Plasmodium fragile: detection of a ring-infected erythrocyte surface antigen (RESA)

A ring-infected erythrocyte surface antigen (RESA) has been detected by modified immunofluorescence assay in erythrocytes infected with the simian malaria parasite, Plasmodium fragile. This RESA, of Mr 95,000, shares many characteristics with the RESA initially found in the human malaria parasite P. falciparum. Both antigens are found in the membrane of erythrocytes infected with young asexual parasite stages, in merozoite-enriched preparations, and in parasite culture supernatant. Since the RESA of P. falciparum has been shown to confer protective immunity and since P. fragile infection of rhesus monkeys mimics P. falciparum infection in humans, the finding of a RESA in P. fragile underlines the importance of this species as an animal model for antimalarial vaccines.     

Short odds for malaria vaccines

The immunology of falciparum malaria, the lethal type of human malaria, has been transformed by two developments. First, a culture system for the asexual blood stages of Plasmodium falciparum.¹ Secondly, the cloning and expression of genes coding for a large number of the protein antigens of this malaria parasite over the past two years. Data on proteins, protein antigens and epitopes of P. falciparum supplied by gene cloning techniques have been supplemented by monoclonal antibody approaches, peptide synthesis, and high-resolution immunochemistry.     

A research agenda for malaria eradication: vaccines

Vaccines could be a crucial component of efforts to eradicate malaria. Current attempts to develop malaria vaccines are primarily focused on Plasmodium falciparum and are directed towards reducing morbidity and mortality. Continued support for these efforts is essential, but if malaria vaccines are to be used as part of a repertoire of tools for elimination or eradication of malaria, they will need to have an impact on malaria transmission. We introduce the concept of "vaccines that interrupt malaria transmission" (VIMT), which includes not only "classical" transmission-blocking vaccines that target the sexual and mosquito stages but also pre-erythrocytic and asexual stage vaccines that have an effect on transmission. VIMT may also include vaccines that target the vector to disrupt parasite development in the mosquito. Importantly, if eradication is to be achieved, malaria vaccine development efforts will need to target other malaria parasite species, especially Plasmodium vivax, where novel therapeutic vaccines against hypnozoites or preventive vaccines with effect against multiple stages could have enormous impact. A target product profile (TPP) for VIMT is proposed and a research agenda to address current knowledge gaps and develop tools necessary for design and development of VIMT is presented.     

The Quantity and Quality of African Children's IgG Responses to Merozoite Surface Antigens Reflect Protection against Plasmodium falciparum Malaria

Background

Antibodies, particularly cytophilic IgG subclasses, with specificity for asexual blood stage antigens of Plasmodium falciparum, are thought to play an important role in acquired immunity to malaria. Evaluating such responses in longitudinal sero-epidemiological field studies, allied to increasing knowledge of the immunological mechanisms associated with anti-malarial protection, will help in the development of malaria vaccines.

Methods and Findings

We conducted a 1-year follow-up study of 305 Senegalese children and identified those resistant or susceptible to malaria. In retrospective analyses we then compared post-follow-up IgG responses to six asexual-stage candidate malaria vaccine antigens in groups of individuals with clearly defined clinical and parasitological histories of infection with P. falciparum. In age-adjusted analyses, children resistant to malaria as well as to high-density parasitemia, had significantly higher IgG1 responses to GLURP and IgG3 responses to MSP2 than their susceptible counterparts. Among those resistant to malaria, high anti-MSP1 IgG1 levels were associated with protection against high-density parasitemia. To assess functional attributes, we used an in vitro parasite growth inhibition assay with purified IgG. Samples from individuals with high levels of IgG directed to MSP1, MSP2 and AMA1 gave the strongest parasite growth inhibition, but a marked age-related decline was observed in these effects.

Conclusion

Our data are consistent with the idea that protection against P. falciparum malaria in children depends on acquisition of a constellation of appropriate, functionally active IgG subclass responses directed to multiple asexual stage antigens. Our results suggest at least two distinct mechanisms via which antibodies may exert protective effects. Although declining with age, the growth inhibitory effects of purified IgG measurable in vitro reflected levels of anti-AMA1, -MSP1 and -MSP2, but not of anti-GLURP IgG. The latter could act on parasite growth via indirect parasiticidal pathways.     

