PbARID-associated chromatin remodeling events are essential for gametocyte development in Plasmodium.

Gametocyte development of the Plasmodium parasite is a key step for transmission of the parasite. Male and female gametocytes are produced from a subpopulation of asexual blood-stage parasites, but the mechanisms that regulate the differentiation of sexual stages are still under investigation. In this study, we investigated the role of PbARID, a putative subunit of a SWI/SNF chromatin remodeling complex, in transcriptional regulation during the gametocyte development of P. berghei. PbARID expression starts in early gametocytes before the manifestation of male and female-specific features, and disruption of its gene results in the complete loss of gametocytes with detectable male features and the production of abnormal female gametocytes. ChIP-seq analysis of PbARID showed that it forms a complex with gSNF2, an ATPase subunit of the SWI/SNF chromatin remodeling complex, associating with the male cis-regulatory element, TGTCT. Further ChIP-seq of PbARID in gsnf2-knockout parasites revealed an association of PbARID with another cis-regulatory element, TGCACA. RIME and DNA-binding assays suggested that HDP1 is the transcription factor that recruits PbARID to the TGCACA motif. Our results indicated that PbARID could function in two chromatin remodeling events and paly essential roles in both male and female gametocyte development.

Differentiation of Plasmodium male gametocytes is initiated by the recruitment of a chromatin remodeler to a male-specific cis-element.

Plasmodium parasites, the causative agents of malaria, possess a complex lifecycle; however, the mechanisms of gene regulation involved in the cell-type changes remain unknown. Here, we report that gametocyte sucrose nonfermentable 2 (gSNF2), an SNF2-like chromatin remodeling ATPase, plays an essential role in the differentiation of male gametocytes. Upon disruption of gSNF2, male gametocytes lost the capacity to develop into gametes. ChIP-seq analyses revealed that gSNF2 is widely recruited upstream of male-specific genes through a five-base, male-specific cis-acting element. In gSNF2-disrupted parasites, expression of over a hundred target genes was significantly decreased. ATAC-seq analysis demonstrated that decreased expression of these genes correlated with a decrease of the nucleosome-free region upstream of these genes. These results suggest that global changes induced in the chromatin landscape by gSNF2 are the initial step in male differentiation from early gametocytes. This study provides the possibility that chromatin remodeling is responsible for cell-type changes in the Plasmodium lifecycle.

PbAP2-FG2 and PbAP2R-2 function together as a transcriptional repressor complex essential for Plasmodium female development.

Gametocyte development is a critical step in the life cycle of Plasmodium. Despite the number of studies on gametocyte development that have been conducted, the molecular mechanisms regulating this process remain to be fully understood. This study investigates the functional roles of two female-specific transcriptional regulators, PbAP2-FG2 and PbAP2R-2, in P. berghei. Knockout of pbap2-fg2 or pbap2r-2 impairs female gametocyte development, resulting in developmental arrest during ookinete development. ChIP-seq analyses of these two factors indicated their colocalization on the genome, suggesting that they function as a complex. These analyses also revealed that their target genes contained a variety of genes, including both male and female-enriched genes. Moreover, differential expression analyses showed that these target genes were upregulated through the disruption of pbap2-fg2 or pbap2r-2, indicating that these two factors function as a transcriptional repressor complex in female gametocytes. Formation of a complex between PbAP2-FG2 and PbAP2R-2 was confirmed by RIME, a method that combines ChIP and MS analysis. In addition, the analysis identified a chromatin regulator PbMORC as an interaction partner of PbAP2-FG2. Comparative target analysis between PbAP2-FG2 and PbAP2-G demonstrated a significant overlap between their target genes, suggesting that repression of early gametocyte genes activated by PbAP2-G is one of the key roles for this female transcriptional repressor complex. Our results indicate that the PbAP2-FG2-PbAP2R-2 complex-mediated repression of the target genes supports the female differentiation from early gametocytes.

Identification of a novel AP2 transcription factor in zygotes with an essential role in Plasmodium ookinete development.

