Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen

Malaria parasites must undergo development within mosquitoes to be transmitted to a new host. Antivector transmission-blocking vaccines inhibit parasite development by preventing ookinete interaction with mosquito midgut ligands. Therefore, the discovery of novel midgut antigen targets is paramount. Jacalin (a lectin) inhibits ookinete attachment by masking glycan ligands on midgut epithelial surface glycoproteins. However, the identities of these midgut glycoproteins have remained unknown. Here we report on the molecular characterization of an Anopheles gambiae aminopeptidase N (AgAPN1) as the predominant jacalin target on the mosquito midgut luminal surface and provide evidence for its role in ookinete invasion. alpha-AgAPN1 IgG strongly inhibited both Plasmodium berghei and Plasmodium falciparum development in different mosquito species, implying that AgAPN1 has a conserved role in ookinete invasion of the midgut. Molecules targeting single midgut antigens seldom achieve complete abrogation of parasite development. However, the combined blocking activity of alpha-AgAPN1 IgG and an unrelated inhibitory peptide, SM1, against P. berghei was incomplete. We also found that SM1 can block only P. berghei, whereas alpha-AgAPN1 IgG can block both parasite species significantly. Therefore, we hypothesize that ookinetes can evade inhibition by two potent transmission-blocking molecules, presumably through the use of other ligands, and that this process further partitions murine from human parasite midgut invasion models. These results advance our understanding of malaria parasite-mosquito host interactions and guide in the design of transmission-blocking vaccines.     

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.     

Induction of crossreactive antibodies against the Plasmodium falciparum variant protein

The variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), present on the surface of P. falciparum-parasitized erythrocytes (PE), plays a central role in naturally acquired immunity, although antibodies to PfEMP1 are predominantly variant specific. To overcome this major limitation for vaccine development, we immunized mice with three cysteine-rich interdomain 1 (CIDR1) domains of PfEMP1 that have the critical function of binding the PE to CD36 on endothelium and thus preventing spleen-dependent killing of the parasite. The immunizations consisted of different combinations of three CIDR1 encoded by DNA followed by recombinant protein boost. Immunizations with a single variant in a prime-boost regimen induced no or low cross-reactivity toward heterologous CIDR1; however, a broad range of crossreactivity was detected in mice that were immunized with all three variants simultaneously. The induced crossreactivity suggests that an anti-PfEMP1 vaccine may be possible.     

Synthetic peptides based on conserved Plasmodium falciparum antigens are immunogenic and protective against Plasmodium yoelii malaria

Two synthetic polypeptides containing multiple B- and T-cell epitopes derived from the conserved regions of two vaccine candidate antigens namely MSA-1 and RESA of human malarial parasite P. falciparum were studied for immunogenicity and protectivity. Both constructs elicited strong antibody and lymphocyte proliferation responses in BALB/c mice immunized with the carrier-free peptides. In an ELISA, these peptides also bound antibodies present in the sera from the P. vivax infected humans as well as from the P. yoelii infected mice. Significantly, our data showed that immunization of mice with these P. falciparum peptide could impart partial protection against P. yoelii challenge infection. Our finding that synthetic peptides representing portions of P. falciparum antigens were capable of stimulating protective immune responses against rodent malaria suggests that murine malaria model P. yoelii may provide a suitable system for primary screening of potentially protective synthetic immunogens.     

Identification of a conserved Plasmodium falciparum var gene implicated in malaria in pregnancy

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family is a highly polymorphic class of variant surface antigens encoded by var genes that play an important role in malaria pathogenesis. This report describes the unexpected finding that 1 of the var genes encoding a PfEMP1 variant that binds to the host receptor chondroitin sulfate A (CSA) and is implicated in malaria in pregnancy is well conserved among P. falciparum isolates worldwide. The N-terminal domains of this PfEMP1 variant are especially highly conserved, whereas the functional CSA binding domain is more variable. Analysis of var gene expression in placental parasites from primigravid women in Malawi did not support a role for this conserved gene in placental infection but identified a second commonly occurring var gene. These results indicate the need for reevaluation of previous assumptions of a minimal overlap between var gene repertoires from different parasite isolates.     