Protection from experimental cerebral malaria with a single dose of radiation-attenuated, blood-stage Plasmodium berghei parasites

Background

Whole malaria parasites are highly effective in inducing immunity against malaria. Due to the limited success of subunit based vaccines in clinical studies, there has been a renewed interest in whole parasite-based malaria vaccines. Apart from attenuated sporozoites, there have also been efforts to use live asexual stage parasites as vaccine immunogens.

Methodology and Results

We used radiation exposure to attenuate the highly virulent asexual blood stages of the murine malaria parasite P. berghei to a non-replicable, avirulent form. We tested the ability of the attenuated blood stage parasites to induce immunity to parasitemia and the symptoms of severe malaria disease. Depending on the mouse genetic background, a single high dose immunization without adjuvant protected mice from parasitemia and severe disease (CD1 mice) or from experimental cerebral malaria (ECM) (C57BL/6 mice). A low dose immunization did not protect against parasitemia or severe disease in either model after one or two immunizations. The protection from ECM was associated with a parasite specific antibody response and also with a lower level of splenic parasite-specific IFN-γ production, which is a mediator of ECM pathology in C57BL/6 mice. Surprisingly, there was no difference in the sequestration of CD8+ T cells and CD45+ CD11b+ macrophages in the brains of immunized, ECM-protected mice.

Conclusions

This report further demonstrates the effectiveness of a whole parasite blood-stage vaccine in inducing immunity to malaria and explicitly demonstrates its effectiveness against ECM, the most pathogenic consequence of malaria infection. This experimental model will be important to explore the formulation of whole parasite blood-stage vaccines against malaria and to investigate the immune mechanisms that mediate protection against parasitemia and cerebral malaria.     

Functional analysis of Plasmodium falciparum merozoite antigens: implications for erythrocyte invasion and vaccine development

Malaria is a major human health problem and is responsible for over 2 million deaths per year. It is caused by a number of species of the genus Plasmodium, and Plasmodium falciparum is the causative agent of the most lethal form. Consequently, the development of a vaccine against this parasite is a priority. There are a number of stages of the parasite life cycle that are being targeted for the development of vaccines. Important candidate antigens include proteins on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to manipulate the genome of Plasmodium species has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. This has provided new information on the role of merozoite antigens in erythrocyte invasion and also allows new approaches to address their potential as vaccine candidates.     

Human immune responses against sexual stages of malaria parasites: considerations for malaria vaccines

Studies on the natural immune responses to the sexual stages of malaria parasites have been reviewed in the context of human malaria transmission-blocking vaccines. Antibodies against the sexual stages of the malaria parasite, gametocytes and gametes, are readily evoked by natural malaria infections. These antibodies that suppress infectivity at high concentrations can, at low concentrations, enhance the development of the parasite in the mosquito; however, because enhancing antibodies are prevalent during natural malaria infections, it is likely that a vaccine would rapidly boost these antibodies to blocking levels. The immunogenicity of sexual stage antigens appears to be constrained in the human host, probably due to T epitope polymorphism and MHC restriction in humans. These constraints apply mainly to those antigens that are sensitive targets of host immunity such as the gamete surface antigens and not to internal gamete antigens, indicating that antigenic polymorphism may have evolved in response to immune selection pressure. Evidence for immunosuppression of the host by exposure to endemic malaria is presented and its consequences on vaccine development are discussed.     

Plasmodium falciparum antigens on the surface of the gametocyte-infected erythrocyte

Background

The asexual blood stages of the human malaria parasite Plasmodium falciparum produce highly immunogenic polymorphic antigens that are expressed on the surface of the host cell. In contrast, few studies have examined the surface of the gametocyte-infected erythrocyte.

