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Toxoplasmosis is a zoonotic disease with worldwide prevalence in humans and warm-blooded animal populations. In livestock Toxoplasma gondii is the causal agent of significant economic losses since it can cause abortions in goats and sheep. It is estimated that one third of the world population is infected. Although there are effective therapies for acute infection, these are sometimes poorly tolerated, teratogenic, and have a long administration time. Considering the deficiencies that exist related to the prevention and treatment of toxoplasmosis, the development of a safe and effective vaccine would be extremely valuable in fighting against this infection. In the present work, we characterize for the first time the adjuvant and immunogenic potential of a recombinant profilin protein (rTgPF), in a vaccine formulation alone or in combination with the well-known GRA7 antigen candidate in a murine toxoplasmosis model. Since TgPF acts as a ligand for TLR11 and 12 inducing innate immune responses that promote type 1 adaptive responses, we first study the capacity of the mix rGRA7 + rTgPF to initiate an immune response by evaluating dendritic cell activation. Both rTgPF and rGRA7 induces activation of mouse BMDCs more efficiently than the single proteins, evidenced by increased expression of CD80 and CD86 co-stimulatory proteins and secretion of IL-6, IL-10 and IL-12 cytokines after in vitro stimulation. The sum of the effects of rGRA7 and rTgPF on BMDCs maturation led us to assay them in a vaccination protocol. BALB/c mice vaccinated with this mix elicited a Th1-biased immunity via the induction of lymphocyte proliferation, activation of CD4+T cells and increased IFN-γ production that resulted in enhanced protection against chronic Toxoplama gondii infection. Profilin per se induce only cellular immunity but augments the effect of rGRA7 immune responses when used together, thus allowing us to postulate rTgPF as a potential adjuvant in a protein vaccine.

Background Supporting the hypothesis thatT. gondii infection protects against allergy in humans we previously demonstrated that this infection can modulate not only the susceptibility to develop respiratory allergies in mice but also suppresses allergic responses at systemic level. This latter finding suggests that T. gondii infection could prevent the onset of other allergic diseases, such as atopic dermatitis. At present, few studies have investigated the modulation of atopic dermatitis by parasite infections. Objective Here, we sought to investigate whether chronic infection with T. gondii is capable of modulating the development of atopic dermatitis. Methods Chronically infected mice were sensitized by repeated epicutaneous ovalbumin administration. Skin histopathology, humoral response, cytokine production and innate type-II lymphoid cells (ILC2) were assessed. Results A marked reduction in epidermal thickness and dermal inflammatory infiltrate along with a reduction in mast cell count was observed in infected mice compared to non-infected mice. These results correlated with a diminished TH2 and TH1 allergen specific response. Reduced type-II IL-4 and IL-5 cytokines were already detected during the first 24 h of allergen sensitization in splenocytes and draining lymph nodes from infected mice. Moreover, this reduced type-II profile in chronically infected animals correlated with diminished ILC2 number in draining lymph nodes. Conclusion Chronic infection withT. gondii prevents the development of atopic dermatitis. The diminished susceptibility seems to result from changes in type-II innate immune response that may lead to the induction of a deficient TH2 response and consequently to a lower susceptibility to develop atopic dermatitis.

Toxoplasma gondii is an intracellular protozoan which is widely distributed. Infection occurs as a result of ingestion of uncooked meat and exposure to cat feces. Immunocompetent individuals are generally asymptomatic, while severe disease may occur in immunocompromised subjects and in congenital toxoplasmosis, which is caused by transplacental acquisition of T. gondii. Genetic diversity of T. gondii has often been studied using a PCR-RFLP scheme based on nine molecular markers. These studies led to the description of a clonal population structure with three main lineages (I, II and III) in North America and Europe and higher genetic diversity in South America. The aim of this study was to develop molecular markers that could allow the discrimination of genetic variants within each clonal lineage. We analyzed the genome of T. gondii to identify genes containing variable number tandem repeats (VNTRs). The coding sequences of T. gondii ME49 genome were processed with Tandem Repeat Finder software. A panel of candidate markers was selected based on the following parameters: the repeat unit size (>9 bp) and composition (to avoid single and dinucleotide runs), the number of copies (

