Plasmodium berghei sporozoites are mitogenic for murine T cells, induce interferon, and activate natural killer cells

Enhanced natural killer (NK) activity was detected in the spleens of mice as early as 24 hr after single i.v. inoculation with gamma-irradiated Plasmodium berghei sporozoites. The activity peaked at 48 hr post-injection, and declined below baseline level by day 8. Reinoculation of mice with irradiated sporozoites produced an increased NK activity significantly smaller than the original activity. Spleen cells sensitized in vivo as well as nonsensitized spleen cells stimulated in vitro with sporozoites produced high levels of interferon (IFN) and displayed enhanced NK activity. Characterization of the IFN through the use of specific antibodies revealed that it was mainly IFN-gamma. The cellular basis for IFN-gamma induction was linked to the mitogenicity of P. berghei sporozoites for T cells. The possibility exists that IFN-gamma may have a regulatory effect on antibody production against P. berghei sporozoites.     

Sporozoites of Rodent and Simian Malaria, Purified by Anion Exchangers, Retain their Immunogenicity and Infectivity

SYNOPSIS. Sporozoites of rodent malaria, Plasmodium berghei , and simian malaria, Plasmodium knowlesi and Plasmodium cynomolgi , were partially separated from mosquito debris and microbial contaminants by passage of Anopheles material through a DEAE-cellulcse column. In addition to eliminating most of the contaminants (80–90%), this simple technic has made it possible to recover rapidly large numbers of viable sporozoites (55–75% yield), which have retained their infectivity, immunogenicity, and capacity to react with known antisera. Mice injected with varying doses of column-purified sporozoites (CS) of P. berghei produced infections which paralleled those seen in the controls. Total protection against challenge with a potentially lethal dose of viable sporozoites was acquired by mice inoculated twice with irradiated CS of P. berghei. CS of P. berghei and P. cynomolgi gave positive circumsporozoite precipitation (CSP) reactions, upon inoculation with the respective immune sera. The preservation of the surface antigens of CS was documented by immunofluorescence.
It was shown that differences in elution behavior exist among sporozoites of certain species of Plasmodium as well as among sporozoites of the same species derived from different organs of the mosquito. These results may be attributed to differences in the surface charge of the sporozoites or conditions in sample media.
Purified sporozoites obtained by the method described in this report provide an adequate source of parasites for a variety of in vitro studies.     

Sporozoites of rodent and simian malaria, purified by anion exchangers, retain their immunogenicity and infectivity.

Sporozoites of rodent malaria, Plasmodium berghei, and simian malaria, Plasmodium knowlesi and Plasmodium cynomolgi, were partially separated from mosquito debris and microbial contaminants by passage of Anopheles material through a DEAE-cellulose column. In addition to eliminating most of the contaminants (80-90%), this simple technic has made it possible to recover rapidly large numbers of viable sporozoites (55-75% yield), which have retained their infectivity, immunogenicity, and capacity to react with known antisera. Mice injected with varying doses of column-purified sporozoites (CS) of P. berghei produced infections which paralleled those seen in the controls. Total protection against challenge with a potentially lethal dose of viable sporozoites was acquired by mice inoculated twice with irradiated CS of P. berghei CS of P. berghei and P. cynomolgi gave positive circumsporozoite precipitation (CSP) reactions, upon inoculation with the respective immune sera. The preservation of the surface antigens of CS was documented by immunofluorescence. It was shown that differences in elution behavior exist among sporozoites of certain species of Plasmodium as well as among sporozoiters of the same species derived from different organs of the mosquito. These results may be attributed to differences in the surface charge of the sporozoites or conditions in sample media. Purified sporozoites obtained by the method described in this report provide an adequate source of parasites for a variety of in vitro studies.     

Common Epitopes In the Circumsporozoite Proteins of Plasmodium Berghei and Plasmodium Gallinaceum Identified By Monoclonal Antibodies to the P. Gallinaceum Circumsporozoite Protein

ABSTRACT. Monoclonal antibodies that react with the circumsporozoite protein of the avian malaria Plasmodium gallinaceum sporozoites also reacted with circumsporozoite protein of the rodent malaria Plasmodium berghei. Two types of reactivity were identified: 1) two monoclonal antibodies reacted with P. berghei sporozoite protein by enzyme-linked immunosorbent assay, Western blot and indirect immunofluorescence antibody, 2) six other monoclonal antibodies reacted with P. berghei sporozoites by ELISA and Western blot only. We studied whether these differences could be explained by reactivity in enzyme-linked immunosorbent assay with different P. berghei circumsporozoite peptides. Although all P. gallinaceum monoclonal antibodies reacted with the P. berghei repeats, the first group reacted with a conserved peptide sequence, N1, whereas the second group did not. These results suggest that circumsporozoite proteins from P. gallinaceum and P. berghei share common epitopes. the biological significance of our finding is not yet clear. Indeed, the cross-reactive monoclonal antibodies giving a positive indirect immunofluorescence antibody with the P. berghei sporozoites only caused a borderline effect on the living P. berghei parasites in vitro as measured by inhibition of sporozoite infectivity.     

