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.     

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.     

Genetic immunization with plasmid DNA coding for the ROP2 protein of Toxoplasma gondii

The ROP2 protein of Toxoplasma gondii has previously been proposed as a vaccine candidate against toxoplasmosis. In this work we characterize the immune response induced by injection of plasmid DNA coding for this protein in three strains of mice (BALB/c, C57BL/6, and CBA/J) displaying different levels of susceptibility to toxoplasmosis and compare it with that obtained by vaccination with the live attenuated ts-4 strain of T. gondii. The ROP2 gene was cloned in the eukaryotic expression vector pcDNA3 and the resulting plasmid, named pcDNA3/ROP2, was used to immunize mice. After three immunizations with the plasmid, mice developed antibodies that could be detected by ELISA using a recombinant truncated form of ROP2; and these antibodies also recognized the natural protein by Western blot. Plasmid immunization generated antibodies against the ROP2 of both of the IgG₁ and IgG_2a isotypes in CBA/J and BALB/c mice and both of the IgG₁ and IgG_2c isotypes in C57BL/6 mice. However, animals vaccinated with the ts-4 strain generated only IgG_2a (in CBA/J and BALB/c mice) or IgG_2c (in C57BL/6 mice) against ROP2. Kinetic studies of the generation of isotypes indicated that both isotypes were generated at the same time. Mice immunized with the plasmid DNA did not resist a challenge with the virulent RH strain of T. gondii, while mice vaccinated with the ts-4 strain resisted the same challenge. However, in pcDNA3/ROP2-immunized BALB/c mice, death was significantly delayed with respect to the pcDNA3-immunized control group. These results suggest that plasmid immunization using the ROP2 gene generates a mixed T_H1/T_H2 response against ROP2, which is different from that obtained by vaccination with live tachyzoites of the ts-4 strain (T_H1 response) and is not protective against the highly virulent RH strain of the parasite. Received: 18 March 2000 / Accepted: 13 July 2000     

Comparative evaluation of immunization with recombinant protein and plasmid DNA vaccines of fusion antigen ROP2 and SAG1 from <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in mice: cellular and humoral immune responses

The aim of this work was to evaluate immune responses in BALB/c mice vaccinated subcutaneously by recombinant protein, or intramuscularly by plasmid DNA with fusion antigen of rhoptry protein 2 (ROP2) and major surface protein 1 (SAG1) from Toxoplasma gondii (T. gondii). BALB/c mice were immunized with one of three different antigen formulations respectively, which were rROP2-SAG1, pcROP2-SAG1, and pcROP2-SAG1 boosted with rROP2-SAG1. The production of IgG, IgG subclasses, lymphoproliferation, and level of gamma interferon (IFN-γ) were detected after vaccination. The animals vaccinated with rROP2-SAG1 quickly developed specific anti-TLA (T. gondii lysate antigen) antibodies, which continued to rise after immunization. However, production of IgG against TLA in mice vaccinated with pcROP2-SAG1 was relatively slow and maintained a high level after reaching plateau. There are more vigorous specific lymphoproliferative responses observed in mice of group rROP2-SAG1 than in pcROP2-SAG1. Immune responses in mice of group pcROP2-SAG1 boosted with rROP2-SAG1 were similar to the protein immunization group. Three immunization procedures resulted in a similar level of IFN-γ production. Our results indicate that BALB/c mice vaccinated by three immunization procedures induce similar humoral and cellular immunity against infection of T. gondii. Mice immunized with recombinant protein rROP2-SAG1 produce more humoral immune responses than mice immunized with other antigen formulations.     

Toxoplasma gondii microneme protein 8 (MIC8) is a potential vaccine candidate against toxoplasmosis

Microneme protein 8 (MIC8) is considered a new essential invasion factor in Toxoplasma gondii. In the present study, a deoxyribonucleic acid vaccine expressing MIC8 of T. gondii was constructed and the immune response it induced in Kunming mice was evaluated. The gene sequence encoding MIC8 was inserted into the eukaryotic expression vector pVAX I, and the pVAX-MIC8 expression plasmid was constructed, and the plasmid diluted with PBS to l00 mg/100?µl was injected into the Kunming mice muscularly. Levels of IgG antibody, gamma-interferon (IFN-γ), interleukin-2 (IL-2), interleukin-4, and interleukin-10 were detected. The mice were challenged with tachyzoites of the virulent T. gondii RH strain at the 14th day after the last immunization to observe the survival time. The high level of IFN-γ, IL-2, and IgG antibody indicated that mice vaccinated with recombinant pVAX-MIC8 plasmid could elicit strong cellular and humoral immune responses and showed a significantly increased survival time (10.3?±?0.9 days) compared with control mice which died within 5 days of challenge infection. These data demonstrate that the T. gondii MIC8 is a potential vaccine candidate against toxoplasmosis.     