The development of an effective and safe vaccine to prevent Toxoplasma gondii infection is an important aim due to the great clinical and economic impact of this parasitosis. We have previously demonstrated that immunization with the serine protease inhibitor-1 (TgPI-1) confers partial protection to C3H/HeN and C57BL/6 mice. In order to improve the level of protection, in this work, we combined this novel antigen with ROP2 and/or GRA4 recombinant proteins (rTgPI-1+rROP2, rTgPI-1+rGRA4, rTgPI-1+rROP2+rGRA4) to explore the best combination against chronic toxoplasmosis in C3H/HeN mice. All tested vaccine formulations, administered following a homologous prime-boost protocol that combines intradermal and intranasal routes, conferred partial protection as measured by the reduction of brain cyst burden following oral challenge with tissue cysts of Me49 T. gondii strain. The highest level of protection was achieved by the mixture of rTgPI-1 and rROP2 proteins with an average parasite burden reduction of 50% compared to the unvaccinated control group. The vaccine-induced protective effect was related to the elicitation of systemic cellular and humoral immune responses that included antigen-specific spleen cell proliferation, the release of Th1/Th2 cytokines, and the generation of antigen-specific antibodies in serum. Additionally, mucosal immune responses were also induced, characterized by secretion of antigen-specific IgA antibodies in intestinal lavages and specific mesenteric lymph node cell proliferation. Our results demonstrate that rTgPI-1+rROP2 antigens seem a promising mixture to be combined with other immunogenic proteins in a multiantigenic vaccine formulation against toxoplasmosis.

The Toxoplasma gondii serin protease inhibitor-1 (TgPI-1) is a dense granule antigen that showed to specifically inhibit trypsin, chymotrypsin and neutrophil elastase, suggesting a possible modulatory role during the parasite invasion process and on the development of the innate immune response. To study the immune-protective value of TgPI-1, C3H/HeN mice were immunized with a recombinant form of the antigen rTgPI-1 combined with alum. All immunized mice produced specific anti-rTgPI-1 immunoglobulins, with high IgG antibody titers and a mixed IgG(1)/IgG(2a) response, with predominance of IgG(1) production. The cellular immune response was associated with the production of IFN-gamma and IL-10 cytokines. Vaccinated mice displayed significant protection against an oral challenge either after a lethal infection with Me49 cysts (90% survival vs. 50%) and also after a non-lethal infection (58% reduction in brain parasite load) compared to the non-vaccinated control group. In conclusion, rTgPI-1 elicits a strong specific immune response providing partial protection against both T. gondii acute and chronic infection, so it would be a good candidate in a vaccine against toxoplasmosis, which could be combined with other relevant parasite antigens.

Subunit-based vaccines are safer than live or attenuated pathogen vaccines, although they are generally weak immunogens. Thus, proper combination of immunization strategies and adjuvants are needed to increase their efficacy. We have previously protected C3H/HeN mice from Toxoplasma gondii infection by immunization with the serine protease inhibitor-1 (TgPI-1) in combination with alum. In this work, we explore an original vaccination protocol that combines administration of recombinant TgPI-1 by intradermal and intranasal routes in order to enhance protection in the highly susceptible C57BL/6 strain. Mice primed intradermally with rTgPI-1 plus alum and boosted intranasally with rTgPI-1 plus CpG-ODN elicited a strong specific Th1/Th2 humoral response, along with a mucosal immune response characterized by specific-IgA in intestinal lavages. A positive cellular response of mesentheric lymph node cells and Th1/Th2 cytokine secretion in the ileon were also detected. When immunized mice were challenged with the cystogenic Me49 T. gondii strain, they displayed up to 62% reduction in brain parasite burden. Moreover, adoptive transfer of mesenteric lymph node cells from vaccinated to naïve mice induced significant protection against infection. These results demonstrate that this strategy that combines the administration of TgPI-1 by two different routes, intradermal priming and intranasal boost, improves protective immunity against T. gondii chronic infection in highly susceptible mice. Fil: Sánchez, Vanesa Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Fenoy, Ignacio Martín. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Picchio, Mariano Sergio. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Soto, Ariadna Soledad. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Arcon, Nadia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Golgman, Alejandra. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Martín, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina

The reactivity values of Toxoplasma gondii ROP2, GRA4, and GRA7 recombinant antigens (rAgs) were analyzed by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) in 23 congenitally infected (I) and 36 noninfected (NI) infants. The reactivity values observed from the serum samples of I versus NI infants for each recombinant protein were 91% versus 67% for rROP2 (P = .05), 86% versus 29% for rGRA4 (P.001), and 56% versus 11% for rGRA7 (P = .003). The follow-up showed that serum samples from NI infants became negative for specific IgG at 5.8 months (95% confidence interval [CI], 4.9-6.7) using a commercial assay; meanwhile, by specific recombinant protein ELISA, the samples became negative at 3.7 months with rROP2 (95% CI, 2.8-4.6), at 1.3 months with rGRA4 (95% CI, 0.8-1.8), and at 0.9 months with rGRA7 (95% CI, 0.5-1.3). Kinetic analysis also showed that serum samples from group I presented different IgG-profiles among rAgs. The rROP2 IgG profile was similar to that of the commercial assay, whereas rGRA4 and rGRA7 profiles showed a gradual decrease along the period of the study. The potential of the utility of rAgs to develop a diagnostic system that discriminates congenitally I infants from NI is discussed.

