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Antibodies against ribosomal phosphoprotein P0 of Plasmodium falciparum protect mice against challenge with Plasmodium yoelii.

Antibodies against the Plasmodium falciparum P0 ribosomal phosphoprotein (PfP0) have been detected exclusively but extensively in malaria-immune persons. Polyclonal rabbit and mice sera were raised against two recombinant polypeptides of P. falciparum P0 protein, PfP0N and PfP0C, covering amino acids 17 to 61 and the remaining amino acids 61 to 316, respectively. Sera against both these domains detected a 35-kDa protein from Plasmodium yoelii subsp. yoelii, a rodent malarial parasite, and stained the surface of merozoites in immunofluorescence assays. Total immunoglobulin G (IgG) purified from rabbit and mouse anti-PfP0 sera by ammonium sulfate and DEAE-cellulose chromatography was used for passive transfer experiments in mice. Mice passively immunized with both anti-PfP0N and anti-PfP0C showed distinctly lower levels of parasitemia than control mice. With immunizations on days -1, 0, 1, 3, and 5, about 50% of both sets of mice receiving anti-PfP0N and anti-PfP0C cleared the lethal 17XL strain of P. yoelii and revived by day 25. All the control mice died by day 10. By extending the immunization schedule, the survival period of the mice could be extended for every mouse that received anti-PfP0 IgG. These data demonstrate the cross-protection of the anti-PfP0 IgG and establish parasite P0 protein as a target for invasion-blocking antibodies.

Animals↗

DNA immunization by Plasmodium falciparum liver-stage antigen 3 induces protection against Plasmodium yoelii sporozoite challenge.

DNA-based immunization of mice by Plasmodium falciparum liver-stage antigen 3 (PfLSA3), a novel highly conserved P. falciparum preerythrocytic antigen, was evaluated. Animals developed a dominant Th1 immune response (high gamma interferon T-cell responses and predominance of immunoglobulin G2a) to each of three recombinant proteins spanning the molecule. We have exploited the immunological cross-reactivity of PfLSA3 with its putative homologue on sporozoites of the rodent parasite Plasmodium yoelii, and we show for the first time that responses induced by PfLSA3 in mice significantly protect against a heterologous challenge by P. yoelii sporozoites. These results support a significant effect of DNA-induced immune responses on preerythrocytic stages.

Animals↗

Monoclonal antibody against the Plasmodium falciparum chitinase, PfCHT1, recognizes a malaria transmission-blocking epitope in Plasmodium gallinaceum ookinetes unrelated to the chitinase PgCHT1.

To initiate invasion of the mosquito midgut, Plasmodium ookinetes secrete chitinases that are necessary to cross the chitin-containing peritrophic matrix en route to invading the epithelial cell surface. To investigate chitinases as potential immunological targets of blocking malaria parasite transmission to mosquitoes, a monoclonal antibody (MAb) was identified that neutralized the enzymatic activity of the sole chitinase of Plasmodium falciparum, PfCHT1, identified to date. This MAb, designated 1C3, previously shown to react with an apical structure of P. falciparum ookinetes, also reacts with a discrete apical structure of P. gallinaceum ookinetes. In membrane feeding assays, MAb 1C3 markedly inhibited P. gallinaceum oocyst development in mosquito midguts. MAb 1C3 affinity isolated an approximately 210-kDa antigen which, under reducing conditions, became a 35-kDa antigen. This isolated 35-kDa protein cross-reacted with an antiserum raised against a synthetic peptide derived from the P. gallinaceum chitinase active site, PgCHT1, even though MAb 1C3 did not recognize native or recombinant PgCHT1 on Western blot. Therefore, this affinity-purified 35-kDa antigen appears similar to a previously identified protein, PgCHT2, a putative second chitinase of P. gallinaceum. Epitope mapping indicated MAb 1C3 recognized a region of PfCHT1 that diverges from a homologous amino acid sequence conserved within sequenced chitinases of P. berghei, P. yoelii, and P. gallinaceum (PgCHT1). A synthetic peptide derived from the mapped 1C3 epitope may be useful as a component of a subunit transmission-blocking vaccine.

Aedes↗

Field evaluation of the ICT malaria P.f/P.v immunochromatographic test for detection of Plasmodium falciparum and Plasmodium vivax in patients with a presumptive clinical diagnosis of malaria in eastern Indonesia.

