Microneme Protein Research Articles

Plasmodium falciparum rhoptry neck protein 5 peptides bind to human red blood cells and inhibit parasite invasion

Plasmodium falciparum malaria parasite invasion of erythrocytes is an essential step in host infection and the proteins involved in such invasion are the main target in developing an antimalarial vaccine. Secretory organelle-derived proteins (micronemal AMA1 protein and the RON2, 4, and 5 rhoptry neck proteins) have been recently described as components of moving junction complex formation allowing merozoites to move into a newly created parasitophorous vacuole. This study led to identifying RON5 regions involved in binding to human erythrocytes by using a highly robust, sensitive and specific receptor–ligand interaction assay; it is further shown that the RON5 protein remains highly conserved throughout different parasite strains. It is shown that the binding peptide–erythrocyte interaction is saturable and sensitive to chymotrypsin and trypsin. Invasion inhibition assays using erythrocyte binding peptides showed that the RON5–erythrocyte interaction could be critical for merozoite invasion of erythrocytes. This work provides evidence (for the first time) suggesting a fundamental role for RON5 in erythrocyte invasion.

Identification and Prioritization of Merozoite Antigens as Targets of Protective Human Immunity to Plasmodium falciparum Malaria for Vaccine and Biomarker Development

The development of effective malaria vaccines and immune biomarkers of malaria is a high priority for malaria control and elimination. Ags expressed by merozoites of Plasmodium falciparum are likely to be important targets of human immunity and are promising vaccine candidates, but very few Ags have been studied. We developed an approach to assess Ab responses to a comprehensive repertoire of merozoite proteins and investigate whether they are targets of protective Abs. We expressed 91 recombinant proteins, located on the merozoite surface or within invasion organelles, and screened them for quality and reactivity to human Abs. Subsequently, Abs to 46 proteins were studied in a longitudinal cohort of 206 Papua New Guinean children to define Ab acquisition and associations with protective immunity. Ab responses were higher among older children and those with active parasitemia. High-level Ab responses to rhoptry and microneme proteins that function in erythrocyte invasion were identified as being most strongly associated with protective immunity compared with other Ags. Additionally, Abs to new or understudied Ags were more strongly associated with protection than were Abs to current vaccine candidates that have progressed to phase 1 or 2 vaccine trials. Combinations of Ab responses were identified that were more strongly associated with protective immunity than responses to their single-Ag components. This study identifies Ags that are likely to be key targets of protective human immunity and facilitates the prioritization of Ags for further evaluation as vaccine candidates and/or for use as biomarkers of immunity in malaria surveillance and control.

Protective immunity induced by a DNA vaccine-encoding Toxoplasma gondii microneme protein 11 against acute toxoplasmosis in BALB/c mice

Toxoplasma gondii is one of the most prevalent intracellular parasites and is threatening the health of both humans and animals, therefore causing incalculable economic losses worldwide. Vaccination is thought to be an efficient way of controlling toxoplasmosis. T. gondii microneme protein 11 (MIC11) is a soluble microneme protein which is presumably considered facilitating the early stage of cell invasion. To evaluate the protective efficacy of T. gondii MIC11, in the present study, a new DNA vaccine-encoding the α-chain of T. gondii MIC11 was constructed using the pcDNA3.1 vector. Expression of MIC11 from this vector was confirmed by indirect immunofluorescence assay following transfection into baby hamster kidney (BHK) cells. Intramuscular immunization of BALB/c mice with pcDNA/MIC11 was carried out to evaluate the immune responses by serum antibodies titers, lymphoproliferation assay, and cytokines assay. The protective efficacy was evaluated by survival rate in mice after challenging with highly virulent strain of T. gondii. The results demonstrated that this vaccination elicited significant humoral responses and T. gondii lysate antigen (TLA)-stimulated lymphoproliferation (p < 0.05). Compared to controls, the pcDNA/MIC11 immunized mice had high production of IFN-γ, IL-12, and IL-2 (p < 0.05), but not IL-4 (p > 0.05), indicating that a predominant Th1 type response was developed. The vaccination also increased the survival rate of immunized mice when they were challenged with a lethal dose of tachyzoites of T. gondii RH strain. These data suggest that T. gondii MIC11 is a reasonable vaccine candidate deserving further studies, and pcDNA/MIC11 is a potential strategy for the control of toxoplasmosis.

