The chemotherapy of rodent malaria. LX. The importance of formulation in evaluating the blood schizontocidal activity of some endoperoxide antimalarials.

The activities of artemisinin (QHS) and a number of its semi-synthetic analogues, as well as Fenozan B07 (B07), a synthetic 1,2,4-trioxane, and arteflene (ATF), a synthetic surrogate of yingzhaosu, were compared in mice infected with drug-sensitive Plasmodium berghei or chloroquine-resistant P. yoelii ssp. NS. The studies were stimulated by the observation that B07, in certain aqueous preparations, appears to be equipotent by the subcutaneous (sc) or oral (po) routes in the rodent model but not in a simian model. In the rodent model, B07 was found to undergo rapid alteration (with a half-life of <24h) in an aqueous stock solution prepared using dimethyl sulphoxide (DMSO) to pre-dissolve the drug. Therefore, for all later experiments with aqueous preparations, the test material was newly formulated each day. In a carboxymethylcellulose formulation used as a 'standard suspending vehicle' (SSV), B07 and dihydroartemisinin (DIHYD) were found to be, respectively, one sixth and one 10th as active po as when the drugs were pre-dissolved in DMSO and then diluted with water. ATF in DMSO given po was less than one 20th as active as when used sc in the rodent model, and this drug in SSV was almost inactive po. The relatively low oral activity of these three compounds (especially DIHYD and ATF) may be attributable to extensive first-pass metabolism in the mouse. Oral beta-artemether (AM) and beta-arteether (AE) were highly active when used in SSV. ATF has been found to have low activity in simian models and clinical trials because of its poor absolute bio-availability. In in-vivo studies of the blood schizontocidal action of anti-malarials, in rodent malaria models, the data collected on the structure-activity relationships (SAR) of the drugs must be viewed critically when selecting specific compounds from a chemical series for further development. A study of the influence of drug formulation on the activity of other, novel antimalarials is crucial to the evaluation of the drugs, and merits high priority.

Antigenic and sequence diversity at the C-terminus of the merozoite surface protein-1 from rodent malaria isolates, and the binding of protective monoclonal antibodies.

Merozoite surface protein-1 (MSP-1) is a major candidate in the development of a vaccine against malaria. Immunisation with a recombinant fusion protein containing the two Plasmodium yoelii MSP-1 C-terminal epidermal growth factor-like domains (MSP-1(19)) can protect mice against homologous but not heterologous challenge, and therefore, antigenic differences resulting from sequence diversity in MSP-1(19) may be crucial in determining the potential of this protein as a vaccine. Representative sequence variants from a number of distinct P. yoelii isolates were expressed in Escherichia coli and the resulting recombinant proteins were screened for binding to a panel of monoclonal antibodies (Mabs) capable of suppressing a P. yoelii YM challenge infection in passive immunisation experiments. The sequence polymorphisms affected the binding of the antibodies to the recombinant proteins. None of the Mabs recognised MSP-1(19) of P. yoelii yoelii 2CL or 33X or P. yoelii nigeriensis N67. The epitopes recognised by the Mabs were further distinguished by their reactivity with the other fusion proteins. The extent of sequence variation in MSP-1(19) among the isolates was extensive, with differences detected at 35 out of the 96 positions compared. Using the 3-dimensional structure of the Plasmodium falciparum MSP-1(19) as a model, the locations of the amino acid substitutions that may affect Mab binding were identified. The DNA sequence of MSP-1(19) from two Plasmodium vinckei isolates was also cloned and the deduced amino acid sequence compared with that in other species.

A subtilisin-like protein in secretory organelles of Plasmodium falciparum merozoites.

