Malaria sporozoite penetration A new approach by double staining

Rénia, Laurent; Miltgen, F; Charoenvit, Yupin; Ponnudurai, T.; Verhave, J.P.; Collins, William E.; Mazier, Dominique

doi:10.1016/0022-1759(88)90358-4

Cited by 98 publications

(82 citation statements)

References 8 publications

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“…Inoculated hepatoma cells were incubated for additional 90 min at 37°C with 5% CO 2 to quantify cell invasion. The protocol was slightly modified from the established two-color invasion assay (24). Briefly, cells were fixed with 4% paraformaldehyde, followed by an immunofluorescence assay using anti-P. berghei CSP antibody (23) to label extracellular sporozoites and anti-GFP antibody after cell permeabilization to detect intracellular parasites.…”

Section: Methodsmentioning

confidence: 99%

Plasmodium berghei Sporozoites Acquire Virulence and Immunogenicity during Mosquito Hemocoel Transit

2014

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bMalaria is a vector-borne disease caused by the single-cell eukaryote Plasmodium. The infectious parasite forms are sporozoites, which originate from midgut-associated oocysts, where they eventually egress and reach the mosquito hemocoel. Sporozoites actively colonize the salivary glands in order to be transmitted to the mammalian host. Whether residence in the salivary glands provides distinct and vital cues for the development of infectivity remains unsolved. In this study, we systematically compared the infectivity of Plasmodium berghei sporozoites isolated from the mosquito hemocoel and salivary glands. Hemocoel sporozoites display a lower proportion of gliding motility but develop into liver stages when added to cultured hepatoma cells or after intravenous injection into mice. Mice infected by hemocoel sporozoites had blood infections similar to those induced by sporozoites liberated from salivary glands. These infected mice display indistinguishable systemic inflammatory cytokine responses and develop experimental cerebral malaria. When used as metabolically active, live attenuated vaccine, hemocoel sporozoites elicit substantial protection against sporozoite challenge infections. Collectively, these findings show that salivary gland colonization does not influence parasite virulence in the mammalian host when sporozoites are administered intravenously. This conclusion has important implications for in vitro sporozoite production and manufacturing of whole-sporozoite vaccines.

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Section: Methodsmentioning

confidence: 99%

Plasmodium berghei Sporozoites Acquire Virulence and Immunogenicity during Mosquito Hemocoel Transit

2014

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“…A total of 5 ϫ 10 4 sporozoites were treated as described above, resuspended in cell medium, and added to wells containing semiconfluent Hepa 1-6 cells. Plates were incubated for 1 h at 37°C, fixed in 4% paraformaldehyde at 4°C, and stained using 3D11 and a double staining technique (36). Intracellular and extracellular sporozoites were differentially stained and counted using a Nikon Eclipse E600 microscope.…”

Section: Methodsmentioning

confidence: 99%

Chemical Attenuation of Plasmodium berghei Sporozoites Induces Sterile Immunity in Mice

et al. 2008

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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.The development of an effective vaccine is critical to curb the significant health, social, and economic impacts caused annually by malaria in countries where the disease is endemic (39). Malaria infection involves injection of Plasmodium sporozoites from a mosquito into humans. The sporozoites migrate to the liver, invade hepatocytes, and transform into exoerythrocytic forms (EEFs) that replicate to produce schizonts containing thousands of merozoites (35). These merozoites are released into the host bloodstream and invade erythrocytes. The blood stages of malaria are responsible for producing the symptoms of the disease. Many attempts have been made in recent years to develop effective subunit vaccines composed of recombinant Plasmodium antigens. Due to the complexity of Plasmodium, these vaccines have been only partially effective (2,12,14,38).Recently, there has been renewed interest in the attenuated whole-organism vaccine strategy (16,22,38,53). The wholeorganism approach has historically used radiation-attenuated sporozoites (RAS) to obtain sterile immunity experimentally in both mice and humans (16,30). The RAS invade hepatocytes in a susceptible host and begin to develop into EEFs, but the majority of parasites fail to undergo nuclear division and do not progress to the merozoite form (44, 45). Using mice, RAS dosing regimens that generate protective immunity have varied, although most regimens require a prime-boost schedule (4,11,32,50). A meta-analysis of 10 years of immunization of human volunteers using irradiated Plasmodium falciparum sporozoites showed a dose response in terms of the immunization dose required for protection (16,22). One key issue with RAS has been the delivery of the correct irradiation dose to ensure adequate attenuation of the parasite (16,23,38,41,51). A strategy to overcome this issue has been to generate genetically attenuated ...

