Looking under the skin: the first steps in malarial infection and immunity

A Corrigendum to this article was published on 08 October 2013

This article has been updated

Key Points

  • Malaria is the most deadly parasitic infection of humans, killing up to 1 million people per year. No vaccine is currently available, and the development of drug-resistant Plasmodium spp. is of increasing concern.

  • The first phase of infection, the pre-erythrocytic (PE) phase, is clinically asymptomatic. Only after parasite replication in the liver and infection of large numbers of erythrocytes do symptoms arise.

  • The PE phase comprises sporozoites (the infectious stage) and the liver stages. Once injected by a mosquito, the sporozoites can remain in the skin, be transported in lymph vessels to draining lymph nodes or travel through the bloodstream to the liver. In the liver, sporozoites undergo an elaborate replication and developmental programme and transform into the merozoites that are released from the liver to infect erythrocytes.

  • The PE phase of infection is a formidable window of opportunity for therapeutic interventions owing to the small number of parasites present. Thus, targeting this 'bottleneck' of Plasmodium spp. infection with vaccines is an attractive strategy.

  • Live attenuated parasites mimicking the PE phase of infection can be used as vaccines. Attenuation is achieved by radiation, genetic alterations or drug-mediated developmental arrest.

Abstract

Malaria, which is caused by Plasmodium spp., starts with an asymptomatic phase, during which sporozoites, the parasite form that is injected into the skin by a mosquito, develop into merozoites, the form that infects erythrocytes. This pre-erythrocytic phase is still the most enigmatic in the parasite life cycle, but has long been recognized as an attractive vaccination target. In this Review, we present what has been learned in recent years about the natural history of the pre-erythrocytic stages, mainly using intravital imaging in rodents. We also consider how this new knowledge is in turn changing our understanding of the immune response mounted by the host against the pre-erythrocytic forms.

Main

Malaria is the most deadly parasitic infection of humans. Although economic development and the implementation of control measures during the twentieth century have eliminated malaria from many areas of the world1, the disease is still rampant in the tropics and in the poorest regions of the globe, affecting 3 billion people and killing up to 1 million annually2. Sub-Saharan Africa pays the heaviest toll, mainly because of the efficiency of Anopheles gambiae mosquitoes (which live in this region) as hosts of Plasmodium falciparum, the Plasmodium species that is most lethal to humans, delivering up to 120 infective bites per person per year3.

The symptoms of malaria are caused by cycles of parasite multiplication inside host erythrocytes, and various complications, including cerebral malaria, result from cytoadherence of infected erythrocytes to endothelia. However, infection with Plasmodium spp. starts when parasites are injected into the skin by a mosquito (Fig. 1). The first, pre-erythrocytic (PE) phase of infection is clinically silent. During this phase, the few parasites that are inoculated into the skin by the mosquito, called sporozoites, reach the liver. They invade and multiply inside hepatocytes into new parasite forms, termed merozoites, which invade erythrocytes. This preclinical parasite metamorphosis in the liver lasts 7–10 days in humans and 2 days in rodents.

Figure 1: The Plasmodium spp. life cycle.
figure1

The life cycle of Plasmodium species that infect mammals. Symptoms of malaria are caused by cycles of parasite multiplication inside erythrocytes. One cycle is initiated as a result of erythrocyte invasion by a merozoite form and consists of 3–4 nuclear divisions that generate 10–30 new merozoites; a cycle typically lasts 24 hours and 48–72 hours in species that infect rodents and humans, respectively. Some intra-erythrocytic parasites transform into male or female gametocytes, which are taken up by a mosquito. Gametocytes egress from erythrocytes, activate into gametes and fuse in the mosquito midgut lumen. The motile zygote, called an ookinete, crosses the gut epithelium to transform into an oocyst, in which thousands of sporozoites develop. Sporozoites are released into the mosquito body cavity and later pass through salivary gland cells to enter the salivary ducts. After transmission into the skin of the mammalian host during a bite by the mosquito, motile sporozoites reach the liver, where they invade hepatocytes. One intra-hepatocytic sporozoite generates tens of thousands of hepatic merozoites, which re-enter the bloodstream and invade erythrocytes. Numbers indicate parasite progeny after multiplication.

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The PE stages (namely, the sporozoite and ensuing liver stages) are present in very small numbers in the host. Therefore, they constitute a transmission bottleneck and are ideal vaccination targets, offering the possibility of preventing progression of the parasite life cycle before clinical illness occurs. PE forms have been the subject of intensive immunological research ever since the first demonstrations, in animal models and humans, that injection of attenuated parasites which do not cause blood infection confers protection against sporozoite challenge4,5,6. Today, this vaccination method is still the most efficient at offering sterilizing immunity against Plasmodium spp. infection.

This Review gives a perspective on our understanding of the PE phase of natural infection in rodents and of host defence against the PE stages in both rodents and humans. We begin by describing recent insights into PE infection. Many approaches have been used to better define sporozoite fate in the host, including powerful intravital imaging techniques that have been adapted for use in laboratory rodents (Box 1). Although numerous molecular insights into the basic biology of the sporozoite and liver stages have also been gained, we restrict our coverage here to a few details relevant to our specific focus on infection at the organismal level. We then deal with the immunobiology of the PE stages, with emphasis on the immune response induced by live attenuated parasites (LAP). Most notable in recent years has been the increasing recognition of the importance of the initial skin step, which affects both infection by, and immunity to, PE parasites.

An historical perspective on PE stages

Defining the PE phase of malaria has been a long and winding road. After the discoveries of Plasmodium sp. parasites in the blood of a patient in 1880 by Laveran7 and of the role of mosquitoes in Plasmodium spp. transmission in the late 1890s by Ross8 and Grassi9, studies on PE stages in the first half of the twentieth century were mainly carried out using avian models of infection. The pioneering work of Raffaele and others in the 1930s revealed the presence of exo-erythrocytic schizonts in macrophages at the inoculation site in the skin, as well as in reticuloendothelial cells in the liver and spleen of infected birds, giving rise to successive PE cycles of infection10. In mammals, evidence for a PE 'tissue phase' was first obtained in 1948: Shortt and colleagues found that when sporozoites of the simian parasite Plasmodium cynomolgi were transmitted by mosquitoes to rhesus monkeys, these parasites developed as schizonts in parenchymal cells, but not other cells, in the liver11. Subsequent work showed that sporozoites of species that infect primates (including humans) undergo a single schizogonic cycle inside hepatocytes, although parasites of some species are thought to also persist inside hepatocytes for weeks to years as singly occurring PE forms called hypnozoites12,13. Recently, it has been speculated that at least some of the malarial recurrences which would conventionally be presumed to have a hypnozoite origin might actually have non-hypnozoite dormant stages as their source14.

Figure 2: The pre-erythrocytic phase in rodents.
figure2

a | Motile sporozoites injected into the skin of rodents during a mosquito bite can leave the bite site via blood or lymphatic vessels, or stay in the skin. Sporozoites drained by lymphatic vessels can invade host cells in the proximal lymph node. Skin sporozoites can invade cells in the epidermis, dermis or associated with the hair follicle (exo-erythrocytic forms (EEFs)). Sporozoites that leave the bite site via the bloodstream stop in the liver, glide in the sinusoids (which are lined by endothelial cells and harbour Kupffer cells), wound Kupffer cells, cross the sinusoidal barrier, traverse several hepatocytes and invade a final hepatocyte within a parasitophorous vacuole. Merozoites, the erythrocyte-infecting parasite form, can be formed inside hepatocytes and skin cells. b | Confocal microscopy images from tissue cryosections (lymph node and epidermis) or intravital imaging (dermis, hair follicle and liver) show EEFs of the parasite (green). In the lymph node, the parasite is seen associated with a podoplanin-expressing cell. In the epidermis, the parasite is seen in the basal layer (cells stained with DAPI). In the dermis, an infected cell (right) releases merosomes (white arrowheads) and merozoites (red arrowheads). In a hair follicle of a Blimp1–GFP mouse, the parasite is shown associated with Blimp1–GFP-expressing cells in the sebaceous glands (SG); the hair shaft (HS) is visible because of autofluorescence. In the liver, a mature liver stage releases several merosomes (m) into the blood circulation, indicated by bovine serum albumin (BSA); the sinusoidal barrier, through which the merosomes are released, is indicated (arrow). Days post-infection are shown; scale bars represent 10 μm. Lymph node image is reproduced, with permission, from Ref. 32 © (2006) Macmillan Publishers Ltd. All rights reserved. Dermis image is reproduced and and hair follicle image is modified, with permission, from Ref. 72 © (2010) National Academy of Sciences USA. Liver image is reproduced, with permission, from Ref. 68 © (2006) American Association for the Advancement of Science.

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The rodent-infecting Plasmodium species Plasmodium berghei was discovered in 1948 in Grammomys surdaster (an African tree rat)15, and in 1965, P. berghei sporozoites were shown to generate schizonts in the liver of laboratory rodents16,17. Since then, P. berghei and its sibling species Plasmodium yoelii, both of which are easily amenable to molecular genetic research18, have become practical and powerful models for investigating the basic biology of the PE stages.