Protective Efficacy of a Toxoplasma gondii Rhoptry Protein 13 Plasmid DNA Vaccine in Mice

Toxoplasma gondii is an obligate intracellular parasite infecting humans and other warm-blooded animals, resulting in serious public health problems and economic losses worldwide. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In the present study, a DNA vaccine expressing rhoptry protein 13 (ROP13) of T. gondii inserted into eukaryotic expression vector pVAX I was constructed, and the immune protection it induced in Kunming mice was evaluated. Kunming mice were immunized intramuscularly with pVAX-ROP13 and/or with interleukin-18 (IL-18). Then, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged with the virulent T. gondii RH strain (type I) and the cyst-forming PRU strain (type II). The results showed that pVAX-ROP13 alone or with pVAX/IL-18 induced a high level of specific anti-T. gondii antibodies and specific lymphocyte proliferative responses. Coinjection of pVAX/IL-18 significantly increased the production of gamma interferon (IFN-γ), IL-2, IL-4, and IL-10. Further, challenge experiments showed that coimmunization of pVAX-ROP13 with pVAX/IL-18 significantly (P < 0.05) increased survival time (32.3 ± 2.7 days) compared with pVAX-ROP13 alone (24.9 ± 2.3 days). Immunized mice challenged with T. gondii cysts (strain PRU) had a significant reduction in the number of brain cysts, suggesting that ROP13 could trigger a strong humoral and cellular response against T. gondii cyst infection and that it is a potential vaccine candidate against toxoplasmosis, which provided the foundation for further development of effective vaccines against T. gondii.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis>: expression and characterization of a recombinant protein containing SAG1 and GRA2 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Toxoplasma gondii is an obligate intracellular protozoan which infects most species of warm-blooded animals and causes toxoplasmosis. Previous immunological and immunization studies have demonstrated the potential role of T. gondii antigens SAG1 and GRA2 as a vaccine candidate. In the present study, we have cloned, expressed, and purified a recombinant protein SAG1–GRA2 in Pichia pastoris. Results showed that P. pastoris was a robust system producing a large amount of highly purified and biological activity protein. BALB/c mice immunized with SAG1–GRA2 elicited stronger humoral and cellular responses in comparison to control groups. This immunization resulted in an enhanced Th1 immune response as measured by IgG2a antibody production and increased splenocyte IFN-γ production, whereas no IL-4 was detected. After a lethal challenge with the highly virulent T. gondii RH strain, a prolonged survival time in SAG1–GRA2-immunized mice was observed in comparison to control groups. Our data demonstrate that SAG1–GRA2 triggered a protective response against toxoplasmosis. Therefore, SAG1–GRA2 protein might be a good candidate for the further development of a multiantigenic vaccine.     

Evaluation of protective effect of multiantigenic DNA vaccine encoding MIC3 and ROP18 antigen segments of Toxoplasma gondii in mice

The high incidence and severe damage caused by Toxoplasma gondii infection clearly indicates the need for the development of a vaccine. In this study, we evaluated the immune responses and protection against toxoplasmosis by immunizing ICR mice with a multiantigenic DNA vaccine. To develop the multiantigenic vaccine, two T. gondii antigens, MIC3 and ROP18, selected on the basis of previous studies were chosen. ICR mice were immunized subcutaneously with PBS, empty pcDNA3.1 vector, pMIC3, pROP18, and pROP18–MIC3, respectively. The results of lymphocyte proliferation assay, cytokine, and antibody determinations showed that mice immunized with pROP18–MIC3 elicited stronger humoral and Th1-type cellular immune responses than those immunized with single-gene plasmids, empty plasmid, or phosphate-buffered saline. After a lethal challenge with the highly virulent T. gondii RH strain, a prolonged survival time in pROP18–MIC3-immunized mice was observed in comparison to control groups. Our study indicates that the introduction of multiantigenic DNA vaccine is more powerful and efficient than single-gene vaccine, and deserves further evaluation and development.     

