Plasmodium Liver-stage Infection Research Articles

Development of an ectopic huLiver model for Plasmodium liver stage infection.

Early Plasmodium falciparum and P. vivax infection requires parasite replication within host hepatocytes, referred to as liver stage (LS). However, limited understanding of infection dynamics in human LS exists due to species-specificity challenges. Reported here is a reproducible, easy-to-manipulate, and moderate-cost in vivo model to study human Plasmodium LS in mice; the ectopic huLiver model. Ectopic huLiver tumors were generated through subcutaneous injection of the HC-04 cell line and shown to be infectible by both freshly dissected sporozoites and through the bite of infected mosquitoes. Evidence for complete LS development was supported by the transition to blood-stage infection in mice engrafted with human erythrocytes. Additionally, this model was successfully evaluated for its utility in testing antimalarial therapeutics, as supported by primaquine acting as a causal prophylactic against P. falciparum. Presented here is a new platform for the study of human Plasmodium infection with the potential to aid in drug discovery.

Cut it out! A CRISPR-Cas9 screen identifies host regulators of the Plasmodium liver stage

In this issue of Cell Chemical Biology, Vijayan and colleagues identify host factors integral for Plasmodium liver-stage infection using a whole-genome CRISPR-Cas9 knockout screen. Their efforts reveal that liver-stage parasites redistribute host microtubules to the parasite membrane in a process dependent on the host Golgi.

Elucidating Spatially-Resolved Changes in Host Signaling During Plasmodium Liver-Stage Infection.

Upon transmission to the human host, Plasmodium sporozoites exit the skin, are taken up by the blood stream, and then travel to the liver where they infect and significantly modify a single hepatocyte. Low infection rates within the liver have made proteomic studies of infected hepatocytes challenging, particularly in vivo, and existing studies have been largely unable to consider how protein and phosphoprotein differences are altered at different spatial locations within the heterogeneous liver. Using digital spatial profiling, we characterized changes in host signaling during Plasmodium yoelii infection in vivo without disrupting the liver tissue. Moreover, we measured alterations in protein expression around infected hepatocytes and identified a subset of CD163+ Kupffer cells that migrate towards infected cells during infection. These data offer the first insight into the heterogeneous microenvironment that surrounds the infected hepatocyte and provide insights into how the parasite may alter its milieu to influence its survival and modulate immunity.

Liver stage malaria infection is controlled by host regulators of lipid peroxidation

The facets of host control during Plasmodium liver infection remain largely unknown. We find that the SLC7a11-GPX4 pathway, which has been associated with the production of reactive oxygen species, lipid peroxidation, and a form of cell death called ferroptosis, plays a critical role in control of Plasmodium liver stage infection. Specifically, blocking GPX4 or SLC7a11 dramatically reduces Plasmodium liver stage parasite infection. In contrast, blocking negative regulators of this pathway, NOX1 and TFR1, leads to an increase in liver stage infection. We have shown previously that increased levels of P53 reduces Plasmodium LS burden in an apoptosis-independent manner. Here, we demonstrate that increased P53 is unable to control parasite burden during NOX1 or TFR1 knockdown, or in the presence of ROS scavenging or when lipid peroxidation is blocked. Additionally, SLC7a11 inhibitors Erastin and Sorafenib reduce infection. Thus, blocking the host SLC7a11-GPX4 pathway serves to selectively elevate lipid peroxides in infected cells, which localize within the parasite and lead to the elimination of liver stage parasites.