Infection by PE stages

The skin as a branch point. Sporozoites can move at high speed by gliding motility19 and can traverse host cells by piercing host membranes20,21. They can also invade host cells inside a parasitophorous vacuole22,23, where they typically differentiate into merozoites. Around 100 sporozoites are injected by a mosquito in an experimental situation24,25, whereas in the wild, infected mosquitoes probably inoculate fewer than 50 sporozoites per bite on average26. In agreement with this, a limited parasite release rate through the mosquito proboscis (1–2.5 sporozoites per second) was reported by counting fluorescent P. berghei sporozoites during Anopheles stephensi salivation27 or transmission to mice28. It has long been considered that sporozoites are injected directly into blood vessels by mosquitoes, and the notion that sporozoites are instead inoculated into the extracellular matrix of the skin received experimental support only recently from experiments using bite site removal29 and interruption of mosquito feeding30. Intravital fluorescence microscopy (Box 1) confirmed that most sporozoites are injected into the skin when the mosquito proboscis probes the skin for blood and ejects saliva31. Sporozoites glide in the skin as fast as on glass slides (1–2 μm per second in the first 30 minutes), display tortuous and apparently random three-dimensional movement patterns with no detectable tactism31,32,33 and can move for more than 1 hour, although their speed gradually decreases with time. Surprisingly, quantitative studies of P. berghei sporozoites injected by mosquitoes into the ear pinna of mice indicate that 25% and 15% of the sporozoites leave the skin by invading blood or lymphatic vessels, respectively, whereas 60% remain at the bite site32 (Fig. 2). Quantitative PCR experiments show a similar tripartite fate for P. yoelii sporozoites inoculated into the skin34. The exact proportions of parasites that follow each route is likely to be influenced by many factors, including the vessel density at the bite site and the parasite species.

Entering the liver. Most of the sporozoites injected into the bloodstream reach and remain in the liver35. Extensive biochemical work in the 1990s implicated circumsporozoite protein (CSP), the 'coat-forming' protein of the sporozoite36, in this process. CSP binds the particularly highly sulphated glycosaminoglycan chains in liver heparan sulphate proteoglycans (HSPGs) produced by hepatocytes and stellate cells37. Which domain of CSP, the amino-terminal domain38,39 or the carboxy-terminal thrombospondin type I repeat (TSR) domain40,41,42 (both of which display HSPG-binding capacity), is involved in CSP binding to liver HSPGs and, thus, in sporozoite homing to the liver has been a long-debated question. A recent genetic study showed that only the N terminus of CSP is exposed on free sporozoites, and the TSR is masked; cleavage of the N terminus was found to expose the TSR and trigger sporozoite invasion43. However, as intravenously injected sporozoites lacking the CSP N terminus infect the liver normally43, it is possible that neither the N terminus (dispensable) nor the TSR (masked) is important for sporozoite arrest in the liver.

Insights into the life of the sporozoite in the liver have come from studies of its aggressive cell traversal (CT) behaviour. Following the first description of sporozoite traversal of host cells20, which had been seen using macrophages in vitro, work focused on traversal of hepatocytes, which was observed both in vitro21 and in vivo21,44. A wild-type sporozoite traverses several hepatocytes before invading a final hepatocyte inside a parasitophorous vacuole, where it differentiates. Initial studies using wild-type sporozoites concluded that hepatocyte CT activates parasite invasion of hepatocytes45, as well as intracellular development46, via the activity of hepatocyte growth factor (HGF) released from the traversed cells. More recently, a 1.5-fold increase in the number of parasites developing inside cultured hepatocytes in the presence of excess HGF was observed for P. berghei but not for P. yoelii, leading to the conclusion that the role of HGF is crucial and species specific47. By contrast, studies using CT-deficient sporozoites paint a different picture, in which CT is important because it allows traversal of cell types other than hepatocytes48,52. CT-deficient sporozoites were initially shown to be highly impaired in liver infection in vivo, but to invade and develop inside hepatocytes normally in vitro48,49,50. The in vivo contributions of CT (Fig. 3) were later revealed by imaging of sporozoites lacking sporozoite protein essential for cell traversal 2 (SPECT2)49, a protein containing a membrane attack complex/perforin-like domain. CT allows sporozoite locomotion and access to blood capillaries in the skin, as 80% of SPECT2-null sporozoites were rapidly immobilized in the skin51. CT then ensures sporozoite survival in the liver sinusoids, because >90% of intravenously injected SPECT2-null sporozoites were rapidly phagocytosed by Kupffer cells52. Furthermore, CT facilitates sporozoite translocation across the liver sinusoidal barrier into the parenchyma, as 75% of the crossing events by wild-type sporozoites occurred by traversal of endothelial cells and/or Kupffer cells52. Intravital imaging showed that wild-type sporozoites use multiple pathways to cross the liver sinusoidal barrier52 (Fig. 3), contradicting the long-held view that sporozoites translocate across the barrier exclusively through Kupffer cells, a concept known as the gateway model44,53,54.

Figure 3: Host cell traversal by sporozoites.
figure3

Images obtained by intravital confocal imaging of cell traversal (CT) protein-expressing (CT+) or CT-deficient (CT) Plasmodium berghei sporozoites. a | CT in the skin. Maximal intensity projections of CT+ (left) or CT (right) sporozoite trajectories in the mouse ear over a 10-minute period. Immobilized CT sporozoites are shown in yellow. Scale bars represent 50 μm. b | CT in the liver sinusoids. Time-lapse microscopy of sporozoites (anterior tip indicated by a yellow star) in the liver of a mouse expressing fetal liver kinase fused to GFP (FLK1–GFP) (labelling endothelial cells) (upper and lower panels); Kupffer cells are labelled with F4/80-specific antibody; fluorescent bovine serum albumin (BSA) is used to label the liver sinusoids (middle panel). In the upper panel, a CT+ sporozoite wounding a Kupffer cell is seen by the incorporation of propidium iodide (PI; arrowheads) into the nucleus of the wounded Kupffer cell. In the middle panel, a CT sporozoite durably interacts with, and is cleared by, a Kupffer cell (arrowheads). In the lower panel, a CT+ sporozoite extravasates (yellow arrowhead) by traversing an endothelial cell, visualized by fading of the fluorescence of the traversed endothelial cell (arrowheads). Times of imaging are shown. Scale bars represent 10 μm. Part b images are reproduced, with permission, from Ref. 52 © (2013) The Rockefeller University Press.

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CT is therefore important for the progression of sporozoites through host tissues and for sporozoite resistance to host innate immunity en route to hepatocytes, but hepatocyte traversal does not seem to play a significant part in the establishment of infection. An important question is how the sporozoite switches from CT to invasion. As mentioned above, cell invasion is associated with cleavage of CSP and exposure of the TSR domain43, possibly corresponding to cell contact-dependent activation of sporozoite invasion. Turning off CT and membrane wounding, to avoid lysis of the parasitophorous vacuole membrane, is also obligatory for cell invasion, and indeed CT-deficient sporozoites readily invade cells51. A related question is, what triggers the switch? One study identified the particularly high levels of sulphation of liver HSPGs as the factor that induces the switch55. This conclusion, however, gives rise to the question of whether HSPGs can both cause arrest of sporozoites in the liver sinusoids40,41,42 and activate sporozoites to invade the final hepatocyte55. In addition, cleavage of glycosaminoglycan chains from HepG2 cell surfaces does not affect sporozoite invasion56. More work is needed to discover what triggers sporozoites to stop traversing cells, and how the switch to invasion operates.

Egressing from hepatocytes. Inside the parasitophorous vacuole, the parasite undergoes a spectacular development involving metamorphosis of the slender sporozoite into a large spherical liver stage, followed by schizogony to generate tens of thousands of merozoites in 2 days57. Molecular insights have been gained into the composition of the parasitophorous vacuole membrane, the strategies for scavenging vital compounds from the host cell, and the contribution of the apicoplast, and these subjects have been reviewed elsewhere58,59,60. Another aspect of cellular parasitism that has been studied is the way the parasite controls host hepatocyte survival. Initially, host cell apoptosis is blocked61, possibly via inhibitor of cysteine proteases (ICP; called falstatin in P. falciparum)62, which has been suggested to inhibit cathepsin L-like host cell proteases but not cathepsin B-type parasite proteases. The P. yoelii liver stage has been shown to modify the levels of several hepatocyte proteins, including p53, which is suppressed to enhance survival of the infected cell63. Later in development, the parasitophorous vacuole membrane is lysed64, probably after activation of cysteine proteases belonging to the serine repeat antigen (SERA) family65, and the merozoites are released into the host cell cytoplasm. The parasite then controls a unique form of host cell death that has some features of apoptosis, such as mitochondrial disintegration and nuclear condensation, but lacks others, such as caspase activation; crucially, this type of cell death preserves the integrity of the host cell membrane66.

The final merozoite egress, which was long assumed to occur by mechanical bursting of the host cell67, is instead a sophisticated process of host cell membrane manipulation68. Real-time in vivo imaging has shown that hundreds of merozoites are packaged together inside vesicles called merosomes, surrounded by the hepatocyte membrane (Fig. 2b). These structures bud off and detach from the cell into the sinusoidal lumen68,69 and have also been seen during P. falciparum infection in immunocompromised mice engrafted with human hepatocytes70. Merosomes are solid enough to pass through the heart and reach the lung circulation, where they have been observed liberating infectious merozoites ex vivo71. Merozoites manipulate the merosome membrane by impeding the exposure of phosphatidylserine on the outer leaflet64,68 (a normal 'eat-me' signal of dead cells), so merosomes are immunologically silent carriers. Therefore, whereas the sporozoite relies on its motile and membrane-wounding capacities to evade host phagocytes, the immotile merozoite has evolved a membrane-wrapping strategy.

