Targeting Plasmodium liver stages: better late than never

Review

Targeting Plasmodium liver stages: better late than never Steffen Borrmann1,2 and Kai Matuschewski3 1

Department of Infectious Diseases, Heidelberg University School of Medicine, 69120 Heidelberg, Germany Kenya Medical Research Institute-Wellcome Trust Research Programme, Kilifi 80108, Kenya 3 Parasitology Unit, Max Planck Institute for Infection Biology, 10117 Berlin, Germany 2

The worldwide burden of malaria can be substantially reduced using existing public health interventions. However, elimination of Plasmodium will require fundamentally different approaches. Novel experimental attenuation strategies for active immunization using knockout strains have recently stimulated renewed interest in whole-parasite vaccine approaches. Preventive drug administration during transmission of wild-type sporozoites is a complementary strategy for eliciting protective immune responses. These whole-cell immunization strategies are based on one fundamental principle: inducing protection by blocking parasite conversion from the clinically silent liver phase to the pathogenic intraerythrocytic replication cycle. Here, we review the basis, evidence and targets for whole-cell-based vaccination strategies against the liver stage bottleneck in Plasmodium infections and discuss preclinical and clinical research opportunities. Naturally acquired and vaccine-induced immunity to malaria: the secretive liver stages Malaria parasites have evolved a complex life cycle between their arthropod and mammalian hosts. In the mammalian host, unicellular parasites of the genus Plasmodium alternate between intracellular population-expansion phases (intrahepatocytic and intraerythrocytic stages) and very transient, extracellular invasive stages during which the parasites can enter new host cells, thereby driving life cycle progression (Figure 1). Host switches are accompanied by parasite population bottlenecks that create potential for successful malaria intervention strategies, such as transmission reduction by long-lasting insecticide-treated bed nets. Five Plasmodium species are known to infect humans: P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi [1]. The clinical symptoms and life-threatening complications associated with Plasmodium infection are caused exclusively by asexual blood-stage parasites (Figure 1). In 2008, an estimated 2.5 billion people in 108 malariaendemic countries were at risk of infection [2]. P. falciparum is the most virulent species and caused an estimated 225 million cases and 0.8 million deaths in 2009, mostly in children under 5 years of age in sub-Saharan Africa [3]. Infections with P. vivax, the second most important human malaria parasite, are characterized by frequent relapses arising from hepatic hypnozoites (see Glossary) [4]. Corresponding author: Borrmann, S. ([email protected])

In individuals living in malaria-endemic areas, the risk of mortality and, later in life, morbidity decreases as a function of repeated exposure to P. falciparum infections [5,6]. It is thought that this naturally acquired and partially protective immunity against disease is directed primarily against the pathogenic blood-stage parasites [7]. Over the course of many repeated infections, the variant-specific antibody Glossary Apicomplexan parasites: protozoan, unicellular parasites of animals belonging to the phylum Apicomplexa. Their name is derived from the characteristic apical complex, a set of specialized cellular structures (microtubules, micronemes secretory rhoptries and a polar ring) that mediate attachment and invasion of host cells. Plasmodium species (particularly P. falciparum) are the most prominent apicomplexan parasites because of their global medical importance. Other medically relevant Apicomplexa are Toxoplasma gondii, Cryptosporidium parvum and Babesia. Apicoplast: non-photosynthetic plastid probably derived by secondary endosymbiosis and found as a single organelle in the majority of apicomplexan parasites. Essential cellular functions of the apicoplast include fatty acid type II synthesis in liver-stage parasites and isoprenoid biosynthesis in asexual bloodstage parasites. The apicoplast contains a small 35-kb circular genome that encodes prokaryotic components essential for protein expression and plastid survival and replication. These prokaryotic and plant-like properties of the apicoplast provide ample targets for drugs, including antibiotics with antiplasmodial activity. Artemisinin: sesquiterpene lactone extracted from Artemisia annua (sweet wormwood) leaves. Preparations of Artemisia have been used in China to treat fever for more than 2000 years. Artemisinin and its derivatives are the most potent antimalarial drugs, with a characteristic broad window of activity throughout the intra-erythrocytic replication phase. Artemisinin-based combinations are used as first-line treatment for uncomplicated P. falciparum malaria in all endemic countries. Chemoprophylaxis: protects individuals against infections with pathogens using the activity of a drug at or exceeding minimum growth-inhibitory concentrations in tissues or blood circulation. Hypnozoites: dormant, non-dividing hepatic forms in P. vivax and P. ovale infections. On reactivation they give rise to repeated relapses in individuals not treated specifically against hypnozoites. Primaquine is the only available drug for prevention of relapses. Immunoprophylaxis: provides protection against pathogenic microorganisms and/or pathogenic effects of infections by induction of protective memory in one or more effector arms of the immune system. This is primarily achieved by vaccination with attenuated pathogens, recombinant pathogenicity factors, or subunit vaccines but it can also be achieved as a secondary benefit by combining with chemoprophylaxis in individuals either naturally or artificially exposed to frequent reinfections. Schizogony: peculiar cell division process in which multiple rounds of nuclear DNA replication precede formation of invasive stage parasites by cytokinesis and, finally, cellular division. Sporozoites: motile, crescent-shaped parasite stages released from oocysts in the mid-gut of infected Anopheles mosquitoes. Mid-gut sporozoites migrate to the mosquito salivary glands, where they mature to hepatocyte-invasive sporozoites that are deposited when the infected mosquitoes prick into skin tissue. Sporozoites passively travel to the liver via the bloodstream. Transformation into liver stages occurs soon after invasion of suitable hepatocytes, which depends on parasite-encoded host remodelling factors released from the apical complex on hepatocyte contact.

