Liver-stage Malaria Research Articles

Improved isolation of murine hepatocytes for in vitro malaria liver stage studies

BackgroundPrimary hepatocyte cultures are a valuable tool for the understanding of cellular and molecular phenomena occurring during malaria liver stage. This paper describes an improved perfusion/dissociation procedure to isolate hepatocytes from mouse liver that is suitable for malaria studies and allows reproducible preparation of primary hepatocytes with consistent cell yields and controlled purity.ResultsThis protocol is a detailed description of a technique to isolate and culture mouse hepatocytes and represents an improvement over previous descriptions of hepatocyte isolation for malaria studies, regarding three technical aspects: (1) dissociation reagents choice; (2) cell separation gradient and (3) cell purity control. Cell dissociation was optimized for a specific collagenase digestion media. The cell dissociation step was improved by using a three-layer discontinuous gradient. A cell purity check was introduced to monitor the expression of CD95 on hepatocytes using flow cytometry methods.ConclusionThe procedure described allows reproducible recovery of one to three million hepatocytes per preparation with cell purity of about 90% as determined by FACS analysis. Completion of the protocol is usually achieved in about four hours per preparation and pooling is suggested for multiple preparations of larger number of cells.

Plasmodium Circumsporozoite Protein Promotes the Development of the Liver Stages of the Parasite

The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFkappaB nuclear import, thus downregulating the expression of many genes controlled by NFkappaB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite.

Elevated basal hepcidin levels in the liver may inhibit the development of malaria infection: Another piece towards solving the malaria puzzle?

Inflammatory cytokines play a crucial role in the human immune response to infection by malaria. During the initial sporozoite infection of the liver the presence of Interleukin-6 (IL-6) can be determinant. IL-6 controls systemic iron homeostasis through hepcidin, which is produced mainly by hepatocytes. An elevated basal hepcidin level in the liver can be induced by chronic inflammatory disease. Hepcidin is also a peptide with antimicrobial properties. We hypothesize that elevated basal hepcidin levels in the liver inhibit the development of malaria infection. When hepcidin is abundant, hepatocytes sequester iron, and this inhibits sporozoite development in liver-stage malaria infection. The validity of our hypothesis can be proven by observing sporozoite growth in hepcidin-treated hepatocytes, or in hepatocytes, stimulated with IL-6 to increase hepcidin levels before incubation with malaria sporozoites and observing the effect the hepcidin knockout function has on the infection. Confirmation of our hypothesis could help to understand the complexity of the malaria infection.

CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes

The success of immunization with irradiated sporozoites is unparalleled among the current vaccination approaches against malaria, but its mechanistic underpinnings have yet to be fully elucidated. Using a model mimicking natural infection by Plasmodium yoelii, we delineated early events governing the development of protective CD8(+) T-cell responses to the circumsporozoite protein. We demonstrate that dendritic cells in cutaneous lymph nodes prime the first cohort of CD8(+) T cells after an infectious mosquito bite. Ablation of these lymphoid sites greatly impairs subsequent development of protective immunity. Activated CD8(+) T cells then travel to systemic sites, including the liver, in a sphingosine-1-phosphate (S1P)-dependent fashion. These effector cells, however, no longer require bone marrow-derived antigen-presenting cells for protection; instead, they recognize antigen on parenchymal cells-presumably parasitized hepatocytes. Therefore, we report an unexpected dichotomy in the tissue restriction of host responses during the development and execution of protective immunity to Plasmodium.

Dissecting in vitro host cell infection by Plasmodium sporozoites using flow cytometry

The study of the liver stage of malaria has been hampered by limitations in the experimental approaches required to effectively dissect and quantify hepatocyte infection by Plasmodium. Here, we report on the use of flow cytometry, in conjunction with GFP-expressing Plasmodium sporozoites, to assess the various steps that constitute a successful malaria liver infection: cell traversal, hepatocyte invasion and intrahepatocyte parasite development. We show that this rapid, efficient and inexpensive method can be used to overcome current limitations in the independent quantification of those steps, facilitating routine or large-scale studies of host-pathogen molecular interactions.

