Liver Stage Malaria Infection Research Articles

Hepcidin in malaria superinfection: can findings be translated to humans?

To the Editor: With great interest we read the paper by Portugal et al.1, who reported that increased production of the iron-regulatory hormone hepcidin is responsible for inhibition of the liver-stage of malaria infection during ongoing blood-stage parasitemia. Febrile malaria in humans is indeed associated with high circulating hepcidin levels2, but, in our opinion, two aspects of this study need further consideration. First, the authors excluded a role for immune responses in preventing malaria superinfection by using mice deficient in components of various pathways of the innate and adaptive immune systems. However, the authors did not report the circulating hepcidin concentrations or hepatic hepcidin expression levels in these mice. Their model predicts that hepatic hepcidin expression would be unchanged in these mice, yet inflammatory cytokines are key inducers of hepatic hepcidin expression3,4. Moreover, confirmation of their findings in hepcidin-knockout mice would have further strengthened their conclusion5. Second, the limited data available in humans are not apparently consistent with the idea that an increase in hepcidin production inhibits liver-stage malaria infection. Chronic hepatitis C virus (HCV) infection is highly prevalent in many malaria-endemic regions, and recent evidence has shown HCV infection to be associated with reduced hepatic hepcidin expression with secondary hepatocyte iron accumulation6,7. According to the model proposed by Portugal et al.1, this would be expected to result in a favorable growth environment for liver-stage parasites. Instead, the emergence of blood-stage malaria parasites was delayed in people with HCV in Gabon8. Of course, other factors related to HCV infection, such as immunosuppression, may negate the effects of hepcidin on the liver-stage infection, and these clinical observations, therefore, do not fully refute a role for hepcidin. In conclusion, Portugal et al.1 have reported intriguing data that suggest a role for hepcidin in malaria pathogenesis beyond anemia and iron dysregulation, but additional clinical studies are needed to substantiate the claims.

CD8+ T Effector Memory Cells Protect against Liver-Stage Malaria

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8(+) T cells into effector, effector memory (T(EM)), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and T(EM) response that requires long intervals for an efficient boost. A preferential T(EM) phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8(+) T(EM) cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Ag-specific T(EM) cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 T(EM) populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design.

Prevalence of a type-I interferon immune response against malaria liver stage infection

Plasmodium sporozoites, transmitted to the mammalian host through a mosquito bite, travel to the liver, where they invade hepatocytes and develop into a form that is then able to infect red blood cells [1]. In spite of the importance of innate immunity in controlling microbial infections, very little is known about its role during the liver stage of malaria infection. To determine which type of innate immune response is triggered by Plasmodium berghei ANKA sporozoites during the liver stage of malaria we investigated transcriptome variations in this organ 40 hours after infection. We identified 69 genes, mainly classical type-I interferon stimulated genes, whose expression was induced at least 2-fold in comparison to non-infected control mice. Strikingly, the expression of all induced genes was reduced in Plasmodium-infected mice lacking a functional type-I interferon receptor (IFNAR1). Furthermore, we observed a 2-3 fold increase of liver parasite load in IFNAR1-deficient mice compared to their wild-type counterparts. Conversely, infection of wild-type mice after pre-stimulation of the type-I interferon response led to a reduction in liver infection. Altogether, our data demonstrate for the first time that Plasmodium berghei sporozoites are able to induce a type-I interferon immune response in the liver and that this innate immune response contributes to the host resistance during the malaria liver stage. Future work will address how this immune response is activated during a liver infection by Plasmodium and how it inhibits the infection.