A skin-draining lymph node step in PE malaria. The sporozoites that leave the skin inoculation site by invading lymphatic vessels remain in the proximal draining lymph node32. Although sporozoites are still motile on arrival in the subcapsular sinuses of the lymph node, most are quickly taken up by CD11c+ dendritic cells (DCs). However, a few parasites invade cells expressing podoplanin (a membrane protein found in lymphatic endothelial cells) (Fig. 2b), and the parasites in these cells do not complete exo-erythrocytic development33. The lymphatic route thus seems to be a dead end in terms of the parasite life cycle.

Surprisingly, the skin itself is a suitable niche for parasite development72,73. Intravital imaging of P. berghei sporozoites inoculated into the skin by mosquitoes revealed that up to 10% of the sporozoites develop in the dermis, the epidermis and in association with hair follicles72 (Fig. 2b). Although most skin parasites die before completing their intracellular development, both P. berghei and P. yoelii sporozoites can multiply to form merozoites inside skin cells (probably in dermal fibroblasts). Ex vivo experiments have shown that skin merozoites can infect erythrocytes72. However, whether skin merozoites can initiate a blood infection in vivo could not be decisively demonstrated72,73, although the observation of merosomes detaching from skin cells and moving within the skin72,73 favours this hypothesis. Sporozoites expressing CSP with a constitutively exposed TSR induce blood infection without leaving the skin43, a finding which further suggests that erythrocyte infection can be initiated by skin merozoites. Another striking revelation was the persistence of a small proportion of parasites for weeks in association with hair follicles72, an immunoprivileged site in the mammalian body74, raising the possibility that parasites associated with hair follicles are a source of infection recurrences in mammalian malaria.

The traditional view that Plasmodium spp. sporozoites travel only from the skin to the liver in mammals should thus be revisited. At least in rodents, after mosquito delivery, a large proportion of sporozoites (>50% in the ear) stay in the skin, where multiplication can occur, whereas other sporozoites end up in the draining lymph node until they are eventually cleared (Fig. 2). Whether the same picture holds for human malaria is still unknown. Nonetheless, the development of avian Plasmodium spp. in the skin75, the similarity between host cell invasion by P. yoelii and P. falciparum sporozoites76, and an early description of Plasmodium vivax in a human lymph node77 all point to the possibility that a skin-draining lymph node component in the PE phase of the Plasmodium spp. life cycle is more conserved than previously appreciated.

Protection against PE stages

The PE stages are attractive targets for immunological intervention. During their journey to the liver, sporozoites are exposed to antibodies in tissue fluids and the bloodstream. Moreover, developing PE parasites in the skin and liver are the only parasite forms that are present in a nucleated cell and so capable of presenting antigens in association with major histocompatibility complex (MHC) class I molecules. There is, however, little evidence that natural exposure to parasites induces even partially protective immunity against the PE stages78. By contrast, immunization with large doses of LAP, which are incapable of completing PE development and causing blood infection, has long been known to induce protection against sporozoite challenge in animal models4,5 as well as in humans6,79 (Fig. 4). Although LAP were initially radiation-attenuated sporozoites (RAS), it has been shown that parasites attenuated by genetic engineering80,81,82,83,84,85,86,87,88,89,90,91, drug treatment92,93,94,95,96,97,98,99 or chemical modification100 also act as potent vaccines in rodents and humans.

Figure 4: Liver-stage maturation and live attenuated parasite-based vaccine strategies.
figure4

a | Sporozoite transformation into merozoites inside a hepatocyte. Within a parasitophorous vacuole, the sporozoite transforms into a spherical liver stage that progressively enlarges and undergoes karyokinesis (involving the formation of up to 30,000 nuclei in 30 hours) without cytokinesis, resulting in a multinucleate mother cell. Schizogony ends by the budding off of uninucleate daughter merozoites inside the vacuole. Merozoites egress from hepatocytes and invade erythrocytes. b | Stages of growth arrest of various live attenuated parasites (LAP). Growth of radiation-attenuated sporozoites (RAS) is arrested when DNA replication starts (at 20–24 hours after infection, midway through development), although depending on the irradiation dose, some nuclear division might continue. For genetically attenuated parasites (GAP), depletion of P36 and/or P36p (called P52 in some Plasmodium species) prevents the formation and/or maintenance of the parasitophorous vacuole membrane (PVM), causing very early liver-stage arrest, whereas depletion of UIS3 or UIS4 (which are lipid scavengers inserted in the PVM) causes rapid liver-stage elimination. Depletion of the apicoplast-localized pyruvate dehydrogenase (PDH; an enzyme that catalyses the conversion of pyruvate to acetyl-CoA) or FAB enzymes (type II fatty acid biosynthesis enzymes; that is, FABB/F, FABG, FABZ/A and FABI) causes mid-to-late liver-stage arrest, before merozoite formation and merozoite surface protein 1 (MSP1) expression. Conditional depletion of cyclic GMP-dependent protein kinase (PKG) in liver stages results in a very late liver-stage block, preventing the egress of fully formed merozoites. For drug-arrested parasites (DAP), the antifolate pyrimethamine and the 8-aminoquinoline primaquine both avert nuclear division of liver stages, causing mid liver-stage arrest. Azithromycin and clindamycin, which are macrolide and lincosamide antibiotics, respectively, inhibit apicoplast biogenesis in the liver stage; development is not arrested, but gives rise to non-infectious merozoites, blocked midway during their intra-erythrocytic growth (called the 'delayed death phenotype'). Chloroquine, a 4-aminoquinoline, and mefloquine, a 4-methanolquinoline, do not affect liver-stage growth and specifically inhibit haemozoin formation in infected erythrocytes, preventing the growth of intra-erythrocytic asexual parasites.

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The basis of protection induced by RAS has been studied extensively in rodents. Protection efficiency seems to depend on the host–parasite combination, varying according to both the susceptibility of the host to infection and the ability of the host to mount effective immune responses101,102. Work in the 1980s first revealed an important role for CSP-specific antibodies, which immobilize sporozoites, in RAS-induced protection103,104. It was subsequently acknowledged that CD8+ T cells, which recognize parasite epitopes associated with MHC class I on the surface of infected hepatocytes, play a major part in killing infected hepatocytes105,106,107,108, although CD4+ T cells have also been implicated109,110. How these protective CD8+ T cells kill liver-stage parasites is still unclear. These cells use various cytotoxic effectors, such as perforin, FAS ligand, granzymes, interferon-γ (IFNγ) and TNF, which are expressed in response to inducible nitric oxide synthase (iNOS), but parasite-specific CD8+ T cells lacking IFNγ, perforin and FAS ligand can still eliminate liver-stage parasites111,112. The recent direct observation of CD8+ T cells killing P. yoelii liver-stage parasites in vivo showed that parasite-specific CD8+ T cells recruit both specific and nonspecific T cells to the infected hepatocyte, leading to multiple phenotypes of parasite death113. Therefore, liver-stage elimination seems to depend on the cooperative activity of multiple CD8+ T cells and probably also other cell types, and to involve diverse and functionally redundant cytotoxic effectors.

Historically, LAP have mainly served as models to guide the development of subunit vaccination approaches. However, the subunit vaccines developed so far have not matched the protective efficacy of LAP; this is also true for the most advanced vaccine, RTS,S, which is composed of B cell and T cell epitopes of P. falciparum CSP in hepatitis B virus-like particles114. The idea that protective immunity against PE stages would require the induction of immune responses to a broad range of parasite antigens has consequently become increasingly accepted, and it might be that only whole-parasite vaccines will be able to achieve this115. In recent years, various LAP have entered small-scale human clinical studies, and an important goal is now to establish whether it is possible to formulate LAP for large-scale vaccination116,117. In early research with RAS in humans, 14 volunteers were immunized via bites from more than 1,000 irradiated infected mosquitoes over more than ten occasions, and 13 of these volunteers were found to be protected against sporozoite challenge79. More encouragingly, volunteers exposed to just 15 infectious mosquito bites on each of three occasions and kept under chloroquine cover (see below) were protected for a prolonged period98,99. This compares somewhat favourably with RTS,S, which gives 32–85% protection under similar laboratory challenge conditions118,119,120. No LAP have yet been tested under stringent field conditions, whereas RTS,S has been reproducibly shown to confer protection against clinical disease, although it has not exhibited any ability to block infection by parasites121,122,123.

Protective forms of LAP. A crucial issue is to determine which forms of the parasite are responsible for inducing LAP-mediated protection. Early evidence indicated a role for the liver stages rather than sporozoites, when it was discovered that RAS invade hepatocytes normally but stop developing at the onset of nuclear division124, roughly midway through liver-stage development (Fig. 4). Reports that protection depends on the sporozoite irradiation dose125,126, with the most protective dose allowing several nuclear divisions, further suggested that late arrest of liver-stage maturation confers better protection than earlier arrest. LAP models based on drug-arrested parasites (DAP) recently confirmed this idea. Intravenous injection of normal sporozoites into mice treated with chloroquine92,93,94 or mefloquine95, which have no effect on parasites in the liver but kill erythrocytic stages, can protect the host; compared with the number of RAS required for protection, fewer of these fully developing DAP are required for protection against subsequent challenge. Similar results have been obtained for treatment with clindamycin or azithromycin96,97, which interfere with the development of liver-stage parasites and render hepatic merozoites non-infective. As mentioned above, protection can be induced by just 45 immunizing mosquito bites delivering infectious sporozoites to humans under chloroquine cover98,99, whereas >1,000 bites delivering RAS alone were required for protection79. Remarkably, protection in normal sporozoite-treated volunteers is long-lasting, with four out of six treated volunteers still protected after 2 years, and this protection seems to be correlated with parasite-specific, multifunctional T effector memory cells99. Although after immunization with wild-type sporozoites under chloroquine cover the parasites eventually reach erythrocytes, which might induce blood-stage immunity127, it was recently shown that this DAP model essentially confers PE immunity, as it protects against sporozoite, but not blood-stage, challenge128.