MIC6 associates with aldolase in host cell invasion by <Emphasis Type="Italic">Toxoplasma gondii</Emphasis>

The transmembrane microneme protein MIC6 and its partner MIC1, MIC4 comprise an adhesive complex that play important roles in host cell attachment by the obligate intracellular parasite Toxoplasma gondii. Successful penetration of host cells by T. gondii depends on coordinated interactions between MICs complex and the parasite's cytoskeleton. We have identified that the carboxy-terminal cytoplasmic domain (C domain) of MIC6 interacts with aldolase and the parasite cytoskeleton. Our finding uncovers new features regarding MIC6–aldolase interactions in host cell invasion by T. gondii.     

Secretory organelle trafficking in Toxoplasma gondii: A long story for a short travel

Toxoplasma gondii (T. gondii) possesses a highly polarized secretory system, which efficiently assembles de novo micronemes (MIC) and rhoptries (ROP) during parasite replication. Pioneer works have studied the sorting motifs within MIC and ROP proteins, required for their trafficking towards their final destination. These studies led to the conclusion that protein processing and protein sorting are inter-dependent activities. More recent works have revealed the trafficking routes taken by the MIC and ROP proteins by examining the functions of the endo-exocytic compartments and identified key molecules involved in protein sorting and transport. These recent findings have suggested that T. gondii has repurposed the evolutionarily conserved regulators of the endosomal system to the secretory pathway. This review reports the pioneer as well as the most recent findings on the molecular mechanisms regulating apical organelle and dense granule biogenesis and portrays the parasite as a remarkable secretory machine that has efficiently remodeled its trafficking system to adapt to an intracellular lifestyle.     

Immunogenic characterization of the chimeric surface antigen 1 and 2 (SAG1/2) of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> expressed in the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

In this study, we successfully expressed a chimerical surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii in Pichia pastoris. Eighty human serum samples, including 60 from confirmed cases of toxoplasmosis, were tested against the purified recombinant SAG1/2 in Western blots. Results of Western blots targeted at Toxoplasma IgG and IgM showed that the recombinant SAG1/2 reacted with all sera from the toxoplasmosis cases but none with the Toxoplasma-negative serum samples. These results showed that the P. pastoris-derived recombinant SAG1/2 was sensitive and specific and suitable for use as antigen for detecting anti-Toxoplasma antibodies. To further investigate the immunological characteristic of the recombinant protein, the recombinant SAG1/2 was injected subcutaneously into BALB/c mice, and their serum was tested against total protein lysate of T. gondii. Mice immunized with the recombinant SAG1/2 reacted specifically with the native SAG1 and SAG2 of T. gondii. Significant proliferation of splenocytes stimulated with tachyzoite total protein lysate was observed in vaccinated BALB/c mice but not in those from negative control mice. Specific production of IFN-γ, the Th1-type cytokines, was also found in stimulated splenocytes from vaccinated mice. These results show that the chimeric protein recombinant SAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice. Finally, vaccinated mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.005), and their survival time increased significantly compared to the negative control.     

Multicomponent DNA vaccine-encoding Toxoplasma gondii GRA1 and SAG1 primes: anti-Toxoplasma immune response in mice

A multicomponent DNA vaccine, encoding Toxoplasma gondii GRA1 and SAG1, was constructed and tested for its ability to confer protection. BALB/c mice were challenged with tachyzoites of the virulent T. gondii RH strain at 4?weeks following the last immunization, and immune responses and survival times were observed. The results show that vaccination by the multicomponent vaccine prolonged survival of mice challenged with the T. gondii RH strain (from average 4.50?±?0.22 to 7.60?±?0.74?days); induced high levels of IgG antibody (from 0.252?±?0.080 to 0.790?±?0.083), IFN-gamma (from 598.74?±?67.50 to 853.77?±?66.74?pg/ml), and IL-2 (from 89.44?±?10.66 to 192.24?±?19.90?pg/ml); changed the CD4⁺/CD8⁺ lymphocyte ratio (from 1.81?±?0.14 to 1.09?±?0.19); and stimulated NK cell-killing activity (from 46.81?±?3.96 to 64.15?±?7.71?%). These findings demonstrate that a multicomponent DNA vaccine, encoding GRA1 and SAG1, primes a strong humoral and cellular immune response and enhances protection against T. gondii challenge. The new, combined DNA vaccine provides another means to combat T. gondii infection.     

