Plasmepsin 4-Deficient Plasmodium berghei Are Virulence Attenuated and Induce Protective Immunity against Experimental Malaria

Spaccapelo, Roberta; Janse, Chris J.; Caterbi, Sara; Franke-Fayard, Blandine; Bonilla, J. Alfredo; Syphard, Luke M.; Cristina, Manlio Di; Dottorini, Tania; Savarino, Andrea; Cassone, Antonio; Bistoni, Francesco; Waters, Andrew P.; Dame, John B.; Crisanti, Andrea

doi:10.2353/ajpath.2010.090504

Cited by 104 publications

(139 citation statements)

References 48 publications

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“…The reporter Pb ANKA parasite line Pb GFP-Luc ama1 (1037m1f1m1cl1) expresses a fusion protein of GFP (mutant3) and fi refl y luciferase (LUC-IAV) under the schizont-specifi c ama-1 promoter and is SM free [ 16 ]. The reporter cassette is integrated into the neutral 230p locus (PBANKA_030600).…”

Section: Standard Parasites Lines Used For Generation Transgenic Paramentioning

confidence: 99%

Generation of Transgenic Rodent Malaria Parasites Expressing Human Malaria Parasite Proteins

Salman

Mogollon

Lin

et al. 2015

Methods in Molecular Biology

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We describe methods for the rapid generation of transgenic rodent Plasmodium berghei (Pb) parasites that express human malaria parasite (HMP) proteins, using the recently developed GIMO-based transfection methodology. Three different genetic modifications are described resulting in three types of transgenic parasites. (1) Additional Gene (AG) mutants. In these mutants the HMP gene is introduced as an "additional gene" into a silent/neutral locus of the Pb genome under the control of either a constitutive or stage-specific Pb promoter. This method uses the GIMO-transfection protocol and AG mutants are generated by replacing the positive-negative selection marker (SM) hdhfr::yfcu cassette in a neutral locus of a standard GIMO mother line with the HMP gene expression cassette, resulting in SM free transgenic parasites. (2) Double-step Replacement (DsR) mutants. In these mutants the coding sequence (CDS) of the Pb gene is replaced with the CDS of the HMP ortholog in a two-step GIMO-transfection procedure. This process involves first the replacement of the Pb CDS with the hdhfr::yfcu SM, followed by insertion of the HMP ortholog at the same locus thereby replacing hdhfr::yfcu with the HMP CDS. These steps use the GIMO-transfection protocol, which exploits both positive selection for Pb orthologous gene-deletion and negative selection for HMP gene-insertion, resulting in SM free transgenic parasites. (3) Double-step Insertion (DsI) mutants. When a Pb gene is essential for blood stage development the DsR strategy is not possible. In these mutants the HMP expression cassette is first introduced into the neutral locus in a standard GIMO mother line as described for AG mutants but under the control elements of the Pb orthologous gene; subsequently, the Pb ortholog CDS is targeted for deletion through replacement of the Pb CDS with the hdhfr::yfcu SM, resulting in transgenic parasites with a new GIMO locus permissive for additional gene-insertion modifications.The different types of transgenic parasites can be exploited to examine interactions of drugs/inhibitors or immune factors with HMP molecules in vivo. Mice either immunized with HMP-vaccines or treated with specific drugs can be infected/challenged with these transgenic mutants to evaluate drug or vaccine efficacy in vivo.

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Section: Standard Parasites Lines Used For Generation Transgenic Paramentioning

confidence: 99%

Generation of Transgenic Rodent Malaria Parasites Expressing Human Malaria Parasite Proteins

Salman

Mogollon

Lin

et al. 2015

Methods in Molecular Biology

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“…First, in human malaria, the expression of several specific PfEMP1 proteins promoted cytoadherence to brain endothelial cells and correlated with the severity of cerebral malaria (12,13), suggesting that these proteins constitute a major virulence factor for cerebral malaria. Second, in rodents, the plasmepsin-4 (pm4) gene, encoding an aspartic protease involved in hemoglobin digestion, was shown to be implicated in experimental cerebral malaria (ECM) (14,15). Finally, several studies also reported a crucial role for ICAM-1 in malaria pathogenesis, and notably, ICAM-1-deficient mice are protected from ECM (16).…”

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Disruption of Parasitehmgb2Gene Attenuates Plasmodium berghei ANKA Pathogenicity