1471-4914/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2011.05.008 Trends in Molecular Medicine, September 2011, Vol. 17, No. 9

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Parasite population size per host

1 trillion

1 billion

1 million

1,000

1

Sporozoite transmission

Liver stages

Liver stage merozoites

Asexual blood stages

Gametocytes

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Figure 1. Peaks of parasite population sizes in individual human Plasmodium falciparum infections. Approximate peaks of parasite population sizes during different phases of the parasite life cycle in a human host: blue, diagnostically inaccessible and clinically silent; red, microscopically detectable pathogenic blood stages; green, nonpathogenic sexual-stage parasites. Note the logarithmic scale for the parasite population sizes. During an infectious mosquito bite, a few dozen sporozoites are typically injected into the human skin. Only a fraction of this inoculum successfully enters a suitable hepatocyte and establishes a liver infection. Some 12–15 rounds of synchronized DNA replication, followed by parasite budding and formation, a process termed schizogony, result in thousands of hepatic merozoites. After egress from the hepatocyte into the bloodstream, these merozoites attach to and invade red blood cells. Repeated cycles of erythrocytic schizogony, typically including four rounds of DNA replication, lead to enormous parasite population densities in both uncomplicated and severe malaria cases. For instance, parasitemia of 10%, sometimes detected even in individuals who develop clinical tolerance, corresponds to 300 million parasites per milliliter of blood. A small fraction of the parasite population (1%) differentiates into gametocytes [91], sexual stages that are ingested during a mosquito blood meal and initiate subsequent development in the mosquito vector on gamete fusion in the mosquito midgut.

repertoire against highly polymorphic parasite proteins on the surfaces of merozoites and infected erythrocytes is progressively expanded and boosted to fence off the high pathogen burden arising from exponential replication cycles (Figure 1). Because of cumulative exposure to infectious mosquito bites, positive correlations with patient age and protection against clinical malaria are typically observed for antisporozoite antibody responses across areas subject to both stable and unstable P. falciparum and P. vivax transmission [8–10]. However, it remains contentious to what degree, if at all, immune responses against the clinically silent sporozoites and liver stages play a role in naturally acquired protective immunity [11]. This is primarily a diagnostic problem because the lack of direct or reliable, indirect detection methods frustrates studies aiming to identify potential immunological correlates of naturally acquired anti-liver-stage immunity [12]. Nonetheless, attacking the parasite pre-emptively before it enters the pathogenic blood stage is an intuitive and appealing strategy for disease prevention (Figure 1) [13]. 528

Immunization with a recombinant anti-sporozoite subunit vaccine, termed RTS,S/AS01(E), has consistently induced partial protection in children during the most critical months in early childhood [14]. If confirmed safe and 50% efficacious for longer than 1 year in ongoing multicenter Phase III trials, RTS,S/ASO1(E) will meet community-agreed benchmarks for efficacy and will head toward licensure as the first anti-malaria vaccine ever [15]. The highly anticipated outcomes would justify inclusion of this first-generation vaccine in existing public health tools, which currently consist of long-lasting insecticide-treated nets, vector control, rapid diagnosis and prompt treatment with artemisinin-based combination therapy. This review focuses on promising avenues for the development of whole-cell vaccines and vaccine-like interventions based on established molecular and cellular targets during stage conversion of the complex eukaryotic pathogen Plasmodium from a non-pathogenic to a pathogenic life-cycle phase. The development of sub-unit vaccines and novel drugs that target pre-erythrocytic parasites and immunological mechanisms of protection have recently

