Chemical attenuation of Plasmodium berghei sporozoites induces sterile immunity in mice

Radiation and genetic attenuation of Plasmodium sporozoites are two approaches for whole-organism vaccines that protect against malaria. We evaluated chemical attenuation of sporozoites as an alternative vaccine strategy. Sporozoites were treated with the DNA sequence-specific alkylating agent centanamycin, a compound that significantly affects blood stage parasitemia and transmission of murine malaria and also inhibits Plasmodium falciparum growth in vitro. Here we show that treatment of Plasmodium berghei sporozoites with centanamycin impaired parasite function both in vitro and in vivo. The infection of hepatocytes by sporozoites in vitro was significantly reduced, and treated parasites showed arrested liver stage development. Inoculation of mice with sporozoites that were treated in vitro with centanamycin failed to produce blood stage infections. Furthermore, BALB/c and C57BL/6 mice vaccinated with treated sporozoites were protected against subsequent challenge with wild-type sporozoites. Our findings demonstrate that chemically attenuated sporozoites could be a viable alternative for the production of an effective liver stage vaccine for malaria.     

A novel protein antigen of the malaria parasite Plasmodium falciparum, located on the surface of gametes and sporozoites

A Plasmodium falciparum cDNA clone was isolated of which the insert is transcribed at high rates as a 1.4-kb mRNA in the sexual stages of the malaria parasite. The cDNA clone contains a copy of a non-interrupted gene which codes for a protein of 157 amino acids (Mr = 16607). This 16-kDa protein does not contain repetitive sequences and is characterised by a putative N-terminal signal sequence, a hydrophobic membrane anchor sequence and a highly hydrophilic C-terminal region suggesting that it is an integral membrane protein. Rabbit antisera raised against a synthetic peptide covering amino acids 31-47 of the 16-kDa protein and against recombinant fusion proteins recognised the 16-kDa antigen in protein extracts of gametocytes, macrogamete/zygotes and sporozoites by Western blot analysis. The rabbit antisera also reacted with gametes, gametocytes and sporozoites in a standard immunofluorescence assay. By immunoelectron microscopy using the protein A-gold method the 16-kDa protein could be clearly visualised on the surface of macrogametes and sporozoites, whereas the antigen was not detectable in the asexual erythrocytic stages of the parasite. The 16-kDa antigen of P. falciparum therefore might have the potential to elicit a dual protective immune response against the sporozoite and sexual stage parasites.     

The role of polymorphisms in the spliced leader addition domain in determining promoter activity in Brugia malayi

The antimalarial activity of the antibiotic thiostrepton has long been attributed to inhibition of apicoplast protein synthesis through binding of apicoplast ribosomal RNA. However, the kinetics of parasite death upon thiostrepton treatment differ from those seen for other inhibitors of apicoplast housekeeping functions. We have analysed global changes in gene expression of the malaria parasite, Plasmodium falciparum, in an attempt to shed light on the responses of the parasite to this drug. Our results indicate a delay in gene expression profiles of thiostrepton-treated parasites. A small number of genes appear to be regulated outside of this trend; our data suggest a response from genes encoding components of the mitochondrial translational machinery, while little response is seen from genes encoding apicoplast-targeted proteins. Our findings are consistent with an effect of thiostrepton on mitochondrial protein synthesis, and thus warrant a re-evaluation of the target of thiostrepton in Plasmodium. They also provide some suggestion of mitochondrion-nucleus signalling in the parasite.     