The direct demonstration that late-blocked liver stages induce better protection than RAS in the absence of any blood-stage contribution came from studies on genetically attenuated parasites (GAP) in mice (Fig. 4). The first-generation GAP lack genes crucial to the biogenesis or maintenance of the parasitophorous vacuole membrane80,81,82,83,84,85,86. These parasites are blocked soon after sporozoite invasion and provide protection similar to or lower than that conferred by RAS. The discovery of the essential role of the apicoplast-resident fatty acid synthesis type II (FAS II)-dependent pathway during late liver-stage development129 inspired the creation of the second-generation GAP, which are deficient in the FAS II pathway87,88,89,90. P. yoelii GAP lacking FABB/F, an enzyme involved in the FAS II pathway, are blocked later than RAS but before merozoite formation90. After intravenous immunization, they induce broader and more protective CD8+ T cell responses than RAS, involving a larger fraction of effector memory CD8+ T cells, as well as some degree of cross-stage protection against challenge with blood-stage parasites90. This superior protection could be due to the expression of antigens specific to late liver-stage parasites, directing stronger anti-liver-stage protection, or due to the expression of antigens shared with erythrocytic merozoites, potentially providing protection against both blood-stage and liver-stage parasites (Fig. 4). A further block in GAP development has been achieved by disruption of the cyclic GMP-dependent protein kinase, specifically preventing merozoite release from the infected hepatocyte91. Given that liver-stage development follows large transcriptomic changes in the parasite, GAP arrested at different developmental stages express different and unique sets of genes; for example, 480 genes are co-expressed by sporozoites and merozoites130, whereas 770 transcripts are differentially expressed in mid (24 hour) versus mid-to-late (40–50 hour) P. yoelii liver-stage infection131 (schematically corresponding to RAS and the FABB/F-null GAP, respectively).

LAP delivery into the skin. Studies involving late-arresting parasites have highlighted a role for late liver-stage antigens in protection after intravenous injection of LAP; however, work using RAS has shown that immune responses following inoculation into the skin are largely induced in skin-draining lymph nodes132,133. The development of a protective CD8+ T cell response after RAS delivery into the skin was dissected using transgenic CD8+ T cells specific for an epitope of P. yoelii CSP (CSP-Tg)134. After RAS injection into the ear via syringe or mosquito, CSP-Tg CD8+ T cells are primed in the draining lymph node of the ear and, to a lesser extent, in the spleen, before migrating to the liver and other lymphoid organs, but are minimally activated in the draining lymph nodes of the liver132. Moreover, surgical ablation of the lymph node draining the injection site drastically decreases the number of activated CD8+ T cells in the liver132. It was also shown that in the draining lymph node, sporozoite antigen is cross-presented by DCs to naive CD8+ T cells to induce the protective T cell response135,136. Interestingly, antigen continues to be presented for up to 2 months following immunization via the skin with RAS or GAP, and this persistent antigen presentation is crucial for the complete development and expansion of protective CD8+ T cell responses90,137.

Which parasite forms induce protective responses in the skin-draining lymph node remains unclear. Because dead (heat-inactivated) sporozoites left in the skin are not protective138, the protective material might be sporozoites that actively reach the draining lymph node of the skin32 or those that develop inside skin cells72, and proteins that are continuously shed by gliding and cell-traversing sporozoites in the skin and/or draining lymph node51 might also play a part. Importantly, if the immunogenic forms are those sporozoites that reach the draining lymph node and develop there to only a limited extent32, then late-blocked GAP might not be more protective than early-blocked GAP when injected into the skin.

Another question concerns the protective efficacy of LAP injection into the skin. As mentioned above, all LAP studies carried out so far in humans have delivered the LAP to the skin via mosquitoes79. One exception has been the use of cryopreserved RAS, which did not protect after intradermal injection116, but provided protection after intravenous injection139. Recent work in rodents has yielded contradictory results. One study reported lower protective efficacy after intradermal compared with intravenous immunization with P. berghei RAS or DAP140. However, other studies using P. yoelii DAP95 and RAS141 have recorded similar protection levels for intradermal and intravenous immunization. Some researchers who used P. yoelii95 or P. berghei140,142 have found the parasite load in the liver to be lower after intradermal versus intravenous injection of RAS, suggesting that protective efficacy is related to the ability of sporozoites to reach the liver116,34. By contrast, others using P. yoelii sporozoites have noted that the parasite load in the liver does not depend on the injection route34.

These discrepancies are likely to be due, at least in part, to different intradermal injection procedures and to the volumes used, as sporozoites display reduced motility in, and reduced capacity to leave, flooded skin. Therefore, more work is needed in experimental models to optimize LAP immunogenicity after inoculation into the skin (Box 2).

Protective antigens. To be protective, vaccine-induced CD8+ T cells must eliminate liver-stage parasites that are developing inside hepatocytes. Liver stages express a distinct, though overlapping, repertoire of genes compared with sporozoites131. Thus, RAS might fail to elicit responses to all the potential protective antigens and, at the same time, might induce responses to irrelevant antigens that are not present in infected hepatocytes. Little is known about parasite epitope presentation by hepatocytes. CSP presentation is proteasome- and TAP-dependent136,143; that is, the antigen must be in the hepatocyte cytosol to be presented. This indicates that proteins which are secreted across the parasitophorous vacuole membrane by developing parasites might be crucial protective antigens, and proteins that are deposited by traversing sporozoites might also be presented by cells that recover from the traversal event. Mechanisms of protein export across the parasitophorous vacuole membrane in the infected erythrocyte are becoming increasingly well defined144,145. By contrast, protein secretion in the hepatocyte cytosol has received less attention. CSP bears a so-called PEXEL motif, which is present in many exported proteins in the erythrocyte; however, although this motif was found to be important for CSP translocation into the hepatocyte cytosol146, it has no effect on the ability of CSP-specific CD8+ T cells to recognize and kill parasites136. The basis of liver-stage protein translocation into the hepatocyte cytosol and entry into the MHC class I presentation pathway are clearly important areas for future investigation.

Conclusions and perspectives

Recent research into the in vivo fate of Plasmodium spp. sporozoites in mice has changed our understanding of the PE phase of mammalian malaria. In mice, the skin injection site and its draining lymph node, where the majority of parasites remain, are integral components of the PE phase and the site of the first parasite encounter with the host immune system. The most pressing question is whether the same tripartite picture of the initial PE phase — in which sporozoites can remain in the skin or exit via the draining lymph node or the bloodstream — holds true in humans. There are many unknown factors concerning sporozoite infection in the rodent host, including the molecular basis of the tripartite fate of sporozoites inoculated into the skin and of sporozoite retention in the liver, the functional importance (if any) of epidermal and hair follicle-associated parasite subpopulations, and the fate and possible subversion of traversed host cells.

Much remains to be done if we are to understand the host responses that are generated by injection of sporozoites into the skin (Box 2). Research in this area will be facilitated by the experimental accessibility of the skin and draining lymph nodes, particularly in respect of the powerful immuno-imaging approaches that are now available, as well as by recent progress in understanding skin immunity147. The initial aims are the identification of the host cell subset (or subsets) involved in antigen capture and in priming the protective CD8+ T cells in the draining lymph node, as well as the determination of which LAP forms, whether actively reaching the draining lymph node or remaining in the skin, cause protection. It will also be important to investigate the interaction with, and potential subversion of, regulatory T cells. These cells are abundant in the epidermis of human skin, and sporozoites glide extensively in the epidermis in rodents. A recent meta-analysis of >1,900 immunization studies led to the prediction that live sporozoites in the skin induce the expansion of antigen-specific regulatory T cells that ensure re-infection and systematically deactivate, rather than boost, vaccine-induced immune responses to skin-derived antigens148.

The ultimate question is whether a PE vaccine could be deployed on a large scale. LAP approaches are efficient, but their implementation still requires major technical and logistical hurdles to be overcome149 (Box 3). Regardless, LAP-based studies remain valuable and might lead to the identification of specific sets of protective antigens. In the meantime, efforts to develop subunit vaccines, primarily virus vectored, have yielded promising results150. For example, immunization against epitopes from two sporozoite proteins using prime–boost strategies based on adenovirus and modified vaccinia Ankara (MVA) virus confers potent protection and elicits a prolonged effector memory response in the liver151. Until now, subunit vaccinology has focused on just a few antigens, and mainly CSP. Although CSP is clearly a major protective antigen following RAS vaccination152, it is also obvious that CD8+ T cell-mediated protection can be mounted independently of CSP153. An important priority for future research is therefore to identify new protective T cell antigens that possibly elicit greater protection than CSP. The recent technical breakthroughs in the malaria research field, from genome sequencing to cellular imaging, now make it possible to envisage systematic approaches towards achieving this goal154.

Change history

  • 08 October 2013

    In Box 3 of the above article (page 709) the sentence “Recently, it was found that five intravenous injections of >105 cryopreserved RAS protected all six tested patients against a challenge by bites of five mosquitoes 3 weeks after the last immunization dose, whereas four intravenous immunizations protected only three of nine patients139” should have read “Recently, it was found that five intravenous injections of >105 cryopreserved RAS protected all six tested patients against a challenge by bites of five mosquitoes 3 weeks after the last immunization dose, whereas four intravenous immunizations protected only six of nine patients139.” The authors apologize to readers for any misunderstanding caused. This has now been corrected online.