In the vertebrate host, the malaria parasite invades and replicates asexually within circulating erythrocytes. Parasite proteolytic enzymes play an essential but poorly understood role in erythrocyte invasion. We have identified a Plasmodium falciparum gene, denoted pfsub-1, encoding a member of the subtilisin-like serine protease family (subtilases). The pfsub-1 gene is expressed in asexual blood stages of P. falciparum, and the primary gene product (PfSUB-1) undergoes post-translational processing during secretory transport in a manner consistent with its being converted to a mature, enzymatically active form, as documented for other subtilases. In the invasive merozoite, the putative mature protease (p47) is concentrated in dense granules, which are secretory organelles located toward the apical end of the merozoite. At some point following merozoite release and completion of erythrocyte invasion, p47 is secreted from the parasite in a truncated, soluble form. The subcellular location and timing of secretion of p47 suggest that it is likely to play a role in erythrocyte invasion. PfSUB-1 is a new potential target for antimalarial drug development.

Passive immunization with antibodies against three distinct epitopes on Plasmodium yoelii merozoite surface protein 1 suppresses parasitemia.

We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an approximately 42-kDa fragment (MSP-142) that is derived from the C terminus of MSP-1. MSP-142 is further cleaved to an N-terminal approximately 33-kDa polypeptide (MSP-133) and a C-terminal approximately 19-kDa polypeptide (MSP-119) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-142 but not with either of the constituents MSP-133 and MSP-119, B4 recognized an epitope within the N terminus of MSP-133, and B6, B10, F5, and G3 bound to MSP-119. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes on P. yoelii MSP-1 are recognized by antibodies that suppress parasitemia in vivo.

The chemotherapy of rodent malaria. LIII. 'Fenozan B07' (Fenozan-50F), a difluorinated 3,3'-spirocyclopentane 1,2,4-trioxane: comparison with some compounds of the artemisinin series.

Fenozan B07, a difluorinated 3,3'-spirocyclopentane, 1,2,4-trioxane, is a novel, second-generation antimalarial endoperoxide which is a potent blood schizontocide against strains of rodent malaria that are highly resistant to a wide spectrum of classical antimalarials. Like compounds of the artemisinin series, its action is limited to the intra-erythrocytic stages, both asexual and sexual, and it is devoid of causal prophylactic activity. Both Fenozan B07 and the artemisinins are potent gametocytocides. In contrast to arteether, in a model using synchronous infection with Plasmodium vinckei petteri, Fenozan B07 inhibits the development of all asexual stages except preschizonts, as well as gametocytes. The activity of the artemisinin series in rodent-malaria models is limited to the rings and young trophozoites. The combined effect of Fenozan B07 with artesunate against P. v. petteri was only additive. A slight degree of potentiation was found in mice infected with asynchronous, drug-sensitive P. berghei but the combination was only additive against CQ-resistant P. yoelli ssp. NS. On the other hand, a significant degree of synergism was observed when mice infected with the artemisinin-resistant ART line of P. yoelii ssp. NS received combinations of Fenozan B07 with artemisinin. The conclusion is drawn from these and other data that there are significant differences between the blood schizontocidal actions of Fenozan B07 and the artemisinins. The basis of these differences remains to be determined.

The chemotherapy of rodent malaria. LIV. Combinations of 'Fenozan B07' (Fenozan-50F), a difluorinated 3,3'-spirocyclopentane 1,2,4-trioxane, with other drugs against drug-sensitive and drug-resistant parasites.

Fenozan B07, a 1,2,4-trioxane endoperoxide with potent blood schizontocidal activity against drug-sensitive and drug-resistant rodent malaria parasites, exerted a modest potentiating action when administered with chloroquine (CQ) to mice infected with parasites of the CQ-resistant P. yoelii ssp. NS, but not when given to mice infected with the CQ-sensitive P. berghei N strain. The reason why this potentiation may be of particular value in the treatment of severe falciparum malaria is discussed. Mefloquine and halofantrine displayed a similar level of potentiation with Fenozan B07 against the CQ-resistant parasites. However, antagonism was shown by combinations of Fenozan B07 with pyronaridine or the 8-aminoquinoline WR 238 605 when used against CQ-resistant parasites. Mefloquine with Fenozan B07 is also antagonistic against a highly mefloquine-resistant line of P. yoelii ssp. NS. The reasons behind such antagonism are not known. The importance is stressed of using carefully selected drug combinations of novel antimalarials, rather than single drugs, in order to impede the selection of drug-resistant parasites, but only after adequate, preclinical, toxicity testing.