Review been extensively reviewed [4,12,16–20], but relevant aspects are discussed in the context of whole-cell vaccine approaches. Whole-cell vaccine strategies against the Plasmodium liver stage Because intrahepatic development is (i) clinically silent, (ii) metabolically extremely active, (iii) essential for lifecycle progression and (iv) characterized by rapidly expanding parasite populations arising from only a small number of sporozoites, the Plasmodium liver stage is an established target for vaccine development [18,19]. Current experimental and candidate approaches for whole-cell vaccine strategies against the Plasmodium liver stage are based on the principle of vaccination using attenuated sporozoites, either aseptic and purified [21], or by the bite of laboratory-reared Anopheles mosquitoes [22]. Attenuation is conventionally achieved by g-irradiation [23] and, more recently, by genetic manipulation leading to targeted disruption of essential liver stage genes [24]. The attenuation principle is akin to vaccination with attenuated viruses except that attenuated parasites need to strictly fulfill two requirements to be both potent and safe: they need to be viable as sporozoites, yet fully arrested before or during initiation of a blood-stage infection. The aim of this sporozoite-based vaccine approach is to elicit sterilizing immunity against pre-erythrocytic stages; therefore, it differs fundamentally from attempts to use fractionated, killed or attenuated blood-stage parasites [25]. The desire to accelerate translation of this concept led to a complementary, yet equally potent, approach referred to as natural immunization [26]. Preemptive administration of drugs, such as antibiotics with anti-liver-stage activities during exposure to wild-type (non-attenuated) live sporozoites induces potent liver-stage-specific immunity in a rodent malaria model [26]. A closely related approach using blood-stage-suppressive drugs (e.g. chloroquine) during exposure to live sporozoites recently demonstrated induction of strong and long-lasting protection in individuals against both challenge and re-challenge with sporozoites [27,28]. A common feature of these experimental protocols designed to induce vaccine-like immunity is that they permit hepatocyte invasion by sporozoites, while either completely blocking subsequent conversion to or fully suppressing replication of pathogenic intra-erythrocytic parasites (blood-stage phase) [29]. Cellular and molecular targets for whole-cell liver-stage vaccine strategies The 23 Mb Plasmodium genome is located on 14 chromosomes that encode an estimated 5400 genes [30]. During the brief intrahepatocytic reproductive phase, which is characterized by exponential replication from single mosquito-transmitted sporozoites into thousands of hepatic merozoites (Figure 1), the parasite differentially expresses several hundred liver-stage-specific genes [31,32]. In a peculiar cell division process, termed schizogony, multiple rounds of nuclear DNA replication precede cytokinesis and cellular division [33]. This process includes coordinated biogenesis and segregation of both a single mitochondrion and a single apicoplast, a plastid-like organelle of prokary-

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otic origin, to daughter cells. Another unique compartment is the so-called parasitophorous vacuole, which consists of two membranes (one derived from the host cell and one derived from the parasite) that separate the parasite from the host cell. Although the precise function of parasiteencoded proteins [24] exported to the so-called parasitophorous vacuole membrane is unknown, it is likely that these proteins play a critical role in facilitating nutrient supply to the parasite during exponential growth. Table 1 summarizes the cellular and molecular targets that are currently being exploited for (i) subunit test vaccines, (ii) the generation of candidate whole-organism vaccines and (iii) alternative drug-based natural immunization strategies. In the following we highlight major advances made by the complementary paths that are moving towards anti-Plasmodium liver-stage vaccine interventions. Whole-organism vaccines: radiation-attenuated sporozoites Active immunization with attenuated, metabolically active whole organisms remains the most common vaccine strategy in humans. This approach includes licensed vaccines against tuberculosis and many viral pathogens, such as measles, influenza, yellow fever and polio. The same principle has been applied to experimental vaccination against malaria with irradiated sporozoites (Box 1). DNA breaks and damage to parasite chromosomes induced by g-irradiation during the extracellular sporozoite stage does not affect sporozoite survival or capacity to invade hepatocytes, but leads to arrest during the early stages of liver-stage development [34]. In very high doses, g-irradiated sporozoites elicited lasting sterile protection in experimental animal and human challenge studies [23,35]. This level of protection has set the benchmark for malaria vaccine development to date. The capacity to manufacture aseptic, purified vialed P. falciparum sporozoites in compliance with regulatory standards [21] has recently paved the way for a series of ongoing clinical trials Box 1. A brief history of the discovery of Plasmodium liver stages and sporozoite immunizations As early as 1883, Camillo Golgi suggested that Plasmodium might undergo a tissue phase prior to the cycles of erythrocyte invasion and burst, the signature feature of malaria infections [83]. Shortly after World War II it was discovered that single sporozoites undergo differentiation into red-blood-cell-invasive merozoites within hepatocytes [84,85], but establishment of a rodent model for malaria transmission was required before systematic work on liver-stage biology could be carried out [86]. It took another two decades until the early liver forms that mark the transition from sporozoites to replicating liver stages [87] and dormant stages, termed hypnozoite and the cause of frequent P. vivax relapses [88], were discovered. In 1910, Sergent and Sergent, French brothers working in Algeria, described a first experiment using in vitro heat-inactivated P. relictum sporozoites for active immunization of birds [89]. These experiments did not show evidence of sterilizing protection. As early as 1881, Pasteur used oxygen-exposed anthrax bacilli for vaccination of livestock to demonstrate the capacity of attenuated whole organisms for induction of protective immunity [90]. This principle of attenuation was successfully transferred, beginning in 1967, to malaria vaccine development using X-irradiated P. berghei sporozoites, which arrest early after hepatocyte invasion [23].