References

  1. 1

    Carter, R. & Mendis, K. N. Evolutionary and historical aspects of the burden of malaria. Clin. Microbiol. Rev. 15, 564–594 (2002).

    PubMed  PubMed Central  Article  Google Scholar 

  2. 2

    WHO. Malaria fact sheet. WHO Media Center [online], (2013).

  3. 3

    Mbogo, C. M. et al. Spatial and temporal heterogeneity of Anopheles mosquitoes and Plasmodium falciparum transmission along the Kenyan coast. Am. J. Trop. Med. Hyg. 68, 734–742 (2003).

    Article  PubMed  Google Scholar 

  4. 4

    Mulligan, H. W., Russell, P. F. & Mohan, B. N. Active immunization of fowls against Plasmodium gallinaceum by injections of killed homologous sporozoites. J. Malaria Inst. India 4, 25–34 (1941).

    Google Scholar 

  5. 5

    Nussenzweig, R. S., Vanderberg, J., Most, H. & Orton, C. Protective immunity produced by the injection of X-irradiated sporozoites of Plasmodium berghei. Nature 216, 160–162 (1967). The first vaccination of mice against sporozoite challenge.

    CAS  Article  PubMed  Google Scholar 

  6. 6

    Clyde, D. F., Most, H., McCarthy, V. C. & Vanderberg, J. P. Immunization of man against sporozoite-induced falciparum malaria. Am. J. Med. Sci. 266, 169–177 (1973). The first vaccination of humans against malaria.

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Laveran, A. Un nouveau parasite trouvé dans le sang des malades atteints de fièvre palustre: origine parasitaire des accidents de l'impaludisme. Bull. Mem. Soc. Med. Hop. Paris 17, 158–164 (in French) (1881).

    Google Scholar 

  8. 8

    Ross, R. On some peculiar pigmented cells found in two mosquitos fed on malaria blood. Br. Med. J. 2, 1786–1788 (1897).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  9. 9

    Grassi, B., Bignami, A. & Bastianelli, G. Ulteriori ricerche sul ciclo dei parassiti malarici umani nel corpo del zanzarone. Atti R. Accad. Lincei 8, 21–28 (1899) (in Italian).

    Google Scholar 

  10. 10

    Huff, C. G. Life cycle of malarial parasites. Annu. Rev. Microbiol. 1, 43–60 (1947).

    Article  Google Scholar 

  11. 11

    Shortt, H. E., Garnham, P. C. C. & Malamos, B. The pre-erythrocytic stage of mammalian malaria. Br. Med. J. 1, 192–194 (1948). The first demonstration that sporozoites of Plasmodium spp. which infect mammals transform in the liver.

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  12. 12

    Krotoski, W. A. et al. Demonstration of hypnozoites in sporozoite-transmitted Plasmodium vivax infection. Am. J. Trop. Med. Hyg. 31, 1291–1293 (1982).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Markus, M. B. The hypnozoite concept, with particular reference to malaria. Parasitol. Res. 108, 247–252 (2011).

    Article  PubMed  Google Scholar 

  14. 14

    Markus, M. B. Dormancy in mammalian malaria. Trends Parasitol. 28, 39–45 (2012).

    Article  PubMed  Google Scholar 

  15. 15

    Vincke, I. H. & Lips, M. Un nouveau Plasmodium d'un rongeur sauvage du Congo: Plasmodium berghei n. sp. Ann. Soc. Belge Med. Trop. 28, 97–104 (1948) (in French).

    CAS  PubMed  Google Scholar 

  16. 16

    Yoeli, M., Most, H. & Boné, G. Plasmodium berghei: cyclical transmissions by experimentally infected Anopheles quadrimaculatus. Science 144, 1580–1581 (1964).

    CAS  Article  PubMed  Google Scholar 

  17. 17

    Yoeli, M., Vanderberg, J., Upmanis, R. S. & Most, H. Primary tissue phase of Plasmodium berghei in different experimental hosts. Nature 208, 903 (1965). The first observation of P. berghei schizonts in the liver of rodents.

    CAS  Article  PubMed  Google Scholar 

  18. 18

    van Dijk, M. R., Janse, C. J. & Waters, A. P. Expression of a Plasmodium gene introduced into subtelomeric regions of Plasmodium berghei chromosomes. Science 271, 662–665 (1996). The first genetic transformation by homologous recombination in P. berghei erythrocytic stages.

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Vanderberg, J. P. Studies on the motility of Plasmodium sporozoites. J. Protozool. 21, 527–537 (1974).

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Vanderberg, J. P., Chew, S. & Stewart, M. J. Plasmodium sporozoite interactions with macrophages in vitro: a videomicroscopic analysis. J. Protozool. 37, 528–536 (1990). The first observation of Plasmodium sp.sporozoites traversing host cells in vitro.

    CAS  Article  PubMed  Google Scholar 

  21. 21

    Mota, M. M. et al. Migration of Plasmodium sporozoites through cells before infection. Science 291, 141–144 (2001). A study that shows the membrane-wounding activity of Plasmodium spp. sporozoites.

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Hollingdale, M. R., Leef, J. L., McCullough, M. & Beaudoin, R. L. In vitro cultivation of the exoerythrocytic stage of Plasmodium berghei from sporozoites. Science 213, 1021–1022 (1981).

    CAS  Article  PubMed  Google Scholar 

  23. 23

    Mazier, D. et al. Complete development of hepatic stages of Plasmodium falciparum in vitro. Science 227, 440–442 (1985).

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Ponnudurai, T., Lensen, A. H., van Gemert, G. J., Bolmer, M. G. & Meuwissen, J. H. Feeding behaviour and sporozoite ejection by infected Anopheles stephensi. Trans. R. Soc. Trop. Med. Hyg. 85, 175–180 (1991).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Medica, D. L. & Sinnis, P. Quantitative dynamics of Plasmodium yoelii sporozoite transmission by infected anopheline mosquitoes. Infect. Immun. 73, 4363–4369 (2005).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  26. 26

    Beier, J. C., Davis, J. R., Vaughan, J. A., Noden, B. H. & Beier, M. S. Quantitation of Plasmodium falciparum sporozoites transmitted in vitro by experimentally infected Anopheles gambiae and Anopheles stephensi. Am. J. Trop. Med. Hyg. 44, 564–570 (1991).

    CAS  Article  PubMed  Google Scholar 

  27. 27

    Frischknecht, F. et al. Imaging movement of malaria parasites during transmission by Anopheles mosquitoes. Cell. Microbiol. 6, 687–694 (2004).

    CAS  Article  PubMed  Google Scholar 

  28. 28

    Jin, Y., Kebaier, C. & Vanderberg, J. Direct microscopic quantification of dynamics of Plasmodium berghei sporozoite transmission from mosquitoes to mice. Infect. Immun. 75, 5532–5539 (2007).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  29. 29

    Sidjanski, S. & Vanderberg, J. P. Delayed migration of Plasmodium sporozoites from the mosquito bite site to the blood. Am. J. Trop. Med. Hyg. 57, 426–429 (1997). Work showing that most sporozoites are deposited into the skin when a mosquito bites.

    CAS  Article  PubMed  Google Scholar 

  30. 30

    Matsuoka, H., Yoshida, S., Hirai, M. & Ishii, A. A rodent malaria, Plasmodium berghei, is experimentally transmitted to mice by merely probing of infective mosquito, Anopheles stephensi. Parasitol. Int. 51, 17–23 (2002).

    Article  PubMed  Google Scholar 

  31. 31

    Vanderberg, J. P. & Frevert, U. Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes. Int. J. Parasitol. 34, 991–996 (2004). The first imaging of sporozoite gliding motility in the skin of mice.

    Article  PubMed  Google Scholar 

  32. 32

    Amino, R. et al. Quantitative imaging of Plasmodium transmission from mosquito to mammal. Nature Med. 12, 220–224 (2006). An article describing the tripartite fate of sporozoites inoculated into the skin.

    CAS  Article  PubMed  Google Scholar 

  33. 33

    Amino, R. et al. Imaging malaria sporozoites in the dermis of the mammalian host. Nature Protoc. 2, 1705–1712 (2007).

    CAS  Article  Google Scholar 

  34. 34

    Yamauchi, L. M., Coppi, A., Snounou, G. & Sinnis, P. Plasmodium sporozoites trickle out of the injection site. Cell. Microbiol. 9, 1215–1222 (2007).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  35. 35

    Xu, W.-Y., Wang, X.-X., Qi, J., Duan, J.-H. & Huang, F.-S. Plasmodium yoelii: influence of immune modulators on the development of the liver stage. Exp. Parasitol. 126, 254–258 (2010).

    CAS  Article  Google Scholar 

  36. 36

    Ellis, J. et al. Cloning and expression in E. coli of the malarial sporozoite surface antigen gene from Plasmodium knowlesi. Nature 302, 536–538 (1983). The first cloning of a Plasmodium spp. gene.

    CAS  Article  PubMed  Google Scholar 

  37. 37

    Pradel, G., Garapaty, S. & Frevert, U. Proteoglycans mediate malaria sporozoite targeting to the liver. Mol. Microbiol. 45, 637–651 (2002).

    CAS  Article  PubMed  Google Scholar 

  38. 38

    Suarez, J. E. et al. Plasmodium falciparum circumsporozoite (CS) protein peptides specifically bind to HepG2 cells. Vaccine 19, 4487–4495 (2001).