Plasmodium berghei-Infected Primary Hepatocytes Process and Present the Circumsporozoite Protein to Specific CD8+ T Cells In Vitro

A substantial and protective response against malaria liver stages is directed against the circumsporozoite protein (CSP) and involves induction of CD8(+) T cells and production of IFN-gamma. CSP-derived peptides have been shown to be presented on the surface of infected hepatocytes in the context of MHC class I molecules. However, little is known about how the CSP and other sporozoite Ags are processed and presented to CD8(+) T cells. We investigated how primary hepatocytes from BALB/c mice process the CSP of Plasmodium berghei after live sporozoite infection and present CSP-derived peptides to specific H-2K(d)-restricted CD8(+) T cells in vitro. Using both wild-type and spect(-/-) P. berghei sporozoites, we show that both infected and traversed primary hepatocytes process and present the CSP. The processing and presentation pathway was found to involve the proteasome, Ag transport through a postendoplasmic reticulum compartment, and aspartic proteases. Thus, it can be hypothesized that infected hepatocytes can contribute in vivo to the elicitation and expansion of a T cell response.

L-FABP is a critical host factor for successful malaria liver stage development

The malaria parasite liver stage produces tens of thousands of red cell-infectious forms within its host hepatocyte. It is thought that the vacuole-enclosed parasite completely depends on the host cell for successful development but the molecular parasite–host cell interactions underlying this remarkable growth have remained elusive. Using a yeast two-hybrid screen and a yeast overexpression system we show that UIS3, a parasite protein essential for liver stage development, interacts directly with liver-fatty acid binding protein, L-FABP. Down-regulation of L-FABP expression in hepatocytes severely impairs parasite growth and overexpression of L-FABP promotes growth. This is the first identified direct liver stage-host cell protein interaction, providing a possible explanation for the importance of UIS3 in liver infection.

A Plant-Derived Morphinan as a Novel Lead Compound Active against Malaria Liver Stages

BackgroundThe global spread of multidrug–resistant malaria parasites has led to an urgent need for new chemotherapeutic agents. Drug discovery is primarily directed to the asexual blood stages, and few drugs that are effective against the obligatory liver stages, from which the pathogenic blood infection is initiated, have become available since primaquine was deployed in the 1950s.Methods and FindingsUsing bioassay-guided fractionation based on the parasite's hepatic stage, we have isolated a novel morphinan alkaloid, tazopsine, from a plant traditionally used against malaria in Madagascar. This compound and readily obtained semisynthetic derivatives were tested for inhibitory activity against liver stage development in vitro (P. falciparum and P. yoelii) and in vivo (P. yoelii). Tazopsine fully inhibited the development of P. yoelii (50% inhibitory concentration [IC50] 3.1 μM, therapeutic index [TI] 14) and P. falciparum (IC50 4.2 μM, TI 7) hepatic parasites in cultured primary hepatocytes, with inhibition being most pronounced during the early developmental stages. One derivative, N-cyclopentyl-tazopsine (NCP-tazopsine), with similar inhibitory activity was selected for its lower toxicity (IC50 3.3 μM, TI 46, and IC50 42.4 μM, TI 60, on P. yoelii and P. falciparum hepatic stages in vitro, respectively). Oral administration of NCP-tazopsine completely protected mice from a sporozoite challenge. Unlike the parent molecule, the derivative was uniquely active against Plasmodium hepatic stages.ConclusionsA readily obtained semisynthetic derivative of a plant-derived compound, tazopsine, has been shown to be specifically active against the liver stage, but inactive against the blood forms of the malaria parasite. This unique specificity in an antimalarial drug severely restricts the pressure for the selection of drug resistance to a parasite stage limited both in numbers and duration, thus allowing researchers to envisage the incorporation of a true causal prophylactic in malaria control programs.