Protective immunity in toxoplasmosis: correlation between antibody response, brain cyst formation, T-cell activation, and survival in normal and B-cell-deficient mice bearing the H-2k haplotype.

Correlations of Toxoplasma gondii-specific immunoglobulin M (IgM) and IgG production, antigen-specific T-cell activation, and the number of brain cysts were compared in immunocompetent CBA/J (H-2k), C3H/He (H-2k), and B-cell-deficient CBA/N (H-2k) mice. Almost all of the C3H/He mice (94%) survived in comparison to CBA/J (71%) and CBA/N (53%) mice following infection with 20 cysts of Me 49, an avirulent strain of T. gondii. The mortality in susceptible mice was reduced by treatment of the animals with sulfadiazine during the acute stage of infection. Decreased mortality in CBA/J and C3H/He mice as well as in B-cell-deficient mice was paralleled by formation of fewer brain cysts. The Toxoplasma-specific T-cell proliferation was markedly enhanced in all three strains at day 15 postinfection but not at day 45 postinfection when compared to animals not treated with the drug. In contrast, Toxoplasma-specific IgM and IgG levels were lower in CBA/J and CBA/N mice treated with sulfadiazine than in untreated mice of these strains. Although CBA/N mice developed almost no humoral response either with or without drug treatment, they produced fewer brain cysts than normal CBA/J mice. The results indicate a major role of cell-mediated immunity in protection against an acute Toxoplasma infection.     

A New MIC1-MAG1 Recombinant Chimeric Antigen Can Be Used Instead of the Toxoplasma gondii Lysate Antigen in Serodiagnosis of Human Toxoplasmosis

This study presents an evaluation of the MIC1 (microneme protein 1)-MAG1 (matrix antigen 1) Toxoplasma gondii recombinant chimeric antigen for the serodiagnosis of human toxoplasmosis for the first time. The recombinant MIC1-MAG1 antigen was obtained as a fusion protein containing His tags at the N- and C-terminal ends using an Escherichia coli expression system. After purification by metal affinity chromatography, the chimeric protein was tested for usefulness in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-T. gondii immunoglobulin G (IgG). One hundred ten sera from patients at different stages of infection and 40 sera from seronegative patients were examined. The results obtained for the MIC1-MAG1 chimeric antigen were compared with those of IgG ELISAs using a Toxoplasma lysate antigen (TLA), a combination of recombinant antigens (rMIC1ex2-rMAG1) and single recombinant proteins (rMIC1ex2 and rMAG1). The sensitivity of the IgG ELISA calculated from all of the positive serum samples was similar for the MIC1-MAG1 chimeric antigen (90.8%) and the TLA (91.8%), whereas the sensitivities of the other antigenic samples used were definitely lower, at 69.1% for the mixture of antigens, 75.5% for the rMIC1ex2, and 60% for rMAG1. This study demonstrates that the MIC1-MAG1 recombinant chimeric antigen can be used instead of the TLA in the serodiagnosis of human toxoplasmosis.     

The Surface of Toxoplasma Tachyzoites Is Dominated by a Family of Glycosylphosphatidylinositol-Anchored Antigens Related to SAG1

Toxoplasma gondii is an Apicomplexan parasite with a complex life cycle that includes a rapidly dividing asexual stage known as the tachyzoite. The tachyzoite surface has been reported to comprise five major antigens, the most abundant of which is designated SAG1 (for surface antigen 1). At least one of the other four (SAG3) and another recently described minor antigen (SRS1 [for SAG1-related sequence 1]) have previously been shown to be structurally related to SAG1. To determine if further SAG1 homologs exist, we searched a Toxoplasma expressed sequence tag (EST) database and found numerous ESTs corresponding to at least three new genes related to SAG1. Like SAG1, these new SRS genes encode apparently glycosylphosphatidylinositol-anchored proteins that share several motifs and a set of conserved cysteine residues. This family appears to have arisen by divergence from a common ancestor under selection for the conservation of overall topology. The products of two of these new genes (SRS2 and SRS3) are shown to be expressed on the surface of Toxoplasma tachyzoites by immunofluorescence. We also identified strain-specific differences in relative expression levels. A total of 10 members of the SAG1 gene family have now been identified, which apparently include three of the five major surface antigens previously described and one antigen expressed only in bradyzoites. The function of this family may be to provide a redundant system of receptors for interaction with host cells and/or to direct the immune responses that limit acute T. gondii infections.     

