High non-protective, long-lasting antibody levels in malaria are associated with haplotype shifting in MHC-peptide-TCR complex formation: a new mechanism for immune evasion

An effective malarial vaccine must contain multiple immunogenic, protection-inducing epitopes able to block and destroy the P. falciparum malaria parasite, the most lethal form of this disease in the world. Our strategy has consisted in using conserved peptides blocking parasite binding to red blood cells; however, these peptides are non-immunogenic and non-protection-inducing. Modifying their critical residues can make them immunogenic. Such peptides induced antibody titers (determined by immunofluorescence antibody test, IFA) and made the latter reactive (determined by Western blot) and protection inducing against experimental challenge with a highly infective Aotus monkey adapted P. falciparum strain. Modified peptides also induce highly non-protective long-lasting antibody levels. Modifications performed might allow them to bind specifically to different HLA-DRbeta purified molecules. These immunological and biological activities are associated with modifications in their three-dimensional structure as determined by (1)H-NMR. It was found that modified, high non-protective long-lasting antibody level peptides bound to HLA-DR molecules from a different haplotype (to which immunogenic, protection-inducers bind) and had 4.6 +/- 1.4 A shorter distances between residues fitting into these molecules' Pocket 1 to Pocket 9, suggesting fitting into an inappropriate HLA-DR molecule. A multi-component, subunit-based, malarial vaccine is therefore feasible if modified peptides are suitably modified for an appropriate fit into the correct HLA-DRbeta1* molecule in order to form a proper MHC-II-peptide-TCR complex.     

A role for carbohydrate moieties in the immune response to malaria

Treatment of antigen prepared from asexual blood stages of the human malarial parasite Plasmodium falciparum with a mixture of glycosidases resulted in a reduction in the ability of the antigen to bind antibodies from immune human and monkey sera in an ELISA assay. Some of the epitopes in the parasite material were heat stable, protease resistant, and sensitive to glycosidases. Proteins of Mr 110,000 and 65,000 from parasitized RBC were shown to have reduced antigenicity in Western blots after glycosidase treatment. The carbohydrate side chains of parasite glycoproteins therefore make a contribution to the total antigenicity of the parasite.     

A carboxyl-terminal fragment of Plasmodium falciparum gp195 expressed by a recombinant baculovirus induces antibodies that completely inhibit parasite growth.

The major merozoite surface Ag (gp195) of Plasmodium falciparum has been shown to protect monkeys against parasite infection, and gp195-based synthetic peptides and recombinant polypeptides have been evaluated as potential malaria vaccines. A major problem in developing a gp195-based recombinant vaccine has been the difficulty in obtaining a recombinant polypeptide that is immunologically equivalent to the native protein. In this study, the carboxyl-terminal processing fragment (p42) of gp195 was produced in yeast and in a baculovirus recombinant system. Immunologic analyses indicated that the secreted baculovirus p42 (BVp42) expressed native, disulfide-dependent conformational epitopes, whereas these epitopes were poorly represented in the intracellular yeast p42. BVp42, but not yeast p42, was also recognized by the majority of gp195-specific antibodies of animals immunized with purified native gp195, indicating that the anti-gp195 response of these animals was focused on conformational determinants of the p42 processing fragment. Sera against native gp195 of congenic mice of diverse H-2 haplotypes recognized the BVp42 polypeptide, demonstrating that a genetically heterogeneous population is capable of responding to p42 epitopes. BVp42 was highly immunogenic and induced high titers of antibodies that were cross-reactive with purified native gp195 in an ELISA and also reacted with schizonts and merozoites by immunofluorescence. Anti-BVp42 antibodies completely inhibited the in vitro growth of the malaria parasite, whereas anti-yeast p42 antibodies had no effect. These results indicate that native, conformational epitopes of p42 are critical for the induction of gp195-specific, parasite growth-inhibitory antibodies and that the BVp42 polypeptide efficiently induces antibodies specific for these native determinants.     

