Liver-stage Infection Research Articles

TREM2 governs Kupffer cell activation and explains belr1 genetic resistance to malaria liver stage infection

Plasmodium liver stage infection is a target of interest for the treatment of and vaccination against malaria. Here we used forward genetics to search for mechanisms underlying natural host resistance to infection and identified triggering receptor expressed on myeloid cells 2 (TREM2) and MHC class II molecules as determinants of Plasmodium berghei liver stage infection in mice. Locus belr1 confers resistance to malaria liver stage infection. The use of newly derived subcongenic mouse lines allowed to map belr1 to a 4-Mb interval on mouse chromosome 17 that contains the Trem2 gene. We show that Trem2 expression in the nonparenchymal liver cells closely correlates with resistance to liver stage infection, implicating TREM2 as a mediator of the belr1 genetic effect. Trem2-deficient mice are more susceptible to liver stage infection than their WT counterparts. We found that Kupffer cells are the principle cells expressing TREM2 in the liver, and that Trem2(-/-) Kupffer cells display altered functional activation on exposure to P. berghei sporozoites. TREM2 expression in Kupffer cells contributes to the limitation of parasite expansion in isolated hepatocytes in vitro, potentially explaining the increased susceptibility of Trem2(-/-) mice to liver stage infection. The MHC locus was also found to control liver parasite burden, possibly owing to the expression of MHC class II molecules in hepatocytes. Our findings implicate unexpected Kupffer-hepatocyte cross-talk in the control Plasmodium liver stage infection and demonstrate that TREM2 is involved in host responses against the malaria parasite.

Density-Dependent Blood Stage Plasmodium falciparum Suppresses Malaria Super-Infection in a Malaria Holoendemic Population

Recent studies of Plasmodium berghei malaria in mice show that high blood-stage parasitemia levels inhibit the development of subsequent liver-stage infections. Whether a similar inhibitory effect on liver-stage Plasmodium falciparum by blood-stage infection occurs in humans is unknown. We have analyzed data from a treatment-time-to-infection cohort of children < 10 years of age residing in a malaria holoendemic area of Kenya where people experience a new blood-stage infection approximately every 2 weeks. We hypothesized that if high parasitemia blocked the liver stage, then high levels of parasitemia should be followed by a "skipped" peak of parasitemia. Statistical analysis of "natural infection" field data and stochastic simulation of infection dynamics show that the data are consistent with high P. falciparum parasitemia inhibiting liver-stage parasite development in humans.

CD8+T Cells Specific for a Malaria Cytoplasmic Antigen Form Clusters around Infected Hepatocytes and Are Protective at the Liver Stage of Infection

Following Anopheles mosquito-mediated introduction into a human host, Plasmodium parasites infect hepatocytes and undergo intensive replication. Accumulating evidence indicates that CD8(+) T cells induced by immunization with attenuated Plasmodium sporozoites can confer sterile immunity at the liver stage of infection; however, the mechanisms underlying this protection are not clearly understood. To address this, we generated recombinant Plasmodium berghei ANKA expressing a fusion protein of an ovalbumin epitope and green fluorescent protein in the cytoplasm of the parasite. We have shown that the ovalbumin epitope is presented by infected liver cells in a manner dependent on a transporter associated with antigen processing and becomes a target of specific CD8(+) T cells from the T cell receptor transgenic mouse line OT-I, leading to protection at the liver stage of Plasmodium infection. We visualized the interaction between OT-I cells and infected hepatocytes by intravital imaging using two-photon microscopy. OT-I cells formed clusters around infected hepatocytes, leading to the elimination of the intrahepatic parasites and subsequent formation of large clusters of OT-I cells in the liver. Gamma interferon expressed in CD8(+) T cells was dispensable for this protective response. Additionally, we found that polyclonal ovalbumin-specific memory CD8(+) T cells induced by de novo immunization were able to confer sterile protection, although the threshold frequency of the protection was relatively high. These studies revealed a novel mechanism of specific CD8(+) T cell-mediated protective immunity and demonstrated that proteins expressed in the cytoplasm of Plasmodium parasites can become targets of specific CD8(+) T cells during liver-stage infection.

