Demonstration of antigenic polymorphism in Plasmodium vivax malaria with a panel of 30 monoclonal antibodies.

A panel of 30 monoclonal antibodies was established against asexual erythrocytic stages of Plasmodium vivax and used to investigate the antigenic composition of the parasite. At least 38 different antigenic polypeptides of P. vivax were characterized by the Western blot technique. The possible location of these antigens, as well as their stage and species specificity, was determined on the basis of the staining patterns produced by these antibodies on air-dried parasites in the indirect immunofluorescence test. Immunofluorescence performed with 30 different monoclonal antibodies on 50 different isolates of P. vivax obtained from patients showed that a high level of antigenic polymorphism prevailed in P. vivax. Only six monoclonal antibodies reacted with epitopes that were represented in more than 80% of parasite isolates, and therefore, appeared to be relatively conserved among different isolates. The other 24 monoclonal antibodies reacted with only 20 to 70% of parasite isolates.     

Monoclonal antibody identifies circumsporozoite protein of Plasmodium malariae and detects a common epitope on Plasmodium brasilianum sporozoites.

We produced a hybridoma secreting an immunoglobulin G1 monoclonal antibody against the circumsporozoite protein of the human malaria parasite Plasmodium malariae (Uganda 1/CDC). The monoclonal antibody produces a circumsporozoite precipitation reaction when incubated with viable sporozoites of P. malariae and reacts at high titers with heat-fixed sporozoites in an indirect immunofluorescent antibody test. Using the purified monoclonal antibody and Western blot analysis, we identified two polypeptides with apparent molecular weights of 60,000 (Pm 60) and 48,000 (Pm 48) in extracts of P. malariae sporozoites. Two-dimensional electrophoretic analysis of Pm 60 and the circumsporozoite protein Pm 48 indicated their isoelectric points to be acidic, with values of 5.3 and 4.7 to 5.0, respectively. A two-site immunoradiometric assay showed that the circumsporozoite protein recognized by the monoclonal antibody contains a repetitive epitope. P. malariae monoclonal antibody also reacted strongly with sporozoites of the simian parasite Plasmodium brasilianum, indicating a shared epitope on sporozoites of the two species. The P. malariae antibody did not bind sporozoite antigens of any other primate malarias, including Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale.     

Expression and serologic activity of a soluble recombinant Plasmodium vivax Duffy binding protein.

Plasmodium vivax Duffy binding protein (DBP) is a conserved functionally important protein. P. vivax DBP is an asexual blood-stage malaria vaccine candidate because adhesion of P. vivax DBP to its erythrocyte receptor is essential for the parasite to continue development in human blood. We developed a soluble recombinant protein of P. vivax DBP (rDBP) and examined serologic activity to it in residents of a region of high endemicity. This soluble rDBP product contained the cysteine-rich ligand domain and most of the contiguous proline-rich hydrophilic region. rDBP was expressed as a glutathione S-transferase (GST) fusion protein and was isolated from GST by thrombin treatment of the purified fusion protein bound on glutathione agarose beads. P. vivax rDBP was immunogenic in rabbits and induced antibodies that reacted with P. vivax and Plasmodium knowlesi merozoites. Human sera from adult residents of a region of Papua New Guinea where malaria is highly endemic or P. vivax-infected North American residents reacted with rDBP in an immunoblot and an enzyme-linked immunosorbent assay. The reactivity to reduced, denatured P. vivax rDBP and the cross-reactivity with P. knowlesi indicated the presence of immunogenic conserved linear B-cell epitopes. A more extensive serologic survey of Papua New Guinea residents showed that antibody response to P. vivax DBP is common and increases with age, suggesting a possible boosting of the antibody response in some by repeated exposure to P. vivax. A positive humoral response to P. vivax DBP correlated with a significantly higher response to P. vivax MSP-1(19). The natural immunogenicity of this DBP should strengthen its usefulness as a vaccine.     

Circumsporozoite protein of the human malaria parasite Plasmodium ovale identified with monoclonal antibodies.

