Antibody responses stimulated in rabbits, guinea-pigs and mice by recombinant and synthetic portions of a 75 kDa malarial merozoite protein.

The 75 kDa heat-shock-related protein (p75) of Plasmodium falciparum is an abundant, highly conserved, merozoite surface protein. A bacterial clone, C7, produces a polypeptide (C7Ag) of approximately 30 kDa representing the C-terminal 40% of p75. In several species of animals, the C7Ag stimulated high titre IgG antibodies which cross-react with p75. Two major portions of the C7Ag, theoretically predicted to have strong secondary structural preferences, were modelled with four synthetic peptides. An alpha-helical, hydrophilic region, modelled with a 28-mer, proved a poor immunogen in guinea-pigs and several strains of inbred mice, even though it had been a strong immunogen in rabbits. A disulphide-bonded region of the C7Ag was modelled with three peptides of increasing length, namely 49-, 64- and 76-amino acid residues. In general, the order of immunogenicity was 49 less than 64 less than 76-mer. Antibodies to the 76-mer and the 64-mer reacted strongly with the native parasite protein. The data also suggested that the 76-mer was a good model for the region of the molecule it was made to represent.

Heat shock response of Babesia divergens and identification of the hsp70 as an immunodominant early antigen during ox, gerbil and human babesiosis.

Using antisera (alpha-R and alpha-C7Ag) directed against the conserved Gly-Gly-Met-Pro-epitope of the hsp70 family, a single antigen was identified in the human Babesia divergens Rouen 1987 isolate by Western immunoblotting and immunoprecipitation experiments. This B divergens hsp70 is highly conserved as shown by the analysis of five other geographical B divergens isolates from different hosts (human and bovine). Indirect immunofluorescence assay performed on the asexual intraerythrocytic stages showed that the hsp70 is mainly cytoplasmic and stage-independent. Heat-shock experiments, with 20 min incubation at 40 degrees C followed by a 10 to 50 min shift to 37 degrees C in the presence of [35S]-methionine, led to an increase of two hsp of 85 and 70 kDa while protein synthesis in general decreased within 10 min. Immunoprecipitations of [35S]-methionine radiolabelled proteins with human, ox and gerbil antisera raised against various B divergens isolates, showed the presence of a B divergens 70 kDa protein which was demonstrated to be a hsp70 by coupling immunoblotting assays with alpha-C7Ag serum on the same immunoprecipitated material. During human babesiosis, the B divergens hsp70 appears as an early antigen during the acute phase. These results are in agreement with the use of the B divergens hsp70 as an essential valence antigen in an anti-babesiosis vaccine.

Plasmodium falciparum: hetero-oligomeric complexes of rhoptry polypeptides.

We have previously described several monoclonal antibodies (McAbs) which specifically recognize antigens in the rhoptries of Plasmodium falciparum and which immunoprecipitate polypeptides of 82, 70, 67, 39, and 37 kDa. We now show that only p82, p70, p67, and a 86-kDa precursor (Pr86) of p82 possessed epitopes for these McAbs. These four proteins were not synthesized until schizogony. These results and proteolysis experiments indicated that Pr86, p82, p70, and p67 were the products of the same gene, whereas the dissimilar digestion patterns of p39 and p37 suggested that p39 was encoded by a second gene and p37 by yet another. Complexes of these proteins (termed RI complexes) are maintained by noncovalent interactions since the ionic detergent SDS was sufficient to dissociate them into individual polypeptides. Sucrose gradient centrifugation demonstrated that RI complex formation was not dependent on the presence of antibody and that these complexes had higher sedimentation rates than the 185-kDa P. falciparum merozoite glycoprotein. Covalent crosslinking with the reversible, homobifunctional, primary amine-specific reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by RI McAb immunoprecipitation resulted in purification of intact complexes which were not dissociable by SDS alone. Immunodepletion experiments with a subtype of RI McAb which does not immunoprecipitate p37 suggested that the binding of p39 and p37 to the other RI proteins was mutually exclusive. Therefore, the minimal composition of the RI complexes is one molecule of Pr86, p82, p70, or p67 and one of p39 or p37. The epitopes of Pr86, p82, p70, and p67 for the RI McAbs were sensitive to disulfide bond reduction. Surprisingly, reduction increased their electrophoretic mobilities. This enhanced mobility could not be accounted for by post-translational glycosylation, phosphorylation, or acylation, or by covalent attachment via the sulfhydryl moiety of cysteine residues to additional parasite proteins. We suggest that, due to an asymmetric distribution of amino acids in the Pr86-class molecules, SDS binding results in a lower charge to mass ratio in the native folded polypeptides and a higher charge to mass ratio upon disulfide bond reduction and unfolding of the polypeptides.

