Zonal human hepatocytes are differentially permissive to Plasmodium falciparum malaria parasites.

Plasmodium falciparum (Pf) is a major cause of human malaria and is transmitted by infected Anopheles mosquitoes. The initial asymptomatic infection is characterized by parasite invasion of hepatocytes, followed by massive replication generating schizonts with blood-infective merozoites. Hepatocytes can be categorized by their zonal location and metabolic functions within a liver lobule. To understand specific host conditions that affect infectivity, we studied Pf parasite liver stage development in relation to the metabolic heterogeneity of fresh human hepatocytes. We found selective preference of different Pf strains for a minority of hepatocytes, which are characterized by the particular presence of glutamine synthetase (hGS). Schizont growth is significantly enhanced by hGS uptake early in development, showcasing a novel import system. In conclusion, Pf development is strongly determined by the differential metabolic status in hepatocyte subtypes. These findings underscore the importance of detailed understanding of hepatocyte host-Pf interactions and may delineate novel pathways for intervention strategies.

Development of Plasmodium falciparum liver-stages in hepatocytes derived from human fetal liver organoid cultures.

Plasmodium falciparum (Pf) parasite development in liver represents the initial step of the life-cycle in the human host after a Pf-infected mosquito bite. While an attractive stage for life-cycle interruption, understanding of parasite-hepatocyte interaction is inadequate due to limitations of existing in vitro models. We explore the suitability of hepatocyte organoids (HepOrgs) for Pf-development and show that these cells permitted parasite invasion, differentiation and maturation of different Pf strains. Single-cell messenger RNA sequencing (scRNAseq) of Pf-infected HepOrg cells has identified 80 Pf-transcripts upregulated on day 5 post-infection. Transcriptional profile changes are found involving distinct metabolic pathways in hepatocytes with Scavenger Receptor B1 (SR-B1) transcripts highly upregulated. A novel functional involvement in schizont maturation is confirmed in fresh primary hepatocytes. Thus, HepOrgs provide a strong foundation for a versatile in vitro model for Pf liver-stages accommodating basic biological studies and accelerated clinical development of novel tools for malaria control.

Mid-Liver Stage Arrest of Plasmodium falciparum Schizonts in Primary Porcine Hepatocytes.

During co-evolution Plasmodium parasites and vertebrates went through a process of selection resulting in defined and preferred parasite-host combinations. As such, Plasmodium falciparum (Pf) sporozoites can infect human hepatocytes while seemingly incompatible with host cellular machinery of other species. The compatibility between parasite invasion ligands and their respective human hepatocyte receptors plays a key role in Pf host selectivity. However, it is unclear whether the ability of Pf sporozoites to mature in cross-species infection also plays a role in host tropism. Here we used fresh hepatocytes isolated from porcine livers to study permissiveness to Pf sporozoite invasion and development. We monitored intra-hepatic development via immunofluorescence using anti-HSP70, MSP1, EXP1, and EXP2 antibodies. Our data shows that Pf sporozoites can invade non-human hepatocytes and undergo partial maturation with a significant decrease in schizont numbers between day three and day five. A possible explanation is that Pf sporozoites fail to form a parasitophorous vacuolar membrane (PVM) during invasion. Indeed, the observed aberrant EXP1 and EXP2 staining supports the presence of an atypical PVM. Functions of the PVM include the transport of nutrients, export of waste, and offering a protective barrier against intracellular host effectors. Therefore, an atypical PVM likely results in deficiencies that may detrimentally impact parasite development at multiple levels. In summary, despite successful invasion of porcine hepatocytes, Pf development arrests at mid-stage, possibly due to an inability to mobilize critical nutrients across the PVM. These findings underscore the potential of a porcine liver model for understanding the importance of host factors required for Pf mid-liver stage development.

Human antibodies against noncircumsporozoite proteins block Plasmodium falciparum parasite development in hepatocytes.

