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.

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.

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.

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.

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.

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.

Immunization with CSP and a RIG-I Agonist is Effective in Inducing a Functional and Protective Humoral Response Against Plasmodium.

Malaria is a major public health concern, as a highly effective human vaccine remains elusive. The efficacy of a subunit vaccine targeting the most abundant protein of the sporozoite surface, the circumsporozoite protein (CSP) has been hindered by difficulties in generating an effective humoral response in both quantity and quality. Using the rodent Plasmodium yoelii model we report here that immunization with CSP adjuvanted with 5'ppp-dsRNA, a RIG-I agonist, confers early and long-lasting sterile protection in mice against stringent sporozoite and mosquito bite challenges. The immunization induced high levels of antibodies, which were functional in targeting and killing the sporozoites and were sustained over time through the accumulation of long-lived plasma cells in the bone marrow. Moreover, 5'ppp-dsRNA-adjuvanted immunization with the CSP of P. falciparum was also significantly protective against challenges using a transgenic PfCSP-expressing P. yoelii parasite. Conversely, using the TLR3 agonist poly(A:U) as adjuvant resulted in a formulation that despite inducing high antibody levels was unable to generate equally functional antibodies and was, consequently, less protective. In conclusion, we demonstrate that using 5'ppp-dsRNA as an adjuvant to vaccines targeting CSP induces effective anti-Plasmodium humoral immunity.

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.

Highly efficient CRISPR/Cas9 system in Plasmodium falciparum using Cas9-expressing parasites and a linear donor template.

The CRISPR/Cas9 system is a powerful genetic engineering technology for Plasmodium falciparum. We here report further improvement of the CRISPR/Cas9 system by combining the Cas9-expressing parasite with a liner donor template DNA. The Cas9-expressing parasite was generated by inserting the cas9 gene in the genome by double crossover recombination. The site-directed mutagenesis and the fusion of fluorescence protein was achieved within two weeks with high efficiency (> 85%), by transfecting the schizonts of the Cas9-expressing parasite with the liner donor template and the plasmid carrying the sgRNAs. Notably, there were neither off-target mutations in the resultant transgenic parasites nor unexpected recombination, that are the technical problems of the current CRISPR/Cas9 system. Furthermore, with our system, two genes on different chromosomes were successfully modified in single transfection. Because of its high efficiency and robustness, our improved CRISPR/Cas9 system will become a standard technique for genetic engineering of P. falciparum, which dramatically advances future studies of this parasite.

Mechanisms of triggering malaria gametocytogenesis by AP2-G.

The transcription factor (TF) AP2-G is essential for gametocytogenesis in the malaria parasite; however, it remains unclear if AP2-G determines commitment to sexual stage development fate in the schizont stage, or whether AP2-G directly initiates sexual stage differentiation and development beginning in the late-trophozoite stage. In this study, we addressed this issue by investigating the expression profile of AP2-G and determining genome-wide target genes in Plasmodium berghei. Fluorescence microscopy showed that AP2-G expression was first observed in the parasite 12 h after erythrocyte invasion and peaked at 18 h when sexual features were first manifested in early gametocytes. Expression of AP2-G decreased with manifestation of sex-specific features. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed at peak AP2-G expression and identified over 1000 binding sites in the genome. The main binding motif of the TF predicted from the binding sites was GTACNY. Predicted targets contained a number of genes related to protein biogenesis, suggesting that AP2-G plays a role in establishing a cellular basis required for sexual differentiation. AP2-G binding sites also existed upstream of gametocyte-specific TFs, namely AP2-G2, AP2-FG, and AP2-G itself. Furthermore, the target contained two AP2 TF-related genes. Disruption of these genes resulted in the arrest of ookinete development. These results suggest another role of AP2-G: activating a transcriptional cascade to promote conversion into early gametocytes. Taken together, AP2-G is involved not in establishing sexual commitment of schizonts, but rather in triggering the initiation of differentiation and the early development of gametocytes in the late trophozoite stage.

Improvement of CRISPR/Cas9 system by transfecting Cas9-expressing Plasmodium berghei with linear donor template.

Malaria is caused by infection with Plasmodium parasites and is a major public health concern. The CRISPR/Cas9 system is a promising technology, but still has technical problems, such as low efficiency and unexpected recombination. Here, we solved these problems by transfecting Cas9-expressing parasites with linear donor templates. The use of a linear donor template prevented unexpected recombination; in addition, constitutive expression of Cas9 enabled immediate cleavage of the target locus after transfection, allowing efficient integration of the donor template. Furthermore, due to the absence of the cNHEJ pathway, there were no off-target mutations in the resultant parasites. In addition, this developed method could be applied for multiple genetic modifications on different chromosomes and for large-scale chromosomal deletion in the subtelomeric region. Because of its robustness, high efficiency, and versatile applicability, we hope this method will be standard in the post-genomic era of Plasmodium species.

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.

CXCR4 regulates Plasmodium development in mouse and human hepatocytes.

