Protective Efficacy of Baculovirus Dual Expression System Vaccine Expressing Plasmodium falciparum Circumsporozoite Protein

We have previously developed a new malaria vaccine delivery system based on the baculovirus dual expression system (BDES). In this system, expression of malaria antigens is driven by a dual promoter consisting of the baculovirus-derived polyhedrin and mammal-derived cytomegalovirus promoters. To test this system for its potential as a vaccine against human malaria parasites, we investigated immune responses against the newly developed BDES-based Plasmodium falciparum circumsporozoite protein vaccines (BDES-PfCSP) in mice and Rhesus monkeys. Immunization of mice with BDES-PfCSP induced Th1/Th2-mixed type immune responses with high PfCSP-specific antibody (Ab) titers, and provided significant protection against challenge from the bites of mosquitoes infected with a transgenic P. berghei line expressing PfCSP. Next, we evaluated the immunogenicity of the BDES-PfCSP vaccine in a rhesus monkey model. Immunization of BDES-PfCSP elicited high levels of anti-PfCSP Ab responses in individual monkeys. Moreover, the sera from the immunized monkeys remarkably blocked sporozoite invasion of HepG2 cells. Taken together with two animal models, our results indicate that this novel vaccine platform (BDES) has potential clinical application as a vaccine against malaria.     

The complexity of protective immunity against liver-stage malaria

Sterile protective immunity against challenge with Plasmodium spp. sporozoites can be induced in multiple model systems and humans by immunization with radiation-attenuated Plasmodium spp. sporozoites. The infected hepatocyte has been established as the primary target of this protection, but the underlying mechanisms have not been completely defined. Abs, CD8+ T cells, CD4+ T cells, cytokines (including IFN-gamma and IL-12), and NO have all been implicated as critical effectors. Here, we have investigated the mechanisms of protective immunity induced by immunization with different vaccine delivery systems (irradiated sporozoites, plasmid DNA, synthetic peptide/adjuvant, and multiple Ag peptide) in genetically distinct inbred strains, genetically modified mice, and outbred mice. We establish that there is a marked diversity of T cell-dependent immune responses that mediate sterile protective immunity against liver-stage malaria. Furthermore, we demonstrate that distinct mechanisms of protection are induced in different strains of inbred mice by a single method of immunization, and in the same strain by different methods of immunization. These data underscore the complexity of the murine host response to a parasitic infection and suggest that an outbred human population may behave similarly. Data nevertheless suggest that a pre-erythrocytic-stage vaccine should be designed to induce CD8+ T cell- and IFN-gamma-mediated immune responses and that IFN-gamma responses may represent an in vitro correlate of pre-erythrocytic-stage protective immunity.     

Host immunity to Plasmodium infection: Contribution of Plasmodium berghei to our understanding of T cell-related immune response to blood-stage malaria

Malaria is a life-threatening disease caused by infection with Plasmodium parasites. The goal of developing an effective malaria vaccine is yet to be reached despite decades of massive research efforts. CD4⁺ helper T cells, CD8⁺ cytotoxic T cells, and γδ T cells are associated with immune responses to both liver-stage and blood-stage Plasmodium infection. The immune responses of T cell-lineages to Plasmodium infection are associated with both protection and immunopathology. Studies with mouse model of malaria contribute to our understanding of host immune response. In this paper, we focus primarily on mouse malaria model with blood-stage Plasmodium berghei infection and review our knowledge of T cell immune responses against Plasmodium infection. Moreover, we also discuss findings of experimental human studies. Uncovering the precise mechanisms of T cell-mediated immunity to Plasmodium infection can be accomplished through further investigations using mouse models of malaria with rodent Plasmodium parasites. Those findings would be invaluable to advance the efforts for development of an effective malaria vaccine.     

The effect of helminth co-infection on malaria in mice: a meta-analysis

The question of how helminths may alter the course of concurrent malaria infection has attracted much interest in recent years. In particular, it has been suggested that by creating an anti-inflammatory immune environment, helminth co-infection may dampen both protective and immunopathological responses to malaria parasites, thus altering malaria infection dynamics and disease severity. Both synergistic and antagonistic interactions are reported in the literature, and the causes of variation among studies are not well understood. Here, meta-analysis of 42 mouse co-infection experiments was used to address how helminths influence malaria parasite replication and host mortality, and explore the factors explaining variation in findings. Most notably, this analysis revealed contrasting effects of helminth co-infection in lethal and resolving malaria models. Whilst co-infection exacerbated mortality and increased peak parasitaemia in ordinarily resolving malaria infections (Plasmodium chabaudi and Plasmodium yoelii), effects among lethal malaria infections (Plasmodium berghei) tended to be in the opposite direction with no change in parasitaemia. In the subset of experiments on cerebral malaria models (P. berghei ANKA strain in a susceptible host), helminth co-infection significantly delayed death. These findings are consistent with the hypothesis that depending on the existing balance of pro- and anti-inflammatory responses mounted against malaria parasites in a given host, immune responses elicited by helminth co-infection may either promote or inhibit malarial disease. However, despite such broad patterns, a prominent feature of this dataset was great heterogeneity in effects across studies. A key future challenge therefore lies in explaining the biological causes of this variation, including a more thorough exploration of non-immunological mechanisms of helminth-malaria interaction.     

