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.     

HIV and malaria: a lesson in immunology?

HIV is now common in many areas of Africa that are also highly endemic for malaria. In this article, Geoff Butcher summarizes the available data on the possible interaction o f HIV and malaria, and shows that the course of falciparum malaria is virtually unaffected by the presence of HIV. This raises significant questions for our understanding of immunity to the asexual blood stages of human malaria and the use of animal models in malaria research.     

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.     

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.     

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's deadly secret: a skin stage

The role skin plays in malaria infection has long been overlooked. Recent analysis, however, suggests skin-infecting sporozoites initiate rapid suppression of immunity, establishing early tolerance to subsequent lifecycle stages. This explains susceptibility to reinfection by mosquito bite, independent of blood stage-induced immunosuppression or semi-immunity. Vaccine trials corroborate skin-initiated immunosubversion due to skin-infecting forms, tightly correlating bite pre-exposure, live parasites in the skin and endemic vaccine failure. Rapidly advancing skin immunobiology and recently described parasite development in host skin further substantiate the proposed model, consolidating a new concept in parasite biology, exemplified by malaria: natural infection has a defined, potently immunosubversive skin stage, crucially affecting vaccine function and vitally relevant to eradication.     

Human T clones reactive to the sexual stages of Plasmodium falciparum malaria. High frequency of gamete-reactive T cells in peripheral blood from nonexposed donors

Malarial gametocytes, which are taken up by mosquitoes during a blood meal, develop in the gut of the mosquito into gametes. Gametes and gametocytes contain the target antigens of transmission-blocking immunity. Here, we show that the peripheral blood of nonexposed donors contains Plasmodium falciparum gamete-reactive T cells at frequencies ranging from 1/300 to 1/4000. Studies on long-term clones demonstrated that these cells often recognized antigens shared between gametes and asexual stage parasites or even between heterologous gametes, although it has been possible to derive a P. falciparum gamete-specific T clone. The T clones examined were T3+, T4+, T8-, and either HLA-DR- or HLA-DQ-restricted. They responded to gametes by both proliferation and the secretion of gamma-interferon. The gamete-specific clone and other asexual cross-reactive clones examined could be stimulated in vitro by a preparation of mature gametocytes within RBC, but not by RBC alone, suggesting that gametocytes are immunogenic or can become immunogenic for T cells in vivo. The significance of these observations to mosquito transmission of malaria and development and application of a gamete vaccine are discussed.     

Immune responses to asexual blood-stages of malaria parasites

The blood stage of the malaria parasite's life cycle is responsible for all the clinical symptoms of malaria. The development of clinical disease is dependent on the interplay of the infecting parasite with the immune status and genetic background of the host. Following repeated exposure to malaria parasites, individuals residing in endemic areas develop immunity. Naturally acquired immunity provides protection against clinical disease, especially severe malaria and death from malaria, although sterilizing immunity is never achieved. Given the absence of antigen processing in erythrocytes, immunity to blood stage malaria parasites is primarily conferred by humoral immune responses. Cellular and innate immune responses play a role in controlling parasite growth but may also contribute to malaria pathology. Here, we analyze the natural humoral immune responses acquired by individuals residing in P. falciparum endemic areas and review their role in providing protection against malaria. In addition, we review the dual potential of cellular and innate immune responses to control parasite multiplication and promote pathology.     

Blood-Stage Malaria Parasite Antigens: Structure,Function, and Vaccine Potential

Plasmodium parasites are the causative agent of malaria, a disease that kills approximately 450,000 individuals annually, with the majority of deaths occurring in children under the age of 5 years and the development of a malaria vaccine is a global health priority. Plasmodium parasites undergo a complex life cycle requiring numerous diverse protein families. The blood stage of parasite development results in the clinical manifestation of disease. A vaccine that disrupts the blood stage is highly desired and will aid in the control of malaria. The blood stage comprises multiple steps: invasion of, asexual growth within, and egress from red blood cells. This review focuses on blood-stage antigens with emphasis on antigen structure, antigen function, neutralizing antibodies, and vaccine potential.     

Target antigens in malaria transmission blocking immunity

Malaria transmission blocking immunity has been found to operate against two distinct phases of development of malaria parasites in the mosquito midgut: (i) against the extracellular gametes and newly fertilized zygotes shortly after ingestion by a mosquito of parasitized blood and (ii) against the zygotes during their subsequent development into ookinetes. Immunity is antibody-mediated and stage-specific. A set of three proteins, synthesized in the gametocytes, expressed on the surface of the gametes and newly fertilized zygotes and subsequently shed during later transformation of the zygotes, has been identified as the target antigens of anti-gamete fertilization blocking antibodies. A single protein, synthesized and expressed on the zygote surface during its development to ookinetes, has been identified as the target of antibodies which block the development of the fertilized parasites in the mosquito. Immunization of human populations against gamete or zygote antigens, while not directly protecting an immunized individual from inflection, would reduce the transfer of malaria within the population. Such immunity, in addition to reducing the overall rate of malaria transmission, would, if combined with a vaccine against the asexual (disease-causing) stages, reduce the chance of selection of parasites that are resistant to the asexual vaccine by preventing their entry into the mosquito population.     