Identification of Targets of CD8+ T Cell Responses to Malaria Liver Stages by Genome-wide Epitope Profiling

CD8+ T cells mediate immunity against Plasmodium liver stages. However, the paucity of parasite-specific epitopes of CD8+ T cells has limited our current understanding of the mechanisms influencing the generation, maintenance and efficiency of these responses. To identify antigenic epitopes in a stringent murine malaria immunisation model, we performed a systematic profiling of H2b-restricted peptides predicted from genome-wide analysis. We describe the identification of Plasmodium berghei (Pb) sporozoite-specific gene 20 (S20)- and thrombospondin-related adhesive protein (TRAP)-derived peptides, termed PbS20318 and PbTRAP130 respectively, as targets of CD8+ T cells from C57BL/6 mice vaccinated by whole parasite strategies known to protect against sporozoite challenge. While both PbS20318 and PbTRAP130 elicit effector and effector memory phenotypes in both the spleens and livers of immunised mice, only PbTRAP130-specific CD8+ T cells exhibit in vivo cytotoxicity. Moreover, PbTRAP130-specific, but not PbS20318-specific, CD8+ T cells significantly contribute to inhibition of parasite development. Prime/boost vaccination with PbTRAP demonstrates CD8+ T cell-dependent efficacy against sporozoite challenge. We conclude that PbTRAP is an immunodominant antigen during liver-stage infection. Together, our results underscore the presence of CD8+ T cells with divergent potencies against distinct Plasmodium liver-stage epitopes. Our identification of antigen-specific CD8+ T cells will allow interrogation of the development of immune responses against malaria liver stages.

Dual role of type I IFN during plasmodium infection (P3056)

Abstract The molecular mechanisms regulating the inflammatory response during malaria are still poorly defined. Inflammatory cytokines and type I IFNs induced when innate immune sensors recognize components of the malaria parasite can contribute to clearance of the parasite and in some circumstances these same effectors lead to experimental cerebral malaria (ECM). Infection of C57BL/6 mice with Plasmodium berghei ANKA (PbA) leads to ECM where animals succumb to infection and die. Upon infection with PbA infected red blood cells (iRBCs), C57/Bl6 mice succumb to death 6-12 days post-infection. Recently, we have found that mice lacking the type I IFN receptor, IFNAR-/-, are protected from ECM-mediated death, implicating an important role of type I IFNs in exacerbating the ECM phenotype. Alternatively, when mice are infected with liver-tropic PbA sporozoites, a type I IFN response is induced while the parasites develop inside hepatocytes. This host response is responsible for upregulating interferon stimulated genes (ISGs) and limit the parasite load in the liver. The expression of ISGs are abrogated in IFNAR-/- mice. This protective phenotype is dependent on IRF3/7 and the adaptor MAVS suggesting that parasite RNA is recognized by host cells. Collectively, these findings reveal a dual role of type I IFNs that contribute to ECM-mediated death during PbA blood stage infections, but are also responsible for reducing parasite load during a liver stage infection.

The impact of IL-4 on the CD8 T cell response to infection (P4483)

Abstract IL-4 influences the development of memory-phenotype, IFNγ-producing, CD8 T cells and plays a critical role in the generation of antigen-specific memory cells during liver-stage malaria infection. Elevated IL-4 levels, which can be efficiently created by treatment with IL-4 cytokine-antibody complexes (IL-4C), enhances the number of CD8 memory T cells displaying high expression of CXCR3 and Eomes, but low levels of T-bet. This is in contrast to treatment with IL-2 cytokine-antibody complexes (IL-2C) that generate T-bet high, Eomes and CXCR3 low, and granzyme B expressing cells. IL-2C and IL-4C also caused differential responses to an acute pathogen. Whereas IL-2C treatment of naïve C57BL/6 mice produced a significant improvement in clearance of Listeria monocytogenes(LM) infection, IL-4C failed to have an effect, despite a large expansion of the CD8 memory pool. Interestingly, BALB/c mice, which are known as a “Type-2” or naturally IL-4 high mouse strain, demonstrated better clearance of LM after IL-4C treatment. This suggests that depending on the mouse strain, there may be intrinsically different CD8 T cell signals generated in response to γc cytokines, with global functional consequences for the host. Future studies will explore both the functional significance of IL-4 at different stages of the CD8 T cell response to LM and malaria and ask whether IL-4C treatment is able to augment immunity.

The role of antibody and CD4 follicular helper cells during malaria liver stage infection (P4485)

Abstract The liver stage malaria provides an excellent target for malaria vaccine: eliminating the parasite at this stage using immunization with attenuated parasites that arrest in the liver results in long-term sterile protection. This immune response has been associated with CD8 T cells, as well as antibodiesdirected against the sporozoite. In this study, we hypothesized that genetically attenuated pasite (GAP) immunization induces T follicular helper cells (Tfh), which in turn induce memory B cells responses, leading to long-lived and high avidity functional antibodies to pre-erythrocytic malaria. Using immunization with GAP that arrest during late liver-stage development, we showed that immunization induces high titer antibodies, and that these antibodies can inhibit parasite growth in liver cells in vitro by 99%. To understand the induction of the antibody response, we characterized both the B cell response and the Tfh response in the liver, the spleen and the lymph nodes. Furthermore, in order to determine the localization of the priming of the response, immunization via mosquito bite and intravenous injection were compared. Understanding the role of Tfh in liver stage malaria and how they are induced will be crucial in designing an effective antibody based malaria vaccine.