Human monoclonal antibodies against Plasmodium falciparum: production, stabilization and characterization

Nine human monoclonal antibodies (MoAbs) recognizing 7 different antigenic structures of blood-stages of the human malarial parasite P. falciparum (Pf) were produced by Epstein-Barr virus transformed B-cell lines (EBV-TCL) with or without fusion to the lymphoblastoid cell line KR4. The peripheral blood B-lymphocytes were obtained from 8 Gambian donors immune to Pf malaria. Two of the EBV-TCL could be expanded and maintained for more than 6 months but neither one could be cloned. Six additional EBV-TCL were stabilized after fusion with the KR4 lymphoblastoid cell line. All resulting hybridomas permitted easy cloning. Some of the MoAbs produced distinct fluorescent staining patterns of asexual Pf blood-stage parasites when using high-resolution digitized video-intensified fluorescence microscopy. Antigens on 195 kD and 155 kD proteins were recognized by 3 and 1 MoAb, respectively, using Western blotting and immunoprecipitation techniques.     

Naturally acquired antibodies to polymorphic and conserved epitopes of Plasmodium falciparum merozoite surface protein 3

Many studies on the role of merozoite surface protein 3 (MSP3) in immunity against malaria have focused on a conserved section of MSP3. New evidence suggests that polymorphic sequences within MSP3 are under immune selection. We report a detailed analysis of naturally-acquired antibodies to allele-specific and conserved parts of MSP3 in a Kenyan cohort. Indirect and competition ELISA to heterologous recombinant MSP3 proteins were used for antibody assays, and parasites were genotyped for msp3 alleles. Antibody reactivity to allele-specific and conserved epitopes of MSP3 was heterogeneous between individuals. Overall, the prevalence of allele-specific antibody reactivity was significantly higher (3D7-specific 54%, K1-specific 41%) than that to a recombinant protein representing a conserved portion of C-terminal MSP3 (24%, P < 0.01). The most abundant IgG subclass was IgG3, followed by IgG1. Allele-specific reactivity to the K1-type of MSP3 was associated with a lower risk of clinical malaria episodes during a 6-month follow-up in individuals who were parasitized at the start of the malaria transmission season (Relative risk 0.41 with 95% confidence interval 0.20-0.81, P = 0.011). The potential importance of allele-specific immunity to MSP3 should be considered in addition to immunity to conserved epitopes, in the development of an MSP3 malaria vaccine.     

Validation of isoleucine utilization targets in Plasmodium falciparum

Intraerythrocytic malaria parasites can obtain nearly their entire amino acid requirement by degrading host cell hemoglobin. The sole exception is isoleucine, which is not present in adult human hemoglobin and must be obtained exogenously. We evaluated two compounds for their potential to interfere with isoleucine utilization. Mupirocin, a clinically used antibacterial, kills Plasmodium falciparum parasites at nanomolar concentrations. Thiaisoleucine, an isoleucine analog, also has antimalarial activity. To identify targets of the two compounds, we selected parasites resistant to either mupirocin or thiaisoleucine. Mutants were analyzed by genome-wide high-density tiling microarrays, DNA sequencing, and copy number variation analysis. The genomes of three independent mupirocin-resistant parasite clones had all acquired either amplifications encompassing or SNPs within the chromosomally encoded organellar (apicoplast) isoleucyl-tRNA synthetase. Thiaisoleucine-resistant parasites had a mutation in the cytoplasmic isoleucyl-tRNA synthetase. The role of this mutation in thiaisoleucine resistance was confirmed by allelic replacement. This approach is generally useful for elucidation of new targets in P. falciparum. Our study shows that isoleucine utilization is an essential pathway that can be targeted for antimalarial drug development.     