TLR-mediated cell signaling by malaria GPIs

Proinflammatory responses to malaria have crucial roles in controlling parasite growth and disease pathogenesis. The glycosylphosphatidylinositol (GPI) of Plasmodium falciparum is thought to be an important factor in the induction of proinflammatory responses. The GPI induces host cellular responses mainly through Toll-like receptor (TLR)2/MyD88-mediated signaling. Knowledge of the parasite-host factors involved in activating and regulating innate immune responses and of the associated signaling mechanisms is likely to provide insights into the modulation of parasite-specific adaptive immunity and offer targets for the development of novel therapeutics or a vaccine for malaria. This article focuses on the malaria GPI-mediated cell-signaling mechanisms.     

Protective immunity induced with malaria vaccine, RTS,S, is linked to Plasmodium falciparum circumsporozoite protein-specific CD4+ and CD8+ T cells producing IFN-gamma

The Plasmodium falciparum circumsporozoite (CS) protein-based pre-erythrocytic stage vaccine, RTS,S, induces a high level of protection against experimental sporozoite challenge. The immune mechanisms that constitute protection are only partially understood, but are presumed to rely on Abs and T cell responses. In the present study we compared CS protein peptide-recalled IFN-gamma reactivity of pre- and RTS,S-immune lymphocytes from 20 subjects vaccinated with RTS,S. We observed elevated IFN-gamma in subjects protected by RTS,S; moreover, both CD4(+) and CD8(+) T cells produced IFN-gamma in response to CS protein peptides. Significantly, protracted protection, albeit observed only in two of seven subjects, was associated with sustained IFN-gamma response. This is the first study demonstrating correlation in a controlled Plasmodia sporozoite challenge study between protection induced by a recombinant malaria vaccine and Ag-specific T cell responses. Field-based malaria vaccine studies are in progress to validate the establishment of this cellular response as a possible in vitro correlate of protective immunity to exo-erythrocytic stage malaria vaccines.     

Successful induction of CD8 T cell-dependent protection against malaria by sequential immunization with DNA and recombinant poxvirus of neonatal mice born to immune mothers

In some parts of Africa, 50% of deaths attributed to malaria occur in infants less than 8 mo. Thus, immunization against malaria may have to begin in the neonatal period, when neonates have maternally acquired Abs against malaria parasite proteins. Many malaria vaccines in development rely upon CD8 cells as immune effectors. Some studies indicate that neonates do not mount optimal CD8 cell responses. We report that BALB/c mice first immunized as neonates (7 days) with a Plasmodium yoelii circumsporozoite protein (PyCSP) DNA vaccine mixed with a plasmid expressing murine GM-CSF (DG) and boosted at 28 days with poxvirus expressing PyCSP were protected (93%) as well as mice immunized entirely as adults (70%). Protection was dependent on CD8 cells, and mice had excellent anti-PyCSP IFN-gamma and cytotoxic T lymphocyte responses. Mice born of mothers previously exposed to P. yoelii parasites or immunized with the vaccine were protected and had excellent T cell responses. These data support assessment of this immunization strategy in neonates/young infants in areas in which malaria exacts its greatest toll.     

The contribution of Plasmodium chabaudi to our understanding of malaria

Malaria kills close to a million people every year, mostly children under the age of five. In the drive towards the development of an effective vaccine and new chemotherapeutic targets for malaria, field-based studies on human malaria infection and laboratory-based studies using animal models of malaria offer complementary opportunities to further our understanding of the mechanisms behind malaria infection and pathology. We outline here the parallels between the Plasmodium chabaudi mouse model of malaria and human malaria. We will highlight the contribution of P. chabaudi to our understanding of malaria in particular, how the immune response in malaria infection is initiated and regulated, its role in pathology, and how immunological memory is maintained. We will also discuss areas where new tools have opened up potential areas of exploration using this invaluable model system.     