In areas such as eastern Indonesia where both Plasmodium falciparum and Plasmodium vivax occur, rapid antigen detection tests for malaria need to be able to detect both species. We evaluated the new combined P. falciparum-P. vivax immunochromatographic test (ICT Malaria P.f/P.v.) in Radamata Primary Health Centre, Sumba, Indonesia, from February to May 1998 with 560 symptomatic adults and children with a presumptive clinical diagnosis of malaria. Blinded microscopy was used as the "gold standard," with all discordant and 20% of concordant results cross-checked blindly. Only 50% of those with a presumptive clinical diagnosis of malaria were parasitemic. The ICT Malaria P.f/P.v immunochromatographic test was sensitive (95. 5%) and specific (89.8%) for the diagnosis of falciparum malaria, with a positive predictive value (PPV) and a negative predictive value (NPV) of 88.1 and 96.2%, respectively. HRP2 and panmalarial antigen line intensities were associated with parasitemia density for both species. Although the specificity and NPV for the diagnosis of vivax malaria were 94.8 and 98.2%, respectively, the overall sensitivity (75%) and PPV (50%) for the diagnosis of vivax malaria were less than the desirable levels. The sensitivity for the diagnosis of P. vivax malaria was 96% with parasitemias of >500/microl but only 29% with parasitemias of <500/microl. Nevertheless, compared with the test with HRP2 alone, use of the combined antigen detection test would reduce the rate of undertreatment from 14.7 to 3.6% for microscopy-positive patients, and this would be at the expense of only a modest increase in the rate of overtreatment of microscopy-negative patients from 7.1 to 15. 4%. Cost remains a major obstacle to widespread use in areas of endemicity.

Adolescent↗

Persistent ICT malaria P.f/P.v panmalarial and HRP2 antigen reactivity after treatment of Plasmodium falciparum malaria is associated with gametocytemia and results in false-positive diagnoses of Plasmodium vivax in convalescence.

A problem with rapid Plasmodium falciparum-specific antigen histidine-rich protein 2 (HRP2) detection tests for malaria is the persistence of antigen in blood after the disappearance of asexual-stage parasitemia and clinical symptoms, resulting in false-positive (FP) test results following treatment. The ICT P.f/P.v immunochromatographic test detects both HRP2 and a panmalarial antigen (PMA) found in both P. falciparum and Plasmodium vivax. To examine posttreatment antigen persistence with this test and whether persistent sexual-stage forms (gametocytes) are a cause of FP tests after treatment, we compared serial antigen test results with microscopy results from patients symptomatic with P. falciparum malaria in Indonesia for 28 days following treatment with chloroquine (CQ; n = 66), sulfadoxine-pyrimethamine (SP; n = 36), and artesunate plus sulfadoxine-pyrimethamine (ART + SP; n = 15). Persistent FP antigenemia following SP treatment occurred in 29% (HRP2) and 42% (PMA) of the patients on day 7 and in 10% (HRP2) and 23% (PMA) on day 14. The high rates of persistent HRP2 and PMA antigenemia following CQ and SP treatment were strongly associated with the presence of gametocytemia, with the proportion with gametocytes on day 7 posttreatment being significantly greater in those with FP results than in those with true-negative PMA and HRP2 results. Gametocyte frequency on day 14 post-SP treatment was also greater in those with FP PMA results. Following SP treatment, PMA persisted longer than HRP2, giving an FP diagnosis of P. vivax in up to 16% of patients on day 14, with all FP P. vivax diagnoses having gametocytemia. In contrast, PMA was rapidly cleared following ART + SP treatment in association with rapid clearance of gametocytemia. Gametocytes appear to be an important cause of persistent posttreatment panmalarial antigenemia in areas of endemicity and may also contribute in part to persistent HRP2 antigenemia following treatment.

Adult↗

Devices for rapid diagnosis of Malaria: evaluation of prototype assays that detect Plasmodium falciparum histidine-rich protein 2 and a Plasmodium vivax-specific antigen.

