A malaria serological map indicating the intersection between parasite antigenic diversity and host antibody repertoires.

A malaria vaccine targeting Plasmodium falciparum remains a strategic goal for malaria control. If a polyvalent vaccine is to be developed, its subunits would probably be chosen based on immunogenicity (concentration of elicited antibodies) and associations of selected antigens with protection. We propose an additional possible selection criterion for the inclusion of subunit antigens; that is, coordination between elicited antibodies. For the quantitative estimation of this coordination, we developed a malaria serological map (MSM). Construction of the MSM was based on three categories of variables: (i) malaria antigens, (ii) total IgG and IgG subclasses, (iii) different sources of plasma. To validate the MSM, in this study, we used four malaria antigens (AMA1, MSP2-3D7, MSP2-FC27 and Pf332-C231) and re-grouped the plasma samples into five pairs of subsets based on age, gender, residence, HbAS and malaria morbidity in 9 years. The plasma total IgG and IgG subclasses to the test antigens were measured, and the whole material was used for the MSM construction. Most of the variables in the MSM were previously tested and their associations with malaria morbidity are known. The coordination of response to each antigens pair in the MSM was quantified as the correlation rate (CR = overall number of significant correlations/total number of correlations × 100 %). Unexpectedly, the results showed that low CRs were mostly associated with variables linked with malaria protection and the antigen eliciting the least CRs was the one associated with protection. The MSM is, thus, of potential value for vaccine design and understanding of malaria natural immunity.

Pattern of pre-existing IgG subclass responses to a panel of asexual stage malaria antigens reported during the lengthy dry season in Daraweesh, Sudan.

The anti-malarial IgG immune response during the lengthy and dry season in areas of low malaria transmission as in Eastern Sudan is largely unknown. In this study, ELISA was used for the measurement of pre-existing total IgG and IgG subclasses to a panel of malaria antigens, MSP2-3D7, MSP2-FC27, AMA-1 and Pf332-C231. The results showed that the antibody responses were predominantly age dependent, antigen specific, and their lifespan was at least 5-6 month long. Generally, the IgG3 was most abundant IgG subclass, and the most recognized antigen was Pf332-C231. Furthermore, the correlation between the levels of IgG subclasses was strongest between IgG1 and IgG3, which were more predictive to the total IgG levels. Finally, the response pattern of each of the IgG subclasses to the different test antigens that were spanning the dry season and the correlation between these responses were described in details for the first time.

Antibodies to the Plasmodium falciparum rhoptry protein RAP-2/RSP-2 in relation to anaemia in Cameroonian children.

Previous studies have implicated reactive antibodies to the low molecular weight rhoptry-associated proteins (RAP-1, RAP-2/RSP-2 and RAP-3) in erythroid cell destruction during Plasmodium falciparum infection. In this pilot study, the frequency, specificity and functional capacity of naturally acquired anti-RAP-2/RSP-2 antibodies were investigated in the sera of anaemic and nonanaemic malaria-infected Cameroonian children. All sera recognized RAP-2/RSP-2 by FACS, irrespective of the clinical status of the subjects. However, the anaemic children showed higher levels of IgG antibodies than the nonanaemic group, while both groups showed similar levels of IgM antibodies. Only few individuals had detectable levels of RAP-2/RSP-2-specific IgG1 and IgG3 subclass antibodies, while no IgG2 and IgG4 subclass antibodies were detected in these subjects. By ELISA, the anaemic group tended to show higher levels of antibodies to RAP-2/RSP-2 regarding all antibody classes tested, except for IgG4 and IgE. Unexpectedly, sera from the nonanaemic group activated complement to a greater extent than those from the anaemic group. These results need to be confirmed in extended studies but indicate that the effector functions of the RAP-2/RSP-2-reactive antibodies may be more important than their amounts. Such antibodies could play a role in both immunity and pathogenesis during P. falciparum infection.