Alternative invasion pathways for Plasmodium berghei sporozoites

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a natural malaria infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that infection by Plasmodium falciparum and Plasmodium yoelii sporozoites depends on CD81 and cholesterol-dependent tetraspanin-enriched microdomains (TEMs) on the hepatocyte surface. Here we have analyzed the role of CD81 and TEMs during infection by sporozoites from the rodent parasite Plasmodium berghei. We found that depending on the host cell type, P. berghei sporozoites can use several distinct pathways for invasion. Infection of human HepG2, HuH7 and HeLa cells by P. berghei does not depend on CD81 or host membrane cholesterol, whereas both CD81 and cholesterol are required for infection of mouse hepatoma Hepa1-6 cells. In primary mouse hepatocytes, both CD81-dependent and -independent mechanisms participate in P. berghei infection and the relative contribution of the different pathways varies, depending on mouse genetic background. The existence of distinct invasion pathways may explain why P. berghei sporozoites are capable of infecting a wide range of host cell types in vitro. It could also provide a means for human parasites to escape immune responses and face polymorphisms of host receptors. This may have implications for the development of an anti-malarial vaccine targeting sporozoites.     

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.     

Interactions of Plasmodium berghei sporozoites and murine Kupffer cells in vitro

Malaria sporozoites must leave the bloodstream and cross a layer of sinusoidal lining cells in order to infect hepatocytes and undergo exoerythrocytic schizogony. To determine whether Kupffer cells (KC) derived from this layer interact with sporozoites, murine KC were isolated from perfused livers of BALB/cJ mice and incubated in vitro with Plasmodium berghei sporozoites. Isolated KC had characteristic macrophage surface Ag and were phagocytic, ingesting both latex particles and Leishmania major amastigotes. In the absence of immune serum, sporozoites associated with fewer than 10% of these KC. By 30 min, 10% of the cell-associated sporozoites were completely ingested, 30% were in the process of being ingested, and 60% were attached to the surface of the cells. Opsonization of sporozoites with monoclonal or polyclonal antibodies directed against P. berghei circumsporozoite protein markedly enhanced sporozoite association with KC. Up to 40% of cells exposed to opsonized sporozoites had parasites inside or attached to their surfaces. Sporozoites attached to or ingested by KC were uniformly destroyed within 240 min in all cultures; there was no evidence of conversion of sporozoites to the exoerythrocytic stage within KC by light microscopy, and there was no evidence of residual sporozoites, either inside or outside of cells, by either light or electron microscopy. These data suggest that under nonimmune conditions, KC play a minor role in resistance to infection by malaria sporozoites. However, when sporozoites are opsonized by circumsporozoite antibodies, phagocytosis by KC may be an important immune mechanism that prevents parasitization of hepatocytes.     

Electron microscopic studies on the interaction of rat Kupffer cells and Plasmodium berghei sporozoites

The interactions between Plasmodium berghei sporozoites and Kupffer cells in rat liver were studied by transmission electron microscopy. Between 10 and 45 min after inoculation, sporozoites were found in the process of entering Kupffer cells and inside phagolysosomes. The sporozoites entered the Kupffer cells by phagocytosis as determined by the presence of pseudopods and local accumulations of aggregated microfilaments and the resulting exclusion of other organelles in the phagocyte cytoplasm beneath the attached parasite. Sporozoites were taken up either with their anterior end first, or backwards. Scanning electron microscopy of in vitro sporozoite Kupffer cell interaction confirmed these observations. It was concluded that sporozoites are taken up in a normal phagocytic way by the Kupffer cells, regardless of their initial place of contact or position. Thirty min after inoculation sporozoites found in phagolysosomes were still morphologically intact but after 45 min we could encounter completely digested sporozoites.     

Role of a serum factor in enhancement of in vitro interactions between Plasmodium berghei sporozoites and hamster peritoneal macrophages

Interactions between Plasmodium berghei sporozoites and hamster peritoneal macrophages were studied. Hamster serum was shown to enhance the percentage of sporozoites tha attached to macrophages, thus confirming previous studies by other workers using mouse macrophages and mouse serum. The enhancement factor within hamster serum was concentrated by a fractionation procedure consisting of ammonium sulfate precipitation followed by Concanavalin A-Sepharose affinity chromatography. This serum fraction has been shown previously to contain component(s) that bind to P. berghei sporozoites.     