Methodology/Principal Findings

We used flow cytometry to detect antibodies recognising the surface of live cultured erythrocytes infected with gametocytes of P. falciparum strain 3D7 in the plasma of 200 Gambian children. The majority of children had been identified as carrying gametocytes after treatment for malaria, and each donated blood for mosquito-feeding experiments. None of the plasma recognised the surface of erythrocytes infected with developmental stages of gametocytes (I–IV), but 66 of 194 (34.0%) plasma contained IgG that recognised the surface of erythrocytes infected with mature (stage V) gametocytes. Thirty-four (17.0%) of 200 plasma tested recognised erythrocytes infected with trophozoites and schizonts, but there was no association with recognition of the surface of gametocyte-infected erythrocytes (odds ratio 1.08, 95% C.I. 0.434–2.57; P = 0.851). Plasma antibodies with the ability to recognise gametocyte surface antigens (GSA) were associated with the presence of antibodies that recognise the gamete antigen Pfs 230, but not Pfs48/45. Antibodies recognising GSA were associated with donors having lower gametocyte densities 4 weeks after antimalarial treatment.

Conclusions/Significance

We provide evidence that GSA are distinct from antigens detected on the surface of asexual 3D7 parasites. Our findings suggest a novel strategy for the development of transmission-blocking vaccines.     

Naturally acquired immune responses against Plasmodium falciparum sporozoites and liver infection

Malaria is a vector-borne infectious disease caused by infection with eukaryotic pathogens termed Plasmodium. Epidemiological hallmarks of Plasmodium falciparum malaria are continuous re-infections, over which time the human host may experience several clinical malaria episodes, slow acquisition of partial protection against infection, and its partial decay upon migration away from endemic regions. To overcome the exposure-dependence of naturally acquired immunity and rapidly elicit robust long-term protection are ultimate goals of malaria vaccine development. However, cellular and molecular correlates of naturally acquired immunity against either parasite infection or malarial disease remain elusive. Sero-epidemiological studies consistently suggest that acquired immunity is primarily directed against the asexual blood stages. Here, we review available data on the relationship between immune responses against the Anopheles mosquito-transmitted sporozoite and exo-erythrocytic liver stages and the incidence of malaria. We discuss current limitations and research opportunities, including the identification of additional sporozoite antigens and the use of systematic immune profiling and functional studies in longitudinal cohorts to look for pre-erythrocytic signatures of naturally acquired immunity.     

Mosquito-based transmission blocking vaccines for interrupting Plasmodium development

Reduction of transmission is critical for effective malaria control. Transmission blocking vaccines, which are intended to prevent the parasites from infecting the mosquito vectors, could target mosquito antigens that are required for the successful development of the parasite in its vector. Here we review recent advances in the identification of promising candidate antigens for a mosquito-based transmission blocking vaccine.     

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.     

Subtilisin-like proteases of the malaria parasite

Proteases play critical roles in the life cycle of the malaria parasite, Plasmodium spp. Within the asexual erythrocytic cycle, responsible for the clinical manifestations of malaria, substantial interest has focused on the role of parasite serine proteases as a result of indications that they are involved in red blood cell invasion. Over the past 6 years, three Plasmodium genes encoding serine proteases of the subtilisin-like clan, or subtilases, have been identified. All are expressed in the asexual blood stages and, in at least two cases, the gene products localize to secretory organelles of the invasive merozoite. They may have potential as novel drug targets. Here, we review progress in our understanding of the maturation, specificity, structure and function of these Plasmodium subtilases.     

Immunity to the liver stage of malaria

The search for subunit vaccines against malaria has concentrated on asexual and sexual blood stage and sporozoite antigens. In recent years the search for the basis of the protection against sporozoite challenge obtained in mice immunized with irradiated sporozoites has focused attention on the liver or exoerythrocytic (EE) stage of the malaria life cycle. Here, Andreas Suhrbier looks at the various immune responses that appear to be active against this stage, which was once thought to be immunologically insignificant. The liver stage of malaria has thus emerged as a legitimate target for vaccine development.     