Conserved group-specific epitopes of the circumsporozoite proteins revealed by antibodies to synthetic peptides

The immunogenic properties of sporozoites are associated mainly with the circumsporozoite (CS) protein that covers the surface of mature sporozoites. This stage-specific protein has an immunodominant region with repetitive epitopes. Rabbits that are repeatedly immunized with sporozoites of Plasmodium knowlesi, a monkey malaria parasite, also recognize two synthetic peptides (N2 and C2) representing other polar domains of the CS protein. We show in this report that antibodies to the N2 and C2 synthetic peptides react not only with P. knowlesi but also with conserved regions of the surface membrane of other human, monkey, and rodent (but not avian) malaria sporozoites. Moreover, antibodies to N2 partially neutralize the infectivity of sporozoites of P. berghei, a rodent malaria parasite. In contrast, antibodies to synthetic peptides representing the repetitive epitope of P. knowlesi were strictly species specific.     

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.     

Antigenic analysis of the repeat domain of the circumsporozoite protein of Plasmodium vivax

In the present study we analyzed the fine specificity of mouse monoclonal and human polyclonal antibodies directed against the repeat domain of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium vivax. Five synthetic peptides, representing monomeric and dimeric repeats of this malarial antigen, were assayed for their capacity to inhibit the binding of these antibodies to a yeast-derived recombinant CS protein. The results revealed the existence of at least two distinct repeated overlapping epitopes in the CS protein of P. vivax. Furthermore, polyclonal sera contain antibodies which recognize additional determinants not represented by the synthetic repeat peptides. Some of these sera contain antibodies recognizing a region flanking the repeat domain (region I). The present findings are in contrast with the antibody response in rodents and humans to the Plasmodium falciparum CS protein, which is directed against a single repeated immunodominant epitope.     

In vitro efficacy, resistance selection, and structural modeling studies implicate the malarial parasite apicoplast as the target of azithromycin

Azithromycin (AZ), a broad-spectrum antibacterial macrolide that inhibits protein synthesis, also manifests reasonable efficacy as an antimalarial. Its mode of action against malarial parasites, however, has remained undefined. Our in vitro investigations with the human malarial parasite Plasmodium falciparum document a remarkable increase in AZ potency when exposure is prolonged from one to two generations of intraerythrocytic growth, with AZ producing 50% inhibition of parasite growth at concentrations in the mid to low nanomolar range. In our culture-adapted lines, AZ displayed no synergy with chloroquine (CQ), amodiaquine, or artesunate. AZ activity was also unaffected by mutations in the pfcrt (P. falciparum chloroquine resistance transporter) or pfmdr1 (P. falciparum multidrug resistance-1) drug resistance loci, as determined using transgenic lines. We have selected mutant, AZ-resistant 7G8 and Dd2 parasite lines. In the AZ-resistant 7G8 line, the bacterial-like apicoplast large subunit ribosomal RNA harbored a U438C mutation in domain I. Both AZ-resistant lines revealed a G76V mutation in a conserved region of the apicoplast-encoded P. falciparum ribosomal protein L4 (PfRpl4). This protein is predicted to associate with the nuclear genome-encoded P. falciparum ribosomal protein L22 (PfRpl22) and the large subunit rRNA to form the 50 S ribosome polypeptide exit tunnel that can be occupied by AZ. The PfRpl22 sequence remained unchanged. Molecular modeling of mutant PfRpl4 with AZ suggests an altered orientation of the L75 side chain that could preclude AZ binding. These data imply that AZ acts on the apicoplast bacterial-like translation machinery and identify Pfrpl4 as a potential marker of resistance.     

Plasmodium falciparum: purification, properties, and immunochemical study of ornithine decarboxylase, the key enzyme in polyamine biosynthesis