Notes about the impact of human seasonal migration on malaria spreading in rural Venezuela

This paper considers the effect of social and cultural factors on malaria spreading in rural Venezuela. It argues that standard vertical malaria control programs are inclined to ignore local workplace and living conditions instead of recruiting traditional practices into the planning scenario for more effective malaria control. An epidemiological survey on people migrating from malaria endemic areas to non-endemic region studied by blood films and a Plasmodium falciparum in vivo test. The results of the survey on people migrating from malaria endemic areas to non endemic regions revealed that 138 (118 males and 20 females) (23.3%) had fever (38.2-39 degrees C), malarial parasites were detected in the blood films of 49 (35.5%) from 138 febrile (parasitaemic) patients, and 45 (91.8%) of the parasitaemic cases were infected with P. falciparum; other four cases carried P. vivax only. Differences in prevalence, parasites load and the density of infection were observed between the three age groups. The asexual parasite load and the density of parasites (asexual and sexual forms) were appreciably higher in older children than in the other two age-groups (P < 0.001 for each). In the Plasmodium falciparum in vivo test, nine (22.5%) patients presented resistance grade III. People with transient jobs in malaria endemic areas could transport the parasites to non-endemic areas establishing a new malaria focus during seasonal migration activities.     

The Dynamics of Naturally Acquired Immunity to Plasmodium falciparum Infection

Severe malaria occurs predominantly in young children and immunity to clinical disease is associated with cumulative exposure in holoendemic settings. The relative contribution of immunity against various stages of the parasite life cycle that results in controlling infection and limiting disease is not well understood. Here we analyse the dynamics of Plasmodium falciparum malaria infection after treatment in a cohort of 197 healthy study participants of different ages in order to model naturally acquired immunity. We find that both delayed time-to-infection and reductions in asymptomatic parasitaemias in older age groups can be explained by immunity that reduces the growth of blood stage as opposed to liver stage parasites. We found that this mechanism would require at least two components – a rapidly acting strain-specific component, as well as a slowly acquired cross-reactive or general immunity to all strains. Analysis and modelling of malaria infection dynamics and naturally acquired immunity with age provides important insights into what mechanisms of immune control may be harnessed by malaria vaccine strategists.     

The effect of partial host immunity on the transmission of malaria parasites.

Experiments were carried out to determine the effect of partial host immunity against the rodent malaria parasite Plasmodium chabaudi on the transmission success of the parasite. There was a fourfold reduction in both the blood-stage, asexually replicating parasite density and the gametocyte (transmissable stage) density in immunized hosts. Some of the reduction in asexual parasite densities was due to strain-specific immunity, but there was no evidence that strain-specific immunity affected gametocyte densities. However, immunity did affect transmission in a strain-specific manner, with a fivefold reduction in gametocyte infectivity to mosquitoes in homologous challenges compared with heterologous challenges or non-immunized controls. This implies the existence of a mechanism of strain-specific infectivity-reducing immunity that does not affect the density of gametocytes circulating in peripheral blood. The proportion of asexual parasites that produced gametocytes increased during the course of infection in both non-immunized and in immunized hosts, but immunity increased gametocyte production early in the infection.     

Modeling transmission dynamics of stage-specific malaria vaccines

Population effects of malaria vaccination programs will depend on a complex interaction of the stage specificity of the vaccine, its duration of effectiveness, whether it is responsive to natural boosting, the strategy implemented, the proportion vaccinated and the pre-existing endemic conditions. In this article, Elizabeth Halloran and Claudio Struchiner review models of malaria transmission that incorporate aspects of immunity relevant to studying the effects of stage-specific malaria vaccination programs. They discuss the difference in the assumptions and applicability of the models and compare their predictions. Experience with malaria has demonstrated the difficulty in eliminating transmission, so emphasis needs to be on the new host-parasite balance that will be induced by the vaccination program. Although Halloran and Struchiner advise caution in interpreting the results of such models, they conclude that quantitative and theoretical analysis will be important in planning and evaluating interventions with malaria vaccines.     

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.     

Cloning and expression in Escherichia coli of a surface antigen of Plasmodium falciparum merozoites.

