Quassinoids Exhibit Greater Selectivity Against Plasmodium Falciparum Than Against Entamoeba Histolytica, Giardia Intestinalis Or Toxoplasma Gondii In Vitro

ABSTRACT. The in vitro activities of a series of quassinoids against Plasmodium falciparum, Entamoeba histolytica, Giardia intestinalis and Toxoplasma gondii have been compared with their in vitro cytotoxic effects against KB cells (human epidermoid carcinoma of the nasopharynx). All of the compounds tested were more toxic to KB cells than to G. intestinalis , but four (ailanthinone, bruceine D, brusatol and glaucarubinone) were slightly less toxic to KB cells than to E. histolytica. Glaucarubinone was similarly more toxic to intracellular T. gondii than to KB cells but ailanthinone was more selective (36 times more toxic to T. gondii than to KB cells). All of the compounds were more toxic to P. falciparum than to KB cells; the most selective quassinoids—glaucarubinone, bruceine D, ailanthinone and brusatoi—were found to have toxicity/activity ratios of 285, 76, 48 and 32 respectively. These results suggest that quassinoids have a selective action on P. falciparum. Further studies to elucidate the basis for this are in progress.     

Potentiation of antimalarial drug action by chlorpheniramine against multidrug-resistant Plasmodium falciparum in vitro

Chlorpheniramine, a histamine H1 receptor antagonist, was assayed for in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum K1 strain and chloroquine-resistant P. falciparum T9/94 clone, by measuring the 3H-hypoxanthine incorporation. Chlorphenirame inhibited P. falciparum K1 and T9/94 growth with IC50 values of 136.0+/-40.2 microM and 102.0+/-22.6 microM respectively. A combination of antimalarial drug and chlorpheniramine was tested against resistant P. falciparum in vitro. Isobologram analysis showed that chlorpheniramine exerts marked synergistic action on chloroquine against P. falciparum K1 and T9/94. Chlorpheniramine also potentiated antimalarial action of mefloquine, quinine or pyronaridine against both of the resistant strains of P. falciparum. However, chlorpheniramine antagonism with artesunate was obtained in both P. falciparum K1 and T9/94. The results in this study indicate that antihistaminic drugs may be promising candidates for potentiating antimalarial drug action against drug resistant malarial parasites.     

Functional conservation of the malaria vaccine antigen MSP-119across distantly related Plasmodium species

The C-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP-119) is at present a leading malaria vaccine candidate. Antibodies against the epidermal growth factor-like domains of MSP-1 19are associated with immunity to P. falciparum and active immunization with recombinant forms of the molecule protect against malaria challenge in various experimental systems. These findings, with the knowledge that epidermal growth factor-like domains in other molecules have essential binding functions, indicate the importance of this protein in merozoite invasion of red blood cells. Despite extensive molecular epidemiological investigations, only limited sequence polymorphism has been identified in P. falciparum MSP-119 (refs. 9-11). This indicates its sequence is functionally constrained, and is used in support of the use of MSP-119 as a vaccine. Here, we have successfully complemented the function of most of P. falciparum MSP-119 with the corresponding but highly divergent sequence from the rodent parasite P. chabaudi. The results indicate that the role of MSP-119 in red blood cell invasion is conserved across distantly related Plasmodium species and show that the sequence of P. falciparum MSP-119 is not constrained by function.     

The Plasmodium vivax rhoptry-associated protein 1

Rhoptries are cellular organelles localized at the apical pole of apicomplexan parasites. Their content is rich in lipids and proteins that are released during target cell invasion. Plasmodium falciparum rhoptry-associated protein 1 (RAP1) has been the most widely studied among this parasite species' rhoptry proteins and is considered to be a good anti-malarial vaccine candidate since it displays little polymorphism and induces antibodies in infected humans. Monoclonal antibodies directed against RAP1 are also able to inhibit target cell invasion in vitro and protection against P. falciparum experimental challenge is induced when non-human primates are immunized with this protein expressed in its recombinant form. This study describes identifying and characterizing RAP1 in Plasmodium vivax, the most widespread parasite species causing malaria in humans, producing more than 80 million infections yearly, mainly in Asia and Latin America. This new protein is encoded by a two-exon gene, is proteolytically processed in a similar manner to its falciparum homologue and, as observed by microscopy, the immunofluorescence pattern displayed is suggestive of its rhoptry localization. Further studies evaluating P. vivax RAP1 protective efficacy in non-human primates should be carried out taking into account the relevance that its P. falciparum homologue has as an anti-malarial vaccine candidate.     