The ParaSight F test was developed as a pioneer industry effort in the large-scale, process-controlled production of a device for the rapid diagnosis of malaria. This device performed well in field settings but was limited to the detection of a single malaria species, Plasmodium falciparum. The ParaSight F+V assay advanced upon the ParaSight F test format by incorporating a monoclonal antibody directed against a proprietary Plasmodium vivax-specific antigen, in addition to the antibody directed against P. falciparum histidine-rich protein 2, which was used in the ParaSight F assay. The modified assay was developed to add the capability to detect P. falciparum and P. vivax in a single-test-strip format. The present study evaluated three distinct ParaSight F+V prototypes with samples from symptomatic patients in regions of Thailand and Peru where malaria is endemic. Over a 2-year enrollment period (1998 and 1999), a total of 4,894 patients consented to participation in the study. Compared with the results for duplicate microscopic examinations of Giemsa-stained blood smears as the reference diagnostic standard, each successive prototype showed substantial improvement in performance. The final ParaSight F+V prototype, evaluated in 1999, had an overall sensitivity for detection of asexual P. falciparum parasites of 98%. The sensitivity of the device was 100% for P. falciparum densities of >500 parasites/ micro l, with a sensitivity of 83% for parasite densities of </=500/ micro l. The specificity for the exclusion of P. falciparum was 93%. For P. vivax, the overall sensitivity was 87% for the final 1999 prototype. The sensitivities calculated for different levels of P. vivax parasitemia were 99% for parasite densities of >5,000/ micro l, 92% for parasite densities of 1,001 to 5,000/ micro l, 94% for parasite densities of 501 to 1,000/ micro l, and 55% for parasite densities of 1 to 500/ micro l. The specificity for the exclusion of P. vivax was 87%. The areas under the receiver operating characteristic curves for the diagnostic performance of the assay for the detection of P. falciparum and P. vivax were 0.8907 and 0.8522, respectively. These findings indicate that assays for rapid diagnosis have the potential to enhance diagnostic capabilities in those instances in which skilled microscopy is not readily available.

Animals↗

Taxol arrests the development of blood-stage Plasmodium falciparum in vitro and Plasmodium chabaudi adami in malaria-infected mice.

Taxol, a natural product used to treat a variety of human cancers, is shown here to be extremely effective against chloroquine- and pyrimethamine-resistant malaria parasites. Addition of Taxol (1.0 microM) for one cycle to cultures of human erythrocytes infected with Plasmodium falciparum prevents the establishment of new infections. Blood parasitemia is eliminated in mice infected with Plasmodium chabaudi adami when they are given a single intraperitoneal injection of Taxol at 150 mg/m2. The majority of the animals treated immediately preceding parasite schizogony remain free of infection after eight replication cycles. The impressive antimalarial activity of Taxol, at a dosage that has been tolerated in humans, establishes its potential utility for treatment of severe, drug-resistant human malaria.

Animals↗

A synthetic tumor necrosis factor-alpha agonist peptide enhances human polymorphonuclear leukocyte-mediated killing of Plasmodium falciparum in vitro and suppresses Plasmodium chabaudi infection in mice.

A peptide corresponding to residues 70-80 of the TNF-alpha polypeptide was synthesized and shown to enhance human PMN-mediated killing of Plasmodium falciparum in vitro and reduced the Plasmodium chabaudi parasitemia in mice. Studies of the mechanism of action showed that the peptide, TNF(70-80), stimulated and primed PMN for an increased respiratory burst and release of granule constituents in response to a second agonist. The PMN-stimulatory activity of the peptide was inhibited by mAbs against the p55 and p75 TNF receptors and a TNF-neutralizing mAb. Analysis of PMN receptor expression showed that CR3 (CD18/CD11b) and Fc gamma RIII were upregulated by TNF(70-80), which was consistent with the peptide's ability to enhance parasite killing by PMN. The peptide, unlike TNF, did not increase the expression of adhesion molecules on endothelial cells and failed to promote binding of P. falciparum-infected erythrocytes to endothelial cells. TNF(70-80) also inhibited the TNF-induced increase in adhesion of P. falciparum-infected erythrocytes to endothelial cells. The results demonstrate that the host-protective effects of TNF can be retained while toxic effects are eliminated using a selected, characterized subunit of the cytokine.

Animals↗

Susceptibility of species A, B, and C of Anopheles culicifacies complex to Plasmodium yoelii yoelii and Plasmodium vinckei petteri infections.

The comparative susceptibilities of colonized species A, B, and C of Anopheles culicifacies complex and Anopheles stephensi were determined for 2 rodent malaria parasites Plasmodium vinckei petteri and Plasmodium yoelii yoelii. All the 3 members of the complex were found to support complete sporogony with varying success. Controls, A. stephensi, become readily infected, with >70% developing oocysts. Of the test groups, species A had the highest percentage of mosquitoes with oocysts (>25%) and sporozoites (>15%). Anopheles culicifacies species B were least susceptible; less than 10% had oocysts and sporozoites in the salivary glands. The results demonstrate that A. culicifacies species A is most susceptible and species B is least susceptible to infections with both the parasites.

Animals↗

The role of IL-18 in blood-stage immunity against murine malaria Plasmodium yoelii 265 and Plasmodium berghei ANKA.