Evaluation of protective effect of pVAX-TgMIC13 plasmid against acute and chronic Toxoplasma gondii infection in a murine model

Toxoplasma gondii is a significant zoonotic parasite which can cause congenital infection and abortion in warm-blooded animals and humans. Microneme protein 13 (MIC13) plays an important role in attachment and penetration of the host cell by T. gondii. In this study, a DNA vaccine expressing mic13 of T. gondii was constructed and its protective efficacy was evaluated in Kunming L615H2k mice. Immunization with pVAX-TgMIC13 induced a strong immune responses demonstrated by significant lymphocyte proliferation, cytokine production and antibody responses. Immunized mice showed increased survival time (21.3±11.3 days) and reduced number of cysts in brain of mice (57.14%) after challenge with tachyzoites of the virulent T. gondii RH strain and cysts of the T. gondii PRU strain, respectively, demonstrating that T. gondii MIC13 is a potential vaccine candidate, worth being included in future vaccine development against acute and chronic T. gondii infection.

Partial protective of chickens against Eimeria tenella challenge with recombinant EtMIC-1 antigen

Eimeria tenella microneme protein 1 (EtMIC-1) is highly conserved with TgMIC-2, which is involved in parasite binding specifically to host cells. Little is known about the immune responses and protective efficacy against E. tenella infection with EtMIC-1 antigen. In the present study, the recombinant proteins of E. tenella mature MIC-1 and adhesive domain (von Willebrand factor type A domain, EtMIC-1-VD) were obtained, protective efficacy against E.tenella infection and the mucosal immune response, which is induced in broilers was evaluated. The antibody levels and the transcription profiles of cytokine of chickens, such as interleukin-12 (IL-12) and interferon-γ (IFN-γ), were detected after being immunized three times with the recombinant EtMIC-1 and EtMIC-1-VD by ELISA assay and quantitative real-time PCR, respectively. The results showed that both groups of chickens, after being immunized with 100μg EtMIC-1 or EtMIC-1-VD antigen, induced about tenfold higher IgG levels compared to the nonimmune groups. The transcription profiles of IL-12 and IFN-γ of the immunized groups were significantly higher than the control groups as well. The anticoccidial index of the group immunized with 100μg EtMIC-1 and the group immunized with 100μg EtMIC-1-VD were 167.2 and 165.5, respectively, which are significantly higher than low-dose immunized groups and challenged control groups. Our data suggests that VD domain is the key functional structure of EtMIC-1 that could trigger a significant humoral and cellular response against E. tenella infection, and EtMIC-1 had the potential in imparting partial protection in chickens against homologous challenge.

Use of a Th1 Stimulator Adjuvant for Vaccination against Neospora caninum Infection in the Pregnant Mouse Model.

Vertical transmission from an infected cow to its fetus accounts for the vast majority of new Neospora caninum infections in cattle. A vaccine composed of a chimeric antigen named recNcMIC3-1-R, based on predicted immunogenic domains of the two microneme proteins NcMIC1 and NcMIC3, the rhoptry protein NcROP2, and emulsified in saponin adjuvants, significantly reduced the cerebral infection in non-pregnant BALB/c mice. Protection was associated with a mixed Th1/Th2-type cytokine response. However, the same vaccine formulation elicited a Th2-type immune response in pregnant mice and did not prevent vertical transmission or disease, neither in dams nor in offspring mice. In this study, an alternative vaccine formulation containing recNcMIC3-1-R emulsified in Freund’s incomplete adjuvant, a stimulator of the cellular immunity, was investigated. No protection against vertical transmission and cerebral infection in the pregnant mice and a very limited protective effect in the non-pregnant mice were observed. The vaccine induced a Th1-type immune response characterized by high IgG2a titres and strong IFN-γ expression, which appeared detrimental to pregnancy.

An Overexpression Screen of Toxoplasma gondii Rab-GTPases Reveals Distinct Transport Routes to the Micronemes

The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA) is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules) that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED) we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.

A thrombospondin structural repeat containing rhoptry protein fromPlasmodium falciparummediates erythrocyte invasion

Host cell invasion by Plasmodium falciparum requires multiple molecular interactions between host receptors and parasite ligands. A family of parasite proteins, which contain the conserved thrombospondin structural repeat motif (TSR), has been implicated in receptor binding during invasion. In this study we have characterized the functional role of a TSR containing blood stage protein referred to as P. falciparum thrombospondin related apical merozoite protein (PfTRAMP). Both native and recombinant PfTRAMP bind untreated as well as neuraminidase, trypsin or chymotrypsin-treated human erythrocytes. PfTRAMP is localized in the rhoptry bulb and is secreted during invasion. Adhesion of microneme protein EBA175 with its erythrocyte receptor glycophorin A provides the signal that triggers release of PfTRAMP from the rhoptries. Rabbit antibodies raised against PfTRAMP block erythrocyte invasion by P. falciparum suggesting that PfTRAMP plays an important functional role in invasion. Combination of antibodies against PfTRAMP with antibodies against microneme protein EBA175 provides an additive inhibitory effect against invasion. These observations suggest that targeting multiple conserved parasite ligands involved in different steps of invasion may provide an effective strategy for the development of vaccines against blood stage malaria parasites.

