Genetically attenuated P36p-deficient Plasmodium berghei sporozoites confer long-lasting and partial cross-species protection

Immunisation with live, radiation-attenuated sporozoites (RAS) or genetically attenuated sporozoites (GAS) of rodent plasmodial parasites protects against subsequent challenge infections. We recently showed that immunisation with Plasmodium berghei GAS that lack the microneme protein P36p protects mice for a period of up to 4 months. Here, we show that the period of full protection induced by p36p(-)-sporozoites lasts 12 and 18 months in C57Bl6 and BALB/c mice, respectively. Full protection is also achieved with three doses of only 1000 p36p(-) (but not RAS) sporozoites. Subcutaneous, intradermal or intramuscular routes of administration also lead to partial protection. In addition, immunisation with either P. berghei RAS- or, to a lesser extent, p36p(-)-sporozoites inhibits parasite intrahepatic development in mice challenged with Plasmodium yoelii sporozoites. Since naturally acquired malaria infections or subunit-based vaccines only induce short-term immune responses, the protection conferred by immunisation with p36p(-)-sporozoites described here further emphasises the potential of GAS as a vaccination strategy for malaria.     

A sporozoite asparagine-rich protein controls initiation of Plasmodium liver stage development

Plasmodium sporozoites invade host hepatocytes and develop as liver stages (LS) before the onset of erythrocytic infection and malaria symptoms. LS are clinically silent, and constitute ideal targets for causal prophylactic drugs and vaccines. The molecular and cellular mechanisms underlying LS development remain poorly characterized. Here we describe a conserved Plasmodium asparagine-rich protein that is specifically expressed in sporozoites and liver stages. Gene disruption in Plasmodium berghei results in complete loss of sporozoite infectivity to rodents, due to early developmental arrest after invasion of hepatocytes. Mutant sporozoites productively invade host cells by forming a parasitophorous vacuole (PV), but subsequent remodelling of the membrane of the PV (PVM) is impaired as a consequence of dramatic down-regulation of genes encoding PVM-resident proteins. These early arrested mutants confer only limited protective immunity in immunized animals. Our results demonstrate the role of an asparagine-rich protein as a key regulator of Plasmodium sporozoite gene expression and LS development, and suggest a requirement of partial LS maturation to induce optimal protective immune responses against malaria pre-erythrocytic stages. These findings have important implications for the development of genetically attenuated parasites as a vaccine approach.     

The importance of human FcgammaRI in mediating protection to malaria

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcgammaRI. This important finding documents the capacity of FcgammaRI to mediate potent antimalaria immunity and supports the development of FcgammaRI-directed therapy for human malaria.     

Alternative invasion pathways for Plasmodium berghei sporozoites

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a natural malaria infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that infection by Plasmodium falciparum and Plasmodium yoelii sporozoites depends on CD81 and cholesterol-dependent tetraspanin-enriched microdomains (TEMs) on the hepatocyte surface. Here we have analyzed the role of CD81 and TEMs during infection by sporozoites from the rodent parasite Plasmodium berghei. We found that depending on the host cell type, P. berghei sporozoites can use several distinct pathways for invasion. Infection of human HepG2, HuH7 and HeLa cells by P. berghei does not depend on CD81 or host membrane cholesterol, whereas both CD81 and cholesterol are required for infection of mouse hepatoma Hepa1-6 cells. In primary mouse hepatocytes, both CD81-dependent and -independent mechanisms participate in P. berghei infection and the relative contribution of the different pathways varies, depending on mouse genetic background. The existence of distinct invasion pathways may explain why P. berghei sporozoites are capable of infecting a wide range of host cell types in vitro. It could also provide a means for human parasites to escape immune responses and face polymorphisms of host receptors. This may have implications for the development of an anti-malarial vaccine targeting sporozoites.     