Malaria sporozoites leave behind trails of circumsporozoite protein during gliding motility

As Plasmodium sporozoites undergo gliding motility in vitro, they leave behind trails of circumsporozoite (CS) protein that correspond to their patterns of movement. This light microscopic observation was made using Plasmodium berghei sporozoites, a monoclonal antibody (MAb H4) directed against the immunodominant repetitive epitope of the CS protein of P. berghei, and an immunogold-silver staining (IGSS) technique. Sporozoites pretreated with agents that inhibit sporozoite motility and invasiveness did not produce trails. Sporozoites that glided on microscope slides coated with MAb H4 left behind considerably longer CS protein trails than those on uncoated slides, and the staining of these trails was more intense. The fact that the CS protein is an exoantigen continuously released as trails by motile sporozoites, together with our previous finding that anti-CS protein antibodies inhibit sporozoite motility, strongly suggests that the CS protein plays a role in gliding motility. The sensitive IGSS technique used in this study may be a useful tool in the study of the translocation of surface proteins during gliding of other apicomplexans, other protists, and bacteria.     

A surface phospholipase is involved in the migration of plasmodium sporozoites through cells

Plasmodium sporozoites, injected by mosquitoes into the skin of the host, traverse cells during their migration to hepatocytes where they continue their life cycle. The mechanisms used by the parasite to rupture the plasma membrane of the host cells are not known. Here we report the presence of a phospholipase on the surface of Plasmodium berghei sporozoites (P. berghei phospholipase; Pb PL) and demonstrate that it is involved in the establishment of a malaria infection in vivo. Pb PL is highly conserved among the Plasmodium species. The protein is about 750 amino acids, with a predicted signal sequence and a carboxyl terminus that is 32% identical to the vertebrate lecithin:cholesterol acyltransferase, a secreted phospholipase. Pb PL contains a motif characteristic of lipases and a catalytic triad of a serine, aspartate, and histidine that is found in several phospholipases. We have verified its lipase and membrane lytic activity in vitro, using recombinant baculovirus-expressed protein. To study its role in vivo, we have disrupted the P. berghei PL open reading frame and generated mutants in its active site. During an infection through mosquito bite, the infectivity of the knock-out parasites in the liver is decreased by approximately 90%. The prepatent period of the resulting blood infection is 1 day longer as compared with wild type. Further, the mutant sporozoites are impaired in their ability to cross epithelial cell layers. Thus, the Pb PL functions as a lipase to damage cell membranes and facilitates sporozoite passage through cells during their migration from the skin to the bloodstream.     

Plasmodium berghei: sporozoites are sensitive to human serum but not susceptible host serum.

Human complement was activated by rodent malaria, Plasmodium berghei, sporozoites through the alternative pathway, as revealed by C3 deposition on sporozoites using the fluorescent antibody technique. Sporozoites exposed to fresh human serum decreased in infectivity to HepG2 cells, but those exposed to heated or C3-deficient human serum showed normal infectivity to HepG2 cells. In contrast, C3 deposition was not observed on the sporozoites treated with mouse or rat serum even in the presence of specific polyclonal anti-sporozoite antibody. However, following treatment with trypsin (250 micrograms/ml), 81% of salivary gland sporozoites and 49% of oocyst sporozoites became reactive with mouse serum, and reactive sporozoites deposited mouse C3 on their surface in the presence of 30 mM EGTA and 1 mM Mg2+ without antibody. Concomitantly some sporozoites lost reactivity to anti-circumsporozoite protein monoclonal antibody. These results suggest that P. berghei sporozoites possibly express surface molecules that regulate the complement activation pathway of susceptible hosts but not of nonhosts, and that the putative structures consist of protease-sensitive molecule(s) which are closely associated with the circumsporozoite protein.     

Use of a DNA probe to measure the neutralization of Plasmodium berghei sporozoites by a monoclonal antibody

A specific DNA probe has been used to quantify the neutralizing effects of monoclonal antibodies (3D11) against the circumsporozoite protein of Plasmodium berghei sporozoites. The amount of parasite DNA was measured in the livers of Norway Brown rats at the peak of proliferation of the exoerythrocytic forms (EEF). In vitro treatment of 1.5 X 10(5) sporozoites with 0.36 microgram/0.5 ml of whole 3D11 IgG neutralized about 90% of the sporozoite infectivity. When the dose was 3.6 micrograms no signal was detected, indicating that less than ten sporozoites developed into EEF in the liver. In contrast, 3.6 micrograms of Fab obtained from 3D11 neutralized sporozoite infectivity by only 60%. Although the neutralizing effect of 3D11 was very marked, the infected rats developed parasitemias after a prolonged delay in patency, suggesting that a small proportion of sporozoites was resistant to the effects of 3D11. The sporozoites were subjected to four cycles of 3D11-mediated selection, each one involving treatment of sporozoites with the antibodies, injection of the mixture into rats, infection of hamsters with blood stage parasites obtained from the rats, feeding of Anopheles stephensi on these hamsters, and obtaining sporozoites from the salivary glands of the infected mosquitoes. After four cycles of selection, the susceptibility of the resulting sporozoites to different concentrations of 3D11 was compared with that of nonselected sporozoites. No differences were detected, indicating that the capacity of a few sporozoites to escape the neutralizing effect of 3D11 antibodies is not inherited.     