Shedding light on Toxoplasma invasion

To date, genome engineering in Toxoplasma gondii has proved difficult owing to inefficient gene targeting. The observation of a mild phenotype in a knockdown experiment always leads to some uncertainty about whether the gene of interest is essential. Now, Andenmatten et al. have used a gene swap approach based on the conditional dimerizable Cre recombinase (DiCre) system to dissect T. gondii gliding motility and host cell invasion. Replacing components of the actin–myosin invasion complex with the gene encoding YFP allowed easy identification and analysis of reliable knockouts. Surprisingly, genes encoding two components of the invasion complex, myosin A (MyoA) and micronemal protein 2 (MIC2), which previously had been described as essential, were dispensable in the gene swap experiments. The isolation of viable MyoA and MIC2 knockout clones suggests the existence of a novel, as-yet-uncharacterized invasion mechanism in T. gondii.

Toxoplasma Invasion Revisited

Cell Biology Apicomplexan parasites actively invade their host cell. Invasion depends on an arsenal of secreted invasion factors and the ability of the parasite to glide. The Myosin A (MyoA) motor complex, also known as the glideosome, is a multisubunit complex localized beneath the plasma membrane of the parasite. It is thought to provide the necessary force to move the parasite forward and is essential for gliding motility in Toxoplasma gondii and Plasmodium . Gliding mutants, however, retain the ability to invade host cells, albeit at reduced efficiency. To establish whether background expression of the respective protein in the knockdown mutants might be sufficient to drive host cell invasion, Andenmatten et al. generated a conditional recombination system based on dimerizable Cre recombinase, which they used to generate complete knockouts of three proteins—MyoA, the micronemal protein MIC2, and actin—each thought to be essential for host cell invasion in T. gondii. Surprisingly, each of the complete knockout mutants could invade host cells, and the MyoA and MIC2 knockouts could be maintained and grown in vitro, suggesting that alternative invasion strategies exist. As expected, parasites lacking MyoA were not able to move by gliding motility and consequently were significantly impaired in host cell egress. Nat. Meth. 10.1038/nmeth.2301 (2012).

Identification of Toxoplasma gondii in-vivo induced antigens by cDNA library immunoscreening with chronic toxoplasmosis sera

Toxoplasmosis is an infection caused by the parasite Toxoplasma gondii. Chronically-infected individuals with a compromised immune system are at risk for reactivation of the disease. In-vivo induced antigen technology (IVIAT) is a promising method for the identification of antigens expressed in-vivo. The aim of the present study was to apply IVIAT to identify antigens which are expressed in-vivo during T. gondii infection using sera from individuals with chronic toxoplasmosis. Forty serum samples were pooled, pre-adsorped against three different preparations of antigens, from each in-vitro grown T. gondii and Escherichia coli XLBlue MRF', and then used to screen a T. gondii cDNA expression library. Sequencing of DNA inserts from positive clones showed eight open reading frames with high homology to T. gondii genes. Expression analysis using quantitative real-time PCR showed that SAG1-related sequence 3 (SRS3) and two hypothetical genes were up-regulated in-vivo relative to their expression levels in-vitro. These three proteins also showed high sensitivity and specificity when tested with individual serum samples. Five other proteins namely M16 domain peptidase, microneme protein, elongation factor 1-alpha, pre-mRNA-splicing factor and small nuclear ribonucleoprotein F had lower RNA expression in-vivo as compared to in-vitro. SRS3 and the two hypothetical proteins warrant further investigation into their roles in the pathogenesis of toxoplasmosis.

Microneme Protein 5 Regulates the Activity of Toxoplasma Subtilisin 1 by Mimicking a Subtilisin Prodomain

Toxoplasma gondii is the model parasite of the phylum Apicomplexa, which contains obligate intracellular parasites of medical and veterinary importance. Apicomplexans invade host cells by a multistep process involving the secretion of adhesive microneme protein (MIC) complexes. The subtilisin protease TgSUB1 trims several MICs on the parasite surface to activate gliding motility and host invasion. Although a previous study showed that expression of the secretory protein TgMIC5 suppresses TgSUB1 activity, the mechanism was unknown. Here, we solve the three-dimensional structure of TgMIC5 by nuclear magnetic resonance (NMR), revealing that it mimics a subtilisin prodomain including a flexible C-terminal peptide that may insert into the subtilisin active site. We show that TgMIC5 is an almost 50-fold more potent inhibitor of TgSUB1 activity than the small molecule inhibitor N-[N-(N-acetyl-L-leucyl)-L-leucyl]-L-norleucine (ALLN). Moreover, we demonstrate that TgMIC5 is retained on the parasite plasma membrane via its physical interaction with the membrane-anchored TgSUB1.