Plasmodium berghei: suppression of antibody response to sporozoite stage by acute blood stage infection

Mice infected with the erythrocytic stages of Plasmodium berghei show an impaired host response to immunization with irradiated sporozoites of the same malarial parasite. The stage-specific anti-sporozoite response was measured by indirect immunofluorescence upon adsorption of the sera with parasitized red blood cells. P. berghei-infected mice, immunized with irradiated sporozoites on the fourth day of a blood-induced malaria infection, developed a normal anti-sporozoite antibody response. However, this antibody response was more short-lived compared with the antibody response in normal mice immunized with a similar dose of irradiated sporozoites. The immune response was severely depressed when the animals were immunized on day 7 or later after malaria infection. None of the sporozoite-immunized animals, including those which responded to the first immunization, developed a secondary antibody response on reinoculation with irradiated sporozoites. A fully established anti-sporozoite immune response, obtained after multiple immunizations with irradiated sporozoites and which resulted in stage-specific protection of the animals, was not affected by a superimposed blood stage malaria infection. The titres of the anti-sporozoite antibodies in these animals were unaltered, in spite of their high parasitaemias. Reduction of the malaria parasitaemia by chloroquine treatment abolished the immunosuppressive effects of the disease.

These observations are discussed in relation to anti-sporozite immunity and immunosuppression in man in malaria endemic areas.

     

Susceptibility of different strains of mice to hepatic infection with Plasmodium berghei.

Despite the low susceptibility of BALB/c mice to hepatic infection by Plasmodium berghei, this animal model is routinely used to investigate the basic biology of the malaria parasite and to test vaccines and the immune response against exoerythrocytic (EE) stages derived from sporozoites. A murine model in which a large number of EE parasites are established would be useful for furthering such investigations. Therefore, we assayed six mouse strains for susceptibility to erythrocytic and hepatic infections. The administration of 50 sporozoites by intravenous inoculation was sufficient to establish erythrocytic infections in five of five C57BL/6 mice compared with 10,000 sporozoites required to infect 100% of BALB/c mice. To assay for hepatic infections, mice received an intravenous inoculum of 10(6) sporozoites, and liver sections for light microscopy and histology were obtained at 29 and 44 h postinoculation. EE parasites were visualized by immunofluorescence, using an antibody to a P. falciparum heat shock protein. The mean number of EE parasites per 100 cm2 for C57BL/6 and A/J strains was significantly higher than that for BALB/c (2,190 +/- 260, 88 +/- 38, and 6 +/- 2, respectively). The proportion of inoculated sporozoites transforming into liver schizonts was 8.2% in C57BL/6 and < 1% in C3H/HeJ, DBA/1, and Swiss CD-1/ICR mice. Nonspecific inflammatory infiltrates around EE parasites were less prevalent in liver sections from C57BL/6 mice than in those from BALB/c mice, which contributed to the decrease in developing EE stages in BALB/c mice. These data indicate that the C57BL/6-P. berghei system is preferable for investigating the biology and immunology of liver stage parasites.     

A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites

Pore-forming proteins are employed by many pathogens to achieve successful host colonization. Intracellular pathogens use pore-forming proteins to invade host cells, survive within and productively interact with host cells, and finally egress from host cells to infect new ones. The malaria-causing parasites of the genus Plasmodium evolved a number of life cycle stages that enter and replicate in distinct cell types within the mosquito vector and vertebrate host. Despite the fact that interaction with host-cell membranes is a central theme in the Plasmodium life cycle, little is known about parasite proteins that mediate such interactions. We identified a family of five related genes in the genome of the rodent malaria parasite Plasmodium yoelii encoding secreted proteins all bearing a single membrane-attack complex/perforin (MACPF)-like domain. Each protein is highly conserved among Plasmodium species. Gene expression analysis in P. yoelii and the human malaria parasite Plasmodium falciparum indicated that the family is not expressed in the parasites blood stages. However, one of the genes was significantly expressed in P. yoelii sporozoites, the stage transmitted by mosquito bite. The protein localized to the micronemes of sporozoites, organelles of the secretory invasion apparatus intimately involved in host-cell infection. MACPF-like proteins may play important roles in parasite interactions with the mosquito vector and transmission to the vertebrate host.     