The sexual phase of Plasmodium represents a crucial step in malaria transmission, during which these parasites fertilize and form ookinetes to infect mosquitoes. Plasmodium development after fertilization is thought to proceed with female-stored mRNAs until the formation of a retort-form ookinete; thus, transcriptional activity in zygotes has previously been considered quiescent. In this study, we reveal the essential role of transcriptional activity in zygotes by investigating the function of a newly identified AP2 transcription factor, AP2-Z, in P. berghei. ap2-z was previously reported as a female transcriptional regulator gene whose disruption resulted in developmental arrest at the retort stage of ookinetes. In this study, although ap2-z was transcribed in females, we show that it was translationally repressed by the DOZI complex and translated after fertilization with peak expression at the zygote stage. ChIP-seq analysis of AP2-Z shows that it binds on specific DNA motifs, targeting the majority of genes known as an essential component of ookinetes, which largely overlap with the AP2-O targets, as well as genes that are unique among the targets of other sexual transcription factors. The results of this study also indicate the existence of a cascade of transcription factors, beginning with AP2-G, that proceeds from gametocytogenesis to ookinete formation.

Female-specific gene regulation in malaria parasites by an AP2-family transcription factor.

The malaria gametocyte, the gamete precursor, is the essential stage for malaria transmission to the mosquito vector. In the vertebrate host's blood, it develops into a mature male or female capable of transforming into a gamete in the mosquito blood meal. Despite the importance of this stage in the malaria life cycle, the genetic regulation of gametocyte development is poorly understood. In particular, transcription factors involved in sex-specific gene expression have not been identified. In this paper, we report that an AP2-family transcription factor, AP2-FG, is responsible for female-specific gene regulation. AP2-FG expression in Plasmodium berghei was observed exclusively in female gametocytes, in the beginning of 4-6 h before sexual dimorphism manifests in developing gametocytes. AP2-FG disruption resulted in the arrest of female maturation, but did not affect the development of males. Chromatin immunoprecipitation sequencing analysis suggested that AP2-FG directly regulates over 700 genes. Its targets include genes for female gametocyte-specific functions, such as gametogenesis, fertilization and zygote development. AP2-FG binding to target gene promoters was associated with a 10 bp sequence motif. These results indicate that AP2-FG plays a role in the differentiation of early gametocytes to mature females by governing a female-specific gene expression repertoire.

Nonspecific CD8+ T Cells and Dendritic Cells/Macrophages Participate in Formation of CD8+ T Cell-Mediated Clusters against Malaria Liver-Stage Infection.

CD8+ T cells are the major effector cells that protect against malaria liver-stage infection, forming clusters around Plasmodium-infected hepatocytes and eliminating parasites after a prolonged interaction with these hepatocytes. We aimed to investigate the roles of specific and nonspecific CD8+ T cells in cluster formation and protective immunity. To this end, we used Plasmodium berghei ANKA expressing ovalbumin as well as CD8+ T cells from transgenic mice expressing a T cell receptor specific for ovalbumin (OT-I) and CD8+ T cells specific for an unrelated antigen, respectively. While antigen-specific CD8+ T cells were essential for cluster formation, both antigen-specific and nonspecific CD8+ T cells joined the clusters. However, nonspecific CD8+ T cells did not significantly contribute to protective immunity. In the livers of infected mice, specific CD8+ T cells expressed high levels of CD25, compatible with a local, activated effector phenotype. In vivo imaging of the liver revealed that specific CD8+ T cells interact with CD11c+ cells around infected hepatocytes. The depletion of CD11c+ cells virtually eliminated the clusters in the liver, leading to a significant decrease in protection. These experiments reveal an essential role of hepatic CD11c+ dendritic cells and presumably macrophages in the formation of CD8+ T cell clusters around Plasmodium-infected hepatocytes. Once cluster formation is triggered by parasite-specific CD8+ T cells, specific and unrelated activated CD8+ T cells join the clusters in a chemokine- and dendritic cell-dependent manner. Nonspecific CD8+ T cells seem to play a limited role in protective immunity against Plasmodium parasites.

Global transcriptional repression: An initial and essential step for Plasmodium sexual development.

Gametocytes are nonreplicative sexual forms that mediate malaria transmission to a mosquito vector. They are generated from asexual blood-stage parasites that proliferate in the circulation. However, little is known about how this transition is genetically regulated. Here, we report that an Apetala2 (AP2) family transcription factor, AP2-G2, regulates this transition as a transcriptional repressor. Disruption of AP2-G2 in the rodent malaria parasite Plasmodium berghei did not prevent commitment to the sexual stage but did halt development before the appearance of sex-specific morphologies. ChIP-seq analysis revealed that AP2-G2 targeted ∼1,500 genes and recognized a five-base motif in their promoters. Most of these target genes are required for asexual proliferation of the parasites in the blood, suggesting that AP2-G2 blocks the program that precedes asexual replication to promote conversion to the sexual stage. Microarray analysis showed that the identified targets constituted ∼70% of the up-regulated genes in AP2-G2-depleted parasites, suggesting that AP2-G2 actually functions as a repressor in gametocytes. A promoter assay using a centromere plasmid demonstrated that the binding motif functions as a cis-acting negative regulatory element. These results suggest that global transcriptional repression, which occurs during the initial phase of gametocytogenesis, is an essential step in Plasmodium sexual development.