A Unique Protein Phosphatase with Kelch-Like Domains (PPKL) in Plasmodium Modulates Ookinete Differentiation, Motility and Invasion

Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl− mutants, with global phosphorylation studies of ookinete differentiation from 1.5–24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.

Toxoplasma gondii Antigens Recognized by IgG Antibodies Differ between Mice with and without Active Proliferation of Tachyzoites in the Brain during the Chronic Stage of Infection

We examined whether tachyzoite proliferation in the brains of immunocompetent hosts during the chronic stage of infection with Toxoplasma gondii induces production of IgG antibodies that recognize parasite antigens different from those recognized by the antibodies of infected hosts that do not have tachyzoite growth. For this purpose, two groups of CBA/J mice, which display continuous tachyzoite growth in their brains during the later stage of infection, were infected, and one group received treatment with sulfadiazine to prevent tachyzoite proliferation during the chronic stage of infection. T. gondii antigens recognized by the IgG antibodies from these two groups of mice were compared using immunoblotting following separation of tachyzoite antigens by two-dimensional gel electrophoresis. Several antigens, including the microneme protein MIC2, the cyst matrix protein MAG1, and the dense granule proteins GRA4 and GRA7, were commonly recognized by IgG antibodies from both groups of mice. There were multiple antigens recognized mostly by IgG antibodies of only one group of mice, either with or without cerebral tachyzoite growth. The antigens recognized only by or mostly by the antibodies of mice with cerebral tachyzoite growth include MIC6, the rhoptry protein ROP1, GRA2, one heat shock protein HSP90, one (putative) HSP70, and the myosin heavy chain. These results indicate that levels of IgG antibody to only selected T. gondii antigens increase in association with cerebral tachyzoite proliferation (reactivation of infection) in immunocompetent hosts with chronic infection.

Identification and Characterization of Eimeria tenella Apical Membrane Antigen-1 (AMA1)

Apical membrane antigen-1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. In this study, a full-length cDNA of AMA1 was identified from Eimeria tenella (Et) using expressed sequence tag and the rapid amplification of cDNA ends technique. EtAMA1 had an open reading frame of 1608 bp encoding a protein of 535 amino acids. Quantitative real-time PCR analysis revealed that EtAMA1 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second-generation merozoites). The ectodomain sequence was expressed as recombinant EtAMA1 (rEtAMA1) and rabbit polyclonal antibodies raised against the rEtAMA1 recognized a 58-kDa native parasite protein by Western Blotting and had a potent inhibitory effect on parasite invasion, decreasing it by approximately 70%. Immunofluorescence analysis and immunohistochemistry analysis showed EtAMA1 might play an important role in sporozoite invasion and development.

Toxoplasma Sortilin-like Receptor Regulates Protein Transport and Is Essential for Apical Secretory Organelle Biogenesis and Host Infection

Apicomplexan parasites have an assortment of unique apical secretory organelles (rhoptries and micronemes), which have crucial functions in host infection. Here, we show that a Toxoplasma gondii sortilin-like receptor (TgSORTLR) is required for the subcellular localization and formation of apical secretory organelles. TgSORTLR is a transmembrane protein that resides within Golgi-endosomal related compartments. The lumenal domain specifically interacts with rhoptry and microneme proteins, while the cytoplasmic tail of TgSORTLR recruits cytosolic sorting machinery involved in anterograde and retrograde protein transport. Ectopic expression of the N-terminal TgSORTLR lumenal domain results in dominant negative effects with the mislocalization of both endogenous TgSORTLR as well as rhoptry and microneme proteins. Conditional ablation of TgSORTLR disrupts rhoptry and microneme biogenesis, inhibits parasite motility, and blocks both invasion into and egress from host cells. Thus, the sortilin-like receptor is essential for protein trafficking and the biogenesis of key secretory organelles in Toxoplasma.

Galactose Recognition by the Apicomplexan Parasite Toxoplasma gondii

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.