CD8+ T Cells Eliminate Liver-Stage Plasmodium berghei Parasites without Detectable Bystander Effect

Immunization with attenuated Plasmodium sporozoites or viral vectored vaccines can induce protective CD8⁺ T cells that can find and eliminate liver-stage malaria parasites. A key question is whether CD8⁺ T cells must recognize and eliminate each parasite in the liver or whether bystander killing can occur. To test this, we transferred antigen-specific effector CD8⁺ T cells to mice that were then coinfected with two Plasmodium berghei strains, only one of which could be recognized directly by the transferred T cells. We found that the noncognate parasites developed normally in these mice, demonstrating that bystander killing of parasites does not occur during the CD8⁺ T cell response to malaria parasites. Rather, elimination of infected parasites is likely mediated by direct recognition of infected hepatocytes by antigen-specific CD8⁺ T cells.     

Carrageenans inhibit the in vitro growth of Plasmodium falciparum and cytoadhesion to CD36

Carbohydrates are implicated in many of the invasive and adhesive interactions that occur between Plasmodium falciparum malaria parasites and human host cells, including invasion of sporozoites into hepatocytes, entry of merozoites into new host erythrocytes during asexual blood-stage replication, adhesion of infected erythrocytes to uninfected erythrocytes (rosetting) and to a number of host endothelial receptors including ICAM-1, CD36 and chondroitin-4-sulphate. In addition to increasing our understanding of host–parasite interactions, the investigation of carbohydrates with differing levels and patterns of sulphation as inhibitors may contribute to the development of novel therapeutics targeting malaria. Here we show that three polysaccharides derived from seaweed (carrageenans) with differing sulphation levels and patterns can inhibit the in vitro erythrocytic invasion and growth of both drug sensitive and drug resistant P. falciparum lines and the adhesion of parasitized erythrocytes to the human glycoprotein CD36.     

Immune-mediated parasite clearance in mice infected with Plasmodium berghei following treatment with manzamine A

Manzamine A, a sponge-derived alkaloid, was recently shown to possess in vivo antimalarial activity against the blood stages of the rodent malaria parasite Plasmodium berghei. A single intraperitoneal dose of 100 micromol/kg of manzamine A suppressed parasite growth but was followed by parasite recrudescence. Forty percent of mice with recrudescing parasites were able to recover and clear the fulminating parasitaemia. Examination of sera from these mice revealed that infected mice treated with manzamine A had a suppressed IFN-gamma production but an increase in their IL-10 and IgG production. The prolonged survival of infected mice treated with manzamine A and the eventual clearance of recrudescing parasites in some of these mice involve a down-regulation of Thl responses and a switch to antibody dependent-Th2 responses.     

Subpopulations of CD4+ (T4+) cells in homosexual/bisexual men with persistent generalized lymphadenopathy.

As tumour necrosis serum kills malarial parasites in vitro and inhibits the multiplication of some species of Plasmodium in mice, we examined the effect of recombinant mouse tumour necrosis factor (rTNF) on P. yoelii both in vitro and in vivo. Parasites incubated overnight with rTNF showed no loss of viability, but repeated injection of rTNF into infected mice reduced parasitaemia and significantly prolonged survival of mice infected with a lethal variant of the parasite. We conclude that TNF acts on blood-stage malaria in vivo via a host cell and that another molecule in tumour necrosis serum is involved in killing the parasite in vitro.     

In vivo studies support the role of trafficking and cytoskeletal-binding motifs in the interaction of MESA with the membrane skeleton of Plasmodium falciparum-infected red blood cells

In red blood cells (RBCs) infected with the malaria parasite Plasmodium falciparum, a 19-residue region of the mature parasite-infected erythrocyte surface antigen (MESA) associates with RBC cytoskeleton protein 4.1R; an interaction essential for parasite survival. This region in MESA is adjacent to a host targeting motif found in other malaria parasite proteins exported to the membrane skeleton. To demonstrate function of these motifs in vivo, regions of MESA fused to a reporter were expressed in malaria parasites. Immunochemical analyses confirmed the requirement for both motifs in the trafficking and interaction of MESA with the cytoskeleton and demonstrates their function in vivo.     

expression of members of the heat-shock protein 70 family in the exoerythrocytic stages ofPlasmodium berghei andPlasmodium falciparum

Exoerythrocytic stages ofPlasmodium berghei cultured in HepG2-A16 hepatoma cells and those ofP. falciparum in human hepatocytes transplanted under the kidney capsule of CB-17/ICr scid/scid mice were used to evaluate expression of heat-shock-related stress proteins. Although undetectable in the sporozoites, the expression of proteins similar in sequence of a heat-shock protein of 70 kDa and a glucose-regulated protein of 78 kDa was markedly induced in the hepatic stages of malaria parasites. Expression of these proteins in the exoerythrocytic stages of the malaria parasite warrants a systematic evaluation of their potential role in eliciting cellular immune responses directed against infected hepatocytes.     