A novel type of Plasmodium sporozoite transcellular migration

During liver-stage malaria infection, Plasmodium sporozoites must invade a hepatocyte in order to produce thousands of merozoites, the stage that infects erythrocytes and causes malarial pathology. One of the first steps of hepatocyte infection is the invagination of the host cell membrane, which shelters the parasite within a parasitophorous vacuole (PV) and allows parasite development inside the host cell. Sporozoites also have another peculiar type of cell invasion, in which the parasite breaches the host cell plasma membrane and transmigrates, free in the cytoplasm, through the host cell. This cell wounding and crossing process is termed cell traversal. Here, using live-cell imaging techniques, fluorescent markers of the host cell plasma membrane and a mutant parasite lacking cell traversal activity, we describe a new type of sporozoite transcellular migration. We have observed wild-type Plasmodium berhgei sporozoites moving through hepatocytes while surrounded by host cell membrane. Additionally, a small percentage of sporozoites drag host cell membrane with them when they leave hepatocytes, forming aggregates of membrane we have termed membrane bobbins. Membrane-surrounded migrating sporozoites and membrane bobbins are also observed during transmigration of primary liver sinusoidal endothelial cells. As cell traversal deficient sporozoites are still able to move across the cell surrounded by host cell plasma membrane, we define this new transcellular migration as being distinct from cell traversal. We hypothesize that this form of transmigration starts with the PV membrane formation similar to invasion with the exception that these sporozoites continue migration, ultimately leaving the cell by membrane fusion or disruption of the vacuole membrane and the plasma membrane.

Functional immunoassays using an in-vitro malaria liver-stage infection model: where do we go from here?

For more than 25 years, the ISI assay and ILSDA have been used to study the development of the malaria parasite in the liver, to discover and characterize sporozoite and liver-stage antigens, to support the development of malaria vaccine candidates, and to search for immunological correlates of protection in animals and in humans. Although both assays have been limited by low sporozoite invasion rates, significant biological variability, and the subjective nature of manually counting hepatocytes containing parasites as the read-out, they have nevertheless been useful tools for exploring parasite biology. This review describes the origin, application and current status of these assays, critically discusses the need for improvements, and explores the roles of these assays in supporting the development of an effective vaccine against Plasmodium falciparum malaria.

Host cell transcriptional profiling during malaria liver stage infection reveals a coordinated and sequential set of biological events

BackgroundPlasmodium sporozoites migrate to the liver where they traverse several hepatocytes before invading the one inside which they will develop and multiply into thousands of merozoites. Although this constitutes an essential step of malaria infection, the requirements of Plasmodium parasites in liver cells and how they use the host cell for their own survival and development are poorly understood.ResultsTo gain new insights into the molecular host-parasite interactions that take place during malaria liver infection, we have used high-throughput microarray technology to determine the transcriptional profile of P. berghei-infected hepatoma cells. The data analysis shows differential expression patterns for 1064 host genes starting at 6 h and up to 24 h post infection, with the largest proportion correlating specifically with the early stages of the infection process. A considerable proportion of those genes were also found to be modulated in liver cells collected from P. yoelii-infected mice 24 and 40 h after infection, strengthening the data obtained with the in vitro model and highlighting genes and pathways involved in the host response to rodent Plasmodium parasites.ConclusionOur data reveal that host cell infection by Plasmodium sporozoites leads to a coordinated and sequential set of biological events, ranging from the initial stage of stress response up to the engagement of host metabolic processes and the maintenance of cell viability throughout infection.

Immunization with Radiation-Attenuated Plasmodium berghei Sporozoites Induces Liver cCD8α+DC that Activate CD8+T Cells against Liver-Stage Malaria