Molecular mechanism of host specificity in Plasmodium falciparum infection: role of circumsporozoite protein

Plasmodium falciparum sporozoites invade liver cells in humans and set the stage for malaria infection. Circumsporozoite protein (CSP), a predominant surface antigen on sporozoite surface, has been associated with the binding and invasion of liver cells by the sporozoites. Although CSP across the Plasmodium genus has homology and conserved structural organization, infection of a non-natural host by a species is rare. We investigated the role of CSP in providing the host specificity in P. falciparum infection. CSP from P. falciparum, P. gallinaceum, P. knowlesi, and P. yoelii species representing human, avian, simian, and rodent malaria species were recombinantly expressed, and the proteins were purified to homogeneity. The recombinant proteins were evaluated for their capacity to bind to human liver cell line HepG2 and to prevent P. falciparum sporozoites from invading these cells. The proteins showed significant differences in the binding and sporozoite invasion inhibition activity. Differences among proteins directly correlate with changes in the binding affinity to the sporozoite receptor on liver cells. P. knowlesi CSP (PkCSP) and P. yoelii CSP (PyCSP) had 4,790- and 17,800-fold lower affinity for heparin in comparison to P. falciparum CSP (PfCSP). We suggest that a difference in the binding affinity for the liver cell receptor is a mechanism involved in maintaining the host specificity by the malaria parasite.     

Expression of human CD81 differently affects host cell susceptibility to malaria sporozoites depending on the Plasmodium species

Plasmodium sporozoites can enter host cells by two distinct pathways, either through disruption of the plasma membrane followed by parasite transmigration through cells, or by formation of a parasitophorous vacuole (PV) where the parasite further differentiates into a replicative exo-erythrocytic form (EEF). We now provide evidence that following invasion without PV formation, transmigrating Plasmodium falciparum and Plasmodium yoelii sporozoites can partially develop into EEFs inside hepatocarcinoma cell nuclei. We also found that rodent P. yoelii sporozoites can infect both mouse and human hepatocytes, while human P. falciparum sporozoites infect human but not mouse hepatocytes. We have previously reported that the host tetraspanin CD81 is required for PV formation by P. falciparum and P. yoelii sporozoites. Here we show that expression of human CD81 in CD81-knockout mouse hepatocytes is sufficient to confer susceptibility to P. yoelii but not P. falciparum sporozoite infection, showing that the narrow P. falciparum host tropism does not rely on CD81 only. Also, expression of CD81 in a human hepatocarcinoma cell line is sufficient to promote the formation of a PV by P. yoelii but not P. falciparum sporozoites. These results highlight critical differences between P. yoelii and P. falciparum sporozoite infection, and suggest that in addition to CD81, other molecules are specifically required for PV formation during infection by the human malaria parasite.     

A malaria scavenger receptor-like protein essential for parasite development

Malaria parasites suffer severe losses in the mosquito as they cross the midgut, haemolymph and salivary gland tissues, in part caused by immune responses of the insect. The parasite compensates for these losses by multiplying during the oocyst stage to form the infectious sporozoites. Upon human infection, malaria parasites are again attenuated by sustained immune attack. Here, we report a single copy gene that is highly conserved amongst Plasmodium species that encodes a secreted protein named PxSR. The predicted protein is composed of a unique combination of metazoan protein domains that have been previously associated with immune recognition/activation and lipid/protein adhesion interactions at the cell surface, namely: (i) scavenger receptor cysteine rich (SRCR); (ii) pentraxin (PTX); (iii) polycystine-1, lipoxygenase, alpha toxin (LH2/PLAT); (iv) Limulus clotting factor C, Coch-5b2 and Lgl1 (LCCL). In our assessment the PxSR molecule is completely novel in biology and is only found in Apicomplexa parasites. We show that PxSR is expressed in sporozoites of both human and rodent malaria species. Disruption of the PbSR gene in the rodent malaria parasite P. berghei results in parasites that form normal numbers of oocysts, but fail to produce any sporozoites. We suggest that, in addition to a role in sporogonic development, PxSR may have a multiplicity of functions.     

Motility and infectivity of Plasmodium berghei sporozoites expressing avian Plasmodium gallinaceum circumsporozoite protein

Avian and rodent malaria sporozoites selectively invade different vertebrate cell types, namely macrophages and hepatocytes, and develop in distantly related vector species. To investigate the role of the circumsporozoite (CS) protein in determining parasite survival in different vector species and vertebrate host cell types, we replaced the endogenous CS protein gene of the rodent malaria parasite Plasmodium berghei with that of the avian parasite P. gallinaceum and control rodent parasite P. yoelii. In anopheline mosquitoes, P. berghei parasites carrying P. gallinaceum and rodent parasite P. yoelii CS protein gene developed into oocysts and sporozoites. Plasmodium gallinaceum CS expressing transgenic sporozoites, although motile, failed to invade mosquito salivary glands and to infect mice, which suggests that motility alone is not sufficient for invasion. Notably, a percentage of infected Anopheles stephensi mosquitoes showed melanotic encapsulation of late stage oocysts. This was not observed in control infections or in A. gambiae infections. These findings shed new light on the role of the CS protein in the interaction of the parasite with both the mosquito vector and the rodent host.     