1,3,5‐Triazepan‐2,6‐diones as Structurally Diverse and Conformationally Constrained Dipeptide Mimetics: Identification of Malaria Liver Stage Inhibitors from a Small Pilot Library

The development of the 1,3,5-triazepane-2,6-dione system as a novel, conformationally restricted, and readily accessible class of dipeptidomimetics is reported. The synthesis of the densely functionalized 1,3,5-triazepane-2,6-dione skeleton was achieved in only four steps from a variety of simple linear dipeptide precursors. To extend the practical value of 1,3,5-triazepane-2,6-diones, a general polymer-assisted solution-phase synthesis approach amenable to library production in a multiparallel format was developed. The conformational preferences of the 1,3,5-triazepane-2,6-dione skeleton were investigated in detail by NMR spectroscopy and X-ray diffraction. The ring exhibits a characteristic folded conformation which was compared to that of related dipeptide-derived scaffolds including the more planar 2,5-diketopiperazine (DKP). Molecular and structural diversity was increased further through post-cyclization appending operations at urea nitrogens. Preliminary biological screens of a small collection of 1,3,5-triazepane-2,6-diones revealed inhibitors of the underexplored malaria liver stage and suggest strong potential for this dipeptide-derived scaffold to interfere with and to modulate biological pathways.

Infection by and protective immune responses against Plasmodium berghei ANKA are not affected in macrophage scavenger receptors A deficient mice

BackgroundScavenger receptors (SRs) recognize endogenous molecules modified by pathological processes as well as components of diverse microorganisms. Mice deficient for both SR-AI and II are more susceptible to infections by a variety of bacterial and viral pathogens.ResultsHere we show that SR-A deficient mice and wild type mice are equally susceptible to malaria infection both during liver and blood stages. Moreover, like wild type mice, SR-A deficient mice are able to mount a protective immune response against radiation attenuated sporozoites.ConclusionOur results do not reveal a function of SR-A I and II receptors in the Plasmodium berghei ANKA infection, both in the development of CM and parasitemia control. Moreover, these receptors appear not to be required for the establishment of a protective immune response against the malaria liver stages.

The epidemiology of Plasmodium falciparum gametocytes: weapons of mass dispersion

Much of the epidemiology of Plasmodium falciparum in Sub-Saharan Africa focuses on the prevalence patterns of asexual parasites in people of different ages, whereas the gametocytes that propagate the disease are often neglected. One expected benefit of the widespread introduction of artemisinin-based combination therapy for malaria is a reduction in gametocyte carriage. However, the factors that affect the transmission of parasites from humans to mosquitoes show complex dynamics in relation to the intensity and seasonality of malaria transmission, and thus such benefits might not be automatic. Here, we review data on gametocyte carriage in the context of the development of naturally acquired immunity and population infectivity.

ESTABLISHMENT OF A HUMAN HEPATOCYTE LINE THAT SUPPORTS IN VITRO DEVELOPMENT OF THE EXO-ERYTHROCYTIC STAGES OF THE MALARIA PARASITES PLASMODIUM FALCIPARUM AND P. VIVAX

Our understanding of the biology of malaria parasite liver stages is limited because of the lack of efficient in vitro systems that support the exo-erythrocytic (EE) development of the parasite. We report the development of a new hepatocyte line (HC-04) from normal human liver cells. The HC-04 cells have proliferated in hormone-free medium for more than 200 passages. The cells were hyperdiploid, resembled liver parenchymal cells, and synthesized major liver-specific proteins and enzymes. Using Plasmodium falciparum and P. vivax sporozoites harvested from salivary glands of infected mosquitoes, we showed that HC-04 cells supported the complete EE development of these two most prevalent human malaria parasites. The EE parasites attained full maturation as shown by their infectivity to human erythrocytes. The infection rates of the liver cells were estimated to be 0.066% and 0.041% for P. falciparum and P. vivax, respectively. As the first human hepatocyte line known to support complete EE development of both P. falciparum and P. vivax, HC-04 will provide an experimental model that can be used for studying the biology of liver stage malaria parasites.

