Plasmodium, the parasite that causes malaria, is transmitted by a mosquito into the dermis and must reach the liver before infecting erythrocytes and causing disease. We present here a quantitative, real-time analysis of the fate of parasites transmitted in a rodent system. We show that only a proportion of the parasites enter blood capillaries, whereas others are drained by lymphatics. Lymph sporozoites stop at the proximal lymph node, where most are degraded inside dendritic leucocytes, but some can partially differentiate into exoerythrocytic stages. This previously unrecognized step of the parasite life cycle could influence the immune response of the host, and may have implications for vaccination strategies against the preerythrocytic stages of the parasite.

The merozoite stage of the malaria parasite that infects erythrocytes and causes the symptoms of the disease is initially formed inside host hepatocytes. However, the mechanism by which hepatic merozoites reach blood vessels (sinusoids) in the liver and escape the host immune system before invading erythrocytes remains unknown. Here, we show that parasites induce the death and the detachment of their host hepatocytes, followed by the budding of parasite-filled vesicles (merosomes) into the sinusoid lumen. Parasites simultaneously inhibit the exposure of phosphatidylserine on the outer leaflet of host plasma membranes, which act as "eat me" signals to phagocytes. Thus, the hepatocyte-derived merosomes appear to ensure both the migration of parasites into the bloodstream and their protection from host immunity.

CD8 + T cells are specialized cells of the adaptive immune system capable of finding and eliminating pathogen-infected cells. To date it has not been possible to observe the destruction of any pathogen by CD8 + T cells in vivo. Here we demonstrate a technique for imaging the killing of liver-stage malaria parasites by CD8 + T cells bearing a transgenic T cell receptor specific for a parasite epitope. We report several features that have not been described by in vitro analysis of the process, chiefly the formation of large clusters of effector CD8 + T cells around infected hepatocytes. The formation of clusters requires antigen-specific CD8 + T cells and signaling by G protein-coupled receptors, although CD8 + T cells of unrelated specificity are also recruited to clusters. By combining mathematical modeling and data analysis, we suggest that formation of clusters is mainly driven by enhanced recruitment of T cells into larger clusters. We further show various death phenotypes of the parasite, which typically follow prolonged interactions between infected hepatocytes and CD8 + T cells. These findings stress the need for intravital imaging for dissecting the fine mechanisms of pathogen recognition and killing by CD8 + T cells.Plasmodium | immunity | lymphocytes

The malaria sporozoite, the parasite stage transmitted by the mosquito, is delivered into the dermis and differentiates in the liver. Motile sporozoites can invade host cells by disrupting their plasma membrane and migrating through them (termed cell traversal), or by forming a parasite-cell junction and settling inside an intracellular vacuole (termed cell infection). Traversal of liver cells, observed for sporozoites in vivo, is thought to activate the sporozoite for infection of a final hepatocyte. Here, using Plasmodium berghei, we show that cell traversal is important in the host dermis for preventing sporozoite destruction by phagocytes and arrest by nonphagocytic cells. We also show that cell infection is a pathway that is masked, rather than activated, by cell traversal. We propose that the cell traversal activity of the sporozoite must be turned on for progression to the liver parenchyma, where it must be switched off for infection of a final hepatocyte.

Highlights d The subdominant NVDP minor repeats of PfCSP are neutralizing epitopes d Potent mAbs with junctional and NANP cross-reactivity bind PfCSP in two steps d Cytotoxic PfCSP mAbs kill sporozoites in the liver d PfCSP mAbs prevent sporozoites from exiting liver sinusoids to infect hepatocytes

Malaria sporozoites cross the liver sinusoidal barrier, target Kupffer cells and endothelial cells with cell traversal inhibiting sporozoite clearance.

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.

The first step of Plasmodium development in vertebrates is the transformation of the sporozoite, the parasite stage injected by the mosquito in the skin, into merozoites, the stage that invades erythrocytes and initiates the disease. The current view is that, in mammals, this stage conversion occurs only inside hepatocytes. Here, we document the transformation of sporozoites of rodentinfecting Plasmodium into merozoites in the skin of mice. After mosquito bite, ∼50% of the parasites remain in the skin, and at 24 h ∼10% are developing in the epidermis and the dermis, as well as in the immunoprivileged hair follicles where they can survive for weeks. The parasite developmental pathway in skin cells, although frequently abortive, leads to the generation of merozoites that are infective to erythrocytes and are released via merosomes, as typically observed in the liver. Therefore, during malaria in rodents, the skin is not just the route to the liver but is also the final destination for many inoculated parasites, where they can differentiate into merozoites and possibly persist.intravital imaging | Plasmodium | schizogony M alarial infection starts with the inoculation of Plasmodium sporozoites by mosquitoes probing the vertebrate skin for blood. The highly motile sporozoites eventually invade host target cells where they differentiate and divide into numerous merozoites, the parasite form that invades erythrocytes and initiates the pathogenic phase of malarial infection. The host cell type in which sporozoites transform into merozoites, however, differs between Plasmodium species. In species that infect birds, sporozoites differentiate inside macrophages primarily in the skin but also in the spleen, liver, and bone marrow (1). In species that infect mammals, sporozoites are known to differentiate only inside hepatocytes in the liver (2-4).The first demonstration that sporozoites of mammal-infecting Plasmodium species develop inside hepatocytes was made in 1948 after i.v. inoculation of sporozoites of P. cynomologi into rhesus monkeys (2). In addition to reporting fully mature parasites inside hepatocytes, the authors also documented the persistence of immature and dormant forms of the parasite in the liver several months after the initial inoculation, which they proposed to be the cause of relapses (5), and were later called hypnozoites (6). Subsequent work indicated that sporozoites of species that infect humans (7) also undergo complete development inside hepatocytes.Since these early studies, P. berghei and the related P. yoelii species, which infect rodents, have been used as practical and safe models for studying the pre-erythrocytic phase of malaria. These parasites were shown to differentiate in the liver of laboratory rodents (8), and the P. berghei/rodent system was used to demonstrate that the majority of sporozoites were inoculated by mosquitoes in the skin rather than directly into the blood circulation (9), as traditionally assumed. More recently, the generation of fluorescent P. berghei parasites, alo...