Genetic engineering of attenuated malaria parasites for vaccination

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Highlights? Genetically attenuated parasites (GAPs) as potential vaccines against malaria. ? Attenuated Plasmodium sporozoites confer sustained sterile immunity against malaria. ? Rodent malaria models are an important in vivo platform to delineate GAP safety and potency. ? Late liver-arresting GAPs elicit superior protective immunity than early arresting parasites. ? P. falciparum GAP vaccine candidates need ultimately to be validated in clinical studies.     

Genome comparison of human and non-human malaria parasites reveals species subset-specific genes potentially linked to human disease

Genes underlying important phenotypic differences between Plasmodium species, the causative agents of malaria, are frequently found in only a subset of species and cluster at dynamically evolving subtelomeric regions of chromosomes. We hypothesized that chromosome-internal regions of Plasmodium genomes harbour additional species subset-specific genes that underlie differences in human pathogenicity, human-to-human transmissibility, and human virulence. We combined sequence similarity searches with synteny block analyses to identify species subset-specific genes in chromosome-internal regions of six published Plasmodium genomes, including Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. To improve comparative analysis, we first revised incorrectly annotated gene models using homology-based gene finders and examined putative subset-specific genes within syntenic contexts. Confirmed subset-specific genes were then analyzed for their role in biological pathways and examined for molecular functions using publicly available databases. We identified 16 genes that are well conserved in the three primate parasites but not found in rodent parasites, including three key enzymes of the thiamine (vitamin B1) biosynthesis pathway. Thirteen genes were found to be present in both human parasites but absent in the monkey parasite P. knowlesi, including genes specifically upregulated in sporozoites or gametocytes that could be linked to parasite transmission success between humans. Furthermore, we propose 15 chromosome-internal P. falciparum-specific genes as new candidate genes underlying increased human virulence and detected a currently uncharacterized cluster of P. vivax-specific genes on chromosome 6 likely involved in erythrocyte invasion. In conclusion, Plasmodium species harbour many chromosome-internal differences in the form of protein-coding genes, some of which are potentially linked to human disease and thus promising leads for future laboratory research.     

Cutting edge: a new tool to evaluate human pre-erythrocytic malaria vaccines: rodent parasites bearing a hybrid Plasmodium falciparum circumsporozoite protein

Malaria vaccines containing the Plasmodium falciparum Circumsporozoite protein repeat domain are undergoing human trials. There is no simple method to evaluate the effect of vaccine-induced responses on P. falciparum sporozoite infectivity. Unlike the rodent malaria Plasmodium berghei, P. falciparum sporozoites do not infect common laboratory animals and only develop in vitro in human hepatocyte cultures. We generated a recombinant P. berghei parasite bearing P. falciparum Circumsporozoite protein repeats. These hybrid sporozoites are fully infective in vivo and in vitro. Monoclonal and polyclonal Abs to P. falciparum repeats neutralize hybrid parasite infectivity, and mice immunized with a P. falciparum vaccine are protected against challenge with hybrid sporozoites.     

Transgenic parasites: improving our understanding of innate immunity to malaria

Clinical immunity to Plasmodium falciparum malaria takes years to develop and is never complete. One explanation for these observations is that antigenic variation enables malaria parasites to evade humoral immunity; another is that P. falciparum induces immune dysregulation, which inhibits the development of protective cellular immunity. Research described by D'Ombrain et al. in this Cell Host & Microbe issue probes how the parasite's main virulence factor PfEMP-1 might significantly alter human innate immune responses.     