Suppression of Host p53 Is Critical for Plasmodium Liver-Stage Infection

Plasmodium parasites infect the liver and replicate inside hepatocytes before they invade erythrocytes and trigger clinical malaria. Analysis of host signaling pathways affected by liver-stage infection could provide critical insights into host-pathogen interactions and reveal targets for intervention. Using protein lysate microarrays, we found that Plasmodium yoelii rodent malaria parasites perturb hepatocyte regulatory pathways involved in cell survival, proliferation, and autophagy. Notably, the prodeath protein p53 was substantially decreased in infected hepatocytes, suggesting that it could be targeted by the parasite to foster survival. Indeed, mice that express increased levels of p53 showed reduced liver-stage parasite burden, whereas p53 knockout mice suffered increased liver-stage burden. Furthermore, boosting p53 levels with the use of the small molecule Nutlin-3 dramatically reduced liver-stage burden in vitro and in vivo. We conclude that perturbation of the hepatocyte p53 pathway critically impacts parasite survival. Thus, host pathways might constitute potential targets for host-based antimalarial prophylaxis.

Plasmodium falciparum synthetic LbL microparticle vaccine elicits protective neutralizing antibody and parasite-specific cellular immune responses

Epitopes of the circumsporozoite (CS) protein of Plasmodium falciparum, the most pathogenic species of the malaria parasite, have been shown to elicit protective immunity in experimental animals and human volunteers. The mechanisms of immunity include parasite-neutralizing antibodies that can inhibit parasite motility in the skin at the site of infection and in the bloodstream during transit to the hepatocyte host cell and also block interaction with host cell receptors on hepatocytes. In addition, specific CD4+ and CD8+ cellular mechanisms target the intracellular hepatic forms, thus preventing release of erythrocytic stage parasites from the infected hepatocyte and the ensuing blood stage cycle responsible for clinical disease. An innovative method for producing particle vaccines, layer-by-layer (LbL) fabrication of polypeptide films on solid CaCO3 cores, was used to produce synthetic malaria vaccines containing a tri-epitope CS peptide T1BT* comprising the antibody epitope of the CS repeat region (B) and two T-cell epitopes, the highly conserved T1 epitope and the universal epitope T*. Mice immunized with microparticles loaded with T1BT* peptide developed parasite-neutralizing antibodies and malaria-specific T-cell responses including cytotoxic effector T-cells. Protection from liver stage infection following challenge with live sporozoites from infected mosquitoes correlated with neutralizing antibody levels. Although some immunized mice with low or undetectable neutralizing antibodies were also protected, depletion of T-cells prior to challenge resulted in the majority of mice remaining resistant to challenge. In addition, mice immunized with microparticles bearing only T-cell epitopes were not protected, demonstrating that cellular immunity alone was not sufficient for protective immunity. Although the microparticles without adjuvant were immunogenic and protective, a simple modification with the lipopeptide TLR2 agonist Pam3Cys increased the potency and efficacy of the LbL vaccine candidate. This study demonstrates the potential of LbL particles as promising malaria vaccine candidates using the T1BT* epitopes from the P. falciparum CS protein.

Elimination challenges in the Pacific: vivax and submicroscopic parasitemia

Major progress has been made in reducing the burden of malaria in the Solomon Islands and Vanuatu. This has been achieved by improving health systems, increasing deployment of ITN, improving diagnostics, and by deployment of more effective antimalarials, including ACT. These advances have highlighted the challenges in achieving elimination of malaria in these countries, and may provide useful lessons for other settings where control activities are less advanced. Specific challenges include the significant prevalence of submicroscopic parasitemia that can only be detected by molecular methods such as PCR. This poses significant challenges to active case detection and to programmatic needs to monitor progress in elimination. As well, the significant prevalence of asymptomatic submicroscopic parasitemia in an epidemiological setting where herd immunity is waning raises questions regarding the nature of anti-disease immunity in such settings. Further, the relative prevalence of P. vivax infection is increasing compared to P. falciparum. A specific challenges this poses is the need to address the issue of latent liver stage infection with hypnozoites in a setting where the prevalence of clinically significant G6PD deficiency exceeds 10%. In this presentation these challenges all be discussed along with research underway to address them.