Monoclonal antibodies (MAbs) have been produced against Plasmodium ovale sporozoites and used to characterize the circumsporozoite (CS) protein. Six MAbs were produced, and all were species specific. By using Western blot (immunoblot) analysis, three polypeptides were detected: a predominant 51,000-Mr polypeptide and two presumed precursor 57,000- and 67,000-Mr molecules. The presence of a repeating epitope in the CS protein of P. ovale was demonstrated by using one of the MAbs in a single-antibody two-site enzyme immunoassay. Three MAbs recognized epitopes on the surfaces of sporozoites; the presence of at least one other epitope within the CS protein, but not on the surfaces of P. ovale sporozoites, was also demonstrated.     

Enhancement or inhibition of Plasmodium falciparum erythrocyte reinvasion in vitro by antibodies to an asparagine rich protein

A clone encoding a recombinant protein which reacted strongly with human antibodies from a donor clinically immune to malaria, was isolated from a genomic Plasmodium falciparum library. Mice injected with this protein, designated 10b, produced antibodies which reacted with all developmental stages of erythrocytic asexual parasites in indirect immunofluorescence. In immunoblotting, the same antibodies recognized two P. falciparum polypeptides of 36 kDa and 33 kDa. Of three monoclonal antibodies raised against the 10b recombinant protein, two inhibited parasite reinvasion of erythrocytes in an isolate specific manner. Surprisingly, however, the third was found to significantly enhance reinvasion of erythrocytes and also to induce a more rapid maturation of intraerythrocytic parasites in all isolates tested. Nucleotide sequence analysis of the 1124 bp insert revealed that it encodes a protein which consists of 30% asparagine and contains three asparagine rich, imperfect tandem repeats: Lys-Lys-Asn-Asn (3x), Met-Asn-His/Gln-Pro-Asn-Asn (14x), and Lys-Asn-Asn-Asn-Asn (7x).     

A panel of monoclonal antibodies targeting the rabies virus phosphoprotein identifies a highly variable epitope of value for sensitive strain discrimination

A recombinant rabies virus phosphoprotein fusion product (GST-P) was used to generate a series of monoclonal antibodies (MAbs) with anti-P reactivity. Competitive binding assays classified 27 of these MAbs into four groups (I to IV), and 24 of them were deemed to recognize linear epitopes, as judged by their reaction in immunoblots. The linear epitope recognized in each case was mapped by using two series of N- and C-terminally deleted recombinant phosphoproteins. Assessment of the reactivities of representative MAbs to a variety of lyssavirus isolates by an indirect fluorescent antibody test indicated that group I MAbs, which recognized a highly conserved N-terminal epitope, were broadly cross-reactive with all lyssaviruses assayed, while group III MAbs, which reacted with a site overlapping that of group I MAbs, exhibited variable reactivities and group IV MAbs reacted with most isolates of genotypes 1, 6, and 7 only. In contrast, group II MAbs, which recognized an epitope located within a highly divergent central portion of the protein, were exquisitely strain specific. These anti-P MAbs are potentially useful tools for lyssavirus identification and discrimination.     

Identification and characterization of a conserved,stage-specific gene product of Plasmodium falciparum recognized by parasite growth inhibitory antibodies

We have identified a novel conserved protein of Plasmodium falciparum, designated D13, that is stage-specifically expressed in asexual blood stages of the parasite. The predicted open reading frame (ORF) D13 contains 863 amino acids with a calculated molecular mass of 99.7 kDa and displays a repeat region composed of pentapeptide motives. Northern blot analysis with lysates of synchronized blood stage parasites showed that D13 is highly expressed at the mRNA level during schizogony. The first N'-terminal 138 amino acids of D13 were expressed in Escherichia coli and the purified protein was used to generate anti-D13 monoclonal antibodies (MAbs). Using total lysates of blood stage parasites and Western blot analysis, these MAbs stained one single band of approximately 100 kDa, corresponding to the predicted molecular mass of ORF D13. Western blot analysis demonstrated further that D13 is expressed during schizogony, declines rapidly in early ring stages and is undetectable in trophozoites. D13 protein is localized in individual merozoites in a distinct area, as demonstrated by indirect immunofluorescence analysis. After subcellular fractionation, D13 was confined to the pelleted fraction of the parasite lysate and its extraction by alkaline carbonate buffer treatment indicated that D13 is not a membrane-integral protein. Inclusion of certain anti-D13 MAbs into in vitro cultures of blood stage parasites resulted in considerable reduction in parasite growth. The N'-terminal domain encompassing 158 amino acids is 94 and 95%, respectively, identical at the amino acid level between Plasmodium knowlesi, Plasmodium yoelii, and P. falciparum. In summary, we describe a novel stage-specifically expressed, highly conserved gene product of P. falciparum that is recognized by parasite growth inhibitory antibodies.     