Cross-reactive asparagine-rich determinants shared between several blood-stage antigens of Plasmodium falciparum and the circumsporozoite protein.

From a Plasmodium falciparum cDNA expression library derived from mRNA of the asexual blood stages, we isolated and sequenced five different cDNA clones whose predicted protein products were unusually rich in asparagine (Asn). Two of the clones, R5 and G5, contain tandem imperfectly repeated sequences based on Asn-Asn-Thr (NNT) and Asn-Asn-Met (NNM) respectively. The other three, E4, C5 and R13, as well as G5, contain stretches of polyasparagine varying in length from 2 to 26 residues. Results of DNA blotting experiments with the individual cDNA sequences as probes suggest that each of the five clones corresponds to a different P. falciparum gene. The fragments of P. falciparum proteins expressed by the cDNA clones shared cross-reactive antigenic determinants which were present on multiple P. falciparum proteins. In immunoblotting experiments, owl monkey antibodies selected for binding to the polypeptide expressed by clone E4, C5 or G5 reacted with the expressed proteins from all 5 clones, and with at least 10 proteins from schizont infected erythrocytes. The cross-reactive epitopes could be modeled by two Asn-rich peptide structures: (1) (NNT)8, whose sequence was based on the R5 repeat; and (2) (NPNA)6, whose sequence was based on the Asn-rich repeat of the P. falciparum circumsporozoite protein (CSP). Antibodies that bound to each peptide were selected from sera of immune monkeys that had never been exposed to sporozoites. The selected antibodies bound all 5 expressed proteins in immunoblotting assays and also bound to several proteins from parasitized erythrocytes. Such cross reactivity between the CSP repeating unit and several blood-stage antigens has not been previously reported.

Plasmodium falciparum antigens associated with membrane structures in the host erythrocyte cytoplasm.

Hybridomas were made from mice immunized with plasma membranes from erythrocytes infected with Plasmodium falciparum. Among the monoclonal antibodies produced, a series reacted with antigens in the host cell cytoplasm. Immunoelectron microscopy, along with indirect fluorescent antibody double labeling experiments, were used to further localize the antigens to membrane structures (presumably Maurer's clefts) in the erythrocyte cytoplasm. The epitopes thus localized are found on three parasite proteins (20 kDa, 29 kDa, and 45 kDa) and one parasite glycoprotein (45 kDa). They are likely to be part of a transport system for the parasite.

Peptide mapping of conformational epitopes in a human malarial parasite heat shock protein.

A protein of 75 kDa is found in large quantities throughout the blood stages of the human malarial parasite, Plasmodium falciparum. Based on a partial amino acid sequence for p75, previously deduced from a cDNA clone encoding approximately 40% of the molecule, secondary structural predictions were made. The potential role of long range effects on the tertiary structure of the protein stabilized by disulfide bridges was determined by reduction and alkylation of the fusion protein. Five regions were then chosen for peptide modeling. Peptides of 16, 28, 49, 64, and 76 residues were synthesized and used to immunize rabbits. All but the 16-residue peptides were capable of stimulating boostable IgG antibody responses in rabbits, but the antibody produced against the 49 mer did not react with the native parasite protein. Thus, the 28, 64, and 76 residue peptides represent good immunologic models for portions of the P. falciparum 75-kDa protein capable of stimulating both T and B cells in rabbits. The peptides were also used to probe whether any of the selected regions contain epitopes which react with antibodies from owl monkeys immune to P. falciparum. Of these peptides, two were found to be consistently recognized in ELISA by four owl monkey antisera raised in response to malarial infection. Because these two peptides model a cysteine-containing region of the protein, owl monkey sera were also used as probes of the importance of disulfide bonding in maintaining the native structure. The results obtained were consistent with a folding pattern for p75 that incorporates a disulfide bond between cysteines 161 and 194. These results also suggest that most of the epitopes recognized in this part of p75 by the immune system of the monkey are created by folding of the molecule.