The effect of atovaquone (566C80) on the maturation and viability of Plasmodium falciparum gametocytes in vitro.

Atovaquone (566C80), a hydroxynaphthoquinone, was investigated for activity against Plasmodium falciparum gametocytes (NF54 strain) in vitro. After 96 h of continuous exposure to the drug at 1.4 x 10(-7) M (a concentration achievable in humans 14 d after administration of a therapeutic dose of 10 mg/kg) reductions of 75%, 54% and 20% in the number of gametocyte stages 2, 3 and 4, respectively, were achieved. A small increase (14%) in stage 5 gametocytes was seen. At the same concentration, atovaquone showed greater activity against the asexual stages of P. falciparum, reductions of 93%, 96% and 43% in the number of rings, schizonts and trophozoites, respectively, being achieved. These data are consistent with inhibition of maturation of trophozoites. The observed effect on maturation of gametocytes is similarly consistent with blockade of gametocyte recruitment from merozoites produced by the preceding schizogony, or to stasis of intraerythrocytic sexual development before the formation of stage 2 gametocytes.

Specific and nonspecific responses to Plasmodium falciparum blood-stage parasites and observations on the gametocytemia in schoolchildren living in a malaria-endemic area of Mozambique.

We have observed specific and nonspecific reactivities to the asexual states and gametocytes of Plasmodium falciparum and examined the effect of chloroquine and Fansidar (pyrimethamine/sulfadoxine) on the dynamics of gametocytemia. Schoolchildren peripheral blood films positive for P. falciparum gametocytes were identified in a malaria-endemic area of Mozambique. The children were randomly allocated into two groups to receive chloroquine or pyrimethamine/sulfadoxine, and were followed for 28 days after treatment. In patients harboring drug-sensitive parasites, asexual parasitemias were cleared by day 4, but gametocytes persisted for an additional 17 days. The prevalence of the asexual parasites was 67.6% in the chloroquine-treated group at day 0 and 61.1% at day 28, whereas in the pyrimethamine/sulfadoxine treated group, the initial parasite prevalence of 70.7% was reduced to 2.4% at day 28, suggesting a high prevalence of chloroquine-resistant parasites. On day 0, gametocyte prevalence was 59.5% in the chloroquine-treated group and in 68.3% in the pyrimethamine/sulfadoxine-treated group; these values were reduced to 5.6% and 2.4%, respectively, at day 28. Our results suggest strongly that there is no induction of gametocytogenesis by either course of chemotherapy.

Characterization of the modes of action of anti-Pbs21 malaria transmission-blocking immunity: ookinete to oocyst differentiation in vivo.

The impact of immune sera, and peripheral blood cells (PBC) from mice immunized with Plasmodium berghei ookinetes; and of purified immunoglobulin or Fab fragments from anti-Pbs21 monoclonal antibody 13.1, upon establishment of oocyst infections in the mosquito was studied. Infections were initiated either from gametocyte-infected mice, or membrane feeders which contained either gametocytes or mature ookinetes. PBC from ookinete-immunized mice presented with non-immune serum failed to show any transmission-blocking activity. Anti-ookinete serum, intact anti-Pbs21 monoclonal antibody 13.1 or its Fab fragments, all inhibited oocyst formation significantly. When gametocyte-infected mice or gametocytes in membrane feeds were used, inhibition did not directly correlate with antibody concentration. In membrane feeders that contained ookinetes and antibody, concentration-dependent inhibition usually occurred. The efficacy of purified 13.1 IgG was dependent upon the ookinete concentration. The ookinete plasmalemma and cytoplasm were significantly disturbed after 12 h in bloodmeals that contained antibody 13.1, but not in the isotype controls. These changes may have caused the observed failure of the ookinete to migrate as rapidly as the controls from the destructive environment of the bloodmeal.

Plasmodium berghei: serum-mediated inhibition of infectivity of infected mice to Anopheles stephensi mosquitoes.