Humanized mouse model for liver-stage malaria infection

Humanized mouse model for liver-stage malaria infection

Liver-stage malaria parasites vulnerable to diverse chemical scaffolds

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. All high-throughput malaria drug discovery efforts have focused on the cyclic blood stage, which has limited potential for the prophylaxis, transmission blocking, and eradication efforts that will be needed in the future. To address these unmet needs, a high-throughput phenotypic liver-stage Plasmodium parasite screen was developed to systematically identify molecules with liver-stage efficacy. The screen recapitulates liver-stage infection by isolating luciferase-expressing Plasmodium berghei parasites directly from the salivary glands of infected mosquitoes, adding them to confluent human liver cells in 384-well plates, and measuring luciferase activity after a suitable incubation period. Screening 5,375 known bioactive compounds identified 37 liver-stage malaria inhibitors with diverse modes of action, as shown by inhibition time course experiments. Further analysis of the hits in the Food and Drug Administration-approved drug subset revealed compounds that seem to act specifically on the liver stage of infection, suggesting that this phase of the parasite's life cycle presents a promising area for new drug discovery. Notably, many active compounds in this screen have molecular structures and putative targets distinctly different from those of known antimalarial agents.

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.

The crucial role of hepatocyte growth factor receptor during liver-stage infection is not conserved among Plasmodium species

The crucial role of hepatocyte growth factor receptor during liver-stage infection is not conserved among Plasmodium species

The crucial role of hepatocyte growth factor receptor during liver-stage infection is not conserved among Plasmodium species

The crucial role of hepatocyte growth factor receptor during liver-stage infection is not conserved among Plasmodium species

Combining Liver- and Blood-Stage Malaria Viral-Vectored Vaccines: Investigating Mechanisms of CD8+ T Cell Interference

Replication-deficient adenovirus and modified vaccinia virus Ankara (MVA) vectors expressing single pre-erythrocytic or blood-stage Plasmodium falciparum Ags have entered clinical testing using a heterologous prime-boost immunization approach. In this study, we investigated the utility of the same immunization regimen when combining viral vectored vaccines expressing the 42-kDa C terminus of the blood-stage Ag merozoite surface protein 1 and the pre-erythrocytic Ag circumsporozoite protein in the Plasmodium yoelii mouse model. We find that vaccine coadministration leads to maintained Ab responses and efficacy against blood-stage infection, but reduced secondary CD8(+) T cell responses against both Ags and efficacy against liver-stage infection. CD8(+) T cell interference can be minimized by coadministering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy in mice immunized against both Ags compared with just one. CD8(+) T cell interference (following MVA coadministration as a mixture) may be caused partly by a lack of physiologic space for high-magnitude responses against multiple Ags, but is not caused by competition for presentation of Ag on MHC class I molecules, nor is it due to restricted T cell access to APCs presenting both Ags. Instead, enhanced killing of peptide-pulsed cells is observed in mice possessing pre-existing T cells against two Ags compared with just one, suggesting that priming against multiple Ags may in part reduce the potency of multiantigen MVA vectors to stimulate secondary CD8(+) T cell responses. These data have important implications for the development of a multistage or multicomponent viral vectored malaria vaccine for use in humans.

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.

Host-mediated regulation of superinfection in malaria

In regions of high rates of malaria transmission, mosquitoes repeatedly transmit liver-tropic Plasmodium sporozoites to individuals who already have blood-stage parasitemia. This manifests itself in semi-immune children (who have been exposed since birth to Plasmodium infection and as such show low levels of peripheral parasitemia but can still be infected) older than 5 years of age by concurrent carriage of different parasite genotypes at low asymptomatic parasitemias. Superinfection presents an increased risk of hyperparasitemia and death in less immune individuals but counterintuitively is not frequently observed in the young. Here we show in a mouse model that ongoing blood-stage infections, above a minimum threshold, impair the growth of subsequently inoculated sporozoites such that they become growth arrested in liver hepatocytes and fail to develop into blood-stage parasites. Inhibition of the liver-stage infection is mediated by the host iron regulatory hormone hepcidin, whose synthesis we found to be stimulated by blood-stage parasites in a density-dependent manner. We mathematically modeled this phenomenon and show how density-dependent protection against liver-stage malaria can shape the epidemiological patterns of age-related risk and the complexity of malaria infections seen in young children. The interaction between these two Plasmodium stages and host iron metabolism has relevance for the global efforts to reduce malaria transmission and for evaluation of iron supplementation programs in malaria-endemic regions.

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.

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.