Priming of CD8+ T cell responses following immunization with heat‐killed Plasmodium sporozoites

Protective immune responses against malaria are induced by immunization with radiation-attenuated Plasmodium sporozoites. In contrast, non-viable, heat-killed sporozoites do not induce protection, emphasizing the requirement for live parasites to achieve effective immune responses. Using an experimental system with CD8+ T cells from T cell receptor-transgenic mice, we analyzed the primary CD8+ T cell responses elicited by heat-killed inactivated sporozoites. We found that the numbers of specific CD8+ T cells induced were much lower compared to when immunizing with attenuated sporozoites; however, the kinetics of activation and the phenotype of these T cells were similar in both groups. Despite their low frequency after priming, high numbers of specific CD8+ T cells were observed after boosting with a recombinant vaccinia virus. Upon induction of the recall response, the same level of protection was observed when either heat-killed or attenuated sporozoites were used for priming. We propose that live parasites are not critical for the induction of memory T cell populations against the malaria liver stages.

Enhanced T cell-mediated protection against malaria in human challenges by using the recombinant poxviruses FP9 and modified vaccinia virus Ankara

Malaria is a major global health problem for which an effective vaccine is required urgently. Prime-boost vaccination regimes involving plasmid DNA and recombinant modified vaccinia virus Ankara-encoding liver-stage malaria antigens have been shown to be powerfully immunogenic for T cells and capable of inducing partial protection against experimental malaria challenge in humans, manifested as a delay in time to patent parasitemia. Here, we report that substitution of plasmid DNA as the priming vector with a specific attenuated recombinant fowlpox virus, FP9, vaccine in such prime-boost regimes can elicit complete sterile protection that can last for 20 months. Protection at 20 months was associated with persisting memory but not effector T cell responses. The protective efficacy of various immunization regimes correlated with the magnitude of induced immune responses, supporting the strategy of maximizing durable T cell immunogenicity to develop more effective liver-stage vaccines against Plasmodium falciparum malaria.

Invariant Vα14 Chain NKT Cells PromotePlasmodium bergheiCircumsporozoite Protein-Specific Gamma Interferon- and Tumor Necrosis Factor Alpha-Producing CD8+T Cells in the Liver after Poxvirus Vaccination of Mice

Understanding the protective mechanism in the liver induced by recombinant vaccines against the pre-erythrocytic stages of malaria is important for vaccine development. Most studies in mice have focused on splenic and peripheral blood T cells and identified gamma interferon (IFN-gamma)-producing CD8+ T cells as correlates of protection, which can be induced by prime-boost vaccination with recombinant poxviruses. Invariant natural killer T (Valpha14iNKT) cells can also protect against liver stage malaria, when activated, and are abundant in the liver. Since poxviruses have nonspecific immunomodulating effects, which are incompletely understood, we investigated whether recombinant poxviruses affect the protective properties of hepatic Valpha14iNKT cells and thus vaccine efficacy. We show that intradermal vaccination with recombinant poxviruses activated Valpha14iNKT cells and NK cells in the livers of BALB/c mice while inducing IFN-gamma- and tumor necrosis factor alpha (TNF-alpha)-producing pre-erythrocytic stage antigen-specific CD8+ T cells. Greater numbers of hepatic Valpha14iNKT cells secreted interleukin-4 than IFN-gamma. Vaccinated Valpha14iNKT-cell-deficient mice had lower, but still protective levels of hepatic and splenic IFN-gamma+ and TNF-alpha+ CD8+ T cells and better protection rates later after challenge with Plasmodium berghei sporozoites. Therefore, vaccine-activated hepatic Valpha14iNKT cells help in generating specific T cells but are not required for protection induced by recombinant poxviruses. Furthermore, double-positive INF-gamma+/TNF-alpha+ CD8+ T cells were enriched in protected livers, suggesting that cells expressing both of these cytokines may be most relevant for protection.

Effector and memory CD8+ T cells as seen in immunity to malaria.

