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.

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 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.

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.

Protective immunity against lethal infection is developed when BALB/c or C57BL/6 mice are immunized with plasmids containing genes from the protozoan parasite Trypanosoma cruzi. However, genetic vaccination of the highly susceptible mouse strain A/Sn promoted limited survival after challenge. This observation questioned whether this type of vaccination would be appropriate for highly susceptible individuals. Here, we compared the protective efficacy and the immune response after individual or combined genetic vaccination of A/Sn mice with genes encoding trans-sialidase (TS) or the amastigote surface protein-2 (ASP-2). After challenge, a significant proportion of A/Sn mice immunized with either the asp-2 gene or simultaneously with asp-2 and ts genes, survived infection. In contrast, the vast majority of mice immunized with the ts gene or the vector alone died. Parasitological and histological studies performed in the surviving mice revealed that these mice harbored parasites; however, minimal inflammatory responses were seen in heart and striated muscle. We used this model to search for an in vitro correlation for protection. We found that protective immunity correlated with a higher secretion of interferon- by spleen cells on in vitro restimulation with ASP-2 and the presence of ASP-2-specific CD8 cells. Depletion of either CD4 or CD8 or both T-cell subpopulations prior to the challenge rendered the mice susceptible to infection demonstrating the critical contribution of both cell types in protective immunity. Our results reinforce the prophylactic potential of genetic vaccination with asp-2 and ts genes by describing protective immunity against lethal T. cruzi infection and chronic tissue pathology in a highly susceptible mouse strain.