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.     

Intranasal immunization with Toxoplasma gondii SAG1 induces protective cells into both NALT and GALT compartments

Intranasal (i.n.) immunization with the SAG1 protein of Toxoplasma gondii plus cholera toxin (CT) provides protective immunity. The aim of this study was to analyze the cellular activation of several mucosal compartments after i.n. immunization. Cervical and mesenteric lymph node (CLN and MLN, respectively) lymphoid cell and intraepithelial lymphocyte (IEL) passive transfer experiments were performed with CBA/J mice immunized i.n. with SAG1 plus CT. CLN and MLN cells and IEL isolated 42 days after immunization conferred protective immunity on naive recipient mice challenged with strain 76K T. gondii, as assessed by the reduction in the number of brain cysts. There were proliferative specific responses in nose-associated lymphoid tissue and the CLN and MLN cells from mice immunized with SAG1 plus CT, but no cytokine was detectable. Thus, protective immunity is associated with a specific cellular response in the nasal and mesenteric compartments after i.n. immunization.     

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.     

Highly polymorphic family of glycosylphosphatidylinositol-anchored surface antigens with evidence of developmental regulation in Toxoplasma gondii

The life cycle of the apicomplexan parasite Toxoplasma gondii requires that an infectious cyst develop and be maintained throughout the life of the host. The molecules displayed on the parasite surface are important in controlling the immune response to the parasite. T. gondii has a superfamily of glycosylphosphatidylinositol (GPI)-anchored surface antigens, termed the surface antigen (SAG) and SAG-related surface antigens, that are developmentally regulated during infection. Using a clustering algorithm, we identified a new family of 31 surface proteins that are predicted to be GPI anchored but are unrelated to the SAG proteins, and thus we named these proteins SAG-unrelated surface antigens (SUSA). Analysis of the single nucleotide polymorphism density showed that the members of this family are the most polymorphic genes within the T. gondii genome. Immunofluorescence of SUSA1 and SUSA2, two members of the family, revealed that they are found on the parasite surface. We confirmed that SUSA1 and SUSA2 are GPI anchored by phospholipase cleavage. Analysis of expressed sequence tags (ESTs) revealed that SUSA1 had 22 of 23 ESTs from chronic infection. Analysis of mRNA and protein confirmed that SUSA1 is highly expressed in the chronic form of the parasite. Sera from mice with chronic T. gondii infection reacted to SUSA1, indicating that SUSA1 interacts with the host immune system during infection. This group of proteins likely represents a new family of polymorphic GPI-anchored surface antigens that are recognized by the host's immune system and whose expression is regulated during infection.     

Peptide Microarray Analysis of In Silico-Predicted Epitopes for Serological Diagnosis of Toxoplasma gondii Infection in Humans

Toxoplasma gondii infections occur worldwide in humans and animals. In immunocompromised or prenatally infected humans, T. gondii can cause severe clinical symptoms. The identification of specific epitopes on T. gondii antigens is essential for the improvement and standardization of the serological diagnosis of toxoplasmosis. We selected 20 peptides mimicking linear epitopes on GRA1, GRA2, GRA4, and MIC3 antigenic T. gondii proteins in silico using the software ABCpred. A further 18 peptides representing previously published epitopes derived from GRA1, SAG1, NTPase1, and NTPase2 antigens were added to the panel. A peptide microarray assay was established to prove the diagnostic performance of the selected peptides with human serum samples. Seropositive human serum samples (n = 184) were collected from patients presenting with acute toxoplasmosis (n = 21), latent T. gondii infection (n = 53), and inactive ocular toxoplasmosis (n = 10) and from seropositive forest workers (n = 100). To adjust the cutoff values for each peptide, sera from seronegative forest workers (n = 75) and patients (n = 65) were used. Univariate logistic regression suggested the significant diagnostic potential of eight novel and two previously published peptides. A test based on these peptides had an overall diagnostic sensitivity of 69% (100% in ocular toxoplasmosis patients, 86% in acutely infected patients, 81% in latently infected patients, and 57% in seropositive forest workers). The analysis of seronegative sera performed with these peptides revealed a diagnostic specificity of 84%. The results of our study suggest that the use of a bioinformatic approach for epitope prediction in combination with peptide microarray testing is a powerful method for the selection of T. gondii epitopes as candidate antigens for serological diagnosis.