Malaria parasite pre-erythrocytic stage infection: gliding and hiding

In malaria, the red blood cell-infectious form of the Plasmodium parasite causes illness and the possible death of infected hosts. The initial infection in the liver caused by the mosquito-borne sporozoite parasite stage, however, causes little pathology and no symptoms. Nevertheless, pre-erythrocytic parasite stages are attracting passionate research efforts not least because they are the most promising targets for malaria vaccine development. Here, we review how the infectious sporozoite makes its way to the liver and subsequently develops within hepatocytes. We discuss the factors, both parasite and host, involved in the interactions that occur during this "silent" phase of infection.     

Immunity to malaria.

Malaria remains prevalent throughout tropical and subtropical regions and almost a third of the World's population is exposed to the risk of infection. There is currently a serious resurgence of the disease in Asia and Central America. The failure of global eradication measures based upon the use of insecticides and chemotherapy has resulted from difficulties of practical implementation compounded by the spread of insecticide and drug resistance. Repeated natural infection does not produce detectable resistance to the exo-erythrocytic cycle of malaria in man. Irradiated sporzoite vaccines do, however, induce stage specific immunity in murine malaria and in a proportion of human subjects. Vaccinated individuals remain susceptible to blood stage infection which causes clinical malaria. In addition the vaccine is unstable and must be administered by intravenous inoculation. Since neither sporogonic nor exo-erythrocytic parasite development is cyclical in human malarias, there is little prospect for vaccine production through cultivation of these stages. The inhabitants of hyperendaemic areas become increasingly resistant to malaria during childhood and adolescence, through the slow development of specific, acquired immunity to asexual blood stage parasites. Immunity is mediated by antibody, which blocks merozoite invasion of red cells, as well as by cell mediated mechanisms and non-specific cytotoxic agents. Vaccination with merozoites induces long lasting immunity of broad serological specificity active against the blood-stage of the parasite. Merozoite vaccines can be preserved by freeze drying and harvested from continuous cultures of blood stage parasites. The major problem in development of a human merozoite vaccine concerns the requirement for Freund's complete adjuvant which is not acceptable for man. The effective immunity induced by vaccination contrasts with the slow development of incomplete resistance which follows repeated natural infection. The latter is associated with the generation of immune suppressor cells, lymphoid cell mitogens and soluble antigens, and in some species by the occurrence of antigenic variation--all of which may favour parasite survival. It is probable that vaccination with non-viable antigen of appropriate composition, induces immune effector processes without activating mechanisms which allow parasites to escape the consequences of immunity. Many effective vaccines such as those against measles, poliomyelitis, tetanus and rabies are commercially available but barely used in the developing world. The affected nations cannot afford their purchase, nor do the means exist for their distribution. It follows that if a safe and effective malaria vaccine were to be developed, its bulk manufacture and administration would require massive international support and cooperation.     

Malaria invasion of human erythrocytes

The invasion of human red blood cells (RBC) by plosmodiol merozoites is a key event during malaria infection, and the inhibition o f invasion is regarded as a crucial goal of malaria vaccine development. For Plasmodium falciparum it has been suggested that the red cell sialoglycoproteins, glycophorins A, B and C, are receptors for invasion and that O-linked or N-linked carbohydrate structures may be involved as receptor sites(1-3). However, recent evidence suggests that the role o f these sialoglycoproteins and carbohydrates may have been overestimated. In this article, Peter Hermentin discusses the contradictory findings and presents a revised model for the invasion process.     

Optical imaging techniques for the study of malaria

Malarial infection needs to be imaged to reveal the mechanisms behind malaria pathophysiology and to provide insights to aid in the diagnosis of the disease. Recent advances in optical imaging methods are now being transferred from physics laboratories to the biological field, revolutionizing how we study malaria. To provide insight into how these imaging techniques can improve the study and treatment of malaria, we summarize recent progress on optical imaging techniques, ranging from in vitro visualization of the disease progression of malaria infected red blood cells (iRBCs) to in vivo imaging of malaria parasites in the liver.     

A retrospective evaluation of the role of T cells in the development of malaria vaccine

Due to the fact that the life cycle of malaria parasites is complex, undergoing both an extracellular and intracellular phases in its host, the human immune system has to mobilize both the humoral and cellular arms of immune responses to fight against this parasitic infection. Whereas humoral immunity is directed toward the extracellular stages which include sporozoites and merozoites, cell-mediated immunity (CMI), in which T cells play a major role, targets hepatic stages - liver stages - of the parasites. In this review, the role of T cells in protective immunity against liver stages of the malaria infection is being re-evaluated. Furthermore, this review intends to address how to translate the findings regarding the role of T cells obtained in experimental systems to actual development of malaria vaccine for humans.     

How many pathways for invasion of the red blood cell by the malaria parasite?