The transmission of Plasmodium berghei-infected mice to Anopheles stephensi mosquitoes showed a peak number of oocysts early in the infection prior to the peak of gametocytaemia. This was followed by a precipitous decline on Days 4 and 5 (see also Dearsley et al., Parasitology, 100, 359-368, 1990). By measuring percentage relative infectivity (using membrane feeds with viable gametocytes), we have shown that serum collected daily during the course of a blood-induced infection blocked infectivity from Day 6 postinfection onward. Although there was a correlation between anti-blood stage antibody levels and the loss of infectivity, a comparison of the infectivity pattern of P. berghei-infected BALB/c and SCID mice (the latter being incapable of antibody production) revealed the same pattern of inhibition in both mouse strains, suggesting that antibody alone is not responsible for this suppression. Sera taken from SCID mice late in the infection tested in vitro demonstrated a decline in infectivity similar to that observed in vivo, suggesting that a non-antibody serum factor(s) is responsible for the sustained decline in infectivity of P. berghei to A. stephensi mosquitoes.

Factors regulating natural transmission of Plasmodium berghei to the mosquito vector, and the cloning of a transmission-blocking immunogen.

Naturally occurring factors that regulate the infectivity of P. berghei infected rodent hosts to the mosquito vector in vivo have been compared in T.O., Balb/C and immunodeficient SCID mice. No detectable differences in infectivity were observed suggesting B and T cell mediated factors are not involved. Further studies investigated roles for macrophage colony stimulating factors, the cytokines IFN gamma and TNF alpha, of neutrophils, and of nitric oxide in the SCID mouse, but have failed to demonstrate an important role in vivo for any factor examined. Differences between these results and those obtained in vitro on the human and primate parasites must therefore be explained by biological differences between the parasite/host combinations, or by technical differences in experimental designs. Induced immunity to the ookinete surface antigen Pbs 21 of P. berghei can totally block the transmission of the parasite from the gametocyte infected host to the vector. We have cloned the gene encoding Pbs 21 and shown it bears striking structural similarities to Pfs 25, Pgs 25 and more particularly Pgs 28 in that it has a high cysteine content (9.5%), 4 EGF-like domains and hydrophobic amino-'signal'--and carboxyl-'anchor' sequences. The encoding gene is on chromosome 5 and is found also in P. chabaudi, P. yoelii and P. vinckei.

Immunological detection of cytoskeletal proteins in the exoerythrocytic stages of malaria by fluorescence and confocal laser scanning microscopy.

Using monospecific antibodies, the presence and distribution of tubulin, actin, myosin, intermediate filaments, and lamins were examined in the exoerythrocytic liver schizont of Plasmodium berghei by conventional indirect fluorescent antibody methods and confocal laser scanning microscopy. The binding reactivity of the antibodies to parasite proteins was determined by Western blot analysis. The localisation of all antibodies in control host hepatocytes followed expected distributions in both uninfected and infected hepatocytes; by contrast, reactivity to the exoerythrocytic schizont was variable. The parasite reacted positively with selected anti-tubulin, -actin, and -myosin antibodies in both fluorescence and Western blot analysis. Anti-lamin antibodies were positive by confocal indirect fluorescent antibody labelling, but no labelling was detected with anti-intermediate filament antibody. Within the technical limits of resolution of the methods as applied to asynchronous parasite infections, not one of the antibodies reacting positively with the parasite by the indirect fluorescent antibody technique could be shown to identify unequivocally the classic architectural features associated with their respective target organelles, i.e. microtubules, stress-fibres or the nuclear envelope.

Plasmodium berghei: ionic regulation and the induction of gametogenesis.