A possible protective role of IL-18 in host defense against blood-stage murine malarial infection was studied in BALB/c mice using a nonlethal strain, Plasmodium yoelii 265, and a lethal strain, Plasmodium berghei ANKA. Infection induced an increase in mRNA expression of IL-18, IL-12p40, IFN-gamma, and TNF-alpha in the case of P. yoelii 265 and an increase of IL-18, IL-12p40, and IFN-gamma in the case of P. berghei ANKA. The timing of mRNA expression of IL-18 in both cases was consistent with a role in the induction of IFN-gamma protein expression. Histological examination of spleen and liver tissues from infected controls treated with PBS showed poor cellular inflammatory reaction, massive necrosis, a large number of infected parasitized RBCs, and severe deposition of hemozoin pigment. In contrast, IL-18-treated infected mice showed massive infiltration of inflammatory cells consisting of mononuclear cells and Kupffer cells, decreased necrosis, and decreased deposition of the pigment hemozoin. Treatment with rIL-18 increased serum IFN-gamma levels in mice infected with both parasites, delayed onset of parasitemia, conferred a protective effect, and thus increased survival rate of infected mice. Administration of neutralizing anti-IL-18 Ab exacerbated infection, impaired host resistance and shortened the mean survival of mice infected with P. berghei ANKA. Furthermore, IL-18 knockout mice were more susceptible to P. berghei ANKA than were wild-type C57BL/6 mice. These data suggest that IL-18 plays a protective role in host defense by enhancing IFN-gamma production during blood-stage infection by murine malaria.

Animals↗

The indirect hemagglutination test for malaria. Evaluation of antigens prepared from Plasmodium falciparum and Plasmodium vivax.

Soluble antigens were prepared from Plasmodium falciparum and P. vivax and were evaluated in the indirect hemagglutination test. These antigens, attached to aldehyde-fixed type "O" erythrocytes, detected antibodies in more than 91% of infections with the homologous Plasmodium species. Detection rates in infections caused by the heterologous species ranged from 72% to 76%. Positive reactions occurred in less than 2% of sera from persons without malaria infection.

Animals↗

Plasmodium falciparum and Plasmodium vivax infections in the owl monkey (Aotus trivirgatus). I. The courses of untreated infections.

This study, the first of three designed to determine the feasibility of using owl monkeys infected with human plasmodia in the search for new, more broadly active antimalarial drugs, dealt with the characteristics of untreated infections with eight strains of Plasmodium falciparum and two strains of P. vivax. Such infections, induced by standardized inocula of these strains in 1,733 monkeys, all Aotus trivirgatus griseimembra, were followed from day of inoculation to death of self-cure. The virulence of the various strains differed strikingly. Incidences of fatal reactions, ranging from 24.4--89.4% and 8.1--45.8%, respectively, in infections with strains of P. falciparum and P. vivax, were closely related to the rate at which parasitemia evolved, the height of parasitemia in the primary attack, and/or the time period over which a high parasite level was sustained. Antemortem symptom complexes and gross tissue and organ reactions in infections with P. falciparum varied with survival time, but within that boundary, were the same for infections with all eight strains of this plasmodium. Morbidity in both fatal and self-limited infections with both plasmodial species was related to height of parasitemia; however, at comparable parasite levels, symptoms exhibited in infections with P. vivax were more severe than in infections with P. falciparum. Overall, the characteristics of infections with these plasmodia in owl monkeys were remarkably similar to those of human infections. With respect to biological features, infections with P. falciparum and P. vivax in this simian host appear to have much to offer in the search for new antimalarial drugs.

Animals↗

Concentration of Plasmodium ovale- and Plasmodium vivax-infected erythrocytes from nonhuman primate blood using Percoll gradients.

Plasmodium vivax and Plasmodium ovale schizont-infected erythrocytes were separated from peripheral blood by centrifugation using discontinuous Percoll (colloidal silica) gradients. Infected Aotus monkey or chimpanzee blood was diluted and placed on a discontinuous gradient containing 30%, 40%, 45%, and 50% Percoll (v/v in media) layers before centrifugation at 1,450 X g. Parasitized erythrocytes were concentrated to greater than 95% schizont-infected cells in two bands that contained an average of one leukocyte per 500 infected cells. Mononuclear cells and trophozoites were isolated in another band and noninfected red cells, ring-infected cells, and granulocytes were pelleted to the bottom. The yield of parasitized erythrocytes ranged from 50% to close to 100% of the estimated number of infected cells in the original whole blood. Use of this Percoll procedure results in a high yield of concentrated parasitized erythrocytes and separation of these cells from host white blood cells.