529

Immunization approach

Recombinant subunit

Biological target in the parasite

Immunological mechanism

Molecular or cellular target a CSP

Cellular function

Mode of action

Immunological strategy

Major sporozoite surface coat protein, sporozoite egress, motility and invasion, host cell remodeling Sporozoite motility and hepatocyte invasion

Blockage or neutralization of functional epitopes involved in parasite– host interactions Killing of infected hepatocytes

To elicit high anti-CSP titers to decrease sporozoite burden and/or virulence To induce memory T-cell responses against multiple epitopes To induce broad memory T-cell responses

TRAP

DNA

–

Induction of DNA double-strand breaks or chromosome damage

GAPs

UIS3/4

Parasite-host interface, exact biological function unknown Hepatocyte invasion

Liver-stage-specific vital role

To induce broad memory T-cell responses

Liver-stage-specific vital role

To induce broad memory T-cell responses

Unresolved molecular target in digestive vacuole of blood stages Unknown

Heme detoxification

Suppression of blood-stage replication

–

Killing of liver stages

To induce broad memory T-cell responses against pre-erythrocytic stages and anti-blood-stage responses To induce broad memory T-cell responses

Live sporozoites and pyrimethamine

DHFR

Purine biosynthesis or DNA replication

Inhibition of DNA synthesis resulting in early arrest of liver stages

To induce broad memory T-cell responses

Live sporozoites and azithromycin or antibiotics

Rpl4

Protein translation in apicoplast

Inhibition of apicoplast biogenesis and replication leads to delayed death of fully mature liver-stage merozoites

To induce very broad memory T-cell responses

P36/p36p

Natural immunization Live sporozoites and chloroquine

Live sporozoites and primaquine

Benefits b

Limitations b

Refs.

Phase III

Safe, large-scale production

Partial protection

[14,81,82]

Phase II

Safe, scale-up possible

No protection

[92,93]

Developmental arrest in the liver; no or delayed blood-stage parasitemia No blood stage development, efficient killing of early liver stages No blood stage development, efficient killing of early liver stages

Phase II

Sterile protection demonstrated

Challenging production and delivery

[35]

No or delayed blood-stage parasitemia; normal liver-stage development

Phase II

No blood stage development, efficient killing of early liver stages No blood stage development, efficient killing of early liver stages No blood stage development, efficient killing of early liver stages

Preclinical

Preclinical and clinical protective efficacy Delay of malaria episodes, reduction of severe malaria risk

Preclinical

[24,37]

Risk of breakthrough infections c

[38,44,94]

New benchmark; long-lasting sterilizing protection demonstrated

Ubiquitous parasite resistance to chloroquine

[27,28,72,73]

Preclinical

No drug resistance reported

-

[95]

Preclinical

Potential additional benefits in population-based chemoprophylactic interventions Potential additional benefits in population-based chemoprophylactic interventions

Widespread drug resistance

[61,62]

-

[26]

Phase I

a Abbreviations: CSP, circumsporozoite protein [96]; DHFR, dihydrofolate reductase [97]; P36, protein with a size of 36 kDa; p36p, p36 paralog; Rpl4, ribosomal protein L4 [98]; TRAP, thrombospondin-related anonymous protein [99]; UIS3,4, upregulated in infectious sporozoite genes 3 and 4. b c

Benefits and limitations are highlighted only for advanced clinical challenge (Phase II) trials, multicenter (Phase III) trials, or advanced treatment (Phase II) trials in case of antimalarial drugs.