Plasmodium Sporozoite Interactions with Macrophages In Vitro: a Videomicroscopic Analysis

ABSTRACT. Studies of in vitro interactions between Plasmodium berghei sporozoites and peritoneal macrophages from mice and rats were performed. A videomicroscopic analysis was made of interactions observed by phase-contrast microscopy. Our results showed a diversity of dynamic interactions between sporozoites and macrophages that included no interaction, surface interaction without sporozoite interiorization, active sporozoite penetration, active penetration with subsequent sporozoite escape, macrophage destruction, and the formation of "tethers" or web-like structures by sporozoites that had actively invaded macrophages. Sporozoites are thus clearly capable of actively invading host macrophages and are not restricted to being phagocytosed for interiorization. The formation of "tethers" by the moving sporozoite might function in vivo by anchoring the sporozoite to the cells lining the lumen of the liver sinusoid. Active sporozoite motility appears to be a functional phenomenon involved in sporozoite invasion of host liver cells.     

Immune response gene regulation of immunity to Plasmodium berghei sporozoites and circumsporozoite protein vaccines. Overcoming genetic restriction with whole organism and subunit vaccines

We conducted a series of experiments to define Ir gene regulation of the immune response to Plasmodium berghei sporozoites and circumsporozoite (CS) protein-derived subunit vaccines. The studies demonstrated that there is no apparent genetic restriction of the capacity to develop protective immunity against a large sporozoite challenge after immunization with irradiation-attenuated P. berghei sporozoites; that the Th response to (Asp-Pro-Ala-Pro-Pro-Asn-Ala-Asn)n, the predominant protective B epitope on the P. berghei CS protein, is genetically restricted and regulated by Class II genes (I-Ab) and by genes in the Class I region (H-2Dk) or telomeric to this region; and that this restriction can be overcome by immunization with a r protein including the entire P. berghei CS protein. The results support the development of full length human CS protein vaccines to take advantage of all potential T epitopes on this protein.     

Plasmodium sporozoite interactions with macrophages in vitro: a videomicroscopic analysis

Studies of in vitro interactions between Plasmodium berghei sporozoites and peritoneal macrophages from mice and rats were performed. A videomicroscopic analysis was made of interactions observed by phase-contrast microscopy. Our results showed a diversity of dynamic interactions between sporozoites and macrophages that included no interaction, surface interaction without sporozoite interiorization, active sporozoite penetration, active penetration with subsequent sporozoite escape, macrophage destruction, and the formation of "tethers" or web-like structures by sporozoites that had actively invaded macrophages. Sporozoites are thus clearly capable of actively invading host macrophages and are not restricted to being phagocytosed for interiorization. The formation of "tethers" by the moving sporozoite might function in vivo by anchoring the sporozoite to the cells lining the lumen of the liver sinusoid. Active sporozoite motility appears to be a functional phenomenon involved in sporozoite invasion of host liver cells.     

Imaging movement of malaria parasites during transmission by Anopheles mosquitoes

Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.     

Plasmodium falciparum: effect of radiation on levels of gene transcripts in sporozoites

Humans immunized by the bites of irradiated Plasmodium falciparum (Pf) sporozoite-infected mosquitoes are protected against malaria. Radiation attenuates the sporozoites preventing them from fully developing and replicating in hepatocytes, but the effects of radiation on gene expression in sporozoites are unknown. We used RT-PCR (35 cycles of PCR followed by densitometry) to assess the expression of ten genes in Pf sporozoites, and in sporozoites irradiated with 15,000cGy. Irradiation reduced expression substantially (>60%) of two DNA repair genes; moderately (30-60%) of PfUIS3, the Pf orthologue of PbUIS3, a gene up-regulated in Plasmodium berghei sporozoites and of a third DNA repair gene; and minimally (<30%) of the Pf18S ribosomal RNA, PfCSP, PfSSP2/TRAP, and PfCELTOS genes. Irradiation increased expression of PfSPATR minimally. PfLSA1 RNA was not detectable in sporozoites. These results establish that radiation of sporozoites affects gene expression levels and provide the foundation for studies to identify specific genes involved in attenuation and protective immunity.