    CAS  Article  PubMed  Google Scholar 

  39. 39

    Rathore, D., Sacci, J. B., de la Vega, P. & McCutchan, T. F. Binding and invasion of liver cells by Plasmodium falciparum sporozoites. Essential involvement of the amino terminus of circumsporozoite protein. J. Biol. Chem. 277, 7092–7098 (2002).

    CAS  Article  PubMed  Google Scholar 

  40. 40

    Cerami, C. et al. The basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of Plasmodium falciparum sporozoites. Cell 70, 1021–1033 (1992).

    CAS  Article  PubMed  Google Scholar 

  41. 41

    Frevert, U. et al. Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes. J. Exp. Med. 177, 1287–1298 (1993).

    CAS  Article  PubMed  Google Scholar 

  42. 42

    Sinnis, P. et al. Structural and functional properties of region II-plus of the malaria circumsporozoite protein. J. Exp. Med. 180, 297–306 (1994).

    CAS  Article  PubMed  Google Scholar 

  43. 43

    Coppi, A. et al. The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host. J. Exp. Med. 208, 341–356 (2011).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  44. 44

    Frevert, U. et al. Intravital observation of Plasmodium berghei sporozoite infection of the liver. PLoS Biol. 3, e192 (2005).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  45. 45

    Mota, M. M., Hafalla, J. C. R. & Rodriguez, A. Migration through host cells activates Plasmodium sporozoites for infection. Nature Med. 8, 1318–1322 (2002).

    CAS  Article  PubMed  Google Scholar 

  46. 46

    Carrolo, M. et al. Hepatocyte growth factor and its receptor are required for malaria infection. Nature Med. 9, 1363–1369 (2003).

    CAS  Article  PubMed  Google Scholar 

  47. 47

    Kaushansky, A. & Kappe, S. H. I. The crucial role of hepatocyte growth factor receptor during liver-stage infection is not conserved among Plasmodium species. Nature Med. 17, 1180–1181 (2011).

    CAS  Article  PubMed  Google Scholar 

  48. 48

    Ishino, T., Yano, K., Chinzei, Y. & Yuda, M. Cell-passage activity is required for the malarial parasite to cross the liver sinusoidal cell layer. PLoS Biol. 2, e4 (2004). The generation of the first Plasmodium spp. sporozoites deficient in CT.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  49. 49

    Ishino, T., Chinzei, Y. & Yuda, M. A. Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection. Cell. Microbiol. 7, 199–208 (2005).

    CAS  Article  PubMed  Google Scholar 

  50. 50

    Yuda, M. & Ishino, T. Liver invasion by malarial parasites — how do malarial parasites break through the host barrier? Cell. Microbiol. 6, 1119–1125 (2004).

    CAS  Article  PubMed  Google Scholar 

  51. 51

    Amino, R. et al. Host cell traversal is important for progression of the malaria parasite through the dermis to the liver. Cell Host Microbe 3, 88–96 (2008).

    CAS  Article  PubMed  Google Scholar 

  52. 52

    Tavares, J. et al. Role of host cell traversal by the malaria sporozoite during liver infection. J. Exp. Med. 210, 905–915 (2013).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  53. 53

    Pradel, G. & Frevert, U. Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion. Hepatology 33, 1154–1165 (2001).

    CAS  Article  PubMed  Google Scholar 

  54. 54

    Baer, K. et al. Kupffer cells are obligatory for Plasmodium yoelii sporozoite infection of the liver. Cell. Microbiol. 9, 397–412 (2007).

    CAS  Article  PubMed  Google Scholar 

  55. 55

    Coppi, A. et al. Heparan sulfate proteoglycans provide a signal to Plasmodium sporozoites to stop migrating and productively invade host cells. Cell Host Microbe 2, 316–327 (2007).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  56. 56

    Frevert, U., Sinnis, P., Esko, J. D. & Nussenzweig, V. Cell surface glycosaminoglycans are not obligatory for Plasmodium berghei sporozoite invasion in vitro. Mol. Biochem. Parasitol. 76, 257–266 (1996).

    CAS  Article  PubMed  Google Scholar 

  57. 57

    Coppens, I., Sullivan, D. J. & Prigge, S. T. An update on the rapid advances in malaria parasite cell biology. Trends Parasitol. 26, 305–310 (2010).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  58. 58

    Spielmann, T., Montagna, G. N., Hecht, L. & Matuschewski, K. Molecular make-up of the Plasmodium parasitophorous vacuolar membrane. Int. J. Med. Microbiol. 302, 179–186 (2012).

    CAS  Article  PubMed  Google Scholar 

  59. 59

    Graewe, S., Stanway, R. R., Rennenberg, A. & Heussler, V. T. Chronicle of a death foretold: Plasmodium liver stage parasites decide on the fate of the host cell. FEMS Microbiol. Rev. 36, 111–130 (2012).

    CAS  Article  PubMed  Google Scholar 

  60. 60

    Lim, L. & McFadden, G. I. The evolution, metabolism and functions of the apicoplast. Phil. Trans. R. Soc. 365, 749–763 (2010).

    CAS  Article  Google Scholar 

  61. 61

    Van de Sand, C. et al. The liver stage of Plasmodium berghei inhibits host cell apoptosis. Mol. Microbiol. 58, 731–742 (2005).

    CAS  Article  PubMed  Google Scholar 

  62. 62

    Rennenberg, A. et al. Exoerythrocytic Plasmodium parasites secrete a cysteine protease inhibitor involved in sporozoite invasion and capable of blocking cell death of host hepatocytes. PLoS Pathog. 6, e1000825 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  63. 63

    Kaushansky, A. et al. Suppression of host p53 is critical for Plasmodium liver-stage infection. Cell Rep. 3, 630–637 (2013).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  64. 64

    Sturm, A. et al. Alteration of the parasite plasma membrane and the parasitophorous vacuole membrane during exo-erythrocytic development of malaria parasites. Protist 160, 51–63 (2009).

    Article  PubMed  Google Scholar 

  65. 65

    Schmidt-Christensen, A., Sturm, A., Horstmann, S. & Heussler, V. T. Expression and processing of Plasmodium berghei SERA3 during liver stages. Cell. Microbiol. 10, 1723–1734 (2008).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  66. 66

    Graewe, S. et al. Hostile takeover by Plasmodium: reorganization of parasite and host cell membranes during liver stage egress. PLoS Pathog. 7, e1002224 (2011).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  67. 67

    Frevert, U. Sneaking in through the back entrance: the biology of malaria liver stages. Trends Parasitol. 20, 417–424 (2004).

    CAS  Article  PubMed  Google Scholar 

  68. 68

    Sturm, A. et al. Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids. Science 313, 1287–1290 (2006). The first observation of hepatic merozoite release via merosomes.

    CAS  Article  PubMed  Google Scholar 

  69. 69

    Thiberge, S. et al. In vivo imaging of malaria parasites in the murine liver. Nature Protoc. 2, 1811–1818 (2007).

    CAS  Article  Google Scholar 

  70. 70

    Vaughan, A. M. et al. Complete Plasmodium falciparum liver-stage development in liver-chimeric mice. J. Clin. Invest. 122, 3618–3628 (2012).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  71. 71

    Baer, K., Klotz, C., Kappe, S. H. I., Schnieder, T. & Frevert, U. Release of hepatic Plasmodium yoelii merozoites into the pulmonary microvasculature. PLoS Pathog. 3, e171 (2007).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  72. 72

    Gueirard, P. et al. Development of the malaria parasite in the skin of the mammalian host. Proc. Natl Acad. Sci. USA 107, 18640–18645 (2010). The first observation of merozoite formation in the skin of mice.

    CAS  Article  PubMed  Google Scholar 

  73. 73

    Voza, T., Miller, J. L., Kappe, S. H. I. & Sinnis, P. Extrahepatic exoerythrocytic forms of rodent malaria parasites at the site of inoculation: clearance after immunization, susceptibility to primaquine, and contribution to blood-stage infection. Infect. Immun. 80, 2158–2164 (2012).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  74. 74

    Mellor, A. L. & Munn, D. H. Immune privilege: a recurrent theme in immunoregulation? Immunol. Rev. 213, 5–11 (2006).

    Article  Google Scholar 

  75. 75

    Frevert, U., Späth, G. F. & Yee, H. Exoerythrocytic development of Plasmodium gallinaceum in the White Leghorn chicken. Int. J. Parasitol. 38, 655–672 (2008).

    CAS  Article  PubMed  Google Scholar 

  76. 76

    Silvie, O. et al. Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity. Nature Med. 9, 93–96 (2003).

    CAS  Article  PubMed  Google Scholar 

  77. 77

    Boyd, M. F. & Kitchen, S. F. The demonstration of sporozoites in human tissues. Am. J. Trop. Med. Hyg. 19, 27–31 (1939).

    Article  Google Scholar 

  78. 78

    Offeddu, V., Thathy, V., Marsh, K. & Matuschewski, K. Naturally acquired immune responses against Plasmodium falciparum sporozoites and liver infection. Int. J. Parasitol. 42, 535–548 (2012).

    CAS  Article  PubMed  Google Scholar 

  79. 79

    Hoffman, S. L. et al. Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J. Infect. Dis. 185, 1155–1164 (2002).

    Article  PubMed  Google Scholar 

  80. 80

    Mueller, A.-K., Labaied, M., Kappe, S. H. I. & Matuschewski, K. Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature 433, 164–167 (2005). The first GAP-induced protection against sporozoite challenge in mice.

    CAS  Article  PubMed  Google Scholar 

  81. 81

    Mueller, A.-K. et al. Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface. Proc. Natl Acad. Sci. USA 102, 3022–3027 (2005).