Malaria transmission-blocking immunity induced by natural infections of Plasmodium vivax in humans.

Immunity to malarial infections in human populations is known to affect the development of the asexual blood stages of the parasites in the human host and to be capable of conferring significant protection against morbidity and mortality due to the disease. In this study we show that during acute infection with Plasmodium vivax malaria, one of the two main malarial pathogens of humans, most individuals also develop immunity that suppresses the infectivity of the sexual stages of the parasite to mosquitoes. The immunity is antibody mediated and is directed against the parasites in the mosquito midgut shortly after ingestion of blood by a mosquito. This immunity could be expected to have significant effects on the natural transmission of P. vivax malaria.     

Antibodies to lactate dehydrogenase of Plasmodium knowlesi are specific to Plasmodium species

Polyclonal immune monkey serum raised against schizonts of Plasmodium knowlesi (H-strain) showed the presence of antibodies to lactate dehydrogenase (LDH) of P. knowlesi by immunodot enzyme staining method. The anti-LDH antibodies are most probably directed towards an epitope distinct from the catalytic site as shown by the specific enzyme staining of LDH after binding with antibody on nitrocellulose paper. These antibodies showed reactivity with LDH from different strains (H, P and W1 strains of P. knowlesi) and species (P. cynomolgi B, P. berghei, P. yoelii, P. falciparum and P. vivax) of malarial parasites but did not cross-react with three isoenzymic forms of mammalian LDH (A4, B4 and C4) as well as with LDH from some protozoan and helminth parasites. These findings suggest that the anti-LDH antibodies have defined specificity to Plasmodium spp.     

Plasmodium ovale: parasite and disease

Humans are infected by four recognized species of malaria parasites. The last of these to be recognized and described is Plasmodium ovale. Like the other malaria parasites of primates, this parasite is only transmitted via the bites of infected Anopheles mosquitoes. The prepatent period in the human ranges from 12 to 20 days. Some forms in the liver have delayed development, and relapse may occur after periods of up to 4 years after infection. The developmental cycle in the blood lasts approximately 49 h. An examination of records from induced infections indicated that there were an average of 10.3 fever episodes of > or = 101 degrees F and 4.5 fever episodes of > or = 104 degrees F. Mean maximum parasite levels were 6,944/microl for sporozoite-induced infections and 7,310/microl for trophozoite-induced infections. Exoerythrocytic stages have been demonstrated in the liver of humans, chimpanzees, and Saimiri monkeys following injection of sporozoites. Many different Anopheles species have been shown to be susceptible to infection with P. ovale, including A. gambiae, A. atroparvus, A. dirus, A. freeborni, A. albimanus, A. quadrimaculatus, A. stephensi, A. maculatus, A. subpictus, and A. farauti. An enzyme-linked immunosorbent assay has been developed to detect mosquitoes infected with P. ovale using a monoclonal antibody directed against the circumsporozoite protein. Plasmodium ovale is primarily distributed throughout sub-Saharan Africa. It has also been reported from numerous islands in the western Pacific. In more recent years, there have been reports of its distribution on the Asian mainland. Whether or not it will become a major public health problem there remains to be seen. The diagnosis of P. ovale is based primarily on the characteristics of the blood stages and its differentiation from P. vivax. The sometimes elliptical shape of the infected erythrocyte is often diagnostic when combined with other, subtler differences in morphology. The advent of molecular techniques, primarily PCR, has made diagnostic confirmation possible. The development of techniques for the long-term frozen preservation of malaria parasites has allowed the development diagnostic reference standards for P. ovale. Infections in chimpanzees are used to provide reference and diagnostic material for serologic and molecular studies because this parasite has not been shown to develop in other nonhuman primates, nor has it adapted to in vitro culture. There is no evidence to suggest that P. ovale is closely related phylogenetically to any other of the primate malaria parasites that have been examined.     

Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.     

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.     

Antibodies to the ring-infected erythrocyte surface antigen of Plasmodium falciparum elicited by infection with Plasmodium malariae.

The ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum (RESA-P), found in the membrane of erythrocytes infected with young asexual stages of P. falciparum, is a promising vaccine candidate. Antibodies to RESA-P were inducible by infection with another human malaria species, P. malariae. Of 298 serum samples from inhabitants of three isolated localities in Peru where P. vivax and P. malariae were endemic and P. falciparum had never been reported, 26% had anti-RESA-P antibodies as evidenced by a modified immunofluorescent-antibody assay and confirmed by Western blot (immunoblot) analysis. These seroepidemiologic observations were corroborated by the fact that of six chimpanzees infected with P. malariae, three developed anti-RESA-P antibodies after infection. The modified immunofluorescent-antibody-reactive antibodies, purified by adsorption and elution on monolayers of glutaraldehyde-fixed and air-dried P. falciparum-infected erythrocytes, reacted in an immunofluorescent-antibody assay with both parasite structures and erythrocyte membrane in P. falciparum antigen preparations, but only with parasite structures in P. malariae antigen preparations. This serologic cross-reactivity between P. falciparum and P. malariae is of interest in view of the importance of RESA-P as a vaccine candidate and because the two species are coendemic in many areas.     

Regulation of the immune response in Plasmodium falciparum malaria: IV. T cell dependent production of immunoglobulin and anti-P. falciparum antibodies in vitro.

T cells from patients with acute Plasmodium falciparum malaria were investigated for induction of immunoglobulin- or anti-malaria antibody secretion in vitro. Stimulation of autologous T/B cell mixtures (2T:1B) with low concentrations of P. falciparum antigen and cultured for 12 days gave rise to a T-dependent IgG secretion which was significantly elevated over that in medium controls. This was achieved with both a crude P. falciparum antigen and a partially purified preparation enriched in Pf 155, a merozoite-derived antigen deposited in the red cell membrane at invasion (Perlmann et al., 1984). Control antigen (RBC ghosts) induced IgG secretion only when added at high concentrations (greater than 10 micrograms/ml). Neither of the antigens induced IgG secretion at concentrations of less than or equal to 10 micrograms/ml in control cultures of lymphocytes from patients with P. vivax malaria. Supernatants from cultures of P. falciparum patients frequently contained anti-P. falciparum antibodies when nanogram quantities (10-100 ng/ml) of either one of the two malaria antigen preparations was used for stimulation. No anti-P. falciparum antibodies were induced by the control antigen at any concentration. The induced anti-P. falciparum antibodies were directed to intracellular parasites and. at lower frequencies, to Pf 155 as detected on the surface of infected erythrocytes. The induction in vitro of anti-P. falciparum antibodies appeared to be correlated with the presence of such antibodies in the sera of the lymphocyte donors. The lymphocytes of only one out of eight P. vivax patients responded to antigen stimulation by secreting anti-P. falciparum antibodies. However, this donor (but not any of the others), was also P. falciparum seropositive. Taken together, these results indicate that the induction of anti-P. falciparum antibody secretion reflects a secondary response in vitro of cells primed in vivo. The present experimental system should be well suited to map parasite antigen for their capacity to induce T cell dependent responses in P. falciparum malaria.     

Conserved and variant epitopes of Plasmodium vivax Duffy binding protein as targets of inhibitory monoclonal antibodies

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains.     

Real-time PCR for detection and identification of Plasmodium spp

Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We developed and used a real-time PCR assay to detect and distinguish four Plasmodium spp. that cause human disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on nucleotide variations within the amplicons provided a basis for accurate differentiation of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. For assay validation, 358 patient blood samples from the National University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76 blinded patient samples with a microscopic diagnosis of P. falciparum, P. vivax, or P. ovale infection, 74 (97.4%) were detected by real-time PCR, including three specimens containing mixed P. falciparum-P. vivax infections. No Plasmodium DNA was amplified in any of the 82 specimens sent for malaria testing but that were microscopically negative for Plasmodium infection. In addition, 200 blood samples from patients whose blood was collected for reasons other than malaria testing were also determined to be negative by real-time PCR. Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of Giemsa-stained blood smears and may replace microscopy following further validation.     