Immunization with radiation (γ)-attenuated Plasmodia sporozoites (γ-spz) confers sterile and long-lasting immunity against malaria liver-stage infection. In the P. berghei γ-spz model, protection is linked to liver CD8+ T cells that express an effector/memory (TEM) phenotype, (CD44hiCD45RBloCD62Llo ), and produce IFN-γ. However, neither the antigen presenting cells (APC) that activate these CD8+ TEM cells nor the site of their induction have been fully investigated. Because conventional (c)CD8α+ DC (a subset of CD11c+ DC) are considered the major inducers of CD8+ T cells, in this study we focused primarily on cCD8α+ DC from livers of mice immunized with Pb γ-spz and asked whether the cCD8α+ DC might be involved in the activation of CD8+ TEM cells. We demonstrate that multiple exposures of mice to Pb γ-spz lead to a progressive and nearly concurrent accumulation in the liver but not the spleen of both the CD11c+NK1.1− DC and CD8+ TEM cells. Upon adoptive transfer, liver CD11c+NK1.1− DC from Pb γ-spz-immunized mice induced protective immunity against sporozoite challenge. Moreover, in an in vitro system, liver cCD8α+ DC induced naïve CD8+ T cells to express the CD8+ TEM phenotype and to secrete IFN-γ. The in vitro induction of functional CD8+ TEM cells by cCD8α+ DC was inhibited by anti-MHC class I and anti-IL-12 mAbs. These data suggest that liver cCD8α+ DC present liver-stage antigens to activate CD8+ TEM cells, the pre-eminent effectors against pre-erythrocytic malaria. These results provide important implications towards a design of anti-malaria vaccines.

Liver cCD8a+DC from Mice Immunized with Gamma-irradiated Plasmodium berghei Sporozoites Mediate the Induction of Intrahepatic Effector CD8+ T cells against Liver-Stage Malaria (45.30)

Abstract Immunization with radiation (γ)-attenuated Plasmodia sporozoites (γ-spz) confers sterile and long-lasting immunity against malaria liver-stage infection. In the P. berghei γ-spz model, protection is linked to liver CD8+ T cells that express an effector/memory (TEM) phenotype, (CD44hiCD45RBloCD62Llo ), and produce IFN-γ. However, neither the antigen presenting cells (APC) that activate these CD8+ TEM cells nor the site of their induction have been fully investigated. Because conventional (c)CD8α+ DC (a subset of CD11c+ DC) are considered the major inducers of CD8+ T cells, in this study we focused primarily on cCD8α+ DC from livers of mice immunized with Pb γ-spz and asked whether the cCD8α+ DC might be involved in the activation of CD8+ TEM cells. We demonstrate that multiple exposures of mice to Pb γ-spz lead to a progressive and nearly concurrent accumulation in the liver but not the spleen of both the CD11c+NK1.1- DC and CD8+ TEM cells. Upon adoptive transfer, liver CD11c+NK1.1- DC from Pb γ-spz-immunized mice induced protective immunity against sporozoite challenge. Moreover, in an in vitro system, liver cCD8α+ DC induced naïve CD8+ T cells to express the CD8+ TEM phenotype and to secrete IFN-γ. The in vitro induction of functional CD8+ TEM cells by cCD8α+ DC was inhibited by anti-MHC class I and anti-IL-12 mAbs. These data suggest that liver cCD8α+ DC present liver-stage antigens to activate CD8+ TEM cells, the pre-eminent effectors against pre-erythrocytic malaria. These results provide important implications towards a design of anti-malaria vaccines. Supported by a grant from NIH and USAMRMC.

CD1d-restricted NK T cells are dispensable for specific antibody responses and protective immunity against liver stage malaria infection in mice.

Immunization with a single dose of irradiated sporozoites is sufficient to induce protection against malaria in wild-type mice. Although this protection is classically attributed to conventional CD4+ and CD8+ T cells, several recent reports have suggested an important role for CD1-restricted NK T cells in immunity to malaria. In this study, we directly compared the ability of C57BL/6 wild-type and CD1-deficient mice to mount a protective immune response against Plasmodium berghei sporozoites. Our data indicate that CD1-restricted NK T cells are not required for protection in this model system. Moreover, specific IgG antibody responses to the P. berghei circumsporozoite repeat sequence were also unaffected by CD1 deficiency. Collectively, our data demonstrate that CD1-restricted NK T cells are dispensable for protective immunity to liver stage P. berghei infection.