High-level breakthrough infections were consistently described in rodent models [38,44].

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g-Irradiated sporozoites

Clinical trial status

Review

530

Table 1. Cellular and molecular targets currently being exploited for vaccine or vaccine-like interventions against liver-stage Plasmodium parasites.

Review to assess the safety, immunogenicity and protective efficacy of attenuated P. falciparum sporozoite vaccine (PfSPZ vaccine), first by the intradermal and subcutaneous routes and next by the intravenous (IV) route (Stephen Hoffman, Sanaria Inc., personal communication). In previous proof-of-principle studies of g-irradiated sporozoites in humans, protection was observed after repeated exposure to between 500 and 3000 bites by infected irradiated mosquitoes [35]. Because each mosquito might not inject more than 100 sporozoites [36], an individual P. falciparum sporozoite vaccine dose may need to consist of up to 100,000 sporozoites. This requirement for large immunization doses possibly reflects the lack of normal intrahepatocytic differentiation from single attenuated sporozoites into thousands of hepatic merozoites, a process that is expected to result in an expanded antigen repertoire and thus to drive qualitatively and quantitatively distinct and overall superior immune responses. This hypothesis could be tested not only by comparative studies in animal models but also by contrasting dose requirements for induction of protection by g-irradiated sporozoites with a natural immunization protocol using wild-type sporozoites and concurrent administration of antibiotics (see below). Because antibiotics permit full-liver stage development but fully block life-cycle conversion to blood-stage infections, such studies could provide fruitful insights into potential targets of protective immune responses. So far, the duration of protection observed in small-scale human studies employing chloroquine prophylaxis during sporozoite exposure has reached up to 2 years [28]. It remains to be seen whether this period can be further extended, which would add tremendous public health value and increase the attractiveness of a candidate PfSPZ vaccine for policy makers. Whole-organism vaccines: genetically arrested parasites Plasmodium genes that fulfill essential functions only during the brief liver stage of development can be successfully targeted by gene deletion, which is performed in cultured asexual blood stages. A small number of developmentally regulated genes that are abundantly transcribed in infectious sporozoites were the earliest candidates for the first generation of genetically arrested parasites (GAPs) that could be tested as experimental whole-organism vaccines in rodent malaria models (Table 1) [24,37,38]. Generation and selection of P. berghei knockout parasite lines for two upregulated in infectious sporozoites genes (UIS3 and UIS4) revealed that these parasite lines progressed normally through the Plasmodium life cycle, including in the mosquito vector, but were arrested after hepatocyte invasion [24,37]. Persistence of uis3–/uis4– parasites in hepatocytes in mice was associated with induction of strong, interferon-g-dependent CD8+ T-cell responses that protected against subsequent homologous challenge with lethal sporozoite doses [24,37,39,40]. Independent work on a member of the 6-cysteine family of Plasmodium surface proteins, termed p36p, demonstrated lasting and complete immunity against reinfection [38]. In comparison to irradiated sporozoites, p36p– GAPs were more potent in inducing protection against a homologous