Atypical forms of Plasmodium vivax. Apropos of a case]

In one case of human malaria imported from Gabon, abnormal forms of Plasmodium vivax are described; severe multiple infections of the host erythrocytes are noticed (up to 6 amoeboid trophozoites in a single red blood cell). Attention is drawn to the numerous morphologically abnormal intra-erythrocytic stages of P. vivax: severe multiple infections of red cells, parasites "ressembling" P. ovale, mature schizonts of small size, with reduced number of merozoites... The geographical distribution of P. vivax in Central Africa is discussed, with reflections on the factors allowing the importation of few P. vivax cases from this area.     

Epitope-specific humoral immunity to Plasmodium vivax Duffy binding protein

Erythrocyte invasion by Plasmodium vivax is completely dependent on binding to the Duffy blood group antigen by the parasite Duffy binding protein (DBP). The receptor-binding domain of this protein lies within a cysteine-rich region referred to as region II (DBPII). To examine whether antibody responses to DBP correlate with age-acquired immunity to P. vivax, antibodies to recombinant DBP (rDBP) were measured in 551 individuals residing in a village endemic for P. vivax in Papua New Guinea, and linear epitopes mapped in the critical binding region of DBPII. Antibody levels to rDBP(II) increased with age. Four dominant linear epitopes were identified, and the number of linear epitopes recognized by semi-immune individuals increased with age, suggesting greater recognition with repeated infection. Some individuals had antibodies to rDBP(II) but not to the linear epitopes, indicating the presence of conformational epitopes. This occurred in younger individuals or subjects acutely infected for the first time with P. vivax, indicating that repeated infection is required for recognition of linear epitopes. All four dominant B-cell epitopes contained polymorphic residues, three of which showed variant-specific serologic responses in over 10% of subjects examined. In conclusion, these results demonstrate age-dependent and variant-specific antibody responses to DBPII and implicate this molecule in partial acquired immunity to P. vivax in populations in endemic areas.     

Cloning and characterization of Plasmodium vivax thioredoxin peroxidase-1

Reactive oxygen species produced from hemoglobin digestion and the host immune system could have adverse effects on malaria parasites. To protect themselves, malaria parasites are highly dependent on the antioxidant enzymes, including superoxide dismutases and thioredoxin-dependent peroxidases. To date, several thioredoxin peroxidases (TPx) have been characterized in Plasmodium falciparum, but the TPx in Plasmodium vivax has not yet been characterized. The complete sequence of gene coding for thioredoxin peroxidase-1 of P. vivax (PvTPx-1) was amplified by PCR and cloned. Using the recombinant PvTPx-1 (rPvTPx-1), polyclonal antibody was produced in mice for immunolocalization of the enzyme in the parasite. The antioxidant activity of rPvTPx-1 was evaluated by mixed-function oxidation assay. PvTPx-1 has two conserved cysteine residues in the amino acid sequence at the positions 50 and 170 which formed a dimer under a non-reducing condition. Using a thiol mixed-function oxidation assay, the antioxidant activity of rPvTPx-1 was revealed. Indirect immunofluorescence microscopy with the specific antibody indicated that PvTPx-1 was expressed in the cytoplasm of the erythrocytic stage of the parasite in a dots-like pattern. The results suggest that P. vivax uses TPx-1 to reduce and detoxify hydrogen peroxides in order to maintain their redox homeostasis and proliferation in the host body.     

Genetic restriction of the murine humoral response to a recombinant Plasmodium vivax circumsporozoite protein

The antibody response of inbred and congenic strains of mice immunized with a recombinant Plasmodium vivax circumsporozoite protein was investigated. Only mice of the H-2k and H-2a haplotypes developed antibodies that react with P. vivax sporozoite in an indirect immunofluorescence assay and with the recombinant protein in enzyme-linked immunosorbent assay. Congenic recombinant strains were used to map Ir gene control to the I-Ak locus. A similar pattern of genetic restriction was found when congenic strains were immunized with a carrier-free synthetic peptide corresponding to the repeat region of the P. vivax circumsporozoite protein. The results suggest that the ability of T helper cells to recognize a T cell epitope(s) in the repeat region of the P. vivax circumsporozoite protein is genetically restricted.