The liver stage of the etiological agent of malaria, Plasmodium, is obligatory for successful infection of its various mammalian hosts. Differentiation of the rod-shaped sporozoites of Plasmodium into spherical exoerythrocytic forms (EEFs) via bulbous expansion is essential for parasite development in the liver. However, little is known about the host factors regulating the morphological transformation of Plasmodium sporozoites in this organ. Here, we show that sporozoite differentiation into EEFs in the liver involves protein kinase C ζ-mediated NF-κB activation, which robustly induces the expression of C-X-C chemokine receptor type 4 (CXCR4) in hepatocytes and subsequently elevates intracellular Ca2+ levels, thereby triggering sporozoite transformation into EEFs. Blocking CXCR4 expression by genetic or pharmacological intervention profoundly inhibited the liver-stage development of the Plasmodium berghei rodent malaria parasite and the human Plasmodium falciparum parasite. Collectively, our experiments show that CXCR4 is a key host factor for Plasmodium development in the liver, and CXCR4 warrants further investigation for malaria prophylaxis.

Cytotoxic anti-circumsporozoite antibodies target malaria sporozoites in the host skin.

The circumsporozoite protein (CSP) is the major surface protein of malaria sporozoites (SPZs), the motile and invasive parasite stage inoculated in the host skin by infected mosquitoes. Antibodies against the central CSP repeats of different plasmodial species are known to block SPZ infectivity1-5, but the precise mechanism by which these effectors operate is not completely understood. Here, using a rodent Plasmodium yoelii malaria model, we show that sterile protection mediated by anti-P. yoelii CSP humoral immunity depends on the parasite inoculation into the host skin, where antibodies inhibit motility and kill P. yoelii SPZs via a characteristic 'dotty death' phenotype. Passive transfer of an anti-repeat monoclonal antibody (mAb) recapitulates the skin inoculation-dependent protection, in a complement- and Fc receptor γ-independent manner. This purified mAb also decreases motility and, notably, induces the dotty death of P. yoelii SPZs in vitro. Cytotoxicity is species-transcendent since cognate anti-CSP repeat mAbs also kill Plasmodium berghei and Plasmodium falciparum SPZs. mAb cytotoxicity requires the actomyosin motor-dependent translocation and stripping of the protective CSP surface coat, rendering the parasite membrane susceptible to the SPZ pore-forming-like protein secreted to wound and traverse the host cell membrane6. The loss of SPZ fitness caused by anti-P. yoelii CSP repeat antibodies is thus a dynamic process initiated in the host skin where SPZs either stop moving7, or migrate and traverse cells to progress through the host tissues7-9 at the eventual expense of their own life.

CRISPR/Cas9 system in Plasmodium falciparum using the centromere plasmid.

The CRISPR/Cas9 nuclease system is a powerful method to genetically modify the human malarial parasite, Plasmodium falciparum. Currently, this method is carried out by co-transfection with two plasmids, one containing the Cas9 nuclease gene, and another encoding the sgRNA and the donor template DNA. However, the efficiency of modification is currently low owing to the low frequency of these plasmids in the parasites. To improve the CRISPR/Cas9 nuclease system for P. falciparum, we developed a novel method using the transgenic parasite, PfCAS9, which stably expresses the Cas9 nuclease using the centromere plasmid. To examine the efficiency of genetic modification using the PfCAS9 parasite, we performed site-directed mutagenesis of kelch13 gene, which is considered to be involved in artemisinin resistance. Our results demonstrated that the targeted mutation could be introduced with almost 100% efficiency when the transfected PfCAS9 parasites were treated with two drugs to maintain both the centromere plasmid containing the Cas9 nuclease and the plasmid having the sgRNA. Therefore, the PfCAS9 parasite is a useful parasite line for the genetic modification of P. falciparum.

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.

A potent malaria vaccine based on adenovirus with dual modifications at Hexon and pVII.

Adenovirus (Ad) is thought to be one of the most promising platforms for a malaria vaccine targeted against its liver stages, because of its ability to induce a strong T-cell response against a transgene. However, a further improvement of this platform is needed in order to elicit another arm of the immunity, i.e. humoral response, against malaria. In order to augment immunogenicity and protective efficacy of Ad-based malaria vaccine, we inserted B-cell, as well as CD4+ T-cell, epitopes of Plasmodium falciparum circumsporozoite protein (PfCSP) into the capsid protein, Hexon, and the core protein, VII (pVII), of Ad, respectively, in addition to the PfCSP transgene. Insertion of PfCSP-derived B cell epitope to Hexon significantly enhanced the epitope-specific antibody response compared to AdPfCSP, an Ad vaccine expressing only PfCSP transgene. PfCSP-derived CD4+ T-cell epitope insertion into pVII augmented not only PfCSP-specific CD4+ T-cell response but also anti-PfCSP antibody response. Finally, mice immunized with AdPfCSP having both Hexon and pVII modifications were more protected than AdPfCSP or Hexon-modified AdPfCSP against challenge with transgenic rodent malaria parasites expressing the PfCSP. Overall, this study has demonstrated that Hexon and pVII-modified AdPfCSP vaccine is a promising malaria vaccine which induces strong PfCSP-specific humoral, CD4+ T-cell, and CD8+ T-cell responses and protects against infection with transgenic malaria parasites expressing the PfCSP.