et al. 2015

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h Eukaryotic high-mobility-group-box (HMGB) proteins are nuclear factors involved in chromatin remodeling and transcription regulation. When released into the extracellular milieu, HMGB1 acts as a proinflammatory cytokine that plays a central role in the pathogenesis of several immune-mediated inflammatory diseases. We found that the Plasmodium genome encodes two genuine HMGB factors, Plasmodium HMGB1 and HMGB2, that encompass, like their human counterparts, a proinflammatory domain. Given that these proteins are released from parasitized red blood cells, we then hypothesized that Plasmodium HMGB might contribute to the pathogenesis of experimental cerebral malaria (ECM), a lethal neuroinflammatory syndrome that develops in C57BL/6 (susceptible) mice infected with Plasmodium berghei ANKA and that in many aspects resembles human cerebral malaria elicited by P. falciparum infection. The pathogenesis of experimental cerebral malaria was suppressed in C57BL/6 mice infected with P. berghei ANKA lacking the hmgb2 gene (⌬hmgb2 ANKA), an effect associated with a reduction of histological brain lesions and with lower expression levels of several proinflammatory genes. The incidence of ECM in pbhmgb2-deficient mice was restored by the administration of recombinant PbHMGB2. Protection from experimental cerebral malaria in ⌬hmgb2 ANKA-infected mice was associated with reduced sequestration in the brain of CD4؉ and CD8 ؉ T cells, including CD8 ؉ granzyme B ؉ and CD8 ؉ IFN-␥ ؉ cells, and, to some extent, neutrophils. This was consistent with a reduced parasite sequestration in the brain, lungs, and spleen, though to a lesser extent than in wild-type P. berghei ANKA-infected mice. In summary, Plasmodium HMGB2 acts as an alarmin that contributes to the pathogenesis of cerebral malaria.

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“…The P. berghei ANKA reporter line 1037cl1 (ANKA-GFPLucschiz [Luc]; mutant RMgm-32) (http://www.pberghei.eu), which contains the gfp-luc fusion gene under the control of the schizontspecific ama1 promoter integrated into the silent 230p gene locus (PBANKA_0306000) and does not contain a drug-selectable marker (21), was used to obtain P. berghei-VAR. P. berghei ANKA-GFP-Luc (1037cl1) parasites (P. berghei) were also used in all experiments.…”

Section: Methodsmentioning

confidence: 99%

Murine Model for Preclinical Studies of Var2CSA-Mediated Pathology Associated with Malaria in Pregnancy

et al. 2016

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g Plasmodium falciparum infection during pregnancy leads to abortions, stillbirth, low birth weight, and maternal mortality. Infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulfate A (CSA) via var2CSA protein exposed on the P. falciparum IE membrane. Plasmodium berghei IE infection in pregnant BALB/c mice is a model for severe placental malaria (PM). Here, we describe a transgenic P. berghei parasite expressing the full-length var2CSA extracellular region (domains DBL1X to DBL6) fused to a P. berghei exported protein (EMAP1) and characterize a var2CSA-based mouse model of PM. BALB/c mice were infected at midgestation with different doses of P. berghei-var2CSA (P. berghei-VAR) or P. berghei wild-type IEs. Infection with 10 4 P. berghei-VAR IEs induced a higher incidence of stillbirth and lower fetal weight than P. berghei. At doses of 10 5 and 10 6 IEs, P. berghei-VAR-infected mice showed increased maternal mortality during pregnancy and fetal loss, respectively. Parasite loads in infected placentas were similar between parasite lines despite differences in maternal outcomes. Fetal weight loss normalized for parasitemia was higher in P. berghei-VAR-infected mice than in P. berghei-infected mice. In vitro assays showed that higher numbers of P. berghei-VAR IEs than P. berghei IEs adhered to placental tissue. Immunization of mice with P. berghei-VAR elicited IgG antibodies reactive to DBL1-6 recombinant protein, indicating that the topology of immunogenic epitopes is maintained between DBL1-6 -EMAP1 on P. berghei-VAR and recombinant DBL1-6 (recDBL1-6). Our data suggested that impairments in pregnancy caused by P. berghei-VAR infection were attributable to var2CSA expression. This model provides a tool for preclinical evaluation of protection against PM induced by approaches that target var2CSA. Malaria is a major health concern in countries where it is endemic. Recent reports show an incidence of 198 million cases in 2013 (82% in Africa) and 584,000 estimated deaths, 74% of which were children under 5 years of age (1). In areas of endemicity, pregnant women are under a greater risk of malaria than nonpregnant (NP) women due to immunological and hormonal changes and higher attractiveness to mosquitoes (2). Placental infection or clinical malaria during pregnancy has been shown to have a negative impact on infant health, increasing the risk of clinical malaria and mortality in infants (3).In areas of unstable malaria transmission, pregnant women can develop severe malaria due to low immunity to the parasite, which has been associated with increased mortality during pregnancy (4, 5), miscarriages, preterm deliveries, low birth weight, stillbirth, and death of neonates (6). These features can be recapitulated in a mouse model of severe placental malaria (PM) (7), which consists of infection of BALB/c mice syngeneically pregnant with parasites of the ANKA strain of the rodent malaria parasite Plasmodium berghei.Apart from physiological changes associated with gestation, increased suscep...

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Plasmepsin 4-Deficient Plasmodium berghei Are Virulence Attenuated and Induce Protective Immunity against Experimental Malaria

Cited by 104 publications

References 48 publications

Generation of Transgenic Rodent Malaria Parasites Expressing Human Malaria Parasite Proteins

Generation of Transgenic Rodent Malaria Parasites Expressing Human Malaria Parasite Proteins

Disruption of Parasitehmgb2Gene Attenuates Plasmodium berghei ANKA Pathogenicity

Murine Model for Preclinical Studies of Var2CSA-Mediated Pathology Associated with Malaria in Pregnancy

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