Genome-Wide Identification of the Target Genes of AP2-O, a Plasmodium AP2-Family Transcription Factor.

Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors.

A highly infectious Plasmodium yoelii parasite, bearing Plasmodium falciparum circumsporozoite protein.

BACKGROUND: Plasmodium circumsporozoite protein (CSP) is a major surface antigen present in the sporozoite (Spz) stage of a malaria parasite. RTS, S vaccine, the most clinically advanced malaria vaccine, consists of a large portion of Plasmodium falciparum CSP (PfCSP). A highly infectious, recombinant rodent malaria, Plasmodium yoelii parasite bearing a full-length PfCSP, PfCSP/Py Spz, was needed as a tool to evaluate the role of PfCSP in mediating, protective, anti-malaria immunity in a mouse model. METHODS: A transgenic parasite, PfCSP/Py Spz, was generated by inserting a construct expressing the PfCSP at the locus of the P. yoelii CSP gene by double cross-over homologous recombination. Then the biological and protective properties of PfCSP/Py Spz were determined. RESULTS: This PfCSP/Py parasite produced up to 30,000 Spz in mosquito salivary glands, which is equal or even higher than the number of Spz produced by wild-type P. yoelii parasites. Five bites of PfCSP/Py-infected mosquitoes could induce blood infection in BALB/c mice. CONCLUSIONS: The current study has demonstrated a successful establishment of a transgenic P. yoelii parasite clone that is able to express a full-length PfCSP, PfCSP/Py parasite. Importantly, this PfCSP/Py parasite can be as infectious as the wild-type P. yoelii parasite both in mosquito vector and in mouse, a mammalian host. A new transgenic parasite that expresses a full-length PfCSP may become a useful tool for researchers to investigate immunity against PfCSP in a mouse model.

A high-coverage artificial chromosome library for the genome-wide screening of drug-resistance genes in malaria parasites.

The global spread of drug-resistant parasites is a serious problem for the treatment of malaria. Although identifying drug-resistance genes is crucial for the efforts against resistant parasites, an effective approach has not yet been developed. Here, we report a robust method for identifying resistance genes from parasites by using a Plasmodium artificial chromosome (PAC). Large genomic DNA fragments (10-50 kb) from the drug-resistant rodent malaria parasite Plasmodium berghei were ligated into the PAC and directly introduced into the drug-sensitive (i.e., wild-type) parasite by electroporation, resulting in a PAC library that encompassed the whole genomic sequence of the parasite. Subsequently, the transformed parasites that acquired resistance were selected by screening with the drug, and the resistance gene in the PAC was successfully identified. Furthermore, the drug-resistance gene was identified from a PAC library that was made from the pyrimethamine-resistant parasite Plasmodium chabaudi, further demonstrating the utility of our method. This method will promote the identification of resistance genes and contribute to the global fight against drug-resistant parasites.

Liver-specific protein 2: a Plasmodium protein exported to the hepatocyte cytoplasm and required for merozoite formation.

The liver stage is the first stage of the malaria parasite that replicates in the vertebrate host. However, little is known about the interplay between the parasite liver stage and its host cell, the hepatocyte. In this study, we identified an exported protein that has a critical role in parasite development in host hepatocytes. Expressed sequence tag analysis of Plasmodium berghei liver-stage parasites indicated that transcripts encoding a protein with an N-terminal signal peptide, designated liver-specific protein 2 (LISP2), are highly expressed in this stage. Expression of LISP2 was first observed 24 h after infection and rapidly increased during the liver-stage schizogony. Immunofluorescent staining with anti-LSP2 antibodies showed that LISP2 was carried to the parasitophorous vacuole and subsequently transported to the cytoplasm and nucleus of host hepatocytes. Gene targeting experiments demonstrated that majority of the LISP2-mutant liver-stage parasites arrested their development during formation of merozoites. These results indicate that exported LISP2 is involved in parasite-host interactions required for the development of liver-stage parasites inside hepatocytes. This study demonstrated that mid-to-late liver-stage malarial parasites have a system for exporting proteins to the host cell as intraerythrocytic stages do and presumably to use the proteins to modify the host cell and improve the environment.