The structural and functional diversity of Hsp70 proteins from Plasmodium falciparum

It is becoming increasingly apparent that heat shock proteins play an important role in the survival of Plasmodium falciparum against temperature changes associated with its passage from the cold-blooded mosquito vector to the warm-blooded human host. Interest in understanding the possible role of P. falciparum Hsp70s in the life cycle of the parasite has led to the identification of six HSP70 genes. Although most research attention has focused primarily on one of the cytosolic Hsp70s (PfHsp70-1) and its endoplasmic reticulum homolog (PfHsp70-2), further functional insights could be inferred from the structural motifs exhibited by the rest of the Hsp70 family members of P. falciparum. There is increasing evidence that suggests that PfHsp70-1 could play an important role in the life cycle of P. falciparum both as a chaperone and immunogen. In addition, P. falciparum Hsp70s and Hsp40 partners are implicated in the intracellular and extracellular trafficking of proteins. This review summarizes data emerging from studies on the chaperone role of P. falciparum Hsp70s, taking advantage of inferences gleaned from their structures and information on their cellular localization. The possible associations between P. falciparum Hsp70s with their cochaperone partners as well as other chaperones and proteins are discussed.     

Identification and characterisation of the Plasmodium vivax rhoptry-associated protein 2

Plasmodium vivax is currently the most widespread of the four parasite species causing malaria in humans around the world. It causes more than 75 million clinical episodes per year, mainly on the Asian and American continents. Identifying new antigens to be further tested as anti-P. vivax vaccine candidates has been greatly hampered by the difficulty of maintaining this parasite cultured in vitro. Taking into account that one of the most promising vaccine candidates against Plasmodium falciparum is the rhoptry-associated protein 2, we have identified the P. falciparum rhoptry-associated protein 2 homologue in P. vivax in the present study. This protein has 400 residues, having an N-terminal 21 amino-acid stretch compatible with a signal peptide and, as occurs with its falciparum homologue, it lacks repeat sequences. The protein is expressed in asexual stage P. vivax parasites and polyclonal sera raised against this protein recognised a 46 kDa band in parasite lysate in a Western blot assay.     

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.     

Antimalarial benzo[c]phenanthridines

Analogues of the antimalarial alkaloid nitidine have been prepared with high potency against both chloroquine-sensitive and -resistant strains of Plasmodium falciparum in vitro. Simple modifications, using an established synthetic route, resulted in an analogue with IC(50) below 5ng/mL against a chloroquine-sensitive strain of P. falciparum. N-Ethylethoxidine had IC(50) below 30ng/mL against both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum.     

Low antibodies against Plasmodium falciparum and imbalanced pro-inflammatory cytokines are associated with severe malaria in Mozambican children: a case-control study

ABSTRACT: BACKGROUND: The factors involved in the progression from Plasmodium falciparum infection to severe malaria (SM) are still incompletely understood. Altered antibody and cellular immunity against P. falciparum might contribute to increase the risk of developing SM. METHODS: To identify immune responses associated with SM, a sex- and age-matched case-control study was carried out in 134 Mozambican children with SM (cerebral malaria, severe anaemia, acidosis and/or respiratory distress, prostration, hypoglycaemia, multiple seizures) or uncomplicated malaria (UM). IgG and IgM against P. falciparum lysate, merozoite antigens (MSP-119, AMA-1 and EBA-175), a Duffy binding like (DBL)-alpha rosetting domain and antigens on the surface of infected erythrocytes were measured by ELISA or flow cytometry. Plasma concentrations of IL-12p70, IL-2, IFN-gamma, IL-4, IL-5, IL-10, IL-8, IL-6, IL- 1beta, TNF, TNF-beta and TGF-beta1 were measured using fluorescent bead immunoassays. Data was analysed using McNemar's and Signtest. RESULTS: Compared to UM, matched children with SM had reduced levels of IgG against DBLalpha (P < 0.001), IgM against MSP-119 (P = 0.050) and AMA-1 (P = 0.047), TGF-beta1 (P <0.001) and IL-12 (P = 0.039). In addition, levels of IgG against P. falciparum lysate and IL-6 concentrations were increased (P = 0.004 and P = 0.047, respectively). Anti-DBLalpha IgG was the only antibody response associated to reduced parasite densities in a multivariate regression model (P = 0.026). CONCLUSIONS: The lower levels of antibodies found in children with SM compared to children with UM were not attributable to lower exposure to P. falciparum in the SM group. IgM against P. falciparum and specific IgG against a rosetting PfEMP1 domain may play a role in the control of SM, whereas an imbalanced pro-inflammatory cytokine response may exacerbate the severity of infection. A high overlap in symptoms together with a limited sample size of different SM clinical groups reduced the power to identify immunological correlates for particular forms of SM.     