Removal of heterologous sequences from Plasmodium falciparum mutants using FLPe-recombinase

Genetically-modified mutants are now indispensable Plasmodium gene-function reagents, which are also being pursued as genetically attenuated parasite vaccines. Currently, the generation of transgenic malaria-parasites requires the use of drug-resistance markers. Here we present the development of an FRT/FLP-recombinase system that enables the generation of transgenic parasites free of resistance genes. We demonstrate in the human malaria parasite, P. falciparum, the complete and efficient removal of the introduced resistance gene. We targeted two neighbouring genes, p52 and p36, using a construct that has a selectable marker cassette flanked by FRT-sequences. This permitted the subsequent removal of the selectable marker cassette by transient transfection of a plasmid that expressed a 37°C thermostable and enhanced FLP-recombinase. This method of removing heterologous DNA sequences from the genome opens up new possibilities in Plasmodium research to sequentially target multiple genes and for using genetically-modified parasites as live, attenuated malaria vaccines.     

Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity

Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans.     

The Plasmodium Apicoplast Genome: Conserved Structure and Close Relationship of P. ovale to Rodent Malaria Parasites

Apicoplast, a nonphotosynthetic plastid derived from secondary symbiotic origin, is essential for the survival of malaria parasites of the genus Plasmodium. Elucidation of the evolution of the apicoplast genome in Plasmodium species is important to better understand the functions of the organelle. However, the complete apicoplast genome is available for only the most virulent human malaria parasite, Plasmodium falciparum. Here, we obtained the near-complete apicoplast genome sequences from eight Plasmodium species that infect a wide variety of vertebrate hosts and performed structural and phylogenetic analyses. We found that gene repertoire, gene arrangement, and other structural attributes were highly conserved. Phylogenetic reconstruction using 30 protein-coding genes of the apicoplast genome inferred, for the first time, a close relationship between P. ovale and rodent parasites. This close relatedness was robustly supported using multiple evolutionary assumptions and models. The finding suggests that an ancestral host switch occurred between rodent and human Plasmodium parasites.     

Early transcriptional responses of HepG2-A16 liver cells to infection by Plasmodium falciparum sporozoites

Invasion of hepatocytes by Plasmodium sporozoites deposited by Anopheles mosquitoes, and their subsequent transformation into infective merozoites is an obligatory step in the initiation of malaria. Interactions between the sporozoites and hepatocytes lead to a distinct, complex and coordinated cellular and systemic host response. Little is known about host liver cell response to sporozoite invasion, or whether it is primarily adaptive for the parasite, for the host, or for both. Our present study used gene expression profiling of human HepG2-A16 liver cells infected with Plasmodium falciparum sporozoites to understand the host early cellular events and factors influencing parasite infectivity and sporozoite development. Our results show that as early as 30 min following wild-type, non-irradiated sporozoite exposure, the expressions of at least 742 genes was selectively altered. These genes regulate diverse biological functions, such as immune processes, cell adhesion and communications, metabolism pathways, cell cycle regulation, and signal transduction. These functions reflect cellular events consistent with initial host cell defense responses, as well as alterations in host cells to sustain sporozoites growth and survival. Irradiated sporozoites gave very similar gene expression pattern changes, but direct comparative analysis between liver gene expression profiles caused by irradiated and non-irradiated sporozoites identified 29 genes, including glypican-3, that were specifically up-regulated only in irradiated sporozoites. Elucidating the role of this subset of genes may help identify the molecular basis for the irradiated sporozoites inability to develop intrahepatically, and their usefulness as an immunogen for developing protective immunity against pre-erythrocytic stage malaria.     

Superior antimalarial immunity after vaccination with late liver stage-arresting genetically attenuated parasites

While subunit vaccines have shown partial efficacy in clinical trials, radiation-attenuated sporozoites (RAS) remain the "gold standard" for sterilizing protection against Plasmodium infection in human vaccinees. The variability in immunogenicity and replication introduced by the extensive, random DNA damage necessary to generate RAS could be overcome by genetically attenuated parasites (GAP) designed via gene deletion to arrest at defined points during liver-stage development. Here, we demonstrate the principle that late liver stage-arresting GAP induce larger and broader CD8 T cell responses that provide superior protection in inbred and outbred mice compared to RAS or early-arresting GAP immunizations. Late liver stage-arresting GAP also engender high levels of cross-stage and cross-species protection and complete protection when administered by translationally relevant intradermal or subcutaneous routes. Collectively, our results underscore the potential utility of late liver stage-arresting GAP as broadly protective next-generation live-attenuated malaria vaccines and support their potential as a powerful model for identifying antigens to generate cross-stage protection.     

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.