Clindamycin activity against chloroquine-resistant Plasmodium falciparum

The clindamycin dose-response curves observed with both chloroquine-resistant and chloroquine-susceptible strains of Plasmodium falciparum in vitro demonstrated a plateau region that extended from 10(-2) to 10(1) micrograms/ml of drug (22 nM to 22 microM). Similar dose-response curves were also observed with the three major metabolites of clindamycin (clindamycin sulfoxide, de-N-methyl clindamycin, and de-N-methyl clindamycin sulfoxide). The position of this plateau was time dependent and rose from 20%-25% to 90%-95% inhibition of parasite growth between 24 and 72 hr of exposure to the drug. Clinidamycin treatment reduced plasmodial protein and nucleic acid synthesis (as measured by the incorporation of [3H]isoleucine and [3H]hypoxanthine, respectively) but did not interfere with knob formation. The combination of quinine plus a fixed concentration of clindamycin (0.1 microgram/ml) inhibited growth of the quinine-resistant Indochina I strain, although most of the antiplasmodial activity observed at quinine concentrations less than 50 ng/ml (154 nM) could be attributed to clindamycin alone.     

Immunoprecipitating antibodies against blood stage antigens of Plasmodium falciparum in cerebral and non-cerebral malaria patients

Immunoprecipitating antibodies were determined in paired sera of 31 patients with cerebral malaria (CM), of whom 14 had complicated cerebral malaria (CCM) and 17 had uncomplicated cerebral malaria (UCCM), 15 single specimens of patients with acute uncomplicated (AM) malaria taken on the day of admission and 8 healthy controls. All but one patient were admitted within the first three days of the onset of fever. More than 20 precipitating bands were observed, of which the predominating molecules were the Mr greater than 200, 180, 157, 135, 130, 115, 103, 96, 91, 73, 71, 61, 49, 45, 43, 41 and 14.3 Kd. In general, there were no significant differences in the positive rates among the AM, CCM and UCCM patients except for the pf135 Kd molecule which was more frequently reactive in UCCM patients than the AM and CCM patients. If immunological naiveness in term of protective immunity is the feature in CM patients, the immunoprecipitation test used is inadequate to demonstrate the fundamental differences in immune responses between CM and AM patients.     

Natural infection of Plasmodium falciparum induces inhibitory antibodies against gametocyte development in human hosts

We identified naturally induced antibodies from malaria patients in Thailand and clarified the effect of the antibodies on gametocyte development. Fifty-nine percent of the Plasmodium falciparum-infected blood samples (17 of 29) fed to female Anopheles mosquitoes showed no oocyst infection. Seventeen percent of the samples (5 of 29) distorted the morphology and hampered the maturity of the gametocytes. A possible mechanism for the gametocyte inhibitory activity was shown by the binding of the plasma antibodies to live, immature, intraerythrocytic gametocytes during the incubation period. One hundred fifty-seven proteins specific to different gametocyte stages were explored to find the targets of the antisera that bound to the live gametocytes. However, no additional gametocyte transmission-blocking vaccine candidate was detected. Therefore, the development of alternative transmission-blocking vaccines in high-transmission areas should focus on the identification of more gametocyte antigens-inducing inhibitory antibodies that reduce gametocytemia.     

Isolation and characterization of parasite-inhibitory Plasmodium falciparum monoclonal antibodies

Three mouse hybridomas were isolated that produced IgM monoclonal antibodies (Mab) which reacted with erythrocytic stages of Plasmodium falciparum and inhibited the invasion of erythrocytes in vitro. Those Mab, initially identified by an ELISA screening of hybridoma culture medium, exhibited a strong binding to trophozoite and schizont but not to ring or merozoite stage parasites or to erythrocytes in an indirect immunofluorescence assay. All inhibited the parasite's ability to infect erythrocytes in an in vitro invasion inhibition assay. Western blot analysis of the binding of the Mab to SDS-PAGE-separated parasite antigens isolated from the ring, trophozoite, schizont or spontaneously released merozoite stages, indicated that two of the Mab bound to a Mr 105,000 antigen in trophozoites and schizonts while the third Mab did not. All three Mab also bound to Mr 30,000-40,000 antigens in all stages, however, in all instances binding to these antigens was enhanced in merozoites. It was further observed that the two Mabs that bound to a Mr 105,000 antigen: exhibited a markedly reduced binding to the Mr 105,000 antigen in merozoite preparations; exhibited different relative intensities of binding to the trophozoite and schizont antigens; both bound to the same Mr 105,000 antigen as demonstrated through Western blot analysis of antigens separated by two-dimensional gel electrophoresis. The findings suggest that the inhibitory Mab bound to different epitopes of the same antigen and that the antigen may either be processed or degraded at about the time of merozoite release and erythrocyte invasion.     