Plasmodium falciparum: identification and purification of the phosphoglycerate kinase of the malaria parasite.

Multiplication of the human malaria parasite Plasmodium falciparum within red blood cells is an energy-dependent process and glucose consumption increases dramatically in infected red blood cells (IRBC) versus normal red blood cells (NRBC). The major pathway for glucose metabolism in P. falciparum IRBC is anaerobic glycolysis. Phosphoglycerate kinase (PGK) is one of the key enzymes of this pathway as it generates ATP. We found that the PGK specific activity in P. falciparum IRBC is seven times higher than that in NRBC. The parasitic origin of the increase in PGK activity is confirmed by isoelectric focusing. Indeed, two P. falciparum isoenzymes with neutral isoelectric points were detected. P. falciparum PGK in purified form has a molecular mass of 48 kDa. Antiserum raised against purified P. falciparum PGK specifically recognizes the 48-kDa protein band in P. falciparum and also reacts with P. berghei and P. yoelii IRBC lysates but does not cross-react with PGK associated with NRBC.     

Cutting edge: a new tool to evaluate human pre-erythrocytic malaria vaccines: rodent parasites bearing a hybrid Plasmodium falciparum circumsporozoite protein

Malaria vaccines containing the Plasmodium falciparum Circumsporozoite protein repeat domain are undergoing human trials. There is no simple method to evaluate the effect of vaccine-induced responses on P. falciparum sporozoite infectivity. Unlike the rodent malaria Plasmodium berghei, P. falciparum sporozoites do not infect common laboratory animals and only develop in vitro in human hepatocyte cultures. We generated a recombinant P. berghei parasite bearing P. falciparum Circumsporozoite protein repeats. These hybrid sporozoites are fully infective in vivo and in vitro. Monoclonal and polyclonal Abs to P. falciparum repeats neutralize hybrid parasite infectivity, and mice immunized with a P. falciparum vaccine are protected against challenge with hybrid sporozoites.     

A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins.

An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial infection.     

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.     

Antibody to P. falciparum in pregnancy varies with intermittent preventive treatment regime and bed net use

BACKGROUND: Antibodies towards placental-binding P. falciparum are thought to protect against pregnancy malaria; however, environmental factors may affect antibody development. Methods and Findings: Using plasma from pregnant Malawian women, we measured IgG against placental-binding P. falciparum parasites by flow cytometry, and related results to intermittent preventive treatment (IPTp) regime, and bed net use. Bed net use was associated with decreased antibody levels at mid-pregnancy but not at 1 month post partum (1 mpp). At 1 mpp a more intensive IPTp regime was associated with decreased antibody levels in primigravidae, but not multigravidae. CONCLUSIONS/SIGNIFICANCE: Results suggest bed nets and IPTp regime influence acquisition of pregnancy-specific P. falciparum immunity.     

Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization

This study was conducted to compare the expression of three constructs of a multistage candidate vaccine (FALVAC-1) against Plasmodium falciparum in an Escherichia coli system: a synthetic gene with P. falciparum codons, a synthetic gene with optimized E. coli codons, and a synthetic gene with P. falciparum codons co-transformed with a RIG plasmid, which encodes three tRNAs (AG(A/G), ATA, GGA) that recognize rare E. coli codons. The expression of the protein increased at least threefold with codon optimization. The presence of the RIG plasmid in the co-transforming cells did not significantly increase the expression level of the gene with P. falciparum codons. The growth of cells transformed by the construct with P. falciparum codons was significantly slower than that of cells transformed by the construct with optimized E. coli codons after induction of protein expression with IPTG. The cells containing the non-codon optimized gene co-expressed with RIG plasmid had the slowest growth at all time points in culture. Thus, codon optimization significantly increases the yield of P. falciparum candidate vaccines in the E. coli expression system.     

Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens

The human mAb 33G2 has high capacity to inhibit in vitro invasion of erythrocytes by Plasmodium falciparum merozoites and, thus, is of special interest with regard to protective immunity against the parasite. In order to obtain more information about asexual blood stage Ag of P. falciparum that are seen by this antibody, material from synchronized P. falciparum cultures was studied by immunofluorescence, immunoelectron microscopy, and immunoblotting. Reactivity was mainly confined to the membrane of infected erythrocytes. Soon after merozoite invasion the antibody stained the erythrocyte membrane. This membrane-associated staining faded during intracellular development of the parasites. Beginning about 18 h after invasion, a dotted pattern appeared which increased in strength with time and persisted to schizont rupture. Pf155/RESA was the major Ag recognized in immunoblots of parasites collected throughout the entire erythrocytic cycle, although other polypeptides also bound the antibody. Among these was a 260-kDa polypeptide found in late trophozoites and schizonts. The specificity of the antibody was analyzed with synthetic peptides corresponding to repeated sequences in the P. falciparum Ag Pf155/RESA, Pf11.1, and Ag332. Synthetic peptides related to Ag332 were the most efficient inhibitors of antibody binding in immunofluorescence studies and cell ELISA. A beta-galactosidase-Ag332 fusion protein was also efficient in reversing reinvasion inhibition caused by 33G2. These results define a family of cross-reactive P. falciparum Ag recognized by mAb 33G2 and suggest that Ag332 was its original target.     

Immunogenicity of C-terminus of Plasmodium falciparum merozoite surface protein 1 expressed as a non-glycosylated polypeptide in yeast

The C-terminal region of the merozoite surface protein 1 (MSP1_(19)) is one of the mostpromising vaccine candidates against the erythrocytic forms of malaria.In the present study,a gene encodingPlasmodium falciparum MSP1_(19) was expressed in yeast Pichia pastoris.A non-glycosylated form of therecombinant protein MSP1_(19) was purified from culture medium.This recombinant protein maintains itsantigenicity.Significant immune responses were seen in C57BL/6 mice after the second immunization.Moreover,the specific antibodies recognized the native antigens of P.falciparum,The prevailing isotypesof immunoglobulin (Ig)G associated with immunization were IgG1,IgG2a and IgG2b.The antibodiesisolated from mouse sera immunized with MSP1_(19) can inhibit parasite growth in vitro.Based on theseimmunological studies,we concluded that MSP1_(19) deserves further evaluation in pre-clinical immunizationsagainst P.falciparum.     

Serum concentrations of granulocyte-colony stimulating factor in complicated Plasmodium falciparum malaria

Involvement of neutrophils in the control of blood parasites in malaria has been reported. Both, mononuclear phagocytes and neutrophils are known to be stimulated by cytokines such as TNF-alpha in order to augment the defence potency against the parasites. Previously, it has been shown that serum-G-CSF concentrations are increased in patients with bacterial sepsis. In vitro studies have shown that P. falciparum - infected erythrocytes induce the release of G-CSF by several cells such as endothelial cells and monocytes, however, nothing is known about G-CSF serum concentrations during the clinical course of severe P. falciparum malaria. Thus, it was the aim of the present study to investigate the time course for G-CSF serum concentrations in patients with complicated P. falciparum malaria, and to correlate these values with other mediators of inflammation and hematopoesis. Twenty-six patients suffering from complicated P. falciparum malaria were included in the study, and 20, age and sex matched, healthy volunteers were used as the negative control group. Serum samples for determination of G-CSF were taken on day 0, 7 and 14, and measured by ELISA. We found significantly increased serum concentrations of G-CSF in patients with complicated P. falciparum malaria on day 0, values decreasing to within the normal range by day 7. A significant correlation was found between G-CSF (d0) and procalcitonin, the parasite count, erythropoietin and macrophage inflammatory protein, however no correlation could be shown for the neutrophil count. In conclusion, on the day of hospital admission, elevated serum concentrations of G-CSF were detected in patients with complicated P. falciparum malaria, which might indicate a role of G-CSF in the acute defence mechanism against the parasites.