Antibodies in malarial sera to parasite antigens in the membrane of erythrocytes infected with early asexual stages of Plasmodium falciparum.

Monolayers of human erythrocytes (E) infected with Plasmodium falciparum were briefly fixed with 1% glutaraldehyde and air dried. They were then exposed to sera from patients with P. falciparum malaria or from donors immune to this parasite and tested in an indirect immunofluorescence assay (IFA). Parasites in infected E were made visible by counterstaining with ethidium bromide. Immunofluorescence (IF) was restricted to the surface of infected E. No antibody binding was detected unless the E were dried, suggesting that the relevant antigens were not available on the outer layers of the E surface. Staining over large parts of the E surface was seen already when the merozoite penetrated noninfected cells and was strong in E containing early stages of the parasite (rings, trophozoites). It was weak or absent from E containing schizonts. Antibodies in sera from different parts of Africa, Colombia, or Sweden reacted similarly with E infected with a Tanzanian P. falciparum strain kept in culture for many years and with parasitized E freshly drawn from African, Swedish, or Colombian patients. All sera from residents of a holoendemic area (Liberia) were IFA positive. In contrast, some sera from Colombian or Swedish patients with primary infection gave negative results. The results of the IFA and of an enzyme-linked immunosorbent assay in which fixed and dried E were the targets were well-correlated, suggesting that the same antibodies were detected by these assays. The antigens involved in the IFA were susceptible to pronase but not to trypsin or neuraminidase. E surface IF was inhibited by lysates of infected E, merozoite extracts, or soluble antigens present in P. falciparum culture supernatants but not by lysates of normal E or ghost extracts. The inhibitory antigens were heat stable (100 degrees C, 5 min). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting of either antigen-enriched preparations from culture supernatants or merozoite extracts showed that antibodies eluted from monolayers of infected E reacted consistently with a predominant polypeptide of Mr 155,000 and two to four minor polypeptides of lower molecular weights. Metabolic labeling of the parasites with 75Se-methionine indicated that these antigens were parasite derived. We conclude that the antigens involved in these reactions are released from bursting schizonts or merozoites and are deposited in the E membrane in the course of invasion.(ABSTRACT TRUNCATED AT 400 WORDS)

Plasmodium falciparum-infected erythrocytes form spontaneous erythrocyte rosettes.

Erythrocytes infected with trophozoites or schizonts of Plasmodium falciparum bind uninfected erythrocytes, leading to rosette formation. Both established laboratory strains and fresh isolates from patients form such rosettes, but at widely different frequencies. IgG preparations from the serum of some P. falciparum-immune donors and heparin inhibited rosette formation. The results indicate that cytoadherence of infected erythrocytes to endothelial cells and rosetting represent distinct genetic traits.

The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition.

A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of methods of Davis and Bloom (21, 22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resoltuion SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nuclei acids, and very little phospholipids being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 percent that of a mitochondrial fraction) and a small amount of 5'- nucleotidase activity (of a specific activity between 6 and 7 percent that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures approximately 400nm long and approximately 40nm wide, made up of apparent particles 13-28nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter approximately 400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 or more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing (125)I-labeled myelin, synaptic vesicle, and mitochondrial fraction proteins with synaptosomes, and then isolating the density fraction from the mixture, it was concluded that a major 26,000 mol wt density fraction protein was common to both mitochondria and density, that none of the proteins of the density were contaminants from the mitochondrial fraction, that a minor approximately 150,000 band was a contaminant from the synaptic vesicle fraction, and that the moderately staining PSD fraction protein of 17,000 mol wt band was the result of contamination by the major basic protein of myelin. On the basis of the marker enzymatic assays and the mixing experiments, it is considered that the density fraction is moderately pure biochemically, and that its protein composition, aside from a few exceptions noted above, reflects its in situ character.

Subcellular distribution in cerebral cortex of two proteins phosphorylated by a cAMP-dependent protein kinase.