Ornithine decarboxylase, the rate-limiting enzyme in the polyamine biosynthetic pathway has been purified 7,600 fold from Plasmodium falciparum by affinity chromatography on a pyridoxamine phosphate column. The partially purified enzyme was specifically tagged with radioactive DL-alpha-difluoromethylornithine and subjected to polyacrylamide gel electrophoresis under denaturing conditions. A major protein band of 49 kilodalton was obtained while with the purified mouse enzyme, a typical 53 kilodalton band, was observed. The catalytic activity of parasite enzyme was dependent on pyridoxal 5'-phosphate and was optimal at pH 8.0. The apparent Michaelis constant for L-ornithine was 52 microM. DL-alpha-difluoromethylornithine efficiently and irreversibly inhibited ornithine decarboxylase activity from P. falciparum grown in vitro or Plasmodium berghei grown in vivo. The Ki of the human malarial enzyme for this inhibitor was 16 microM. Ornithine decarboxylase activity in P. falciparum cultures was rapidly lost upon exposure to the direct product, putrescine. Despite the profound inhibition of protein synthesis with cycloheximide in vitro, parasite enzyme activity was only slightly reduced by 75 min of treatment, suggesting a relatively long half-life for the malarial enzyme. Ornithine decarboxylase activity from P. falciparum and P. berghei was not eliminated by antiserum prepared against purified mouse enzyme. Furthermore, RNA or DNA extracted from P. falciparum failed to hybridize to a mouse ornithine decarboxylase cDNA probe. These results suggest that ODC from P. falciparum bears some structural differences as compared to the mammalian enzyme.     

Glutathione reductase of the malarial parasite Plasmodium falciparum: crystal structure and inhibitor development

The malarial parasite Plasmodium falciparum is known to be sensitive to oxidative stress, and thus the antioxidant enzyme glutathione reductase (GR; NADPH+GSSG+H(+) <==> NADP(+)+2 GSH) has become an attractive drug target for antimalarial drug development. Here, we report the 2.6A resolution crystal structure of P.falciparum GR. The homodimeric flavoenzyme is compared to the related human GR with focus on structural aspects relevant for drug design. The most pronounced differences between the two enzymes concern the shape and electrostatics of a large (450A(3)) cavity at the dimer interface. This cavity binds numerous non-competitive inhibitors and is a target for selective drug design. A 34-residue insertion specific for the GRs of malarial parasites shows no density, implying that it is disordered. The precise location of this insertion along the sequence allows us to explain the deleterious effects of a mutant in this region and suggests new functional studies. To complement the structural comparisons, we report the relative susceptibility of human and plasmodial GRs to a series of tricyclic inhibitors as well as to peptides designed to interfere with protein folding and dimerization. Enzyme-kinetic studies on GRs from chloroquine-resistant and chloroquine-sensitive parasite strains were performed and indicate that the structure reported here represents GR of P.falciparum strains in general and thus is a highly relevant target for drug development.     

Plasmodium falciparum: analysis of B epitopes of a polypeptide antigen expressed in Escherichia coli, using monoclonal antibodies

Monoclonal antibodies were raised against a recombinant molecule corresponding to the polypeptide 72 kDa, previously described as possibly related to protection in Plasmodium falciparum infection. Selection of hybridoma cell lines was done by immunofluorescence to guarantee the reactivity of the monoclonal antibodies both against the recombinant and the native molecule of the parasite. Monoclonal antibodies were characterized by serological and immunochemical techniques. Competitive binding assays between monoclonal antibodies defined four different B epitopes. One epitope is specific for P. falciparum, a second is also present in P. vivax, while the two others seem to be ubiquitous and are also present in the rodent parasite P. chabaudi. The ubiquitous epitope 72.C is apparently the only one recognized by squirrel monkey sera presenting protective antibodies against the asexual blood infection by P. falciparum.     

"Universal" T helper cell determinants enhance immunogenicity of a Plasmodium falciparum merozoite surface antigen peptide.

Synthetic peptide constructs containing a limited number of epitopes are being currently investigated as subunit vaccines against a variety of pathogens. However, because of widespread nonresponsiveness to most such constructs, possibly attributable to MHC restriction, the choice of appropriate carrier molecules to enhance immunogenicity of peptides constitutes an important and essential aspect of designing synthetic immunogens for human use. Widely used vaccines such as tetanus toxoid (TT) have not been uniformly effective as carrier proteins because of the phenomenon of epitope-specific suppression in which induction of an immune response against a synthetic peptide conjugated to TT is prevented by preexisting immunity to TT. Recently, T cell determinants that can be recognized in the context of several class II MHC molecules have been identified in tetanus toxin as well as in the circumsporozoite protein of a human malarial parasite, Plasmodium falciparum. Such determinants can be potentially used to circumvent the problem of epitope-specific suppression. In the present study we evaluated two such T cell determinants, viz., tt830-844 from tetanus toxin and CST3 from the malarial parasite, for their ability to help induce a boostable antibody response and to overcome genetic nonresponsiveness to a synthetic 20-residue construct containing a B cell and an overlapping T cell epitope from a major merozoite surface protein of P. falciparum. Our data provide support for the view that widely recognized T cell determinants may be used as universal carrier molecules for general vaccination.     

Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.     

Production and characteristics of human tissue plasminogen-activator antibodies with region-oriented synthetic peptides.

We selected six peptide sequences as belonging to potential epitopes of tissue plasminogen activator (tPA) using, as the main criterion for their choice, the location of the peptide sequences on the surface of the protein molecule. The six peptides (corresponding to amino acids 4-8, 11-16, 96-101, 272-277, 371-376 and 514-519) were synthesized, coupled to carrier proteins and injected into rabbits. All of these peptides elicited antibodies and 15-75% binding of the corresponding iodinated peptide was obtained with a 1:100 dilution of antiserum. Only two anti-(peptide) sera [anti-(tPA96-101) and anti-(tPA272-277)] reacted with intact tPA and its heavy chain in Western immunoblotting analysis. These two peptides sequences and fragment tPA11-16 appear to be involved in the structure of native antigenic epitopes of tPA, since they were recognized and antibodies present in antisera raised against native tPA. There was no interaction between anti-(tPA4-8) and anti-(tPA371-376) sera with intact one-chain or two-chain tPA. In the case of anti-(tPA4-8) cleavage of one-chain tPA to two-chain tPA and reduction of disulfide bonds exposed this epitope.     

Identification and Characterization of B-Cell Epitopes in the DBL4ε Domain of VAR2CSA

Malaria during pregnancy in Plasmodium falciparum endemic regions is a major cause of mortality and severe morbidity. VAR2CSA is the parasite ligand responsible for sequestration of Plasmodium falciparum infected erythrocytes to the receptor chondroitin sulfate A (CSA) in the placenta and is the leading candidate for a placental malaria vaccine. Antibodies induced in rats against the recombinant DBL4ε domain of VAR2CSA inhibit the binding of a number of laboratory and field parasite isolates to CSA. In this study, we used a DBL4ε peptide-array to identify epitopes targeted by DBL4ε-specific antibodies that inhibit CSA-binding of infected erythrocytes. We identified three regions of overlapping peptides which were highly antigenic. One peptide region distinguished itself particularly by showing a clear difference in the binding profile of highly parasite blocking IgG compared to the IgG with low capacity to inhibit parasite adhesion to CSA. This region was further characterized and together these results suggest that even though antibodies against the synthetic peptides which cover this region did not recognize native protein, the results using the mutant domain suggest that this linear epitope might be involved in the induction of inhibitory antibodies induced by the recombinant DBL4ε domain.     

Translation in vitro of RNA from the human malarial parasitePlasmodium falciparum

RNA has been isolated from the human malarial parasite, Plasmodium falciparum, and translated in vitro using two systems: rabbit-reticulocyte lysate and Xenopus laevis oocytes. Polypeptides are synthesized that are characteristic of the parasite isolate and the developmental stage from which the RNA was purified. Many of these polypeptides are precipitated by antibody from an immune monkey. Analysis of the primary structure of these polypeptides should give the information necessary to produce a vaccine against this parasite.     

Comparative studies on dihydrofolate reductases from Plasmodium falciparum and Aotus trivirgatus.

Dihydrofolate reductase (E.C. 1.5.1.3) from Plasmodium falciparum and from its host, the owl monkey (Aotus trivirgatus), were partially purified and characterized. The molecular weight of the parasite enzyme was estimated to be over 10 times as high as that of the host enzyme. The host enzyme had 2 pH optima whereas the parasite enzyme only one. The activity of the host enzyme was greatly stimulated by KCl and urea, while that of the parasite enzyme was inhibited at high concentrations of such chaotropic agents. Km of the parasite enzyme was significantly higher than that of the host enzyme. The parasite enzyme had much lower Ki for pyrimethamine than the host enzyme. Dihydrofolate reductases isolated from pyrimethamine-resistant and pyrimethamine sensitive strains of P. falciparum were found to be similar.     