Development of memory CD8+ T cells and their recall responses during blood-stage infection with Plasmodium berghei ANKA.

Conditions required for establishing protective immune memory vary depending on the infecting microbe. Although the memory immune response against malaria infection is generally thought to be relatively slow to develop and can be lost rapidly, experimental evidence is insufficient. In this report, we investigated the generation, maintenance, and recall responses of Ag-specific memory CD8(+) T cells using Plasmodium berghei ANKA expressing OVA (PbA-OVA) as a model system. Mice were transferred with OVA-specific CD8(+) T (OT-I) cells and infected with PbA-OVA or control Listeria monocytogenes expressing OVA (LM-OVA). Central memory type OT-I cells were maintained for >2 mo postinfection and recovery from PbA-OVA. Memory OT-I cells produced IFN-γ as well as TNF-α upon activation and were protective against challenge with a tumor expressing OVA, indicating that functional memory CD8(+) T cells can be generated and maintained postinfection with P. berghei ANKA. Cotransfer of memory OT-I cells with naive OT-I cells to mice followed by infection with PbA-OVA or LM-OVA revealed that clonal expansion of memory OT-I cells was limited during PbA-OVA infection compared with expansion of naive OT-I cells, whereas it was more rapid during LM-OVA infection. The expression of inhibitory receptors programmed cell death-1 and LAG-3 was higher in memory-derived OT-I cells than naive-derived OT-I cells during infection with PbA-OVA. These results suggest that memory CD8(+) T cells can be established postinfection with P. berghei ANKA, but their recall responses during reinfection are more profoundly inhibited than responses of naive CD8(+) T cells.

Coordinated regulation of gene expression in Plasmodium female gametocytes by two transcription factors.

Gametocytes play key roles in the Plasmodium lifecycle. They are essential for sexual reproduction as precursors of the gametes. They also play an essential role in parasite transmission to mosquitoes. Elucidation of the gene regulation at this stage is essential for understanding these two processes at the molecular level and for developing new strategies to break the parasite lifecycle. We identified a novel Plasmodium transcription factor (TF), designated as a partner of AP2-FG or PFG. In this article, we report that this TF regulates the gene expression in female gametocytes in concert with another female-specific TF AP2-FG. Upon the disruption of PFG, majority of female-specific genes were significantly downregulated, and female gametocyte lost the ability to produce ookinetes. ChIP-seq analysis showed that it was located in the same position as AP2-FG, indicating that these two TFs form a complex. ChIP-seq analysis of PFG in AP2-FG-disrupted parasites and ChIP-seq analysis of AP2-FG in PFG-disrupted parasites demonstrated that PFG mediates the binding of AP2-FG to a ten-base motif and that AP2-FG binds another motif, GCTCA, in the absence of PFG. In promoter assays, this five-base motif was identified as another female-specific cis-acting element. Genes under the control of the two forms of AP2-FG, with or without PFG, partly overlapped; however, each form had target preferences. These results suggested that combinations of these two forms generate various expression patterns among the extensive genes expressed in female gametocytes.

CD8+ T cells specific for a malaria cytoplasmic antigen form clusters around infected hepatocytes and are protective at the liver stage of infection.

Following Anopheles mosquito-mediated introduction into a human host, Plasmodium parasites infect hepatocytes and undergo intensive replication. Accumulating evidence indicates that CD8(+) T cells induced by immunization with attenuated Plasmodium sporozoites can confer sterile immunity at the liver stage of infection; however, the mechanisms underlying this protection are not clearly understood. To address this, we generated recombinant Plasmodium berghei ANKA expressing a fusion protein of an ovalbumin epitope and green fluorescent protein in the cytoplasm of the parasite. We have shown that the ovalbumin epitope is presented by infected liver cells in a manner dependent on a transporter associated with antigen processing and becomes a target of specific CD8(+) T cells from the T cell receptor transgenic mouse line OT-I, leading to protection at the liver stage of Plasmodium infection. We visualized the interaction between OT-I cells and infected hepatocytes by intravital imaging using two-photon microscopy. OT-I cells formed clusters around infected hepatocytes, leading to the elimination of the intrahepatic parasites and subsequent formation of large clusters of OT-I cells in the liver. Gamma interferon expressed in CD8(+) T cells was dispensable for this protective response. Additionally, we found that polyclonal ovalbumin-specific memory CD8(+) T cells induced by de novo immunization were able to confer sterile protection, although the threshold frequency of the protection was relatively high. These studies revealed a novel mechanism of specific CD8(+) T cell-mediated protective immunity and demonstrated that proteins expressed in the cytoplasm of Plasmodium parasites can become targets of specific CD8(+) T cells during liver-stage infection.