Merozoites of the malaria parasite Plasmodium falciparum use several receptors for cellular engagement when they invade human red blood cells. Recently, a merozoite erythrocyte-binding protein, EBA-140, has been identified that specifically binds to glycophorin C on red blood cells. Up to 50% of Melanesians have a deletion in this gene, and the resultant Gerbich-negative red blood cells are relatively resistant to invasion. While discovery of multiple pathways for invasion could confound the search for suitable vaccine targets, they could also be considered in the design of therapeutic interventions that prevent malaria parasites entering red blood cells.     

The Plasmodium falciparum-infected red blood cell

Plasmodium falciparum, the most virulent of the human malaria parasites, causes up to one million deaths per year. The parasite spends part of its lifecycle inside the red blood cells (RBCs) of its host. As it grows it ingests the RBC cytoplasm, digesting it in an acidic vacuole. Free haem released during haemoglobin digestion is detoxified by conversion to inert crystals of haemozoin. Malaria pathology is evident during the blood stage of the infection and is exacerbated by adhesion of infected RBCs to blood vessel walls, which prevents splenic clearance of the infected cells. Cytoadherence is mediated by surface-exposed virulence proteins that bind to endothelial cell receptors. These 'adhesins' are exported to the RBC surface via an exomembrane system that is established outside the parasite in the host cell cytoplasm. Antimalarial drugs that interfere with haem detoxification, or target other parasite-specific processes, have been effective in the treatment of malaria, but their use has been dogged by the development of resistance. Similarly, efforts to develop an effective blood vaccine are hindered by the variability of surface-exposed antigens.     

DNA vaccine against malaria: a long way to go

Vaccination is the attempt to mimic certain aspects of an infection for the purpose of causing an immune response that will protect the individual from that infection. Malaria, a disease responsible for immense human suffering, is caused by infection with Plasmodium spp. parasites, which have a very complex life cycle--antigenically unique stages infect different tissues of the body. It is a parasitic disease for which no successful vaccine has been developed so far, despite considerable efforts to develop a subunit vaccine that offers protective immunity. Due to the spread of drug-resistant malaria, efforts to develop an effective vaccine have become increasingly critical. DNA vaccination provides a stable and long-lived source of protein vaccine capable of inducing both antibody- and cell-mediated immune responses to a wide variety of antigens. Injected DNA enters the cells of the host and makes the protein, which triggers the immune response. According to present needs, the flexibility of DNA vaccine technology permits the combination of multiple antigens from both the preerythrocytic and erythrocytic stages of malaria parasite. DNA vaccines with genes coding for different antigenic parts of malaria proteins have been created and presently some of these are undergoing field trials. The results from these trials will help to determine the likelihood of success of this technology in humans. This review presents an update of the studies carried out in malaria using DNA vaccine approach, the challenges, and the future prospects.     

Malaria parasite pre-erythrocytic infection: preparation meets opportunity

For those stricken with malaria, the classic clinical symptoms are caused by the parasite's cyclic infection of red blood cells. However, this erythrocytic phase of the parasite's life cycle initiates from an asymptomatic pre-erythrocytic phase: the injection of sporozoites via the bite of a parasite-carrying Anopheline mosquito, and the ensuing infection of the liver. With the increased capabilities of studying liver stages in mice, much progress has been made elucidating the cellular and molecular basis of the parasite's progression through this bottleneck of its life cycle. Here we review relevant findings on how sporozoites prepare for infection of the liver and factors crucial to liver stage development as well as key host/parasite interactions.     

Low red cell production may protect against severe anemia during a malaria infection—Insights from modeling

The malaria parasite causes lysis of red blood cells, resulting in anemia, a major cause of mortality and morbidity. Intuitively, one would expect the production of red blood cells to increase in order to compensate for this loss. However, it has been observed that this response is weaker than would be expected. Furthermore, iron supplementation for iron deficient children in malaria endemic regions can paradoxically adversely affect the clinical outcome of malaria infection. A possible explanation may lie in the preference that some malaria parasites show for infecting immature red blood cells (reticulocytes). In the presence of a parasite preference for immature red cells, a rise in red cell production can ‘fuel the fire’ of infection by increasing the availability of the parasite's preferred target cell.We present a mathematical model of red blood cell production and infection in order to explore this hypothesis. We assess the effect of varying the reticulocyte replacement rate and preference of the parasite for reticulocytes on four key outcome measures assessing anemia and parasitemia.For a given level of parasite preference for reticulocytes we uncover an optimal erythropoietic response which minimizes disease severity. Increasing red blood cell production much above this optimum confers no benefit to the patient, and in fact can increase the degree of anemia and parasitemia. These conclusions are consistent with epidemiological studies demonstrating that both iron deficiency and anemia are protective against severe malaria, whilst iron supplementation in malaria endemic regions is with an increased number of malaria related adverse effects. Thus, suppression of red blood cell production, rather than being an unfortunate side effect of inflammation, may be a host protective effect against severe malarial anemia.     