Biology of gammadelta T cells in tuberculosis and malaria

Tuberculosis and malaria remain the leading causes of mortality among human infectious diseases in the world. It is estimated that 3 to 5 million people die from tuberculosis and malaria each year. Although it is traditionally believed that CD4 and CD8 alphabeta T lymphocytes are mandatory for protective immune responses against Mycobacterium tuberculosis and Plasmodium falciparum (the ethiologic agents of tuberculosis and the most severe form of malaria, respectively), there is still incomplete understanding of the mechanisms of immune protection and of the causes of its failure in the affected patients. Several studies in humans and animal models have suggested that Vgamma9/Vdelta2 T cells may play an important role in the immune responses against Mycobacterium tuberculosis and Plasmodium falciparum. Vgamma9/Vdelta2 T cells represent about 75% of all circulating gammadelta T cells while they can be greatly expanded during the acute phase of Mycobacterium tuberculosis and Plasmodium falciparum malaria. Vgamma9/Vdelta2 T recognize a new class of antigenic molecules which are nonpeptidic in nature and contain critical phosphate moieties (phosphoantigens). Interestingly, phosphoantigens isolated from Mycobacterium tuberculosis and Plasmodium falciparum share strong structural homology and are probably identical. However, despite a large body of data reported in the literature, it is not yet clear whether Vgamma9/Vdelta2 T cells play a protective or pathogenic role in immune responses against Mycobacterium tuberculosis and Plasmodium falciparum. In this review we summarize our current knowledge of the biology of Vgamma9/Vdelta2 T cells in response to the two pathogens, Mycobacterium tuberculosis and Plasmodium falciparum, and provide evidence suggesting definition of a novel and important protective role through which Vgamma9/Vdelta2 T cells can contribute to the killing of microorganisms residing in intracellular compartments.     

A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine

A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective efficacy of adenoviral based malaria vaccines.     

Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans

In animals, effective immune responses against malignancies and against several infectious pathogens, including malaria, are mediated by T cells. Here we show that a heterologous prime-boost vaccination regime of DNA either intramuscularly or epidermally, followed by intradermal recombinant modified vaccinia virus Ankara (MVA), induces high frequencies of interferon (IFN)-gamma-secreting, antigen-specific T-cell responses in humans to a pre-erythrocytic malaria antigen, thrombospondin-related adhesion protein (TRAP). These responses are five- to tenfold higher than the T-cell responses induced by the DNA vaccine or recombinant MVA vaccine alone, and produce partial protection manifest as delayed parasitemia after sporozoite challenge with a different strain of Plasmodium falciparum. Such heterologous prime-boost immunization approaches may provide a basis for preventative and therapeutic vaccination in humans.     

Pre-erythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine

A vaccine is urgently needed to stem the global resurgence of Plasmodium falciparum malaria. Vaccines targeting the erythrocytic stage are often viewed as an anti-disease strategy. By contrast, infection might be completely averted by a vaccine against the liver stage, a pre-erythrocytic stage during which the parasite multiplies 10000-fold within hepatocytes. Sterilizing immunity can be conferred by inoculating humans with irradiated pre-erythrocytic parasites, and a recombinant pre-erythrocytic vaccine partially protects humans from infection. Liver-stage antigen-1, one of a few proteins known to be expressed by liver-stage parasites, holds particular promise as a vaccine. Studies of naturally exposed populations have consistently related immune responses against this antigen to protection.     

Multi-gene vaccination against malaria: A multistage, multi-immune response approach

An ideal malaria vaccine will induce immune responses against each stage of the Plasmodium spp life cycle. During its complicated life cycle, the parasite exists extracellularly in the host's bloodstream, within cells that express major histocompatibility complex (MHC) molecules (hepatocytes), within cells that do not express MHC molecules (erythrocytes) and within the mosquito vector. Different arms of the immune system are required to attack the parasite at the different stages. Therefore, a multistage vaccine must be a multi-immune response vaccine. In addition, given the unique antigenicities of the different stages of the life cycle, implicit in this definition is that the vaccine be multivalent. Here, Denise Doolan and Stephen Hoffman present the rationale for developing a multistage, multivalent, multi-immune response malaria vaccine and explain why, among currently available technologies, DNA vaccines may offer the best prospect for success.     