Inhibition of malaria parasite development in mosquitoes by anti-mosquito-midgut antibodies.

The mosquito midgut plays a central role in the development and subsequent transmission of malaria parasites. Using a rodent malaria parasite, Plasmodium berghei, and the mosquito vector Anopheles stephensi, we investigated the effect of anti-mosquito-midgut antibodies on the development of malaria parasites in the mosquito. In agreement with previous studies, we found that mosquitoes that ingested antimidgut antibodies along with infectious parasites had significantly fewer oocysts than mosquitoes in the control group. We also found that the antimidgut antibodies inhibit the development and/or translocation of the sporozoites. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission-blocking vaccine.     

Genetically attenuated Plasmodium berghei liver stages persist and elicit sterile protection primarily via CD8 T cells

Live-attenuated Plasmodium liver stages remain the only experimental model that confers complete sterile protection against malaria. Irradiation-attenuated Plasmodium parasites mediate protection primarily by CD8 T cells. In contrast, it is unknown how genetically attenuated liver stage parasites provide protection. Here, we show that immunization with uis3(-) sporozoites does not cause breakthrough infection in T and B-cell-deficient rag1(-/-) and IFN-gamma(-/-) mice. However, protection was abolished in these animals, suggesting a crucial role for adaptive immune responses and interferon-gamma. Although uis3(-) immunization induced Plasmodium-specific antibodies, B- cell-deficient mice immunized with uis3(-) sporozoites were completely protected against wild-type sporozoite challenge infection. T-cell depletion experiments before parasite challenge showed that protection is primarily mediated by CD8 T cells. In good agreement, adoptive transfer of total spleen cells and enriched CD8 T cells from immunized animals conferred sterile protection against malaria transmission to recipient mice, whereas adoptive transfer of CD4 T cells was less protective. Importantly, primaquine treatment completely abolished the uis3(-)-mediated protection, indicating that persistence of uis3(-)-attenuated liver stages is crucial for their protective action. These findings establish the basic immune mechanisms underlying protection induced by genetically attenuated Plasmodium parasites and substantiate their use as vaccines against malaria.     

Apical organelle discharge by Cryptosporidium parvum is temperature, cytoskeleton, and intracellular calcium dependent and required for host cell invasion

The apical organelles in apicomplexan parasites are characteristic secretory vesicles containing complex mixtures of molecules. While apical organelle discharge has been demonstrated to be involved in the cellular invasion of some apicomplexan parasites, including Toxoplasma gondii and Plasmodium spp., the mechanisms of apical organelle discharge by Cryptosporidium parvum sporozoites and its role in host cell invasion are unclear. Here we show that the discharge of C. parvum apical organelles occurs in a temperature-dependent fashion. The inhibition of parasite actin and tubulin polymerization by cytochalasin D and colchicines, respectively, inhibited parasite apical organelle discharge. Chelation of the parasite's intracellular calcium also inhibited apical organelle discharge, and this process was partially reversed by raising the intracellular calcium concentration by use of the ionophore A23187. The inhibition of parasite cytoskeleton polymerization by cytochalasin D and colchicine and the depletion of intracellular calcium also decreased the gliding motility of C. parvum sporozoites. Importantly, the inhibition of apical organelle discharge by C. parvum sporozoites blocked parasite invasion of, but not attachment to, host cells (i.e., cultured human cholangiocytes). Moreover, the translocation of a parasite protein, CP2, to the host cell membrane at the region of the host cell-parasite interface was detected; an antibody to CP2 decreased the C. parvum invasion of cholangiocytes. These data demonstrate that the discharge of C. parvum sporozoite apical organelle contents occurs and that it is temperature, intracellular calcium, and cytoskeleton dependent and required for host cell invasion, confirming that apical organelles play a central role in C. parvum entry into host cells.     

Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells.

Exoerythrocytic (EE) stages of Plasmodium berghei derived from irradiated sporozoites were cultured in vitro in HepG2 cells. They synthesized several antigens, predominantly but not exclusively those expressed by normal early erythrocytic schizonts. After invasion, over half the intracellular sporozoites, both normal and irradiated, appeared to die. After 24 h, in marked contrast to the normal parasites, EE parasites derived from irradiated sporozoites continued to break open, shedding their antigens into the cytoplasm of the infected host cells. Increasing radiation dosage, which has previously been shown to reduce the ability of irradiated sporozoites to protect animals, correlated with reduced de novo antigen synthesis by EE parasites derived from irradiated sporozoites.     

Plasmodium chabaudi-infected erythrocytes adhere to CD36 and bind to microvascular endothelial cells in an organ-specific way

Adherence of erythrocytes infected with Plasmodium falciparum to microvascular endothelial cells (sequestration) is considered to play an important role in parasite virulence and pathogenesis. However, the real importance of sequestration for infection and disease has never been fully assessed. The absence of an appropriate in vivo model for sequestration has been a major barrier. We have examined the rodent malaria parasite Plasmodium chabaudi chabaudi AS in mice as a potential model. Erythrocytes infected with this parasite adhere in vitro to purified CD36, a critical endothelium receptor for binding P. falciparum-infected erythrocytes. P. c. chabaudi-infected erythrocytes adhere in vitro to endothelial cells in a gamma interferon-dependent manner, suggesting the involvement of additional adhesion molecules in the binding process, as is also the case with P. falciparum-infected cells. Furthermore, plasma or sera from infected and hyperimmune mice, respectively, have the ability to block binding of infected erythrocytes to endothelial cells. In vivo, erythrocytes containing mature P. c. chabaudi parasites are sequestered from the peripheral circulation. Sequestration is organ specific, occurring primarily in the liver, although intimate contact between infected erythrocytes and endothelial cells is also observed in the spleen and brain. The results are discussed in the context of the use of this model to study (i) the relationship between endothelial cell activation and the level of sequestration and (ii) the primary function of sequestration in malaria infection.     

Anti-malarial activity of leaf-extract of hydrangea macrophylla, a common Japanese plant

To find a new anti-malarial medicine derived from natural resources, we examined the leaves of 13 common Japanese plants in vitro. Among them, a leaf-extract of Hydrangea macrophylla, a common Japanese flower, inhibited the parasitic growth of Plasmodium falciparum. The IC50 of Hydrangea macrophylla leaf extract to Plasmodium falciparum was 0.18 microg/ml. The IC50 to NIH 3T3-3 cells, from a normal mouse cell line, was 7.2 microg/ml. Thus, selective toxicity was 40. For the in vivo test, we inoculated Plasmodium berghei, a rodent malaria parasite, to ddY mice and administered the leaf-extract of Hydrangea macrophylla (3.6 mg/0.2 ml) orally 3 times a day for 3 days. Malaria parasites did not appear in the blood of in the treated mice, but they did appear in the control group on day 3 or 4 after inoculation with the parasites. When leaf extract was administered to 5 mice 2 times a day for 3 days, malaria parasites did not appear in 4 of the mice but did appear in 1 mouse. In addition, the leaf-extract was administered orally 3 times a day for 3 days to Plasmodium berghei infected mice with a parasitemia of 2.7%. In the latter group, malaria parasites disappeared on day 3 after initiating the treatment, but they appeared again after day 5 or 6. Although we could not cure the mice entirely, we confirmed that the Hydrangea macrophylla leaf extract did contain an anti-malarial substance that can be administered orally.