Experimental cerebral malaria is suppressed by disruption of nucleoside transporter 1 but not purine nucleoside phosphorylase.

Protozoan parasites rely on purine nucleosides supplied by the host because they are unable to synthesise purine rings denovo. Nucleoside transporter 1 (NT1) and purine nucleoside phosphorylase (PNP) play an essential role in purine salvage in Plasmodium. It is unclear whether severe pathology, such as cerebral malaria (CM), develops in hosts infected with Plasmodium parasites that lack activity of NT1 or PNP. Plasmodium berghei (Pb) ANKA-infected mice show features similar to human CM, such as cerebral paralysis and cerebral haemorrhage. Therefore, Pb ANKA infection in mice is a good experimental model of CM. In this study, we generated pbnt1-disrupted Pb ANKA (Δpbnt1 parasites) and pbpnp-disrupted Pb ANKA (Δpbpnp parasites), and investigated the effect of pbnt1 or pbpnp disruption on the outcome of infection with Pb ANKA. We showed that the rapid increase of wild-type Pb ANKA (WT parasites) in mice early in infection was significantly inhibited by disruption of pbnt1. Moreover, Δpbnt1 parasite-infected mice showed neither cerebral paralysis nor cerebral haemorrhage, and all mice spontaneously recovered from infection. By contrast, mice infected with Δpbpnp parasites showed features similar to those of mice infected with WT parasites. In this study, we demonstrated that the high virulence of Pb ANKA in the asexual phase is suppressed by disruption of pbnt1 but not pbpnp.

Targetome Analysis of Malaria Sporozoite Transcription Factor AP2-Sp Reveals Its Role as a Master Regulator.

Malaria transmission to humans begins with sporozoite infection of the liver. The elucidation of gene regulation during the sporozoite stage will promote the investigation of mechanisms of liver infection by this parasite and contribute to the development of strategies for preventing malaria transmission. AP2-Sp is a transcription factor (TF) essential for the formation of sporozoites or sporogony, which takes place in oocysts in the midguts of infected mosquitoes. To understand the role of this TF in the transcriptional regulatory system of this stage, we performed chromatin immunoprecipitation sequencing (ChIP-seq) analyses using whole mosquito midguts containing late oocysts as starting material and explored its genome-wide target genes. We identified 697 target genes, comprising those involved in distinct processes parasites experience during this stage, from sporogony to development into the liver stage and representing the majority of genes highly expressed in the sporozoite stage. These results suggest that AP2-Sp determines basal patterns of gene expression by targeting a broad range of genes directly. The ChIP-seq analyses also showed that AP2-Sp maintains its own expression by a transcriptional autoactivation mechanism (positive-feedback loop) and induces all TFs reported to be transcribed at this stage, including AP2-Sp2, AP2-Sp3, and SLARP. The results showed that AP2-Sp exists at the top of the transcriptional cascade of this stage and triggers the formation of this stage as a master regulator. IMPORTANCE The sporozoite stage plays a central role in malaria transmission from a mosquito to vertebrate host and is an important target for antimalarial strategies. AP2-Sp is a candidate master transcription factor for the sporozoite stage. However, study of its role in gene regulation has been hampered because of difficulties in performing genome-wide studies of gene regulation in this stage. Here, we conquered this problem and revealed that AP2-Sp has the following prominent features as a master transcription factor. First, it determines the repertory of gene expression during this stage. Second, it maintains its own expression through a transcriptional positive-feedback loop and induces all other transcription factors specifically expressed in this stage. This study represents a major breakthrough in fully understanding gene regulation in this important malarial stage.

Plasmodium 6-cysteine proteins determine the commitment of sporozoites to liver-infection.