The role of ionic regulation in the induction of gametogenesis of Plasmodium berghei at 20 degrees C was investigated. A potent inhibitor of Na+/H+ exchange, amiloride, strongly inhibited exflagellation and subsequent ookinete formation induced by RPMI 1640 with 10% fetal calf serum at pH 8.0, whereas Na+ or K+ channel inhibitors, H(+)-ATPase inhibitors, and a protonophore had no significant effect. Amiloride-treated 'activated' microgametocytes synthesized DNA to levels consistent with the expected 8C, but failed to develop further. These results may suggest that an increase in intracellular pH induced by Na+/H+ exchange plays an important role in the induction of gametogenesis by cultivating at pH 8.0 and 20 degrees C. Cultivation at pH 8.0 and 37 degrees C did not induce the development, and microgametocytes remained as nonactivated forms, having the DNA content of 1.5C. By culturing at pH 7.3 and 20 degrees C, however, most of microgametocytes finished synthesis of DNA up to the 8C level, but ceased development at various stages. Additionally, exflagellation occurred in a simple medium composed of buffered saline with 10 mM glucose. Glucose was indispensable for exflagellation, presumably acting as an energy source. Exflagellation induced by this solution was also inhibited by amiloride. It is therefore suggested that the induction of microgametogenesis may be composed of two distinct mechanisms, one is a temperature-dependent DNA synthesis and the other is a pH-dependent control of developmental events leading to microgamete assembly and exflagellation.

Possible roles of Ca2+ and cGMP as mediators of the exflagellation of Plasmodium berghei and Plasmodium falciparum.

The roles of Ca2+ and cyclic nucleotides as secondary, intracellular messengers for exflagellation of Plasmodium berghei and Plasmodium falciparum were investigated. Treatment with Ca2+ antagonists such as TMB-8 (an inhibitor of intracellular Ca2+ release) or W-7 (a calmodulin inhibitor) strongly inhibited exflagellation induced by alkaline medium at pH 8.0 whereas EGTA (a Ca2+ chelator) or nicardipine and nifedipine (Ca2+ channel inhibitors) had no effect. These results may indicate that mobilization of parasites' internal resources of Ca2+ is a prerequisite for exflagellation. Agents which increase cAMP levels did not induce exflagellation at the non-permissive pH of 7.3, and had no significant inhibitory effect at the permissive pH of 8.0. IBMX (cAMP/cGMP-phosphodiesterase inhibitor), however, enhanced exflagellation at pH 7.3, indicating the possibility that cGMP, but not cAMP, may be involved in the induction of exflagellation. Furthermore, cGMP or agents which increase cGMP levels such as nitroprusside (a potent activator of guanylate cyclase), enhanced exflagellation at pH 7.3, whereas N-methyl-hydroxylamine (guanylate cyclase inhibitor) inhibited the exflagellation at pH 8.0. From these results, it may be concluded that the induction of exflagellation requires both Ca2+ mobilization and an increase in cGMP levels.

The development and routine application of high-density exoerythrocytic-stage cultures of Plasmodium berghei.

Methods are reviewed for the culture of the exoerythrocytic stages of Plasmodium berghei wherein development reproducibly reflects growth observed in vivo in laboratory rodents. The combination of these methods with the culture of both asexual and sexual blood stages has allowed the completion of the entire vertebrate phase of malaria development in vitro. The development of new methods for high-density exoerythrocytic-stage culture combined with robust statistical analysis of parasite growth by morphological (light microscopy), or DNA probe methods now allows the critical and precise evaluation of chemotherapeutic or immunological treatments. These methods are illustrated by data obtained on pyrimethamine, primaquine and a hydroxynaphthoquinone. Some of the new avenues of research made feasible by the high-density cultures, e.g., direct immunization to produce monoclonal antibodies and biochemical studies are discussed.

The complete development in vitro of the vertebrate phase of the mammalian malarial parasite Plasmodium berghei.

All three 'vertebrate' stages of the rodent malarial parasite Plasmodium berghei berghei were grown in vitro in the absence of the vertebrate host. The parasite was introduced into culture from infected mosquitoes and 2 in vitro culture methods were used sequentially to complete the 'vertebrate' phases of development in hepatoma and erythrocyte host cells. The resultant blood infection produced mature schizonts and male and female gametocytes. The protocol, which is now being extended to the human pathogen P. falciparum, may assist future studies on this important group of parasites.