Activity of selected phytochemicals against Plasmodium falciparum

According to the WHO, in 2008, there were 247 million reported cases of malaria and nearly one million deaths from the disease. Parasite resistance against first-line drugs, including artemisinin and mefloquine, is increasing. In this study the plant-derived compounds aglafolin, rocaglamid, kokusaginine, arborine, arborinine and tuberostemonine were investigated for their anti-plasmodial activity in vitro. Fresh Plasmodium falciparum isolates were taken from patients in the area of Mae Sot, north-western Thailand in 2008 and the inhibition of schizont maturation was determined for the respective compounds. With inhibitory concentrations effecting 50%, 90% and 99% inhibition (IC(50), IC(90) and IC(99)) of 60.95 nM, 854.41 nM and 7351.49 nM, respectively, rocaglamid was the most active of the substances, closely followed by aglafoline with 53.49 nM, 864.55 nM and 8354.20 nM. The activity was significantly below that of artemisinin, but moderately higher than that of quinine. Arborine, arborinine, tuberostemonine and kokusaginine showed only marginal activity against P. falciparum characterized by IC(50) and IC(99) values higher than 350 nM and 180 μM, respectively, and regressions with relatively shallow slopes S>14.38. Analogues of rocaglamid and aglafoline merit further exploration of their anti-plasmodial activity.     

Multiplicity of Plasmodium falciparum infection in pregnancy.

Little is known about the distribution and disease association of multiple Plasmodium falciparum infections in pregnant women. Genotyping of the merozoite surface protein-1 region was performed in 332 P. falciparum infected pregnant women in Ghana, and clinical and epidemiologic data were obtained. Overall, 68% of the women were infected with more than one strain (mean number of strains per carrier = 2.9). The multiplicity of infection decreased significantly with an increasing number of pregnancies, and infection with multiple P. falciparum strains was significantly associated with anemia. In logistic regression, women infected with four or more strains were 2.3 times more likely to be anemic than women harboring fewer strains. This association, however, was only observed in women with up to three pregnancies. The results suggest that with increasing gravidity and subsequent infections with multiple strains effective immune mechanisms against more and more strains develop. In pregnant women, the multiplicity of infection may be an important factor for the acquisition and maintenance of immunity against malaria.     

Predicted essential proteins of Plasmodium falciparum for potential drug targets

ObjectiveTo identify novel drug targets for treatment of Plasmodium falciparum.MethodsLocal BLASTP were used to find the proteins non-homologous to human essential proteins as novel drug targets. Functional domains of novel drug targets were identified by InterPro and Pfam, 3D structures of potential drug targets were predicated by the SWISS-MODEL workspace. Ligands and ligand-binding sites of the proteins were searched by Ef-seek.ResultsThree essential proteins were identified that might be considered as potential drug targets. AAN37254.1 belonged to 1-deoxy-D-xylulose 5-phosphate reductoisomerase, CAD50499.1 belonged to chorismate synthase, CAD51220.1 belonged to FAD binging 3 family, but the function of CAD51220.1 was unknown. The 3D structures, ligands and ligand-binding sites of AAN37254.1 and CAD50499.1 were successfully predicated.ConclusionsTwo of these potential drug targets are key enzymes in 2-C-methyl-d-erythritol 4-phosphate pathway and shikimate pathway, which are absent in humans, so these two essential proteins are good potential drug targets. The function and 3D structures of CAD50499.1 is still unknown, it still need further study.