Towards a blood-stage vaccine for malaria: are we following all the leads?

Although the malaria parasite was discovered more than 120 years ago, it is only during the past 20 years, following the cloning of malaria genes, that we have been able to think rationally about vaccine design and development. Effective vaccines for malaria could interrupt the life cycle of the parasite at different stages in the human host or in the mosquito. The purpose of this review is to outline the challenges we face in developing a vaccine that will limit growth of the parasite during the stage within red blood cells--the stage responsible for all the symptoms and pathology of malaria. More than 15 vaccine trials have either been completed or are in progress, and many more are planned. Success in current trials could lead to a vaccine capable of saving more than 2 million lives per year.     

DNA Vaccine Against Malaria: A Long Way To Go

Vaccination is the attempt to mimic certain aspects of an infection for the purpose of causing an immune response that will protect the individual from that infection. Malaria, a disease responsible for immense human suffering, is caused by infection with Plasmodium spp. parasites, which have a very complex life cycle — antigenically unique stages infect different tissues of the body. It is a parasitic disease for which no successful vaccine has been developed so far, despite considerable efforts to develop a subunit vaccine that offers protective immunity. Due to the spread of drug-resistant malaria, efforts to develop an effective vaccine have become increasingly critical. DNA vaccination provides a stable and long-lived source of protein vaccine capable of inducing both antibody- and cell-mediated immune responses to a wide variety of antigens. Injected DNA enters the cells of the host and makes the protein, which triggers the immune response. According to present needs, the flexibility of DNA vaccine technology permits the combination of multiple antigens from both the preerythrocytic and erythrocytic stages of malaria parasite. DNA vaccines with genes coding for different antigenic parts of malaria proteins have been created and presently some of these are undergoing field trials. The results from these trials will help to determine the likelihood of success of this technology in humans. This review presents an update of the studies carried out in malaria using DNA vaccine approach, the challenges, and the future prospects.     

A clash to conquer: the malaria parasite liver infection

All mammalian malaria parasite species have an initial tissue stage in liver cells. The liver stage produces new parasite forms that can enter and live inside red blood cells. Accordingly, the first place of residence provides parasites with a radically different cellular and molecular environment from their subsequent red blood cell home. Liver stages have remained refractory to reveal their secrets, yet the last few years have seen several advances in elucidating their biology. This review looks at the more recent findings concerning the liver stage-host hepatocyte association, some of which may become powerful weapons in the prevention of malaria infection. We also outline areas of liver stage research and technological development that provide promising foci to accelerate a better understanding of this most elusive of the parasites many life cycle stages.     

The exported Plasmodium berghei protein IBIS1 delineates membranous structures in infected red blood cells

The importance of pathogen-induced host cell remodelling has been well established for red blood cell infection by the human malaria parasite Plasmodium falciparum. Exported parasite-encoded proteins, which often possess a signature motif, termed Plasmodium export element (PEXEL) or host-targeting (HT) signal, are critical for the extensive red blood cell modifications. To what extent remodelling of erythrocyte membranes also occurs in non-primate hosts and whether it is in fact a hallmark of all mammalian Plasmodium parasites remains elusive. Here we characterize a novel Plasmodium berghei PEXEL/HT-containing protein, which we term IBIS1. Temporal expression and spatial localization determined by fluorescent tagging revealed the presence of IBIS1 at the parasite/host interface during both liver and blood stages of infection. Targeted deletion of the IBIS1 protein revealed a mild impairment of intra-erythrocytic growth indicating a role for these structures in the rapid expansion of the parasite population in the blood in vivo. In red blood cells, the protein localizes to dynamic, punctate structures external to the parasite. Biochemical and microscopic data revealed that these intra-erythrocytic P. berghei-induced structures (IBIS) are membranous indicating that P. berghei, like P. falciparum, creates an intracellular membranous network in infected red blood cells.     

An exported kinase (FIKK4.2) that mediates virulence-associated changes in Plasmodium falciparum-infected red blood cells