The subcellular distribution of Proteins Ia and Ib, two proteins which serve as specific substrates for protein kinases present in mammalian brain, was studied in the dog cerebral cortex. Proteins Ia and Ib were found to be most highly enriched in synaptic vesicle fractions; they were also present in postsynaptic density and synaptic membrane fractions in significant amounts. Proteins Ia and Ib present in the synaptic vesicle fraction appear to be similar, if not identical, to those present in the postsynaptic density fraction as judged by several criteria: (a) the ability to serve as substrate for cAMP-dependent protein kinase, (b) electrophoretic mobility in the presence of sodium dodecyl sulfate, (c) extractability with NH4Cl or EGTA, and (d) fragmentation to electrophoretically similar peptides by a purified Staphylococcus aureus protease. In addition, the postsynaptic density fraction has been found to contain cAMP-dependent Protein Ia and Protein Ib kinase activity. The subcellular localization of Proteins Ia and Ib suggests a role for these proteins in the physiology of the synapse.

Human monoclonal antibodies to Pf 155, a major antigen of malaria parasite Plasmodium falciparum.

Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

Antibody responses to a C-terminal fragment of the Plasmodium falciparum blood-stage antigen Pf332 in Senegalese individuals naturally primed to the parasite.

Previous studies have shown that antibodies from humans exposed continuously to malaria recognize the Plasmodium falciparum asexual blood-stage antigen Pf332. Here we analysed the antibody responses to a C-terminal fragment of Pf332, designated C231, in individuals from Senegal, by measuring the serum levels of immunoglobulin M (IgM), IgG class and subclass and IgE antibodies. IgG antibody reactivity with crude P. falciparum antigen was detected in all the donors, while many of the children lacked or had low levels of such antibodies against C231. The antibody levels increased significantly with age for both crude P. falciparum antigen and C231, and in the older age groups most of the donors displayed antibodies to C231. This was also true for IgM, IgE and IgG subclass reactivity against C231. Moreover, the ratio of IgG1/IgG2 was considerably lower for C231 than for crude P. falciparum antigen, and in age groups 10-14 and 15-19 years the levels of IgG2 against C231 even exceeded that of IgG1. The IgG2/IgG3 ratios suggest that C231 gives similar levels of IgG2 and IgG3, except for children aged 4-9 years, where IgG3 was higher. Raw IgM, IgG class and subclass and IgE antibody levels to C231 tended to be higher in those who did not experience a malaria attack, but following linear multivariate analysis the trends were not significant.

Analysis of T-cell responses in malaria-exposed and non-exposed donors using Plasmodium falciparum asexual blood stages enriched by a simple centrifugation method.

Several studies have reported on similar in vitro cellular responses to different malaria-antigen preparations in both malaria-primed and un-primed donors. Whether intact live parasites can exert a distinct type of response in either of the two groups is not well known. In this study, we developed a simple three-step centrifugation method for simultaneous enrichment of early and late blood stages from Plasmodium falciparum cultures. Such enriched P. falciparum fractions and other antigen preparations were used to stimulate lymphocytes from malaria-exposed and non-exposed individuals to examine the proliferative activity and expansion of CD3+, gammadelta+, CD4+, and CD8+ T cells. While lymphocytes from malaria non-exposed donors proliferated relatively higher than those from malaria-exposed donors in response to most antigens tested, the enriched fractions of live parasites exerted higher proliferative responses on cells from the latter donors. This suggests the existence of memory cells in the malaria-exposed donors, but not in the non-exposed ones. Flow cytometric analysis revealed a higher percentage expansion of CD4+ T cells in the responding cells of the exposed donors than the non-exposed ones. Taken together, this study reports on a simple method that simultaneously enriches for intact live early and late blood stages of P. falciparum parasites. Moreover, the study revealed higher expansion CD4+ T cells in the exposed individuals than the non-exposed in response to live malaria parasites and not to other parasite-antigen preparations.

Sex differences in the pattern of esterase-active antigens in microsomes from various rat strains.