γδ-T cells promote IFN-γ–dependent Plasmodium pathogenesis upon liver-stage infection

Cerebral malaria (CM) is a major cause of death due to Plasmodium infection. Both parasite and host factors contribute to the onset of CM, but the precise cellular and molecular mechanisms that contribute to its pathogenesis remain poorly characterized. Unlike conventional αβ-T cells, previous studies on murine γδ-T cells failed to identify a nonredundant role for this T cell subset in experimental cerebral malaria (ECM). Here we show that mice lacking γδ-T cells are resistant to ECM when infected with Plasmodium berghei ANKA sporozoites, the liver-infective form of the parasite and the natural route of infection, in contrast with their susceptible phenotype if challenged with P. berghei ANKA-infected red blood cells that bypass the liver stage of infection. Strikingly, the presence of γδ-T cells enhanced the expression of Plasmodium immunogenic factors and exacerbated subsequent systemic and brain-infiltrating inflammatory αβ-T cell responses. These phenomena were dependent on the proinflammatory cytokine IFN-γ, which was required during liver stage for modulation of the parasite transcriptome, as well as for downstream immune-mediated pathology. Our work reveals an unanticipated critical role of γδ-T cells in the development of ECM upon Plasmodium liver-stage infection.

OC 8415 A TRANSLATIONAL PRECLINICAL PLATFORM TO ASSESS THE CHEMOPROPHYLAXIS AND CHEMOPREVENTION DOSE-RELATIONSHIP OF MALARIA DRUGS: THE CASE STUDY OF M5717

Major progress has been made in the control of malaria leading to significantly reduce the number of cases and deaths. However, to reach the elimination stage, new tools will be needed, including combination of drugs capable of blocking the spread of malaria through chemoprophylaxis.M5717 is a first-in-class compound that targets the Plasmodium Eukaryotic translation Elongation Factor 2, essential for protein synthesis. M5717 is highly potent against all developing stages of Plasmodium parasites and has a long half-life suggesting that a single dose development may be possible for cure, prophylaxis and transmission blocking activities. In vivo preclinical PK/PD data indicates an increased exposure in the portal vein compared to peripheral circulation translating into a prophylactic activity at a lower dose than the curative one. M5717 is currently completing first-in-man studies with the objective of initiating clinical prophylactic development in 2019. Additional data to model the human prophylactic dose will be needed prior to initiate the studies to demonstrate clinical efficacy (phase 2).We recently established a human cell-based platform for drug screening against Plasmodium liver-stage infection relying on human hepatic 3D cultures in bioreactors supporting rodent P. berghei infection with similar rates of infection and parasite development compared to existing models. The platform was validated by assessing the activity of currently used antimalarial drugs.Here we report the data of a dose response experiment to establish the liver stage efficacious concentration of M5717 compared to atovaquone in Plasmodium-infected hepatic 3D cultures. The data obtained with this new model were confirmed using the in vivo model of P. berghei sporozoite-induced infection in mice, demonstrating the validity of the Plasmodium-infected hepatic 3D cultures as an enabling technology for malaria drug development. These data are also providing additional evidence that M5717 should be developed as a chemoprophylactic agent for the prevention of malaria.

Plasmodium parasite exploits host aquaporin-3 during liver stage malaria infection.

Within the liver a single Plasmodium parasite transforms into thousands of blood-infective forms to cause malaria. Here, we use RNA-sequencing to identify host genes that are upregulated upon Plasmodium berghei infection of hepatocytes with the hypothesis that host pathways are hijacked to benefit parasite development. We found that expression of aquaporin-3 (AQP3), a water and glycerol channel, is significantly induced in Plasmodium-infected hepatocytes compared to uninfected cells. This aquaglyceroporin localizes to the parasitophorous vacuole membrane, the compartmental interface between the host and pathogen, with a temporal pattern that correlates with the parasite’s expansion in the liver. Depletion or elimination of host AQP3 expression significantly reduces P. berghei parasite burden during the liver stage and chemical disruption by a known AQP3 inhibitor, auphen, reduces P. falciparum asexual blood stage and P. berghei liver stage parasite load. Further use of this inhibitor as a chemical probe suggests that AQP3-mediated nutrient transport is an important function for parasite development. This study reveals a previously unknown potential route for host-dependent nutrient acquisition by Plasmodium which was discovered by mapping the transcriptional changes that occur in hepatocytes throughout P. berghei infection. The dataset reported may be leveraged to identify additional host factors that are essential for Plasmodium liver stage infection and highlights Plasmodium’s dependence on host factors within hepatocytes.