    CAS  Article  PubMed  Google Scholar 

  82. 82

    van Dijk, M. R. et al. Genetically attenuated, 36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells. Proc. Natl Acad. Sci. USA 102, 12194–12199 (2005).

    CAS  Article  PubMed  Google Scholar 

  83. 83

    Labaied, M. et al. Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection. Infect. Immun. 75, 3758–3768 (2007).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  84. 84

    Jobe, O. et al. Genetically attenuated Plasmodium berghei liver stages induce sterile protracted protection that is mediated by major histocompatibility complex class I-dependent interferon-γ-producing CD8+ T cells. J. Infect. Dis. 196, 599–607 (2007).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  85. 85

    Mueller, A.-K. et al. Genetically attenuated Plasmodium berghei liver stages persist and elicit sterile protection primarily via CD8 T cells. Am. J. Pathol. 171, 107–115 (2007).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  86. 86

    Tarun, A. S. et al. Protracted sterile protection with Plasmodium yoelii pre-erythrocytic genetically attenuated parasite malaria vaccines is independent of significant liver-stage persistence and is mediated by CD8+ T cells. J. Infect. Dis. 196, 608–616 (2007).

    CAS  Article  PubMed  Google Scholar 

  87. 87

    Vaughan, A. M. et al. Type II fatty acid synthesis is essential only for malaria parasite late liver stage development. Cell. Microbiol. 11, 506–520 (2009).

    CAS  Article  PubMed  Google Scholar 

  88. 88

    Pei, Y. et al. Plasmodium pyruvate dehydrogenase activity is only essential for the parasite's progression from liver infection to blood infection. Mol. Microbiol. 75, 957–971 (2010).

    CAS  Article  PubMed  Google Scholar 

  89. 89

    Haussig, J. M., Matuschewski, K. & Kooij, T. W. A. Inactivation of a Plasmodium apicoplast protein attenuates formation of liver merozoites. Mol. Microbiol. 81, 1511–1525 (2011).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  90. 90

    Butler, N. S. et al. Superior antimalarial immunity after vaccination with late liver stage-arresting genetically attenuated parasites. Cell Host Microbe 9, 451–462 (2011). Work showing that late-arrested liver-stage GAP protect better than RAS.

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  91. 91

    Falae, A. et al. Role of Plasmodium berghei cGMP-dependent protein kinase in late liver stage development. J. Biol. Chem. 285, 3282–3288 (2010).

    CAS  Article  PubMed  Google Scholar 

  92. 92

    Beaudoin, R. L., Strome, C. P. A., Mitchell, F. & Tubergen, T. A. Plasmodium berghei: immunization of mice against the ANKA strain using the unaltered sporozoite as an antigen. Exp. Parasitol. 42, 1–5 (1977). The first use of DAP to protect against malaria in rodents.

    CAS  Article  PubMed  Google Scholar 

  93. 93

    Orjih, A. U., Cochrane, A. H. & Nussenzweig, R. S. Comparative studies on the immunogenicity of infective and attenuated sporozoites of Plasmodium berghei. Trans. R. Soc. Trop. Med. Hyg. 76, 57–61 (1982).

    CAS  Article  PubMed  Google Scholar 

  94. 94

    Belnoue, E. et al. Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment. J. Immunol. 172, 2487–2495 (2004).

    CAS  Article  PubMed  Google Scholar 

  95. 95

    Inoue, M. & Culleton, R. L. The intradermal route for inoculation of sporozoites of rodent malaria parasites for immunological studies. Parasite Immunol. 33, 137–142 (2011).

    CAS  Article  PubMed  Google Scholar 

  96. 96

    Friesen, F. et al. Natural immunization against malaria: causal prophylaxis with antibiotics. Sci. Transl. Med. 14, 40ra49 (2010).

    Google Scholar 

  97. 97

    Friesen, J. & Matuschewski, K. Comparative efficacy of pre-erythrocytic whole organism vaccine strategies against the malaria parasite. Vaccine 29, 7002–7008 (2011).

    CAS  Article  PubMed  Google Scholar 

  98. 98

    Roestenberg, M. et al. Protection against a malaria challenge by sporozoite inoculation. N. Engl. J. Med. 361, 468–477 (2009). Validation of the DAP concept in humans.

    CAS  Article  PubMed  Google Scholar 

  99. 99

    Roestenberg, M. et al. Long-term protection against malaria after experimental sporozoite inoculation: an open-label follow-up study. Lancet 377, 1770–1776 (2011).

    CAS  Article  PubMed  Google Scholar 

  100. 100

    Purcell, L. A. et al. Chemically attenuated Plasmodium sporozoites induce specific immune responses, sterile immunity and cross-protection against heterologous challenge. Vaccine 26, 4880–4884 (2008).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  101. 101

    Ngonseu, E., Chatterjee, S. & Wery, M. Blocked hepatic-stage parasites and decreased susceptibility to Plasmodium berghei infections in BALB/c mice. Parasitology 117, 419–423 (1998).

    Article  PubMed  Google Scholar 

  102. 102

    Schmidt, N. W., Butler, N. S. & Harty, J. T. Plasmodium–host interactions directly influence the threshold of memory CD8 T cells required for protective immunity. J. Immunol. 186, 5873–5884 (2011).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  103. 103

    Yoshida, N., Nussenzweig, R. S., Potocnjak, P., Nussenzweig, V. & Aikawa, M. Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science 207, 71–73 (1980). The first demonstration that antibodies to CSP neutralize infectivity.

    CAS  Article  PubMed  Google Scholar 

  104. 104

    Stewart, M. J., Nawrot, R. J., Schulman, S. & Vanderberg, J. P. Plasmodium berghei sporozoite invasion is blocked in vitro by sporozoite-immobilizing antibodies. Infect. Immun. 51, 859–864 (1986).

    PubMed  PubMed Central  CAS  Google Scholar 

  105. 105

    Schofield, L. et al. γ-interferon, CD8+ T cells and antibodies required for immunity to malaria sporozoites. Nature 330, 664–666 (1987). The first evidence that CD8+ T cells are important for RAS-induced protection in mice.

    CAS  Article  PubMed  Google Scholar 

  106. 106

    Weiss, W. R., Sedegah, M., Beaudoin, R. L., Miller, L. H. & Good, M. F. CD8+ T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites. Proc. Natl Acad. Sci. USA 85, 573–576 (1988).

    CAS  Article  PubMed  Google Scholar 

  107. 107

    Romero, P. et al. Cloned cytotoxic T cells recognize an epitope in the circumsporozoite protein and protect against malaria. Nature 341, 323–326 (1989).

    CAS  Article  PubMed  Google Scholar 

  108. 108

    Rodrigues, M. M. et al. CD8+ cytolytic T cell clones derived against the Plasmodium yoelii circumsporozoite protein protect against malaria. Int. Immunol. 3, 579–585 (1991).

    CAS  Article  PubMed  Google Scholar 

  109. 109

    Tsuji, M., Romero, P., Nussenzweig, R. S. & Zavala, F. CD4+ cytolytic T cell clone confers protection against murine malaria. J. Exp. Med. 172, 1353–1357 (1990).

    CAS  Article  PubMed  Google Scholar 

  110. 110

    Rénia, L. et al. Effector functions of circumsporozoite peptide-primed CD4+ T cell clones against Plasmodium yoelii liver stages. J. Immunol. 150, 1471–1478 (1993).

    PubMed  Google Scholar 

  111. 111

    Chakravarty, S., Baldeviano, G. C., Overstreet, M. G. & Zavala, F. Effector CD8+ T lymphocytes against liver stages of Plasmodium yoelii do not require γ interferon for antiparasite activity. Infect. Immun. 76, 3628–3631 (2008).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  112. 112

    Butler, N. S., Schmidt, N. W. & Harty, J. T. Differential effector pathways regulate memory CD8 T cell immunity against Plasmodium berghei versus P. yoelii sporozoites. J. Immunol. 184, 2528–2538 (2010).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  113. 113

    Cockburn, I. A. et al. In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages. Proc. Natl Acad. Sci. USA 110, 9090–9095 (2013). The first In vivo imaging of pathogen killing by CD8+ T cells.

    CAS  Article  PubMed  Google Scholar 

  114. 114

    Cohen, J., Nussenzweig, V., Nussenzweig, R., Vekemans, J. & Leach, A. From the circumsporozoite protein to the RTS, S/AS candidate vaccine. Hum. Vaccines 6, 90–96 (2010).

    CAS  Article  Google Scholar 

  115. 115

    Luke, T. C. & Hoffman, S. L. Rationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine. J. Exp. Biol. 206, 3803–3808 (2003).

    Article  PubMed  Google Scholar 

  116. 116

    Epstein, J. E. et al. Live attenuated malaria vaccine designed to protect through hepatic CD8+ T cell immunity. Science 334, 475–480 (2011).

    CAS  Article  PubMed  Google Scholar 

  117. 117

    Nganou-Makamdop, K. & Sauerwein, R. W. Liver or blood-stage arrest during malaria sporozoite immunization: the later the better? Trends Parasitol. 29, 304–310 (2013).

    Article  PubMed  Google Scholar 

  118. 118

    Stoute, J. A. et al. A preliminary evaluation of a recombinant circumsporozoite protein vaccine against Plasmodium falciparum malaria. N. Engl. J. Med. 336, 86–91 (1997).

    CAS  Article  PubMed  Google Scholar 

  119. 119

    Kester, K. E. et al. Phase 2a trial of 0, 1, and 3 month and 0, 7, and 28 day immunization schedules of malaria vaccine RTS,S/AS02 in malaria-naïve adults at the Walter Reed Army Institute of Research. Vaccine 26, 2191–2202 (2008).