Heat shock-like polypeptides of the sporozoites and merozoites of Eimeria bovis.

Heat shock proteins (hsps) are a group of highly conserved polypeptides found in a wide variety of organisms. Polypeptides of sporozoites and merozoites of Eimeria bovis, blotted onto nitrocellulose, were probed with antibodies to artificially constructed peptides representing portions of the cDNA-generated fragment of pf75, the 75K hsp of merozoites of Plasmodium falciparum. Polypeptide antigens of sporozoites and merozoites of E. bovis with molecular weights of 46K, 71-72K, and 75K reacted with antibodies against pf75, indicating that they are hsp70 (the 70K family of hsps) or hsp70 cognates (noninducible proteins homologous to hsps). Radiolabeling with 125I and treatment with antibodies against pf75 detected a 71K antigen on the merozoite surface. Hsps in sporozoites of E. bovis are either constitutive or evoked by treatment at 37 C for in vitro excystation. If hsp70 is mandatory for parasite survival, it may prove to be an appropriate antigen for a vaccine against bovine coccidiosis.

Characterization of a high-molecular-weight phosphoprotein synthesized by the human malarial parasite Plasmodium falciparum.

During its intra-erythrocytic cycle, Plasmodium falciparum synthesizes a protein of apparent Mr 250,000-300,000. Its precise size is dependent on the P. falciparum isolate examined. This protein contains phosphate covalently bound to one or more serine residues and hence is termed PP300. Monoclonal antibody, McAb4-1F, binds to PP300 on immunoblots of protein extracts from all parasite isolates tested, both those exhibiting and those lacking the knob phenotype. Using McAb4-1F, the polypeptide was shown to be physically associated with the plasma membrane in a membrane-isolation procedure. However, in an indirect immunofluorescence assay the McAb appeared to bind to antigen associated with the erythrocyte plasma membrane in parasitized cells. However, it reacted only to fixed, not unfixed, parasitized erythrocytes indicating that the epitope is not normally exposed to extracellular antibodies. Clone 29-2 was isolated by a McAb4-1F immunoscreen of a P. falciparum complementary DNA (cDNA) expression library created in pUC8. Rat anti-clone serum which was raised to the purified protein encoded by the lacZ-29-2 fusion in pUC8 reacted with PP300 in immunoblots of parasite antigen. In Southern-blot analyses of parasite DNA digested with EcoRI, HindIII, or EcoRV, the 29-2 DNA insert hybridized to more than one fragment even though the insert lacked internal sites for these enzymes. In addition, hybridization studies were conducted using two oligodeoxy-nucleotides which were constructed based on the sequence of a cDNA clone which encoded part of a similar high-molecular-weight P. falciparum protein [Coppel et al., Mol. Biochem. Parasitol. 20 (1986) 265-277]. Analysis of these results indicates that the two cDNA sequences are parts of the same gene or a family of related genes.

Immunologic modeling of a 75-kDa malarial protein with carrier-free synthetic peptides.

A protein of 75 kDa is produced in large quantities by the human malarial parasite Plasmodium falciparum and is present on the surface of the merozoite, whose function is to infect erythrocytes. Based on nucleotide sequence coding for 40% of this protein, two nonoverlapping model peptides 13 and 19 residues long were synthesized, coupled to a keyhole limpet hemocyanin carrier, and used to immunize rabbits. Although both antisera had high titers of anti-peptide antibodies, only that raised against the 13-residue peptide showed good reactivity against the original protein. Although the 19-mer adopted the helical secondary structure predicted for the corresponding protein region, antisera against this peptide reacted with the native protein weakly or not at all. Concluding that the poor anti-protein reactivity was due to modification of lysine-containing epitopes by glutaraldehyde conjugation, we used a carrier-free 28-residue peptide presented as a 56-residue disulfide-bonded dimer to model the same region. This peptide, in contrast to the conjugated 19-mer, stimulated the production of IgG antibodies that reacted at high dilution with the authentic protein in immunoblots, ELISA, and radioimmunoprecipitation assays. These data indicate that large carrier-free peptides may be successfully used as immunogens. In addition, our results show that this strategy may greatly improve the ability of conjugation-sensitive peptides to stimulate antibodies reactive with the original protein and therefore has substantial practical application.

cDNA sequence encoding a Plasmodium falciparum protein associated with knobs and localization of the protein to electron-dense regions in membranes of infected erythrocytes.