Animals↗

Detection of Plasmodium vivax and Plasmodium falciparum circumsporozoite antigen in anopheline mosquitoes collected in southern Thailand.

During a 13-month study on the ecology of malaria vectors in five villages in southern Thailand, Anopheles specimens collected on human-bait, bovid-bait, and in light traps were tested for the presence of Plasmodium vivax and P. falciparum circumsporozoite antigen by enzyme-linked immunosorbent assay. Plasmodium vivax antigen was detected in seven specimens and P. falciparum in 21 specimens, together representing 0.4% of the 7,938 specimens tested. In one village, Palao-U, circumsporozoite antigen was detected in 16 (0.7%) of the 2,196 specimens tested. In this village, combined rates of infection with P. falciparum and P. vivax were 0.6% for An. minimus, 1.1% for An. sawadwongporni, and 1.5% for An. maculatus. Circumsporozoite antigen was also detected in An. dirus, An. nivipes, An. barbirostris group, and An. hyrcanus group specimens. Combined P. falciparum and P. vivax entomologic inoculation rates in the wet season (March-October) were 0.05 for An. minimus, An. maculatus, and An. dirus, but 0 for An. sawadwongporni. Rates were higher in the dry season (November-February): 0.26 for An. minimus, 0.13 for An. maculatus, 0.13 for An. sawadwongporni, and 0 for An. dirus. The vectorial capacity, calculated based on human biting rate and rate of survival, of An. minimus during the dry season was more than two-fold higher than that of An. maculatus, the species with the second highest vectorial capacity.

Animals↗

Blood-stage dynamics and clinical implications of mixed Plasmodium vivax-Plasmodium falciparum infections.

We present a mathematical model of the blood-stage dynamics of mixed Plasmodium vivax-Plasmodium falciparum malaria infections in humans. The model reproduces features of such infections found in nature and suggests several phenomena that may merit clinical attention, including the potential recrudescence of a long-standing, low-level P. falciparum infection following a P. vivax infection or relapse and the capacity of an existing P. vivax infection to reduce the peak parasitemia of a P. falciparum superinfection. We simulate the administration of antimalarial drugs, and illustrate some potential complications in treating mixed-species malaria infections. Notably, our model indicates that when a mixed-species infection is misdiagnosed as a single-species P. vivax infection, treatment for P. vivax can lead to a surge in P. falciparum parasitemia.

Animals↗

Chloroquine/doxycycline combination versus chloroquine alone, and doxycycline alone for the treatment of Plasmodium falciparum and Plasmodium vivax malaria in northeastern Irian Jaya, Indonesia.

Combination therapy is one method of overcoming the global challenge of drug-resistant Plasmodium falciparum malaria. We conducted a hospital-based 28-day in vivo test comparing chloroquine/doxycycline to chloroquine or doxycycline alone for treating P. falciparum and Plasmodium vivax malaria in Irian Jaya, Indonesia. Eighty-nine patients with uncomplicated falciparum malaria were randomized to standard dose chloroquine (n = 30), doxycycline (100 mg every 12 hours [7 days], n = 20), or chloroquine with doxycycline (n = 39); corresponding numbers for vivax malaria (n = 63) were 23, 16, 24. Endpoints were parasite sensitivity (S) or resistance (RI/RII/RIII). Of the 105 evaluable patients, chloroquine/doxycycline cured (S) 20/22 (90.9% [95% CI 78.9-100%]) patients with P. falciparum malaria; 2/22 (9.1% [0-21%]) were RIII resistant. Doxycycline cured 11/17 (64.7% [42.0-87.4%]) patients, and chloroquine 4/20 (20% [2.5-37.5%]). Against P. vivax, chloroquine/doxycycline cured (S) 12/17 (70.6% [48.9-92.2%]) patients, doxycycline 4/12 (33.3% [6.6-59.9%]), and chloroquine 5/17 (29.4% [7.7-51.1%]). Chloroquine/doxycycline was effective against P. falciparum but only modestly effective against P. vivax. These findings support the use of chloroquine/doxycycline as an inexpensive alternative to mefloquine for treating chloroquine-resistant P. falciparum but not chloroquine-resistant P. vivax in this setting.

Adult↗

Plasmodium malariae infection boosts Plasmodium falciparum gametocyte production.

We analyzed records of malaria therapy patients sequentially or simultaneously inoculated with Plasmodium falciparum and Plasmodium malariae. Gametocyte production was enhanced in P. falciparum by prior or concurrent P. malariae infection but diminished or unaffected in P. malariae by P. falciparum. Conversely, asexual-form production was diminished in P. malariae but unaffected in P. falciparum.

Animals↗