Esterase-active antigens present in male and female liver microsomes isolated from three different rat strains-(Sprague-Dawley, Wistar and Dark Agouti) were characterized in crossed immunoelectrophoresis in combination with a zymogram method for esterase activity. No qualitative but some quantitative differencies were encountered between the sexes. Thus, two out of ten antigens were present in significantly lower and one in significantly higher concentrations in male than in female microsomes, demonstrating that although the overall esterase activity in liver may be similar for males and females, the concentration of the individual antigens does vary between the sexes. No qualitative or quantitative differences in the pattern of esterase-active antigens were found between the different strains.

Arylamidases of rat liver and chemically induced hepatomas. II. Preparation and characterization of monospecific antisera against two distinct arylamidase-active antigens.

Monospecific antisera were prepared against the most prominent arylamidase (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) active antigen in plasma membranes (the plasma membrane arylamidase) and lysomal content (the lysosomal content arylamidase), respectively. Plasma membrane extract and lysosomal content were allowed to react in crossed immunoelectrophoresis against their homologous antisera. The electrophoretic plates were washed extensively, dried and subsequently stained for arylamidase activity. The particular immunoprecipitates were thus identified and could be excised to be used for immunizations. The two resulting antisera precipitated the arylamidase used for immunization, but failed to be monospecific as they precipitated additional antigens. These antisera with restricted specificity against some plasma membrane and lysosomal content antigens, respectively, were used to produce immunoprecipitates intended for new attempts to prepare monospecific antisera by a second cycle of immunizations. A monospecific antiserum against the plasma membrane arylamidase was thus obtained, while a third cycle of immunizations was needed to get a monospecific anti-lysosomal content antiserum. The plasma membrane arylamidase showed ATPase activity also after precipitation with the monospecific antiserum, thus still retaining its characteristics as a multienzyme complex.

Arylamidases of rat liver and chemically induced hepatomas. 1. Subcellular distribution of L-leucine. 2. Naphthylamidase-active antigens.

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using L-leucine 2-naphthylamide as substrate. Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase). The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal and cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.

T-cell control of immunity to the asexual blood stages of the malaria parasite.

Both antibody-dependent and antibody-independent mechanisms are involved in immune protection against the asexual blood stages of the malaria parasite. It is well established that T cells play a crucial role in both induction and maintenance of this immunity. Of the two T-cell subsets (CD4+, CD8+) carrying alpha/beta T-cell receptors, the CD4+ T cells are of major importance for the development of blood stage immunity in both experimental and human malaria. In mice, CD4+ T cells comprise at least two functionally distinct cell types (TH1, TH2), distinguished on the basis of their lymphokine production. The balance between these subsets is critical for the outcome of an infection. In some rodent malarias, TH1 cells producing IFN-gamma and IL-2 are important for controlling infection in its early phases, while TH2 cells, producing i.a. IL-4 and IL-10, together with antibodies, are important for parasite clearance in later phases of infection. Distinct CD4+ T cells of either TH1 or TH2 type also have regulatory functions in human P. falciparum infection. In contrast to the CD4+ T cells, the role of CD8+ T cells in blood stage infection appears to be limited, but suppression of some CD4+ activities has been reported for both experimental and human malaria. As in other infections, peripheral T cells equipped with gamma/delta receptors are strongly upregulated in malaria and also respond to parasite antigens in vitro by proliferation and lymphokine production. However, the importance of the gamma/delta T cells for protection when compared with pathogenesis is presently unclear. Rapid advances made in recent years in the characterization and cloning of plasmodial antigens eliciting immune protection have made it possible to define some of the antigenic structures involved in T-cell immunity. This, together with an improved understanding of cellular mechanisms, provides some basis for the development of modern malaria vaccines.

Predominance of H-2d- and H-2k-restricted T-cell epitopes in the highly repetitive Plasmodium falciparum antigen Pf332.