Eimeria tenella heat shock protein 70 enhances protection of recombinant microneme protein MIC2 subunit antigen vaccination against E. tenella challenge

Heat shock proteins have been reported to stimulate the immune system via innate receptors. Our study found that the novel immunopotentiator, Eimeria tenella (E. tenella) heat shock protein 70 (HSP70), enhanced protective immunity elicited by E. tenella antigen microneme protein 2 (EtMIC2) against avian coccidiosis. It demonstrated that the expression of TLR2 and TLR4 were strongly upregulated in EtHSP70 and EtMIC2 plus EtHSP70 stimulated chicken embryo fibroblasts (CEF) compared with untreated controls and EtMIC2 alone. In addition, the same treatment induced high levels of interleukin (IL)-12 and interferon (IFN)-γ that are critical cytokines of innate immunity. In vivo experiments involved using broiler chickens subcutaneously immunized with EtMIC2 alone or EtMIC2 plus EtHSP70 at 7 and 14 days post-hatch, which were then orally challenged with live E. tenella at 7 days following secondary immunization. Body weight gains, cecal lesion scores, fecal oocyst shedding, serum antibody responses against MIC2, and intestinal cytokine transcript levels were assessed as measures of protective immunity. Chickens immunized with EtMIC2 plus EtHSP70 showed increased body weight gains, decreased oocyst shedding, increased serum antibody responses, and high levels of IL-12, IFN-γ, and IL-17 compared with the EtMIC2 only or control groups. Moreover, chickens immunized with EtHSP70 alone showed significantly protective effect against E. tenella infection. In summary, this study provides the first evidence of the immunoenhancing activities of EtHSP70 in poultry.

EmaxDB: Availability of a first draft genome sequence for the apicomplexan Eimeria maxima

Apicomplexan parasites are serious pathogens of animals and man that cause diseases including coccidiosis, malaria and toxoplasmosis. The importance of these parasites has prompted the establishment of genomic resources in support of developing effective control strategies. For the Eimeria species resources have developed most rapidly for the reference Eimeria tenella Houghton strain (http://www.genedb.org/Homepage/Etenella). The value of these resources can be enhanced by comparison with related parasites. The well characterised immunogenicity and genetic diversity associated with Eimeria maxima promote its use in genetics-led studies on coccidiosis and recommended its selection for sequencing. Using a combination of sequencing technologies a first draft assembly and annotation has been produced for an E. maxima Houghton strain-derived clone (EmaxDB; http://www.genomemalaysia.gov.my/emaxdb/). The assembly of a draft genome sequence for E. maxima provides a resource for comparative studies with Eimeria and related parasites as demonstrated here through the identification of genes predicted to encode microneme proteins in E. maxima.

Vaccination with a DNA vaccine coding for perforin-like protein 1 and MIC6 induces significant protective immunity against Toxoplasma gondii.

Host cell invasion by Toxoplasma gondii is tightly related to microneme protein 6 (MIC6) and T. gondii perforin-like protein 1 (TgPLP1). In this study, we constructed a DNA vaccine expressing a TgPLP1/MIC6 fusion protein using the pIRESneo vector, and we evaluated the immune response induced by this vaccine in Kunming mice. Levels of IgG antibody, gamma interferon (IFN-γ), interleukin 2 (IL-2), IL-12, IL-4, and IL-10 were examined. Five mice were chosen randomly from every group (vaccinated groups or the nonvaccinated control group) and were challenged intragastrically with 80 cysts of T. gondii strain PRU (genotype II) in order to observe mortality daily. To analyze protection against a less-virulent challenge, eight mice of each group were orally infected with 20 cysts of strain PRU at the 14th day after the last immunization. The brain parasite load was evaluated 6 weeks after infection. The results demonstrated that immunization with pIRESneo/MIC6/PLP1 resulted in the lowest brain cyst count and prolonged the survival time of immunized mice. The levels of Toxoplasma-specific IgG, IFN-γ, IL-2, and IL-12 increased significantly, and the numbers of cysts in brains decreased more obviously, in the group immunized with plasmid pIRESneo/MIC6/PLP1 than in the other groups (P < 0.05). Compared with pIRESneo/MIC6/PLP1, coimmunization with pIRESneo/MIC6/PLP1 and adjuvant murine IL-18 promoted cellular and humoral immune responses but did not contribute significantly to cyst reduction (65.43% versus 61.60%) or the survival of immunized mice (45.0 ± 2.9 days versus 42.8 ± 2.9 days) (P > 0.05). Furthermore, the study also showed that the immune efficacy induced by pIRESneo/MIC6/PLP1 was better than that induced by pVAX/PLP1 or pVAX/MIC6 alone.