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challenge (i.e. with the same parasite species) but considerably less efficacious against P. yoelii challenges [41], which provides evidence of distinct qualitative differences between GAPs and irradiated sporozoites. For parasites lacking p36p and the neighboring paralogous gene, termed p36, the main defects indeed occur in productive hepatocyte invasion, whereas traversal of non-host cells is normal [42], a finding that was confirmed by simultaneous deletion in P. yoelii and P. berghei ([43] and Shahid Khan, Leiden University Medical Center, personal communication). Perhaps counterintuitively, this hepatocyte invasion defect is associated with the observation of occasional blood-stage infections in mice infected with p36p– sporozoites [38] and growth of P. falciparum p36p– liver stages [44], a feature not detected in irradiated sporozoites and other GAP lines such as slarp– parasites [45,46]. It is not clear whether these observations can be explained by the recent description of occasional transformations from sporozoites to merozoites outside the liver [47]. The risk of breakthrough infections apparently increases in late-stage-arrested GAP lines that were generated and tested more recently [48– 51]. Whether a developmental arrest in the late liver stage offers additional benefits to offset the risk of breakthrough infections (e.g. by more potent protective immunity) awaits systematic immunological testing. Most researchers agree that to enter human safety and efficacy trials, a GAP vaccine line should meet the following criteria: (i) it should contain at least two, if not multiple, deletions of non-paralogous and non-redundant parasite genes, and (ii) it should be devoid of foreign DNA, such as positive selection makers. It is attractive to speculate that GAP lines could be even further enhanced by ectopic expression of immunodominant blood-stage and/ or gametocyte antigens to mount additional lines of defense and immunomodulatory molecules, such as bacterial flagellin [52], as a carry-along adjuvant. Despite the appeal of such an all-in-one live attenuated sporozoite vaccine, which could be crafted using advanced genetic engineering, this approach collides with the urgent need for affordable interventions on an entire continent. Natural immunization: targeting Plasmodium liver stages It has been known for some time that malaria can be treated with certain antibiotics, but that clearance of clinical and parasitological parameters of infections is slow [53,54]. More recently, it was demonstrated that antibiotics with anti-parasitic activity target a non-photosynthetic plastid-like organelle, termed the apicoplast, in apicomplexan parasites such as Plasmodium and Toxoplasma, by inhibiting the prokaryotic protein expression machinery inside these biologically fascinating organelles [55,56]. It was shown that in Toxoplasma gondii tachyzoites [55] and Plasmodium blood stages [53,57], intracellular parasites exposed to antibiotics continue to develop normally. Parasite cell death occurs only after invasion of new host cells, even when drug is no longer present; hence, the term ‘delayed death’. This peculiar killing phenotype correlated with disturbed biogenesis and altered patterns of inheritance of the apicoplast in second-cycle parasites [58,59]. Subsequent reverse genetics data also pointed to 531

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Persistence ?

PM

CQ

AZ Complete growth suppression Invasion of red blood cells ?

Transformation

Maturation

Mature schizont

Merozoite release

Cyclic intra-erythrocytic replication

Liver stage development TRENDS in Molecular Medicine

Figure 2. Plasmodium liver-stage development. Characteristic steps of parasite development within host hepatocytes: blue, nuclei of the host cell (large) and the parasite (small); green, Plasmodium cytoplasm; yellow, parasitophorous vacuole (PV), an intracellular niche that is formed during the active invasion process; red, Plasmodium apicoplast, a vestigial organelle of red algal origin and the therapeutic target of macrolide antibiotics such as azithromycin. Transformation (far left): after successful hepatocyte entry, the elongated sporozoite transforms into a round early hepatic form closely associated with the host nucleus. Maturation (center left): during the intracellular growth phase, parasite nuclei divide and the PV membrane forms extensive protrusions into the hepatocyte cytoplasm. Mature schizont (center right): This stage marks the final phase of the intrahepatic differentiation that occupies a large segment of the host cell. The large PV contains thousands of parasite nuclei that are segregated into individual parasites, termed hepatic merozoites. Merozoite release (far right): vesicles, so-called merosomes, containing mature liver-stage merozoites bud off the infected host cell and give rise to the pathogenic phase of the parasite life cycle, erythrocytic schizogony. When liver-stage development occurs during exposure to the antifolate drug pyrimethamine (PM; top), parasites arrest early in development, immediately after transformation. Whether these parasites persist for some time is still under investigation. On prophylactic exposure to antibiotics (bottom), such as azithromycin (AZ), apicoplast biogenesis is dramatically altered. Instead of branching and final segregation into individual merozoites, the apicoplast in antibiotic-exposed liver stages remains compact. Despite full liver-stage merozoite formation, the daughter cells no longer inherit this vital organelle, resulting in non-infectious parasites and hence a ‘delayed death’ phenotype. Chloroquine cover (CQ; center, far right) does not affect liver-stage development, but suppresses the replication of asexual parasites within host erythrocytes. Late-stage arrest of Plasmodium liver-stage development by antibiotic prophylaxis or suppression of emerging blood stages by chloroquine can induce potent protective immune responses against sporozoite reinfections.

the apicoplast as an attractive drug target in late liverstage parasites [50,51,60]. Recent work using antibiotic administration during sporozoite exposure demonstrated that antibiotics specifically inhibit biogenesis and inheritance of the apicoplast in Plasmodium liver stages, resulting in continued liverstage maturation but subsequent failure to establish a blood-stage infection (Figure 2) [26]. The antibiotics tested include azithromycin, a safe and affordable macrolide antibiotic. Exponential expansion of these attenuated liver-stage merozoites from a single sporozoite induced potent CD8+ T-cell and interferon-g-dependent protection against malaria in an immunization dose- and frequency-dependent manner [26]. In an experiment designed to mimic a natural transmission scenario, 30% and 85% of animals were protected against parasitemia and cerebral malaria, respectively, after only two immunizations with mosquito-transmitted P. berghei during antibiotic cover [26]. Analogous to the targeting of DNA replication, and hence intracellular parasite growth, using g-irradiation, an equivalent effect can be achieved by inhibition of nucleotide biosynthesis with antifolate drugs. Exposure of liver stages to pyrimethamine – a component of the widely used antimalarial combination sulfadoxine–pyrimethamine – 532