A complementary DNA library was constructed from mRNA purified from asexual blood forms of Plasmodium falciparum. Among the members of this library we have identified a plasmid (pMC31-1) coding for a polypeptide exposed at the surface of merozoites, the invasive stage of the asexual cycle. This plasmid was identified by direct expression using both polyclonal and monoclonal antibodies specific for a schizont polypeptide of 200 kd which has been shown to be processed to an 83-kd polypeptide expressed at the surface of merozoites. The cDNA portion of the pMC31-1 plasmid hybridizes with DNA from three isolates of P. falciparum. Antisera raised against extracts of Escherichia coli harbouring pMC31-1 react with surface and internal structures of schizonts and with the surface of merozoites from all the isolates of P. falciparum examined. These results suggest that plasmid pMC31-1 encodes an antigen of value for the development of a vaccine against 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.     

Modeling malaria vaccines. II: Population effects of stage-specific malaria vaccines dependent on natural boosting

Population effects of malaria vaccination programs will depend on the stage specificity of the vaccine, its duration of effectiveness, whether it is responsive to natural boosting, the proportion vaccinated, and the preexisting endemic conditions. This paper develops models of infection-blocking (sporozoite), disease-modifying (merozoite), and transmission-blocking (gametic) vaccines. It explores numerically their different effects on prevalence of infection and disease when utilized in different types of immunization programs at various levels of coverage. Simulations show that possible qualitative consequences of malaria vaccination programs include decreased prevalence of infection and disease and decreased prevalence of infection without a corresponding decrease in prevalence of disease. Epidemics, either one-time or cyclical, could occur. These effects could be accompanied by changes in the age distribution of disease. Finally, vaccination could contribute to elimination of transmission. The duration of effectiveness of the malaria vaccine relative to the duration of natural immunity could have important consequences for the unvaccinated. The problem of predicting a threshold for elimination of transmission is discussed.     

Interdependence of CD4+ T cells and malarial spleen in immunity to Plasmodium vinckei vinckei. Relevance to vaccine development

We studied immunity to the blood stage of the rodent malaria, Plasmodium vinckei vinckei, which is uniformly lethal to mice. BALB/c mice develop solid immunity after two infections and drug cure. The following experiments define the basis of this immunity. Transfer of pooled serum from such immune mice renders very limited protection to BALB/c mice and no protection to athymic nu/nu mice. Moreover, B cell-deficient C3H/HeN mice develop immunity to P. vinckei reinfection in the same manner as immunologically intact mice, an observation made earlier. In vivo depletion of CD4+ T cells in immune mice abrogates their immunity. This loss of immunity could be reversed through reconstitution of in vivo CD4-depleted mice with fractionated B-, CD8-, CD4+ immune spleen cells; however, adoptive transfer of fractionated CD4+ T cells from immune spleen into naive BALB/c or histocompatible BALB/c nude mice does not render recipients immune. In vivo depletion of CD8+ T cells did not influence the parasitemia in nonimmune or immune mice. Splenectomy of immune mice completely reverses their immunity. Repletion of splenectomized mice with their own spleen cells does not reconstitute their immunity. We conclude that some feature of the malaria-modified spleen acts in concert with the effector/inducer function of CD4+ T cells to provide protection from P. vinckei. To be consistent with this finding, a malaria vaccine may require a combination of malaria Ag to induce immune CD4+ T cells and an adjuvant or other vaccine vehicle to alter the spleen.     

A Nonintegrative Lentiviral Vector-Based Vaccine Provides Long-Term Sterile Protection against Malaria

Trials testing the RTS,S candidate malaria vaccine and radiation-attenuated sporozoites (RAS) have shown that protective immunity against malaria can be induced and that an effective vaccine is not out of reach. However, longer-term protection and higher protection rates are required to eradicate malaria from the endemic regions. It implies that there is still a need to explore new vaccine strategies. Lentiviral vectors are very potent at inducing strong immunological memory. However their integrative status challenges their safety profile. Eliminating the integration step obviates the risk of insertional oncogenesis. Providing they confer sterile immunity, nonintegrative lentiviral vectors (NILV) hold promise as mass pediatric vaccine by meeting high safety standards. In this study, we have assessed the protective efficacy of NILV against malaria in a robust pre-clinical model. Mice were immunized with NILV encoding Plasmodium yoelii Circumsporozoite Protein (Py CSP) and challenged with sporozoites one month later. In two independent protective efficacy studies, 50% (37.5–62.5) of the animals were fully protected (p = 0.0072 and p = 0.0008 respectively when compared to naive mice). The remaining mice with detectable parasitized red blood cells exhibited a prolonged patency and reduced parasitemia. Moreover, protection was long-lasting with 42.8% sterile protection six months after the last immunization (p = 0.0042). Post-challenge CD8+ T cells to CSP, in contrast to anti-CSP antibodies, were associated with protection (r = −0.6615 and p = 0.0004 between the frequency of IFN-g secreting specific T cells in spleen and parasitemia). However, while NILV and RAS immunizations elicited comparable immunity to CSP, only RAS conferred 100% of sterile protection. Given that a better protection can be anticipated from a multi-antigen vaccine and an optimized vector design, NILV appear as a promising malaria vaccine.