Plasmodium falciparum modifies the host erythrocyte's plasma membrane by the formation of electron-dense structures called knobs. We have produced monoclonal antibodies (McAbs) which specifically bind to the knobs in immunoelectron microscopic experiments with thin sections of parasitized erythrocytes. However, the McAbs fail to bind to the surface of live parasitized erythrocytes. Immunoblotting experiments with these McAbs show the antigen is localized to the erythrocyte plasma membrane. The antigen with which the McAbs react varies in mol. wt from 80 to 95 kd in different knob-producing isolates of P. falciparum and is absent in knobless variants. The McAbs react with the expressed product of a P. falciparum cDNA clone, thus demonstrating that the clone encodes part of this knob-associated protein. The sequence of the cDNA fragment partially overlaps a published cDNA sequence reported to encode the amino-terminal portion of the knob protein, and extends the predicted open reading frame by 190 amino acids. The carboxyl-terminal portion of the predicted amino acid sequence contains a highly charged stretch of approximately 100 amino acid residues. We suggest that this unusual, highly charged region participates in intermolecular salt bridging leading to dense packing of these molecules. This would create the electron-dense regions observed by electron microscopy and might also explain the insolubility of the knob-associated protein in the absence of strong ionic detergents or chaotropic agents.

A 75 kd merozoite surface protein of Plasmodium falciparum which is related to the 70 kd heat-shock proteins.

Proteins on the merozoite surface of the human malarial parasite Plasmodium falciparum are targets of the host's immune response. The merozoite surface location of p75, a 75 kd P. falciparum protein, was established by immunoelectron microscopy using antisera raised to the expressed product of a cDNA clone. Immunoprecipitation from protein extracts biosynthetically labeled during different periods of the asexual cycle showed that p75 is made continuously, although ring-stage parasites appear to synthesize larger quantities. p75 is conserved and invariant in size in eight isolates of P. falciparum. The 880 bp cDNA sequence encoding part of p75 reveals one open reading frame containing a repetitive sequence unit of four amino acids. The predicted reading frame is correct since antisera to a synthetic peptide corresponding to the repetitive region recognize p75 in immunoblots. The sequence of p75 is homologous with the sequences of proteins from the ubiquitous, highly conserved family of 70 kd heat-shock proteins, suggesting an important physiological function for p75. The cDNA fragment encoding part of p75 hybridizes with multiple genomic fragments, whose sizes are identical in DNA from nine P. falciparum strains, suggesting that the gene for p75 is well conserved and may be part of a gene family.

Plasmodium falciparum polypeptides associated with the infected erythrocyte plasma membrane.

Plasmodium falciparum proteins associated with plasma membranes of infected erythrocytes were identified by using three techniques: isolated plasma membranes from infected and uninfected erythrocytes were compared by gel electrophoresis and silver staining; isolated plasma membranes from cells metabolically labeled with [35S]methionine were assayed by gel electrophoresis; and uninfected and infected intact erythrocytes were surface-labeled by lactoperoxidase iodination, and the labeled polypeptides were compared by gel electrophoresis. The results from these experiments indicate that at least six parasite-derived polypeptides (Mr = greater than 240,000, 150,000, 55,000, 45,000, 35,000, and 20,000) are associated with the infected erythrocyte plasma membrane. At least four of these peptides (Mr = 55,000, 45,000, 35,000, and 20,000) may be exposed on the surface of the infected erythrocytes.

Terminal galactose residues and the antigenicity of Plasmodium falciparum glycoproteins.

The presence of terminal alpha-D-galactosyl residues in the carbohydrate chains of glycoprotein antigens from the asexual blood stages of Plasmodium falciparum is demonstrated by the alpha-D-galactosidase sensitivity of particular parasite antigens, the use of specific glycosidases to cleave sugars from parasite glycoproteins radiolabeled with [3H]glucosamine, and the ability of Bandeirea simplicifolia lectin, which has a specificity for terminal alpha-galactosyl residues, to bind to the parasite. The carbohydrate side chains, and in particular the terminal alpha-galactosyl residues, are shown to have an important role in determining the binding of antibodies to parasite glycoproteins.

Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates.

Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Two Plasmodium falciparum merozoite surface polypeptides share epitopes with a single Mr 185 000 parasite glycoprotein.

The malarial parasite Plasmodium falciparum synthesizes a major glycoprotein (gp) of Mr 185 000 during its asexual blood cycle. Immunoprecipitation of [35S]methionine- or [3H]glucosamine-labeled schizont antigens indicated that two groups of polypeptides were distinguished with anti-gp 185 mouse monoclonal antibodies: group A was composed of glycosylated molecules of Mr 185 000, 120 000, 90 000, 88 000, 46 000, and 40 000 while group B contained, in addition to gp 185, polypeptides of Mr 152 000, 106 000 and 83 000. The latter polypeptides lacked detectable amounts of radiolabeled saccharide. The smaller Mr polypeptides were specifically immunoprecipitated and not merely coprecipitated with gp 185. Our results suggest that gp 185 contains at least two structurally distinct domains which may be processed independently into either the group A or group B polypeptides. Although gp 185 may not be a merozoite surface protein, representative group A and group B-specific monoclonal antibodies bound to surface antigens of the merozoite as demonstrated by immunolabeling followed by electron microscopy. Therefore, at least one group A antigen and one group B antigen appeared to be on the extracellular surface of the merozoite. The proteins found in immunoprecipitates after both (1) sonication in aqueous medium and ultracentrifugation and (2) solubilization and phase separation of parasite molecules with Triton X-114 suggested that the group A and group B polypeptides and glycoproteins are either soluble or peripheral membrane proteins. Some of these, therefore, may be components of the surface coat of the merozoite.

Monkey-derived monoclonal antibodies against Plasmodium falciparum.

A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a Mr 95,000 antigen.

Synthesis of small nuclear ribonucleoprotein particles by the malarial parasite Plasmodium falciparum.

Sera from patients with autoimmune diseases have been used to identify small nuclear ribonucleoprotein particles (snRNPs) present in higher eukaryotic cells and also in dinoflagellates. Previously these sera have not detected crossreactive snRNP protein antigens of other lower eukaryotes such as yeast, Tetrahymena, or Dictyostelium. We report that anti-Sm, anti-U1-RNP, and anti-La/SS-B human antisera react with specific snRNP protein antigens synthesized by the protozoan Plasmodium falciparum, the human malarial parasite. These results suggest that the structure and antigenicity (and thus probably the function) of snRNPs have been widely conserved in eukaryote evolution.

Recognition of a Mr 56K glycoprotein on the surface of Plasmodium falciparum merozoites by mouse monoclonal antibodies.

Hybridomas were prepared from mice repeatedly injected with disrupted Plasmodium falciparum (FVO isolate) schizonts and merozoites. Antibodies secreted by two of these hybridomas were shown by immunoelectron microscopy to bind to the surface of merozoites from the FVO isolate. These monoclonal antibodies (McAb) reacted with the FVO and Geneva isolates by an indirect fluorescence antibody test (IFAT) and immunoprecipitated a protein of relative molecular weight (Mr) 56K from both isolates. The 56K protein could be labeled with [35S] methionine and [3H]glucosamine. Glycosidase treatment of the affinity-purified polypeptide proved that the [3H]glucosamine had been incorporated into sugar side chains and that this protein (called gp56) was glycosylated. The anti-gp56 McAb did not react by IFAT or immunoprecipitation with four isolates (Honduras I, Indochina I, Tanzania I, and Kenya) that lack gp56 but contain major glycoproteins of Mr 50K. Antibodies from an Aotus monkey immune to the FVO isolate immunoprecipitated gp56 from both the FVO and Geneva isolates, but did not immunoprecipitate the 50K glycoproteins from the other four isolates. Extraction experiments conducted with the nonionic detergent Triton X-114 indicate that some of the gp56 molecules are hydrophilic and that the others are either hydrophobic or interact with hydrophobic molecules. These results, together with the electron microscopic data, suggest that the hydrophilic gp56 is a component of the extracellular matrix and that the hydrophobic gp56 may be associated with the plasma membrane of the merozoite.

Expression of Plasmodium falciparum surface antigens in Escherichia coli.

The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity.