Pregnancy-associated malaria (PAM) is expressed in a range of clinical complications that include increased disease severity in pregnant women, decreased fetal viability, intra-uterine growth retardation, low birth weight and infant mortality. The physiopathology of malaria in pregnancy is difficult to scrutinize and attempts were made in the past to use animal models for pregnancy malaria studies. Here, we describe a comprehensive mouse experimental model that recapitulates many of the pathological and clinical features typical of human severe malaria in pregnancy. We used P. berghei ANKA-GFP infection during pregnancy to evoke a prominent inflammatory response in the placenta that entails CD11b mononuclear infiltration, up-regulation of MIP-1 alpha chemokine and is associated with marked reduction of placental vascular spaces. Placenta pathology was associated with decreased fetal viability, intra-uterine growth retardation, gross post-natal growth impairment and increased disease severity in pregnant females. Moreover, we provide evidence that CSA and HA, known to mediate P. falciparum adhesion to human placenta, are also involved in mouse placental malaria infection. We propose that reduction of maternal blood flow in the placenta is a key pathogenic factor in murine pregnancy malaria and we hypothesize that exacerbated innate inflammatory responses to Plasmodium infected red blood cells trigger severe placenta pathology. This experimental model provides an opportunity to identify cell and molecular components of severe PAM pathogenesis and to investigate the inflammatory response that leads to the observed fetal and placental blood circulation abnormalities.

Malaria remains one of the greatest burdens to global health, causing nearly 500,000 deaths in 2014. When manifesting in the lungs, severe malaria causes acute lung injury/acute respiratory distress syndrome (ALI/ARDS). We have previously shown that a proportion of DBA/2 mice infected with Plasmodium berghei ANKA (PbA) develop ALI/ARDS and that these mice recapitulate various aspects of the human syndrome, such as pulmonary edema, hemorrhaging, pleural effusion and hypoxemia. Herein, we investigated the role of neutrophils in the pathogenesis of malaria-associated ALI/ARDS. Mice developing ALI/ARDS showed greater neutrophil accumulation in the lungs compared with mice that did not develop pulmonary complications. In addition, mice with ALI/ARDS produced more neutrophil-attracting chemokines, myeloperoxidase and reactive oxygen species. We also observed that the parasites Plasmodium falciparum and PbA induced the formation of neutrophil extracellular traps (NETs) ex vivo, which were associated with inflammation and tissue injury. The depletion of neutrophils, treatment with AMD3100 (a CXCR4 antagonist), Pulmozyme (human recombinant DNase) or Sivelestat (inhibitor of neutrophil elastase) decreased the development of malaria-associated ALI/ARDS and significantly increased mouse survival. This study implicates neutrophils and NETs in the genesis of experimentally induced malaria-associated ALI/ARDS and proposes a new therapeutic approach to improve the prognosis of severe malaria. A deeper understanding of the pathogenesis of malaria-associated ALI/ARDS may help in the development of new therapeutic approaches to improve the prognosis of severe cases of malaria. Using the Plasmodium berghei ANKA-infection mouse model of ALI/ARDS, which resembles the human disease in many aspects, this study reports the critical role of neutrophils in the pathogenesis of this syndrome. Mice developing ALI/ARDS showed abundant lung-infiltrating neutrophils in association with the increased production of neutrophil-attracting chemokines, myeloperoxidase and reactive oxygen species. The parasites Plasmodium falciparum and P. berghei ANKA both induced the formation of neutrophil extracellular traps ex vivo. By targeting neutrophils and neutrophil extracellular traps with specific drugs, we succeeded in preventing the development of malaria-associated ALI/ARDS and significantly increased mouse survival.

The spectrum of the clinical presentation and severity of malaria infections is broad, ranging from uncomplicated febrile illness to severe forms of disease such as cerebral malaria (CM), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pregnancy-associated malaria (PAM) or severe anemia (SA). Rodent models that mimic human CM, PAM and SA syndromes have been established. Here, we show that DBA/2 mice infected with P. berghei ANKA constitute a new model for malaria-associated ALI. Up to 60% of the mice showed dyspnea, airway obstruction and hypoxemia and died between days 7 and 12 post-infection. The most common pathological findings were pleural effusion, pulmonary hemorrhage and edema, consistent with increased lung vessel permeability, while the blood-brain barrier was intact. Malaria-associated ALI correlated with high levels of circulating VEGF, produced de novo in the spleen, and its blockage led to protection of mice from this syndrome. In addition, either splenectomization or administration of the anti-inflammatory molecule carbon monoxide led to a significant reduction in the levels of sera VEGF and to protection from ALI. The similarities between the physiopathological lesions described here and the ones occurring in humans, as well as the demonstration that VEGF is a critical host factor in the onset of malaria-associated ALI in mice, not only offers important mechanistic insights into the processes underlying the pathology related with malaria but may also pave the way for interventional studies. Malaria remains a major source of morbidity and mortality throughout the tropical regions of the world causing up to 1 million deaths every year, mainly in children. Although infection with malaria parasites is common, only 1 to 2% of infections lead to severe life-threatening disease characterized by a range of clinical features including coma, severe anemia, respiratory distress, metabolic acidosis, or multiorgan failure. Animal models of infection are indispensable tools to better understand the dynamic host-parasite interactions that lead to the onset of different infection outcomes. We now show that DBA/2 mice infected with P. berghei ANKA constitute a rodent model for malaria-associated acute lung injury (ALI). Up to 60% of these infected mice develop respiratory problems including dyspnea, airway obstruction and hypoxemia and die soon after. The most common pathological findings were pleural effusion, pulmonary hemorrhage and edema, features common to human malaria patients that show life-threatening respiratory distress. Malaria-associated ALI in this model correlates with high levels of circulating vascular permeability factor, VEGF, and its blockage by different means leads to protection from ALI. The existence of such a model of disease will certainly contribute to a better understanding of malaria-associated pathology and possibly to the design of novel intervention strategies.

Histological evidence of Plasmodium in the placenta is indicative of placental malaria, a condition associated with severe outcomes for mother and child. Histological lesions found in placentas from Plasmodium-exposed women include syncytial knotting, syncytial rupture, thickening of the placental barrier, necrosis of villous tissue and intervillositis. These histological changes have been associated with P. falciparum infections, but little is known about the contribution of P. vivax to such changes. We conducted a cross-sectional study with pregnant women at delivery and assigned them to three groups according to their Plasmodium exposure during pregnancy: no Plasmodium exposure (n = 41), P. vivax exposure (n = 59) or P. falciparum exposure (n = 19). We evaluated their placentas for signs of Plasmodium and placental lesions using ten histological parameters: syncytial knotting, syncytial rupture, placental barrier thickness, villi necrosis, intervillous space area, intervillous leucocytes, intervillous mononucleates, intervillous polymorphonucleates, parasitized erythrocytes and hemozoin. Placentas from P. vivax-exposed women showed little evidence of Plasmodium or hemozoin but still exhibited more lesions than placentas from women not exposed to Plasmodium, especially when infections occurred twice or more during pregnancy. In the Brazilian state of Acre, where diagnosis and primary treatment are readily available and placental lesions occur in the absence of detected placental parasites, relying on the presence of Plasmodium in the placenta to evaluate Plasmodium-induced placental pathology is not feasible. Multivariate logistic analysis revealed that syncytial knotting (odds ratio [OR], 4.21, P = 0.045), placental barrier thickness (OR, 25.59, P = 0.021) and mononuclear cells (OR, 4.02, P = 0.046) were increased in placentas from P. vivax-exposed women when compared to women not exposed to Plasmodium during pregnancy. A vivax-score was developed using these three parameters (and not evidence of Plasmodium) that differentiates between placentas from P. vivax-exposed and unexposed women. This score illustrates the importance of adequate management of P. vivax malaria during pregnancy. Malaria during pregnancy remains a risk for approximately 125 million women each year. Adverse outcomes of malaria during pregnancy include maternal anemia and low infant birth weight. Additionally, the presence of malaria parasites, namely Plasmodium falciparum, has been associated with the occurrence of placental lesions. In the Amazonian region of Brazil Plasmodium vivax is the primary parasite species. To date, little is known about the capacity of this parasite to induce placental lesions. In this study we have used ten histological parameters to evaluate the effect of exposure to Plasmodium vivax during pregnancy on the occurrence of placental lesions when compared to placentas from non-exposed women. Placentas from women exposed to Plasmodium falciparum were used as controls. Placentas from Plasmodium vivax-exposed placentas did not have strong evidence of placental parasites but had increased syncytial knotting, thickness of the placental barrier and mononuclear cells when compared to non-exposed women. We developed a score based on these three parameters and not on the presence of placental parasites that enables us to visualize the effect that Plasmodium vivax has on placentas from women infected during pregnancy.

We previously described that DNA vaccination with the gene encoding amastigote surface protein 2 (ASP-2) protects approximately 65% of highly susceptible A/Sn mice against the lethalTrypanosoma cruziinfection. Here, we explored the possibility that bacterial recombinant proteins of ASP-2 could be used to improve the efficacy of vaccinations. Initially, we compared the protective efficacy of vaccination regimens using either a plasmid DNA, a recombinant protein, or both sequentially (DNA priming and protein boosting). Survival after the challenge was not statistically different among the three mouse groups and ranged from 53.5 to 75%. The fact that immunization with a recombinant protein alone induced protective immunity revealed the possibility that this strategy could be pursued for vaccination. We investigated this possibility by using six different recombinant proteins representing distinct portions of ASP-2. The vaccination of mice with glutathioneS-transferase fusion proteins representing amino acids 261 to 500 or 261 to 380 of ASP-2 in the presence of the adjuvants alum and CpG oligodeoxynucleotide 1826 provided remarkable immunity, consistently protecting 100% of the A/Sn mice. Immunity was completely reversed by the in vivo depletion of CD8⁺T cells, but not CD4⁺T cells, and was associated with the presence of CD8⁺T cells specific for an epitope located between amino acids 320 and 327 of ASP-2. We concluded that a relatively simple formulation consisting of a recombinant protein with a selected portion of ASP-2, alum, and CpG oligodeoxynucleotide 1826 might be used to cross-prime strong CD8⁺-T-cell-dependent protective immunity againstT. cruziinfection.