Infectious Sporozoites of Plasmodium berghei Effectively Activate Liver CD8α+ Dendritic Cells.

Immunization with radiation-attenuated sporozoites (RAS) shown to confer complete sterile protection against Plasmodia liver-stage (LS) infection that lasts about 6 to 9 months in mice. We have found that the intermittent infectious sporozoite challenge to immune mice following RAS vaccination extends the longevity of sterile protection by maintaining CD8+ T cell memory responses to LS infection. It is reported that CD8α+ dendritic cells (DCs) are involved in the induction of LS-specific CD8+ T cells following RAS or genetically attenuated parasite (GAP) vaccination. In this study, we demonstrate that CD8α+ DCs respond differently to infectious sporozoite or RAS inoculation. The higher accumulation and activation of CD8α+ DCs was seen in the liver in response to infectious sporozoite 72 h postinoculation and found to be associated with higher expression of chemokines (CCL-20 and CCL-21) and type I interferon response via toll-like receptor signaling in liver. Moreover, the infectious sporozoites were found to induce qualitative changes in terms of the increased MHCII expression as well as costimulatory molecules including CD40 on the CD8α+ DCs compared to RAS inoculation. We have also found that infectious sporozoite challenge increased CD40L-expressing CD4+ T cells, which could help CD8+ T cells in the liver through “licensing” of the antigen-presenting cells. Our results suggest that infectious sporozoite challenge to prior RAS immunized mice modulates the CD8α+ DCs, which might be shaping the fate of memory CD8+ T cells against Plasmodium LS infection.

Frequent inoculations with radiation attenuated sporozoite is essential for inducing sterile protection that correlates with a threshold level of Plasmodia liver-stage specific CD8+ T cells

Whole sporozoite vaccine (WSV) is shown to induce sterile protection that targets Plasmodium liver-stage infection. There are many underlying issues associated with induction of effective sterile protracted protection. In this study, we have addressed how the alterations in successive vaccine regimen could possibly affect the induction of sterile protection. We have demonstrated that the pattern of vaccination with RAS (radiation attenuated sporozoites) induces varying degrees of protection among B6 mice. Animals receiving four successive doses generated 100% sterile protection. However, three successive doses, though with the same parasite inoculum as four doses, could induce sterile protection in ∼50% mice. Interestingly, mice immunized with the same 3 doses, but with longer gap, could not survive the challenge. We demonstrate that degree of protection correlates with the frequencies of IFN-γ+ and multifunctional (IFN-γ+ CD107a+) CD8+ TEM cells present in liver. The failure to achieve protective threshold frequency of these cells in liver might make the host more vulnerable to parasite infection during infectious sporozoite challenge.

Parasite-induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection.

Upon infection of a mammalian host, Plasmodium parasites first replicate inside hepatocytes, generating thousands of new parasites. Although Plasmodium intra-hepatic development represents a substantial metabolic challenge to the host hepatocyte, how infected cells respond to and integrate this stress remains poorly understood. Here, we present proteomic and transcriptomic analyses, revealing that the endoplasmic reticulum (ER)-resident unfolded protein response (UPR) is activated in host hepatocytes upon Plasmodium berghei infection. The expression of XBP1s--the active form of the UPR mediator XBP1--and the liver-specific UPR mediator CREBH is induced by P. berghei infection in vivo. Furthermore, this UPR induction increases parasite liver burden. Altogether, our data suggest that ER stress is a central feature of P. berghei intra-hepatic development, contributing to the success of infection.

In vitro alterations do not reflect a requirement for host cell cycle progression during Plasmodium liver stage infection.