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“…The flow chamber, cells, and medium were kept on a slide warmer at 37°C; and the room in which the assay was performed was maintained at 30°C since sporozoites do not attach to cells at temperatures below 25°C. 2 Sporozoite Invasion Assay-This assay is identical to the sporozoite attachment assay except that cells were stained with a double-staining procedure that allows differentiation of intracellular and extracellular sporozoites (5,6). Briefly, cells were fixed with 4% paraformaldehyde and stained with mAb 3D11 (directed against the repeat region of P. berghei CS) followed by goat anti-mouse Ig conjugated to colloidal gold 10 nm (Amersham Pharmacia Biotech).…”

Section: Methodsmentioning

confidence: 99%

The Binding of the Circumsporozoite Protein to Cell Surface Heparan Sulfate Proteoglycans Is Required for PlasmodiumSporozoite Attachment to Target Cells

Pinzon-Ortiz¹,

Friedman²,

Esko³

et al. 2001

Journal of Biological Chemistry

118

120

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The major surface protein of malaria sporozoites, the circumsporozoite protein, binds to heparan sulfate proteoglycans on the surface of hepatocytes. It has been proposed that this binding event is responsible for the rapid and specific localization of sporozoites to the liver after their injection into the skin by an infected anopheline mosquito. Previous in vitro studies performed under static conditions have failed to demonstrate a significant role for heparan sulfate proteoglycans during sporozoite invasion of cells. We performed sporozoite attachment and invasion assays under more dynamic conditions and found a dramatic decrease in sporozoite attachment to cells in the presence of heparin. In contrast to its effect on attachment, heparin does not appear to have an effect on sporozoite invasion of cells. When substituted heparins were used as competitive inhibitors of sporozoite attachment, we found that sulfation of the glycosaminoglycan chains at both the Nand O-positions was important for sporozoite adhesion to cells. We conclude that the binding of the circumsporozoite protein to hepatic heparan sulfate proteoglycans is likely to function during sporozoite attachment in the liver and that this adhesion event depends on the sulfated glycosaminoglycan chains of the proteoglycans.Protozoans of the genus Plasmodium are the causative agents of malaria. Malaria infection is initiated when an infected anopheline mosquito injects sporozoites during a blood meal. The injected sporozoites travel to the liver and invade hepatocytes where they develop into exoerythrocytic forms. The speed and specificity of sporozoite localization to hepatocytes suggest a receptor-mediated event. Previous studies have shown that the major sporozoite surface protein, the circumsporozoite protein (CS), 1 binds to heparan sulfate proteogly- (3, 4). However, the inhibitory effect on sporozoite infectivity, while demonstrating that the CS-HSPG interaction is important, does not indicate if the glycan is required for sporozoite attachment, invasion, or subsequent development in hepatocytes.In vitro assays (5, 6) have been used to determine whether the CS-HSPG interaction is critical for cell invasion. Frevert et al. (6) found that removal of the majority of cell surface HSPGs had a minimal inhibitory effect on sporozoite invasion of cells. One interpretation of these data is that the binding of CS to HSPGs does not function during sporozoite invasion. Another possibility, however, is that CS binding to HSPGs functions in the more dynamic conditions found in the blood circulation and leads to arrest of sporozoites in the liver sinusoids. In this paper we modify the standard sporozoite invasion assay and provide evidence that the interaction between CS and cell surface HSPGs functions during the initial attachment of sporozoites to cells under conditions that mimic flow. In addition, we show that the sulfate moieties of the HSPG glycosaminoglycan chains (GAGs) are important for attachment of sporozoites to cells. MATERIALS AND METHODSSpo...

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Malaria sporozoite penetration A new approach by double staining

Cited by 98 publications

References 8 publications

Plasmodium berghei Sporozoites Acquire Virulence and Immunogenicity during Mosquito Hemocoel Transit

Plasmodium berghei Sporozoites Acquire Virulence and Immunogenicity during Mosquito Hemocoel Transit

Chemical Attenuation of Plasmodium berghei Sporozoites Induces Sterile Immunity in Mice

The Binding of the Circumsporozoite Protein to Cell Surface Heparan Sulfate Proteoglycans Is Required for PlasmodiumSporozoite Attachment to Target Cells

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