    CAS  Article  PubMed  Google Scholar 

  120. 120

    Kester, K. E. et al. Randomized, double-blind, phase 2a trial of falciparum malaria vaccines RTS,S/AS01B and RTS, S/AS02A in malaria-naive adults: safety, efficacy, and immunologic associates of protection. J. Infect. Dis. 200, 337–346 (2009).

    CAS  Article  PubMed  Google Scholar 

  121. 121

    Agnandji, S. T. et al. First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children. N. Engl. J. Med. 365, 1863–1875 (2011).

    Article  PubMed  Google Scholar 

  122. 122

    Nussenzweig, V., Good, M. F. & Hill, A. V. S. Mixed results for a malaria vaccine. Nature Med. 17, 1560–1561 (2011).

    Article  CAS  PubMed  Google Scholar 

  123. 123

    Olotu, A. et al. Four-year efficacy of RTS, S/AS01E and its interaction with malaria exposure. N. Engl. J. Med. 368, 1111–1120 (2013).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  124. 124

    Mellouk, S., Lunel, F., Sedegah, M., Beaudoin, R. & Druilhe, P. Protection against malaria induced by irradiated sporozoites. Lancet 335, 721 (1990).

    CAS  Article  PubMed  Google Scholar 

  125. 125

    Chatterjee, S., François, G., Druilhe, P., Timperman, G. & Wéry, M. Immunity to Plasmodium berghei exoerythrocytic forms derived from irradiated sporozoites. Parasitol. Res. 82, 297–303 (1996).

    CAS  Article  PubMed  Google Scholar 

  126. 126

    Silvie, O. et al. Effects of irradiation on Plasmodium falciparum sporozoite hepatic development: implications for the design of pre-erythrocytic malaria vaccines. Parasite Immunol. 24, 221–223 (2002).

    CAS  Article  PubMed  Google Scholar 

  127. 127

    Pombo, D. J. et al. Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum. Lancet 360, 610–617 (2002).

    Article  PubMed  Google Scholar 

  128. 128

    Bijker, E. M. et al. Protection against malaria after immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity. Proc. Natl Acad. Sci. USA 110, 7862–7867 (2013).

    CAS  Article  PubMed  Google Scholar 

  129. 129

    Yu, M. et al. The fatty acid biosynthesis enzyme FabI plays a key role in the development of liver-stage malarial parasites. Cell Host Microbe 4, 567–578 (2008). Work showing an essential role for the parasite apicoplast in the late phase of liver-stage development.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  130. 130

    Lasonder, E. et al. Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity. PLoS Pathog. 4, e1000195 (2008).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  131. 131

    Tarun, A. S. et al. A combined transcriptome and proteome survey of malaria parasite liver stages. Proc. Natl Acad. Sci. USA 105, 305–310 (2008).

    CAS  Article  Google Scholar 

  132. 132

    Chakravarty, S. et al. CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes. Nature Med. 13, 1035–1041 (2007). A study which finds that protective immune responses to sporozoites are generated in skin-draining lymph nodes.

    CAS  Article  PubMed  Google Scholar 

  133. 133

    Obeid, M. et al. Skin-draining lymph node priming is sufficient to induce sterile immunity against pre-erythrocytic malaria. EMBO Mol. Med. 5, 250–263 (2013).

    CAS  Article  PubMed  Google Scholar 

  134. 134

    Sano, G. et al. Swift development of protective effector functions in naive CD8+ T cells against malaria liver stages. J. Exp. Med. 194, 173–179 (2001).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  135. 135

    Jung, S. et al. In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity 17, 211–220 (2002).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  136. 136

    Cockburn, I. A. et al. Dendritic cells and hepatocytes use distinct pathways to process protective antigen from Plasmodium in vivo. PLoS Pathog. 7, e1001318 (2011). The PEXEL motif is shown to be dispensable for presentation of the CSP antigen.

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  137. 137

    Cockburn, I. A. et al. Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites. PLoS Pathog. 6, e1000877 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  138. 138

    Hafalla, J. C. R. et al. Priming of CD8+ T cell responses following immunization with heat-killed Plasmodium sporozoites. Eur. J. Immunol. 36, 1179–1186 (2006).

    CAS  Article  PubMed  Google Scholar 

  139. 139

    Seder, R. A. et al. Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science http://dx.doi.org/10.1126/science.1241800 (2013). The first demonstrated protection of humans by parenteral injection of RAS.

  140. 140

    Nganou-Makamdop, K. et al. Reduced Plasmodium berghei sporozoite liver load associates with low protective efficacy after intradermal immunization. Parasite Immunol. 34, 562–569 (2012).

    CAS  Article  PubMed  Google Scholar 

  141. 141

    Voza, T., Kebaier, C. & Vanderberg, J. P. Intradermal immunization of mice with radiation-attenuated sporozoites of Plasmodium yoelii induces effective protective immunity. Malaria J. 9, 362 (2010).

    Article  Google Scholar 

  142. 142

    Ploemen, I. H. et al. Plasmodium liver load following parenteral sporozoite administration in rodents. Vaccine 31, 3410–3416 (2013).

    Article  PubMed  Google Scholar 

  143. 143

    Bongfen, S. E., Torgler, R., Romero, J. F., Rénia, L. & Corradin, G. Plasmodium berghei-infected primary hepatocytes process and present the circumsporozoite protein to specific CD8+ T cells in vitro. J. Immunol. 178, 7054–7063 (2007).

    CAS  Article  PubMed  Google Scholar 

  144. 144

    Spielmann, T. & Gilberger, T.-W. Protein export in malaria parasites: do multiple export motifs add up to multiple export pathways? Trends Parasitol. 26, 6–10 (2010).

    CAS  Article  PubMed  Google Scholar 

  145. 145

    Grüring, C. et al. Uncovering common principles in protein export of malaria parasites. Cell Host Microbe 12, 717–729 (2012).

    Article  CAS  PubMed  Google Scholar 

  146. 146

    Singh, A. P. et al. Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite. Cell 131, 492–504 (2007).

    CAS  Article  PubMed  Google Scholar 

  147. 147

    Ginhoux, F., Ng, L. G. & Merad, M. Understanding the murine cutaneous dendritic cell network to improve intradermal vaccination strategies. Curr. Top. Microbiol. Immunol. 351, 1–24 (2012).

    CAS  PubMed  Google Scholar 

  148. 148

    Guilbride, D. L., Gawlinski, P. & Guilbride, P. D. L. Why functional pre-erythrocytic and bloodstage malaria vaccines fail: a meta-analysis of fully protective immunizations and novel immunological model. PLoS ONE 5, e10685 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  149. 149

    Druilhe, P. & Barnwell, J. W. Pre-erythrocytic stage malaria vaccines: time for a change in path. Curr. Opin. Microbiol. 10, 371–378 (2007).

    CAS  Article  PubMed  Google Scholar 

  150. 150

    Hill, A. V. S. et al. Prime-boost vectored malaria vaccines: progress and prospects. Hum. Vaccines 6, 78–83 (2010).

    CAS  Article  Google Scholar 

  151. 151

    Reyes-Sandoval, A. et al. CD8+ T effector memory cells protect against liver-stage malaria. J. Immunol. 187, 1347–1357 (2011).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  152. 152

    Kumar, K. A. et al. The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites. Nature 444, 937–940 (2006).

    CAS  Article  PubMed  Google Scholar 

  153. 153

    Mauduit, M. et al. Minimal role for the circumsporozoite protein in the induction of sterile immunity by vaccination with live rodent malaria sporozoites. Infect. Immun. 78, 2182–2188 (2010).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  154. 154

    Murphy, S. C., Kas, A., Stone, B. C. & Bevan, M. J. A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations. Proc. Natl Acad. Sci. USA 110, 6055–6060 (2013).

    CAS  Article  PubMed  Google Scholar 

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Glossary

Sterilizing immunity

Immunity resulting in parasite clearance from the host.

Schizonts

Multinucleate, intracellular parasite stages originating from a single organism that reproduces by schizogony. Schizonts contain many individual merozoites when mature.

Hypnozoites

Dormant, non-dividing, intrahepatocytic forms of certain Plasmodium species, including Plasmodium vivax and Plasmodium ovale, which infect humans.

Recurrences

New phases of parasite multiplication inside erythrocytes. These recurrences originate from intra-erythrocytic parasite forms (recrudescence) or hypnozoites (relapse).

Gliding

A substrate-dependent type of unicellular motility defined by the lack of cell deformation during movement.

Parasitophorous vacuole

The vacuole inside which the parasite resides on host cell entry and throughout intracellular development.

Stellate cells

Pericytes that are located between a hepatocyte and a sinusoidal endothelial cell; also called Ito cells.

Kupffer cell

A resident macrophage in the liver; these cells mostly double-line the endothelial cell wall inside the sinusoid lumen.

Apicoplast

A relict, non-photosynthetic plastid-like organelle of red-algal origin, inherited from secondary endosymbiosis.

Immunoprivileged sites

Sites in the mammalian body that are characterized by the lack of major histocompatibility complex expression, the absence of phagocytic and antigen-presenting cells, and a potent immunosuppressant microenvironment.

Subunit vaccination

Vaccination using material that is distinct from live parasites (typically, recombinant proteins or virus-vectored antigens).

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Ménard, R., Tavares, J., Cockburn, I. et al. Looking under the skin: the first steps in malarial infection and immunity. Nat Rev Microbiol 11, 701–712 (2013). https://doi.org/10.1038/nrmicro3111

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