Prior to invading nonreplicative erythrocytes, Plasmodium parasites undergo their first obligate step in the mammalian host inside hepatocytes, where each sporozoite replicates to generate thousands of merozoites. While normally quiescent, hepatocytes retain proliferative capacity and can readily reenter the cell cycle in response to diverse stimuli. Many intracellular pathogens, including protozoan parasites, manipulate the cell cycle progression of their host cells for their own benefit, but it is not known whether the hepatocyte cell cycle plays a role during Plasmodium liver stage infection. Here, we show that Plasmodium parasites can be observed in mitotic hepatoma cells throughout liver stage development, where they initially reduce the likelihood of mitosis and ultimately lead to significant acquisition of a binucleate phenotype. However, hepatoma cells pharmacologically arrested in S phase still support robust and complete Plasmodium liver stage development, which thus does not require cell cycle progression in the infected cell in vitro. Furthermore, murine hepatocytes remain quiescent throughout in vivo infection with either Plasmodium berghei or Plasmodium yoelii, as do Plasmodium falciparum-infected primary human hepatocytes, demonstrating that the rapid and prodigious growth of liver stage parasites is accomplished independent of host hepatocyte cell cycle progression during natural infection.

CD8 T-cell-mediated protection against liver-stage malaria: lessons from a mouse model.

Malaria is a major global health problem, with severe mortality in children living in sub-Saharan Africa, and there is currently no licensed, effective vaccine. However, vaccine-induced protection from Plasmodium infection, the causative agent of malaria, was established for humans in small clinical trials and for rodents in the 1960s. Soon after, a critical role for memory CD8 T cells in vaccine-induced protection against Plasmodium liver-stage infection was established in rodent models and is assumed to apply to humans. However, these seminal early studies have led to only modest advances over the ensuing years in our understanding the basic features of memory CD8 T cells required for protection against liver-stage Plasmodium infection, an issue which has likely impeded the development of effective vaccines for humans. Given the ethical and practical limitations in gaining mechanistic insight from human vaccine and challenge studies, animal models still have an important role in dissecting the basic parameters underlying memory CD8 T-cell immunity to Plasmodium. Here, we will highlight recent data from our own work in the mouse model of Plasmodium infection that identify quantitative and qualitative features of protective memory CD8 T-cell responses. Finally, these lessons will be discussed in the context of recent findings from clinical trials of vaccine-induced protection in controlled human challenge models.

No Association of the p53 Codon 72 Polymorphism with Malaria in Ghanaian Primiparae and Rwandan Children

The p53 protein is a key cell-signaling mediator integrating host responses to various types of stress. A common polymorphism of the encoding TP53 gene (codon 72, Pro > Arg, rs1042522) is associated with susceptibility to virus-related and other cancers. The p53 has also been shown to be central for successful Plasmodium liver stage infection. We examined whether the polymorphism is associated with P. falciparum infection in Ghanaian primiparae and Rwandan children. The allele frequency of TP53 codon 72 Arg was 0.30 among 314 Ghanaian primiparae and 0.31 among 545 Rwandan children, respectively, and it was not associated with infection prevalence or parasite density. This does not exclude p53 to be of pathophysiological relevance in malaria but argues against a major respective role of the TP53 codon 72 polymorphism.

Malaria Liver Stage Susceptibility Locus Identified on Mouse Chromosome 17 by Congenic Mapping

Host genetic variants are known to confer resistance to Plasmodium blood stage infection and to control malaria severity both in humans and mice. This work describes the genetic mapping of a locus for resistance to liver stage parasite in the mouse. First, we show that decreased susceptibility to the liver stage of Plasmodium berghei in the BALB/c mouse strain is attributable to intra-hepatic factors and impacts on the initial phase of blood stage infection. We used QTL mapping techniques to identify a locus controlling this susceptibility phenotype (LOD score 4.2) on mouse chromosome 17 (belr1 locus). Furthermore, analysis of congenic mouse strains delimited the belr1 locus boundaries distally to the H2 region. Quantification of parasites in the liver of infected congenic mice strongly suggested that the belr1 locus represents a genetic factor controlling the expansion of P. berghei in the hepatic tissue. The mapping of belr1 locus raises the hypothesis that host gene variation is able to control the progression of Plasmodium liver stage infection and opens the possibility that the human genomic region orthologue to belr1 may contain genes that confer resistance to the human malaria liver stage.