Genetic restriction of immune responses to malaria antigens is an important issue for a better comprehension of malaria immunity as well as for development of subunit vaccines. To experimentally define the major histocompatibility complex restriction of immune responses to the highly repetitive Plasmodium falciparum high-molecular-weight antigen Pf332, H-2-congenic mice were immunized with EB200, a recombinant fragment of Pf332 consisting of degenerate repeat motifs. Strong B- and T-cell responses were elicited in H-2d and H-2k mice whereas responses in H-2b, H-2q and H-2s mice were of lower magnitude. The T-cell specificity elicited by EB200 was defined by in vitro proliferative responses to a panel of overlapping peptides spanning EB200. Dominant epitopes were identified for H-2d and H-2k mice, respectively, and an additional epitope was recognized by all five mouse strains. Selected EB200-derived peptides were further investigated for their ability to elicit T-cell help when injected as multiple antigen peptides. Defined H-2d- and H-2k-restricted T-cell epitopes generated high antibody levels in the respective mouse strains, as did several peptides lacking defined epitopes indicating the presence of additional H-2d- and H-2k-restricted, cryptic or subdominant T-cell epitopes in EB200. The biased H-2 restriction pattern of T-cell epitopes in Pf332 and, as previously reported, in structurally related repeats in the malaria antigens Pf11.1 and Pf155/RESA may be explained by a shared motif for H-2d and H-2k class II-restricted T-cell epitopes, as revealed by alignment of these sequences.

A dual expression system for the generation, analysis and purification of antibodies to a repeated sequence of the Plasmodium falciparum antigen Pf155/RESA.

A novel dual expression system for the generation and analysis of immune responses to recombinant protein is described. The two expression systems are based on the IgG-binding domains (ZZ) of staphylococcal protein A (SpA) and the human serum albumin (HSA) binding domains (BB) of streptococcal protein G, respectively. Products of fusions with the ZZ region are used to generate an immune response against the recombinant peptide and the corresponding peptide fused to the BB region is used for analysis and purification of the specific antibodies. The protein A and protein G expression systems were used to produce fusion proteins with the repeated C terminal octapeptide subunit EENVEHDA of the Plasmodium falciparum merozoite derived protein Pf155/RESA. Rabbits were immunized with the protein A-derived fusion protein (designated ZZ-M1) and the antibody response was analyzed using the protein G-derived fusion protein (designated BB-M1). The rabbit antisera reacted with BB-M1 in both ELISA and immunoblotting. In addition, BB-M1 proved to be an efficient ligand for affinity purification of antibodies specific for the malaria peptide. Furthermore, the rabbit antisera reacted with Pf155/RESA both in merozoite extracts and when deposited in the membrane of parasite infected erythrocytes.

The serum albumin-binding region of streptococcal protein G: a bacterial fusion partner with carrier-related properties.

In this study, we have explored the use of the serum albumin-binding region (BB) from streptococcal protein G (SpG) as a bacterial fusion partner for production of peptide immunogens. The fusion protein BB-M3, containing BB and repeated structures from the Plasmodium falciparum malaria antigen Pf155/RESA, was efficiently purified from Escherichia coli culture supernatants by affinity chromatography using BB as an affinity tag. Rabbits immunized with BB-M3 in Freund's adjuvant produced high levels of antibodies which reacted with both M3 and BB in ELISA and stained intact Pf155/RESA in the membrane of infected erythrocytes. These antibody levels were sustained for more than 30 weeks. BB-M3 also induced antibody responses to M3, BB and intact Pf155/RESA in a number of mouse strains, including several strains which are non-responders to the malaria sequences. In the latter mice, however, BB-M3 only activated BB-specific T cells, suggesting that BB has ability to provide carrier-related T cell help for antibody production. Moreover, the minimal albumin-binding motif of SpG, containing only 46 amino acids, was immunogenic in both B10.BR, B10.D2 and C57BL/6 mice (H-2k, H-2d and H-2b, respectively). These results indicate that BB has both affinity tag and carrier-related properties and suggest that fusion proteins containing BB can be efficient tools for the generation of antibody responses to peptides which are weak immunogens.

Definition of the epitope recognized by the Plasmodium falciparum-reactive human monoclonal antibody 33G2.

The human monoclonal antibody 33G2 has earlier been shown to inhibit merozoite reinvasion of red blood cells in Plasmodium falciparum cultures in vitro and to inhibit cytoadherence of infected red blood cells to melanoma cells in vitro. 33G2 cross-reacts with a family of P. falciparum antigens, Ag332, Pf11.1 and Pf155/RESA, sharing a common feature of repeated sequences consisting of regularly spaced pairs of glutamic acid. Peptides corresponding to residues 2-19 of the known amino acid sequence of Ag332 have been shown earlier to have the highest inhibitory capacity of antibody binding to infected red blood cells. Using the PEPSCAN method, overlapping hepta-, hexa-, penta- and tetrapeptides corresponding to residues 1-19 of the known sequence of Ag332 were synthesized. Antibody fine specificity was examined by synthesizing an octapeptide (residues 1-8) and all possible single amino acid substitutions. The monoclonal antibody was shown to react with a linear 5-amino acid-long sequence corresponding to Ag332 residues 3-7: VTEEI. These amino acids were irreplaceable or only partially replaceable in the replacement set analysis. Furthermore, epitope analogs corresponding to sequences contained within the Pf11.1 repeats and overlapping heptapeptides corresponding to Pf155/RESA repeats were synthesized. Reactivity to epitope analogs and Pf155/RESA peptides provided information which may explain antibody cross-reactivity. The defined epitope of this monoclonal antibody is of interest as a potential B cell epitope for the development of a malaria subunit vaccine.

Identification of a Plasmodium chabaudi antigen present in the membrane of ring stage infected erythrocytes.

A novel antigen of asexual blood stages of the rodent malaria parasite Plasmodium chabaudi, was detected by means of a modified indirect immunofluorescence assay (IFA), using glutaraldehyde fixed and air dried monolayers of P. chabaudi infected erythrocytes. P. chabaudi hyperimmune sera gave a distinct surface immunofluorescence of erythrocytes infected with early stages of the parasite. Fixation and drying of the erythrocytes was necessary for the antigenic activity to be exposed. The antigens were species specific as P. chabaudi hyperimmune serum only stained P. chabaudi but not P. yoelii or P. falciparum infected erythrocytes. The antigenic activity involved in the IFA was resistant to trypsin, phospholipases and neuraminidase but not to pronase, suggesting that the antigens were polypeptides. The surface immunofluorescence was inhibited by an extract of parasitized erythrocytes, but not by similar extracts of normal erythrocytes. The inhibitory antigens were soluble and heat stable (100 degrees C, 5 min). For identification and characterization of the antigens, antibodies were isolated by acid elution from monolayers of infected erythrocytes and monoclonal antibodies were produced. Probing in immunoblotting of extracts of parasitized erythrocytes separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the eluted antibodies, showed that they reacted consistently with a polypeptide of Mr 105 000 (Pch105). The Pch105 antigen shares many characteristics with Pf155, a P. falciparum antigen considered as a candidate for a vaccine against that parasite.

Cloning of genomic fragment from Plasmodium chabaudi expressing a 105 kilodalton antigen epitope.

The 105 kDa antigen of Plasmodium chabaudi has many characteristics of the P. falciparum Pf 155 (RESA) molecule. A clone (pPC105e) from a P. chabaudi genomic library was isolated using immune screening with a 105 kDa antigen specific monoclonal antibody (B7E10). Southern, Northern and Western blotting analyses provide evidence for a lack of variability at the protein and DNA levels. A subclone of the insert in the expression vector pEX2, synthesises a fusion peptide which contains the epitope recognized by B7E10. Sequences homologous to the insert were detected in the genome of three other rodent and two primate malarias.