Malaria vaccine development: current status

The development of an effective malaria vaccine represents one of the most important approaches that would provide a cost-effective intervention for addition to currently available malaria control strategies. Here, Howard Engers and Tore Godal review recent advances. Over the past decade there has been considerable progress in the understanding of immune mechanisms involved in conferring protection to malaria and in the identification of vaccine candidate antigens and their genes. Several new vaccines have entered Phase I/II trials recently, new adjuvants have been developed for human use and new approaches, such as DNA vaccines and structural modification of antigens to circumvent some of the strategies the parasite uses to avoid the immune response, are being applied. Thus, from the TDR perspective, global malaria vaccine development is entering a crucial period with unprecedented opportunities.     

Selective accumulation of a novel antimalarial rhodacyanine derivative, SSJ-127, in an organelle of Plasmodium berghei

SSJ-127, a novel antimalarial rhodacyanine derivative, has shown potent antimalarial activity against chloroquine-resistant Plasmodium strains in vitro and subcutaneous administration of SSJ-127 results in a complete cure of a mouse malaria model. SSJ-127 was detected by fluorescence microscopy in the mouse malaria parasites Plasmodium berghei after exposure of infected red blood cells to the compound in vitro and in vivo. Selective accumulation of SSJ-127 in an organelle is observed in all blood stages of live malaria parasites. The organelle is clearly different from the mitochondrion and the nucleus in terms of morphology. The shape of the organelle changed during the asexual blood stages of the parasite. There was always a close association between the organelle and the mitochondrion. These results raised the possibility that SSJ-127 accumulates in an apicoplast of the malaria parasite and affects protozoan parasite-specific pathways.     

The H89 cAMP-dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes.

In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor.Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria.     

Malaria parasites and red blood cells: from anaemia to transmission

Malaria parasites, Plasmodium spp., invade and exploit red blood cells during their asexual expansion within the vertebrate host. The parasite has evolved a suite of adaptive mechanisms enabling optimal exploitation of the host blood cell environment, avoiding host destruction, maintaining a parasite reservoir of infection and producing sexual transmission stages to infect mosquitoes. The highly variable nature of the host blood environment, both over the course of an infection and as a result of other parasitic infections, has selected for the evolution of considerable phenotypic plasticity in the parasite's response to its environment, particularly those phenotypes concerning transmission of the parasite to mosquitoes. With the evolution of human society, human malaria disease is becoming an increasingly urban problem. This imposes different selection pressures on the parasite. The extent to which the parasite is truly plastic over the short term rather than adaptive over the long term will determine the urban epidemiology of malaria and is essential for developing appropriate control methods. Understanding the adaptive nature of malaria parasites is thus vital for anticipating the future visage of urban human malaria.     

Vaccination against malaria: recent advances and the problems of antigenic diversity and other parasite evasion mechanisms

Due to the complexity of the malaria life cycle and the stage-specificity of immunity, a malaria vaccine will most likely be multicomponent, directed against surface epitopes on sporozoites, infected erythrocytes, merozoites and gametes. The CSP antigen of sporozoites is best understood at the structural and immunochemical level and vaccine trials employing peptides derived from this protein are currently underway. To date, no antigenic diversity of the immunodominant repeat epitope of the CSP protein has been uncovered in natural isolates of P. falciparum, raising optimism for eventual applicability of the laboratory trials to a field vaccine. Numerous surface antigens on merozoites and gametes have been identified with monoclonal antibodies and shown to be potential vaccine targets based on in vitro and in vivo studies with these antibodies. The problem of antigenic diversity and parasite lability seems acute in the asexual blood stages, and perhaps also with transmission-blocking antigens of gametes. Ways must be found to identify invariant surface epitopes that are so critical to parasite survival that in the face of a potentially lethal immune response mutant organisms cannot alter the target epitope and evade destruction.     

Lineage-specific expansion of proteins exported to erythrocytes in malaria parasites

Background  

The apicomplexan parasite Plasmodium falciparum causes the most severe form of malaria in humans. After invasion into erythrocytes, asexual parasite stages drastically alter their host cell and export remodeling and virulence proteins. Previously, we have reported identification and functional analysis of a short motif necessary for export of proteins out of the parasite and into the red blood cell.