Plasmodium sporozoites travel a long way from the site where they are released by a mosquito bite to the liver, where they infect hepatocytes and develop into erythrocyte-invasive forms. The success of this infection depends on the ability of the sporozoites to correctly recognize the hepatocyte as a target and change their behavior from migration to infection. However, how this change is accomplished remains incompletely understood. In this paper, we report that 6-cysteine protein family members expressed in sporozoites including B9 are responsible for this ability. Experiments on parasites using double knockouts of B9 and SPECT2, which is essential for sporozoite to migrate through the hepatocyte, showed that the parasites lacked the capacity to stop migration. This finding suggests that interactions between these parasite proteins and hepatocyte-specific cell surface ligands mediate correct recognition of hepatocytes by sporozoites, which is an essential step in malaria transmission to humans.

Chromosome splitting of Plasmodium berghei using the CRISPR/Cas9 system.

Spatial arrangement of chromosomes is responsible for gene expression in Plasmodium parasites. However, methods for rearranging chromosomes have not been established, which makes it difficult to investigate its role in detail. Here, we report a method for splitting chromosome in rodent malaria parasite by CRISPR/Cas9 system using fragments in which a telomere and a centromere were incorporated. The resultant split chromosomes segregated accurately into daughter parasites by the centromere. In addition, elongation of de novo telomeres were observed, indicating its proper function. Furthermore, chromosome splitting had no effect on development of parasites. Splitting of the chromosome is expected to alter its spatial arrangement, and our method will thus be useful for investigating its biological role related with gene expression.

Genome-wide functional screening of drug-resistance genes in Plasmodium falciparum.

The global spread of drug resistance is a major obstacle to the treatment of Plasmodium falciparum malaria. The identification of drug-resistance genes is an essential step toward solving the problem of drug resistance. Here, we report functional screening as a new approach with which to identify drug-resistance genes in P. falciparum. Specifically, a high-coverage genomic library of a drug-resistant strain is directly generated in a drug-sensitive strain, and the resistance gene is then identified from this library using drug screening. In a pilot experiment using the strain Dd2, the known chloroquine-resistant gene pfcrt is identified using the developed approach, which proves our experimental concept. Furthermore, we identify multidrug-resistant transporter 7 (pfmdr7) as a novel candidate for a mefloquine-resistance gene from a field-isolated parasite; we suggest that its upregulation possibly confers the mefloquine resistance. These results show the usefulness of functional screening as means by which to identify drug-resistance genes.

Layer-by-Layer Delivery of Multiple Antigens Using Trimethyl Chitosan Nanoparticles as a Malaria Vaccine Candidate.

Developing a safe and effective malaria vaccine is critical to reducing the spread and resurgence of this deadly disease, especially in children. In recent years, vaccine technology has seen expanded development of subunit protein, peptide, and nucleic acid vaccines. This is due to their inherent safety, the ability to tailor their immune response, simple storage requirements, easier production, and lower expense compared to using attenuated and inactivated organism-based approaches. However, these new vaccine technologies generally have low efficacy. Subunit vaccines, due to their weak immunogenicity, often necessitate advanced delivery vectors and/or the use of adjuvants. A new area of vaccine development involves design of synthetic micro- and nano-particles and adjuvants that can stimulate immune cells directly through their physical and chemical properties. Further, the unique and complex life cycle of the Plasmodium organism, with multiple stages and varying epitopes/antigens presented by the parasite, is another challenge for malaria vaccine development. Targeting multistage antigens simultaneously is therefore critical for an effective malaria vaccine. Here, we rationally design a layer-by-layer (LbL) antigen delivery platform (we called LbL NP) specifically engineered for malaria vaccines. A biocompatible modified chitosan nanoparticle (trimethyl chitosan, TMC) was synthesized and utilized for LbL loading and release of multiple malaria antigens from pre-erythrocytic and erythrocytic stages. LbL NP served as antigen/protein delivery vehicles and were demonstrated to induce the highest Plasmodium falciparum Circumsporozoite Protein (PfCSP) specific T-cell responses in mice studies as compared to multiple controls. From immunogenicity studies, it was concluded that two doses of intramuscular injection with a longer interval (4 weeks) than traditional malaria vaccine candidate dosing would be the vaccination potential for LbL NP vaccine candidates. Furthermore, in PfCSP/Py parasite challenge studies we demonstrated protective efficacy using LbL NP. These LbL NP provided a significant adjuvant effect since they may induce innate immune response that led to a potent adaptive immunity to mediate non-specific anti-malarial effect. Most importantly, the delivery of CSP full-length protein stimulated long-lasting protective immune responses even after the booster immunization 4 weeks later in mice.