Transgenic (Tg) mice carrying a T-cell receptor (TCR) specific for a CD8(+) T-cell epitope expressed in pre-erythrocytic stages of Plasmodium yoelii has proven to be a valuable tool to advance our understanding of this anti-parasite T-cell response, as it occurs in vivo. The visualization of CD8(+) T cells in vivo and ex vivo greatly facilitated research aimed at characterizing basic features of this T-cell response such as the kinetics of differentiation and proliferation and the in vivo antigen presentation. Importantly, this research unveiled the existence of early self-regulatory mechanisms controlling the magnitude of the CD8(+) T-cell response and also identified CD4(+) T cells as critical elements in the development of memory populations. This review discusses our recent research using Tg mice and highlights our progress in understanding the CD8(+) T-cell-mediated immunity against malaria liver stages.

Dendritic cells induce immunity and long-lasting protection against blood-stage malaria despite an in vitro parasite-induced maturation defect.

Dendritic cells (DC) suffer a maturation defect following interaction with erythrocytes infected with malaria parasites and become unable to induce protective malaria liver-stage immunity. Here we show that, by contrast, maturation-arrested DC in vitro are capable of the successful induction of antigen-specific gamma interferon (IFN-gamma) and interleukin 4 (IL-4) T-cell responses, antibody responses, and potent protection against lethal blood-stage malaria challenge in vivo. Similar results were found with DC pulsed with intact parasitized Plasmodium yoelii or Plasmodium chabaudi erythrocytes. Cross-strain protection was also induced. High levels of protection (80 to 100%) against lethal challenge were evident from 10 days after a single immunization and maintained up to 120 days. Interestingly, correlation studies versus blood-stage protection at different time points suggest that the immune effector mechanisms associated with protection could change over time. Antibody-independent, T-cell- and IL-12-associated protection was observed early after immunization, followed by antibody and IL-4-associated, IFN-gamma-independent protection in long-term studies. These results indicate that DC, even when clearly susceptible to parasite-induced maturation defect effects in vitro, can be central to the induction of protection against blood-stage malaria in vivo.

Liver stage antigen and malaria

Liver stage antigen and malaria

Sneaking in through the back entrance: the biology of malaria liver stages.

Malaria infection is caused by sporozoites, the life cycle stage of Plasmodium that is transmitted by female anopheline mosquitoes. The inoculated sporozoites migrate in the skin, enter a capillary and use the bloodstream for the long haul to the liver. Here, the parasites invade hepatocytes and differentiate to thousands of merozoites that specifically infect red blood cells. Hepatocytes, however, are not directly accessible to sporozoites entering the liver sinusoid. The liver phase of the malaria life cycle can occur only if the parasites first cross the layer of sinusoidal cells that line the liver capillaries. Experimental observations show that sporozoite entry into the liver parenchyma involves a complex cascade of events, from binding to extracellular matrix proteoglycans via passage through Kupffer cells and transmigration through several hepatocytes, until the final host cell is found. By choosing the liver as their initial site of replication, Plasmodium sporozoites can exploit the tolerogenic properties of this unique immune organ to evade the host's immune response.

Progress in DNA-based heterologous prime-boost immunization strategies for malaria.

An effective vaccine against malaria is urgently required to relieve the immense human suffering and mortality caused by this parasite. A successful subunit vaccine against the liver stage of malaria will require the induction of high levels of protective T cells. Despite success in small animal models, DNA vaccines fail to induce strong cellular immune responses in humans. However, DNA vaccines can induce a T-cell response that can be strongly boosted by recombinant viral vectors. We have evaluated this heterologous prime-boost approach using the Plasmodium berghei mouse model for immunogenicity and protective efficacy against malaria challenge using combinations of plasmid DNA, recombinant modified vaccinia virus Ankara, fowlpox virus, and non-replicating adenovirus. We have proceeded to test immunogenicity and efficacy of successful heterologous prime-boost vaccines in phase I/IIa trials in malaria naïve subjects in the UK and in semi-immune individuals in The Gambia. In these clinical trials, remarkably high levels of effector T-cell responses have been induced and significant protection documented in a human sporozoite challenge model. We summarize the preclinical design and development of these heterologous prime-boost vaccines and discuss the encouraging results that have been observed in vaccinated humans.