (Cambridge Favourite, Hapil, Red Gauntlet, Siletz and 52AC18) exhibiting different disease responses to the five isolates was demonstrated by Western blotting. Antiserum anti-H, raised against extracts of healthy Cambridge Favourite roots, detected two antigens which were common to all isolates. Concentration of one of these antigens might be related to the pathogenicity of P. fragariae isolates. Antiserum anti-PfM, raised against mycelial suspensions from the five isolates, detected a doublet of 64 and 61 kDa in the soluble extracts of healthy roots from the five cultivars tested. The corresponding root suspensions revealed numerous other antigens which reacted with anti-PfM. In susceptible interactions, the doublet described above and two additional polypeptides of 38 and 31 kda were detected in large concentrations in both the soluble extracts and root suspensions. These four polypeptides were shown to be present in the healthy roots as well as in the mycelial extracts. In resistant interactions, a 116 kDa polypeptide, present in the root suspensions of the healthy host, was detected in the soluble extracts of the infected roots. It is suggested that these antigens might have a role in resistance and susceptibility.     

Structural alteration of the membrane of erythrocytes infected with Plasmodium falciparum

Human erythrocytes infected with five strains of Plasmodium falciparum and Aotus erythrocytes infected with three strains of P. falciparum were studied by thin-section and freeze-fracture electron microscopy. All strains of P. falciparum we studied induced electron-dense conical knobs, measuring 30-40 nm in height and 90-100 nm in diameter on erythrocyte membranes. Freeze-fracture demonstrated that the knobs were distributed over the membrane of both human and Aotus erythrocytes. A distinct difference was seen between the intramembrane particle (IMP) distribution over the knobs of human and Aotus erythrocyte membranes. There was no change in IMP distribution in infected human erythrocyte membranes, but infected Aotus erythrocytes showed an aggregation of IMP over the P face of the knobs with a clear zone at the base. This difference in IMP distribution was related only to the host species and not to parasite strains. Biochemical analysis demonstrated that a higher proportion of band 3 was bound to the cytoskeleton of uninfected Aotus erythrocytes than uninfected human erythrocytes after Triton X-100 extraction. This may account for the different effects of P. falciparum infection on IMP distribution in the two different cell types.