leads to precise arrest of early liver-stage forms [61]. Multiple rounds of intravenous immunization with high doses of live sporozoites during parallel pyrimethamine administration lead to sterile protection in a mouse model [61]. When tested on a P. berghei strain selected for high pyrimethamine resistance during blood-stage infections, pyrimethamine retained partial activity against liver stages. These results appear to support a postulated vaccine-like effect of antifolate compounds when used prophylactically in high transmission settings, an attractive hypothesis initially proposed by Sutherland and co-workers [62]. A large number of human trials have already been conducted to address the chemoprophylactic efficacy of continuous or intermittent preventive treatment (IPT) regimens of pyrimethamine alone or, in the majority of cases, in combination with sulfadoxine [63]. Although some studies reported impressive persistence of protection beyond the presence of inhibitory drug plasma concentrations [64], in line with animal model data [61], the majority of studies did not provide evidence for additional immunoprophylactic effects [65–67]. The lack of a consistent immunoprophylactic benefit of IPT or continuous prophylaxis with pyrimethamine might be directly related to (i) drug resistance [68], (ii) the typically low-level exposure to naturally transmitted sporozoites combined with the

Review possibly limited antigen display during early liver-stage arrest [36,69], (iii) the absence of strain-transcending protective immune responses [22], or a combination of all three factors. Natural immunization: preventing blood stages by continuous suppressive treatment The immunological benefits of full liver-stage maturation was recently demonstrated in healthy human volunteers who were immunized by repeated bites from 12–15 P. falciparum-infected Anopheles stephensi mosquitoes during continuous prophylaxis with the blood-stage suppressive drug chloroquine, which is thought to interfere with heme detoxification in the digestive vacuole of intra-erythrocytic Plasmodium parasites [27,70]. Protection of all 10 participants against a challenge with mosquitoes infected with the homologous NF54 P. falciparum strain was associated with pluripotent memory T-cell production of interferon-g, tumor necrosis factor a and interleukin-2 [27,71]. In a follow-up study conducted 28 months after immunization, continued protection in 4 out of 6 volunteers against re-challenge with bites from 5 mosquitoes infected with the homologous P. falciparum strain was associated with persistence of pluripotent effector memory T-cell responses [28]. This line of investigation was initiated in the 1970s with live sporozoites injected into naı¨ve animal hosts on chloroquine prophylaxis. It demonstrated that suppression of blood-stage parasites emerging from the liver generates strong protection against challenge [72,73]. Interestingly, control animals injected with identical doses of sporozoites but without prophylactic chloroquine administration had very low anti-sporozoite antibody titers and remained susceptible to sporozoite challenge after treatment of ensuing blood-stage infections with chloroquine [74]. Similar results have recently been obtained with mefloquine, another blood-stage suppressive drug also thought to have a target within the digestive vacuole [70,75]. The fact that protection is induced by this chemically unrelated, yet mechanistically equivalent, drug suggests that the immunomodulatory effects of chloroquine might not be a significant confounding factor in these experiments [27,71]. From these and previously discussed experiments, we thus hypothesize that blocking or suppressing parasite life cycle progression may be essential for preventing Plasmodium immune escape mechanisms that otherwise maintain susceptibility to lifelong re-infections. Concluding remarks and future research directions No malaria vaccine has yet been licensed. However, the promising late-stage clinical development of a safe and, most likely, affordable first-generation anti-sporozoite subunit vaccine combined with the proven efficacy of irradiated sporozoites and sporozoite exposure during prophylactic administration of chloroquine in protecting against human challenge infections demonstrate that targeting the clinically silent pre-erythrocytic life-cycle phase may be the best option for designing a malaria vaccine with >90% protection. Most researchers agree that a future secondgeneration malaria vaccine will be more complex and will almost certainly contain a central anti-liver-stage component as the first and major line of defense. Systematic