Bottom‐up regulation of malaria population dynamics in mice co‐infected with lung‐migratory nematodes

When and how populations are regulated by bottom up vs. top down processes, and how those processes are affected by co‐occurring species, are poorly characterised across much of ecology. We are especially interested in the community ecology of parasites that must share a host. Here, we quantify how resources and immunity affect parasite propagation in experiments in near‐replicate ‘mesocosms’’ – i.e. mice infected with malaria (Plasmodium chabaudi) and nematodes (Nippostrongylus brasiliensis). Nematodes suppressed immune responses against malaria, and yet malaria populations were smaller in co‐infected hosts. Further analyses of within‐host epidemiology revealed that nematode co‐infection altered malaria propagation by suppressing target cell availability. This is the first demonstration that bottom‐up resource regulation may have earlier and stronger effects than top‐down immune mechanisms on within‐host community dynamics. Our findings demonstrate the potential power of experimental ecology to disentangle mechanisms of population regulation in complex communities.     

DNA-based vaccines against malaria: status and promise of the Multi-Stage Malaria DNA Vaccine Operation

The introduction of DNA vaccine technology has facilitated an unprecedented multi-antigen approach to developing an effective vaccine against complex pathogens such as the Plasmodium spp. parasites that cause malaria. We have established the capacity of DNA vaccines encoding Plasmodium antigens to induce CD8(+) cytotoxic T lymphocyte and interferon-gamma responses in mice, monkeys and humans. However, like others, we have found that the first or second generation DNA vaccines on their own are not optimal, and have demonstrated the potential of heterologous prime/boost immunisation strategies involving priming with DNA and boosting with poxvirus or recombinant protein in adjuvant. In this review, we summarise the current status and promise of our programmatic efforts to develop a DNA-based vaccine against malaria, our Multi-Stage Malaria DNA Vaccine Operation, and illustrate the transition of promising developments in the laboratory to clinical assessment in humans.     

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.     

Induction of Plasmodium falciparum-specific CD4+ T cells and memory B cells in Gabonese children vaccinated with RTS,S/AS01(E) and RTS,S/AS02(D)

The recombinant circumsporozoite protein (CS) based vaccine, RTS,S, confers protection against Plasmodium falciparum infection in controlled challenge trials and in field studies. The RTS,S recombinant antigen has been formulated with two adjuvant systems, AS01 and AS02, which have both been shown to induce strong specific antibody responses and CD4 T cell responses in adults. As infants and young children are particularly susceptible to malaria infection and constitute the main target population for a malaria vaccine, we have evaluated the induction of adaptive immune responses in young children living in malaria endemic regions following vaccination with RTS,S/AS01(E) and RTS,S/AS02(D). Our data show that a CS-specific memory B cell response is induced one month after the second and third vaccine dose and that CS-specific antibodies and memory B cells persist up to 12 months after the last vaccine injection. Both formulations also induced low but significant amounts of CS-specific IL-2(+) CD4(+) T cells one month after the second and third vaccine dose, upon short-term in vitro stimulation of whole blood cells with peptides covering the entire CS derived sequence in RTS,S. These results provide evidence that both RTS,S/AS01(E) and RTS,S/AS02(D) induced adaptive immune responses including antibodies, circulating memory B cells and CD4(+) T cells directed against P. falciparum CS protein. TRIAL REGISTRATION: ClinicalTrials.gov NCT00307021.     

A sporozoite asparagine-rich protein controls initiation of Plasmodium liver stage development

Plasmodium sporozoites invade host hepatocytes and develop as liver stages (LS) before the onset of erythrocytic infection and malaria symptoms. LS are clinically silent, and constitute ideal targets for causal prophylactic drugs and vaccines. The molecular and cellular mechanisms underlying LS development remain poorly characterized. Here we describe a conserved Plasmodium asparagine-rich protein that is specifically expressed in sporozoites and liver stages. Gene disruption in Plasmodium berghei results in complete loss of sporozoite infectivity to rodents, due to early developmental arrest after invasion of hepatocytes. Mutant sporozoites productively invade host cells by forming a parasitophorous vacuole (PV), but subsequent remodelling of the membrane of the PV (PVM) is impaired as a consequence of dramatic down-regulation of genes encoding PVM-resident proteins. These early arrested mutants confer only limited protective immunity in immunized animals. Our results demonstrate the role of an asparagine-rich protein as a key regulator of Plasmodium sporozoite gene expression and LS development, and suggest a requirement of partial LS maturation to induce optimal protective immune responses against malaria pre-erythrocytic stages. These findings have important implications for the development of genetically attenuated parasites as a vaccine approach.     

Vaccines against malaria - an update

Malaria vaccine discovery and development follow two principal strategies. Most subunit vaccines are designed to mimic naturally acquired immunity that develops over years upon continuous exposure to Plasmodium transmission. Experimental model vaccines, such as attenuated live parasites and transmission-blocking antigens, induce immune responses superior to naturally acquired immunity. The promises and hurdles of the different tracks towards an effective and affordable vaccine against malaria are discussed.