Alteration of the adhesive and mechanical properties of red blood cells caused by infection with the malaria parasite Plasmodium falciparum underpin both its survival and extreme pathogenicity. A unique family of parasite putative exported kinases, collectively called FIKK (Phenylalanine (F) – Isoleucine (I) – Lysine (K) – Lysine (K)), has recently been implicated in these pathophysiological processes, however, their precise function in P. falciparum-infected red blood cells or their likely role in malaria pathogenesis remain unknown. Here, for the first time, we demonstrate that one member of the FIKK family, FIKK4.2, can function as an active kinase and is localised in a novel and distinct compartment of the parasite-infected red blood cell which we have called K-dots. Notably, targeted disruption of the gene encoding FIKK4.2 (fikk4.2) dramatically alters the parasite’s ability to modify and remodel the red blood cells in which it multiplies. Specifically, red blood cells infected with fikk4.2 knockout parasites were significantly less rigid and less adhesive when compared with red blood cells infected with normal parasites from which the transgenic clones had been derived, despite expressing similar levels of the major cytoadhesion ligand, PfEMP1, on the red blood cell surface. Notably, these changes were accompanied by dramatically altered knob-structures on infected red blood cells that play a key role in cytoadhesion which is responsible for much of the pathogenesis associated with falciparum malaria. Taken together, our data identifies FIKK4.2 as an important kinase in the pathogenesis of P. falciparum malaria and strengthens the attractiveness of FIKK kinases as targets for the development of novel next-generation anti-malaria drugs.     

Periodicity and synchronization in blood-stage malaria infection

Malaria fever is highly periodic and is associated with the parasite replication cycles in red blood cells. The existence of periodicity in malaria infection demonstrates that parasite replication in different red blood cells is synchronized. In this article, rigorous mathematical analysis of an age-structured human malaria model of infected red blood cells (Rouzine and McKenzie, Proc Natl Acad Sci USA 100:3473–3478, 2003) is provided and the synchronization of Plasmodium falciparum erythrocytic stages is investigated. By using the replication rate as the bifurcation parameter, the existence of Hopf bifurcation in the age-structured malaria infection model is obtained. Numerical simulations indicate that synchronization with regular periodic oscillations (of period 48 h) occurs when the replication rate increases. Therefore, Kwiatkowski and Nowak’s observation (Proc Natl Acad Sci USA 88:5111–5113, 1991) that synchronization could be generated at modest replication rates is confirmed.     

Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria.

In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weight ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection, in parallel to the parasitaemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced) stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced) triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that the polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.     

Development of full‐field optical spatial coherence tomography system for automated identification of malaria using the multilevel ensemble classifier

Malaria is a life‐threatening infectious blood disease affecting humans and other animals caused by parasitic protozoans belonging to the Plasmodium type especially in developing countries. The gold standard method for the detection of malaria is through the microscopic method of chemically treated blood smears. We developed an automated optical spatial coherence tomographic system using a machine learning approach for a fast identification of malaria cells. In this study, 28 samples (15 healthy, 13 malaria infected stages of red blood cells) were imaged by the developed system and 13 features were extracted. We designed a multilevel ensemble‐based classifier for the quantitative prediction of different stages of the malaria cells. The proposed classifier was used by repeating k‐fold cross validation dataset and achieve a high‐average accuracy of 97.9% for identifying malaria infected late trophozoite stage of cells. Overall, our proposed system and multilevel ensemble model has a substantial quantifiable potential to detect the different stages of malaria infection without staining or expert.      

Induction and maintenance of protective CD8+ T cells against malaria liver stages: implications for vaccine development

CD8+ T cells against malaria liver stages represent a major protective immune mechanism against infection. Following induction in the peripheral lymph nodes by dendritic cells (DCs), these CD8+ T cells migrate to the liver and eliminate parasite infected hepatocytes. The processing and presentation of sporozoite antigen requires TAP mediated transport of major histocompatibility complex class I epitopes to the endoplasmic reticulum. Importantly, in DCs this process is also dependent on endosome-mediated cross presentation while this mechanism is not required for epitope presentation on hepatocytes. Protective CD8+ T cell responses are strongly dependent on the presence of CD4+ T cells and the capacity of sporozoite antigen to persist for a prolonged period of time. While human trials with subunit vaccines capable of inducing antibodies and CD4+ T cell responses have yielded encouraging results, an effective anti-malaria vaccine will likely require vaccine constructs designed to induce protective CD8+ T cells against malaria liver stages.     

Plasmodium berghei XAT: protective 155/160 kDa antigens are located in parasitophorous vacuoles of schizont-stage parasite

Effective blood-stage malaria vaccine candidates have been mainly developed from the proteins in exposed locations on the parasite such as the surface of free merozoites or infected red blood cells. In the present study, we identified and localized novel protective antigens derived from the blood-stage of Plasmodium berghei XAT after establishment of hybridomas producing protective monoclonal antibodies (mAbs) against the parasites. The protective antigens were expressed in schizonts but not in trophozoites, and located in the parasitophorous vacuoles in the infected erythrocyte cytoplasm. The antigens, with molecular weight of 155/160 kDa, were not identical to any merozoite/schizont antigens that have been reported as target molecules recognized by mAbs developed to rodent malaria parasites. The characterization of new malarial antigenic targets of potentially protective antibody responses following infection would give us new insights for the selection of candidate antigens for malaria vaccine.