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improvement of anti-liver-stage efficacy in malaria-endemic areas is, however, particularly challenging owing at least in part to the following diagnostic difficulties: a lack of robust measures of exposure to infective and non-infective mosquito bites, a lack of diagnostic tools for detecting in vivo sporozoites, liver-stage infections and first-wave blood-stage parasites after release from the liver [22], and the absence of other biological or immunological markers that predict an ongoing pre-erythrocytic infection. Therefore, malaria vaccine development will strictly depend on efficacy trials using experimental infections as challenge and blood stage infections as end-points for years to come [22]. A rare and unique research opportunity for malaria vaccine development is the availability of ethically sound protocols for infection and challenge of volunteers with a potentially deadly pathogen [27,76–78]. These experiments are possible because of rigorous clinical surveillance and the use of sporozoites from drug-sensitive P. falciparum strains. They tend to provide clinically relevant information on endpoints such as delay to detectable bloodstage infection or, in best-case scenarios, absence of a blood-stage infection after challenge, but their capacity for investigating immunological and biological endpoints has not yet been fully explored. Moreover, they barely mimic repeated inoculations in endemic areas, which often occur over many consecutive nights. Therefore, new clinical trial settings hosted by institutions in malaria-endemic countries, ideally mimicking local transmission and incorporating investigations on vaccine effects in semi-immune individuals, are desirable [79]. The liver stage also holds unexploited potential for developing innovative stand-alone or adjunct-drug-based interventions. For instance, the potential of antibiotics to permit full liver-stage maturation yet completely block lifecycle progression could be integrated into clinical development programs [71]. These could range from prophylactic regimens during experimental or natural sporozoite exposure to elicit protective immune responses, to an additional safety net for future genetically arrested, whole-parasite vaccine strategies. Although the path for novel liver-stage drugs for prevention of malaria might face a number of obstacles [4], acceleration of the development pipeline for new causal prophylactic drugs is urgently needed and will require innovative funding strategies for public–private partnerships. More generally, there is a great need for affordable, potent and, in particular, safer drugs for causal prophylaxis and for eliminating the dormant stages of P. vivax. It seems that single or polyvalent subunit vaccines directed against pre-erythrocytic stages have one hypothetical advantage compared to equivalent anti-bloodstage vaccines. The latter target parasite proteins that are amply expressed during blood-stage infection, yet anti-blood-stage immunity develops very slowly and often remains incomplete even after repeated infections [16]. By contrast, exposure to pre-erythrocytic parasite loads is very brief and orders of magnitudes lower (Figure 1), which seemingly precludes significant immune responses in nature. If this hypothesis is true, a vaccine that delivers exceedingly higher levels of antigen(s) might provide the 533

Review Box 2. Objectives and questions related to the development of targeted anti-liver-stage intervention strategies  Identify a more complete inventory of essential genes with liverstage-specific functions as candidates generating of gene-depleted, non-pathogenic strains as whole-organism vaccines.  Develop simpler protocols with higher throughput for inhibitor screens against all human malaria parasites.  Is naturally acquired immunity against pre-erythrocytic stages protective? What are the immune effector mechanisms in naturally acquired anti-liver-stage responses in residents of endemic areas? What are their parasite targets? Is there robust evidence for strain-transcending protection?  Is it possible to design interventions to accelerate the slow acquisition of broad anti-sporozoite and anti-liver-stage responses observed in malaria-endemic areas?  Is there a role for safe, affordable and potent causal prophylactic intervention to interrupt mosquito-to-man transmission as a tool in malaria elimination?  Can we develop innovative diagnostic tools for hidden liver stages?

necessary boost [80]. Instructive lessons for future vaccines, whether whole-cell or subunit-based, may also be gained from a better understanding of the mechanisms of protection induced by the pre-erythrocytic subunit vaccine RTS,S, which delays, but does not prevent, blood-stage infections and malaria episodes [14,81,82]. Many puzzling gaps in our understanding of liver-stage biology remain to be addressed before we can fully realize the potential of anti-liver-stage interventions (Box 2). The hidden nature of the pre-erythrocytic life cycle has not made the study of naturally acquired immunity to these stages any easier. Radically different tools will be required to address fundamental questions, such as whether protection against liver-stage parasites can be acquired and maintained by natural exposure and which parasite proteins are targets of these responses. The answers to many of these questions will be important for predicting the impact of an anti-liver-stage intervention as a public health tool to control malaria regionally, to respond to local epidemics, and to facilitate gradual and sustainable contraction of the world malaria map. Acknowledgments We thank Diane Schad for the artwork. S.B. is supported by a German Research Foundation grant (SFB 544, A7) and the Federal Ministry of Research and Education. K.M. is supported by the Max Planck Society, the German Research Foundation, the Federal Ministry of Research and Education, and the EVIMalaR European Union Network of Excellence. This work is published with permission of the Director of KEMRI.

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