Synthetic, immunological and structural studies on repeat unit peptides of Plasmodium falciparum antigens

Using solid phase methodology, we have synthesized five peptides (16-18 residues long) corresponding to repeat sequences of four antigens of a human malarial parasite, Plasmodium falciparum. Three of these antigens (RESA, FIRA, and ABRA) are found in the asexual blood-stages of the parasite, while the remaining one (CSP) is found in the sporozoites. The synthetic peptides, conjugated to bovine serum albumin, elicited high levels of antibodies in rabbits, and these antibodies were found to cross-react with the heterologous peptides. The degree of cross-reactivity, as estimated in an ELISA, was quite remarkable among all the peptides. The peptide corresponding to the RESA tetrapeptide repeat was found to be the most immunogenic and highly cross-reactive. For this reason this tetrapeptide repeat unit, peptide 1, may be a suitable candidate for inclusion in a multiple epitope polypeptide vaccine design. Conformational studies using circular dichroism spectroscopy show that these peptides have similar conformational characteristics with a common feature of approximately 30% and approximately 50% helical content water and TFE respectively. Theoretical predictions regarding conformation using the Chou-Fasman method have also been presented.     

Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding

To minimize ovarian dysfunction subsequent to immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing the antigenic B cell epitopes as immunogens have been proposed. In this study, attempts have been made to clone and express a recombinant chimeric protein encompassing the epitopes corresponding to bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1, amino acid residues 132-147), ZP glycoprotein-2 (bmZP2, amino acid residues 86-113), and ZP glycoprotein-3 (bmZP3, amino acid residues 324-347). The above chimeric recombinant protein (r-bmZP123) was expressed as a polyhistidine fusion protein in Escherichia coli. Immunoblot with murine monoclonal antibody, MA-813, generated against recombinant bmZP1 revealed a major band of approximately 10 kDa. The r-bmZP123 was purified on nickel-nitrilotriacetic acid resin under denaturing conditions. The female rabbits immunized with purified r-bmZP123 conjugated to diphtheria toxoid (DT) generated antibodies that reacted with r-bmZP123 and DT in an ELISA. In addition, the immune sera also reacted with E. coli expressed recombinant bmZP1, bmZP2, and bmZP3. In an indirect immunofluorescence assay, the antibodies against r-bmZP123 recognized native ZP of bonnet monkey as well as human. The immune sera also inhibited, in vitro, the binding of human spermatozoa to the human zona in the hemizona assay (HZA). These studies, for the first time, demonstrate the feasibility of assembling multiple epitopes of different ZP glycoproteins as a recombinant protein that elicit antibodies which are reactive with native zona and also inhibit, in vitro, human sperm-oocyte binding.     

The cysteine bonding in TSH receptor and it function in autoantibodies recognition

The majority of epitopes for TSH receptor (TSHR) stimulating autoantibodies are clustered around the Nterminal region of the TSH receptor. The characteristic feature of this region is the presence of four cysteine residues. It was proposed that cysteines in positions 29 and 41 in the receptor are connected by disulfide bonds and they are the target for receptor stimulating antibodies. The present study was aimed to check this possibility. The synthetic peptides: peptide corresponding to the part of TSHR containing the above 29-41 cysteine bond, the peptide similar to this peptide but without disulfide bond and the control peptide, containing sequence absent in the receptor were used for rabbit immunization. The thyroid status of all immunized rabbits was the same. Rabbits immunized with peptides related to TSHR generated antisera reactive with TSHR in immunoenzymatic assay. To check specificity of this reaction the influence of the peptides and the antisera on TSH binding to the receptor in competitive assay (TRAK) and their influence on adenylate cyclase activity were studied. It was found that neither synthetic peptides nor antiserum from any rabbit influenced TSH binding to the receptor in TRAK. In contrast low, but significant adenylate cyclase stimulating activity was noticed for antisera from two of six rabbit immunized by peptide containing the disulfide bond. We concluded that such a bond between cysteine residues 29 and 41 are present in TSHR in the site of stimulating antibodies epitope.