Sporozoite-based approaches currently represent the most effective vaccine strategies for induction of sterile protection against Plasmodium falciparum (Pf) malaria. Clinical development of subunit vaccines is almost exclusively centered on the circum-sporozoite protein (CSP), an abundantly expressed protein on the sporozoite membrane. Anti-CSP antibodies are able to block sporozoite invasion and development in human hepatocytes and subsequently prevent clinical malaria. Here, we have investigated whether sporozoite-induced human antibodies with specificities different from CSP can reduce Pf-liver stage development. IgG preparations were obtained from 12 volunteers inoculated with a protective immunization regime of whole sporozoites under chloroquine prophylaxis. These IgGs were depleted for CSP specificity by affinity chromatography. Recovered non-CSP antibodies were tested for sporozoite membrane binding and for functional inhibition of sporozoite invasion of a human hepatoma cell line and hepatocytes both in vitro and in vivo. Postimmunization IgGs depleted for CS specificity of 9 of 12 donors recognized sporozoite surface antigens. Samples from 5 of 12 donors functionally reduced parasite-liver cell invasion or development using the hepatoma cell line HC-04 and FRG-huHep mice containing human liver cells. The combined data provide clear evidence that non-CSP proteins, as yet undefined, do represent antibody targets for functional immunity against Pf parasites responsible for malaria.

Generation of Novel Plasmodium falciparum NF135 and NF54 Lines Expressing Fluorescent Reporter Proteins Under the Control of Strong and Constitutive Promoters.

Transgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene function. Different Plasmodium falciparum (Pf ) reporter lines exist, however nearly all have been created in the African NF54/3D7 laboratory strain. Here we describe the generation of novel reporter lines, using CRISPR/Cas9 gene modification, both in the standard Pf NF54 background and in a recently described Cambodian P. falciparum NF135.C10 line. Sporozoites of this line show more effective hepatocyte invasion and enhanced liver merozoite development compared to Pf NF54. We first generated Pf NF54 reporter parasites to analyze two novel promoters for constitutive and high expression of mCherry-luciferase and GFP in blood and mosquito stages. The promoter sequences were selected based on available transcriptome data and are derived from two housekeeping genes, i.e., translation initiation factor SUI1, putative (sui1, PF3D7_1243600) and 40S ribosomal protein S30 (40s, PF3D7_0219200). We then generated and characterized reporter lines in the Pf NF135.C10 line which express GFP driven by the sui1 and 40s promoters as well as by the previously used ef1α promoter (GFP@ef1α, GFP@sui1, GFP@40s). The GFP@40s reporter line showed strongest GFP expression in liver stages as compared to the other two lines. The strength of reporter expression by the 40s promoter throughout the complete life cycle, including liver stages, makes transgenic lines expressing reporters by the 40s promoter valuable novel tools for analyses of P. falciparum.

A double-blind, placebo-controlled phase 1/2a trial of the genetically attenuated malaria vaccine PfSPZ-GA1.

Immunization with attenuated Plasmodium sporozoites can induce protection against malaria infection, as shown by Plasmodium falciparum (Pf) sporozoites attenuated by radiation in multiple clinical trials. As alternative attenuation strategy with a more homogeneous population of Pf sporozoites (PfSPZ), genetically engineered Plasmodium berghei sporozoites (SPZ) lacking the genes b9 and slarp induced sterile protection against malaria in mice. Consequently, PfSPZ-GA1 Vaccine, a Pf identical double knockout (Pf∆b9∆slarp), was generated as a genetically attenuated malaria parasite vaccine and tested for safety, immunogenicity, and preliminary efficacy in malaria-naïve Dutch volunteers. Dose-escalation immunizations up to 9.0 × 105 PfSPZ of PfSPZ-GA1 Vaccine were well tolerated without breakthrough blood-stage infection. Subsequently, groups of volunteers were immunized three times by direct venous inoculation with cryopreserved PfSPZ-GA1 Vaccine (9.0 × 105 or 4.5 × 105 PfSPZ, N = 13 each), PfSPZ Vaccine (radiation-attenuated PfSPZ, 4.5 × 105 PfSPZ, N = 13), or normal saline placebo at 8-week intervals, followed by exposure to mosquito bite controlled human malaria infection (CHMI). After CHMI, 3 of 25 volunteers from both PfSPZ-GA1 groups were sterilely protected, and the remaining 17 of 22 showed a patency ≥9 days (median patency in controls, 7 days; range, 7 to 9). All volunteers in the PfSPZ Vaccine control group developed parasitemia (median patency, 9 days; range, 7 to 12). Immunized groups exhibited a significant, dose-related increase in anti-Pf circumsporozoite protein (CSP) antibodies and Pf-specific interferon-γ (IFN-γ)-producing T cells. Although no definite conclusion can be drawn on the potential strength of protective efficacy of PfSPZ-GA1 Vaccine, the favorable safety profile and induced immune responses by PfSPZ-GA1 Vaccine warrant further clinical evaluation.

A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle.

Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1.IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.