The efficacy of vaccination with Toxoplasma gondii recombinant GRA4 (rGRA4) and ROP2 (rRPO2) proteins and a mix of both combined with alum were evaluated in C57BL/6 and C3H mice. In C57BL/6 mice, rGRA4 and rGRA4-rROP2 immunizations generated similar levels of immunoglobulin G1 (IgG1) and IgG2a isotypes against GRA4, whereas immunizations with rROP2 and the mix induced a predominant IgG1 production against ROP2. All groups of C3H vaccinated mice exhibited higher levels of IgG1 than IgG2a. rGRA4-stimulated splenocytes from vaccinated mice produced primarily gamma interferon while those stimulated with rROP2 produced interleukin-4. Challenge of rGRA4- or rGRA4-rROP2-vaccinated mice from both strains with ME49 cysts resulted in fewer brain cysts than the controls, whereas vaccination with rROP2 alone only conferred protection to C3H mice. Immunization with a plasmid carrying the entire open reading frame of GRA4 showed a protective level similar to that of rGRA4 combined with alum. These results suggest that GRA4 can be a good candidate for a multiantigen anti-T. gondii vaccine based on the use of alum as an adjuvant. Fil: Martin, Valentina. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina. Fil: Supanitsky, Alicia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina. Fil: Echeverria, Pablo C. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina. Fil: Litwin, Silvana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina. Fil: Tanos, Tamara. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina. Fil: de Roodt, Adolfo R. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina. Fil: Guarnera, Eduardo. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina. Fil: Angel, Sergio O. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.

The increased prevalence of allergies in developed countries has been attributed to a reduced exposure to some microbes. In agreement with epidemiological studies, we previously showed that Toxoplasma gondii infection prevents allergic airway inflammation. The mechanisms would be related to the strong Th1 response induced by the parasite and to regulatory cell induction. Herein we further characterized whether T. gondii allergy modulation extents to a systemic level or if it is limited to the lung. Parasite infection before allergic sensitization resulted in a diminished Th2 cytokine response and, when sensitized during acute infection, an increased in TGF-β production was detected. Allergen specific T cell proliferation was also reduced. Sensitization during both acute and chronic phases of infection resulted in a decreased anaphylaxis reaction. Our results extend earlier work and show that, in addition to lung airway inflammation, T. gondii infection can suppress allergic responses at systemic level. These results open the possibility that this protozoan infection could modulate other allergic disorders such as atopic dermatitis or oral allergies. Understanding the mechanisms by which different microorganisms regulate inflammation may potentially lead to the development of strategies aimed to control atopic diseases. Fil: Fenoy, Ignacio Martín. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sánchez, Vanesa Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Soto, Ariadna Soledad. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Picchio, Mariano Sergio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Martín, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Goldman, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina

The increased prevalence of allergies in developed countries has been attributed to a reduction of some infections. Supporting epidemiological studies, we previously showed that both acute and chronic Toxoplasma gondii infection can diminish allergic airway inflammation in BALB/c mice. The mechanisms involved when sensitization occurs during acute phase would be related to the strong Th1 response induced by the parasite. Here, we further investigated the mechanisms involved in T. gondii allergy protection in mice sensitized during acute T. gondii infection. Adoptive transference assays and ex vivo co-cultures experiments showed that not only thoracic lymph node cells from infected and sensitized mice but also from non-sensitized infected animals diminished both allergic lung inflammation and the proliferation of effector T cells from allergic mice. This ability was found to be contact-independent and correlated with high levels of CD4+FoxP3+ cells. IL-10 would not be involved in allergy suppression since IL-10-deficient mice behaved similar to wild type mice. Our results extend earlier work and show that, in addition to immune deviation, acute T. gondii infection can suppress allergic airway inflammation through immune suppression. Fil: Fenoy, Ignacio Martín. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sánchez, Vanesa Roxana. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soto, Ariadna Soledad. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Picchio, Mariano Sergio. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Maglioco, Andrea Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Corigliano, Mariana Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Dran, Graciela Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Martín, Valentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Goldman, Alejandra. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Background & Aims: Acute inflammatory ileitis occurs in susceptible (C57BL/6) mice after oral infection with Toxoplasma gondii . Overproduction of interferon (IFN)-γ and synthesis of nitric oxide mediate the inflammation. We evaluated the role of transforming growth factor (TGF)-β produced by intraepithelial lymphocytes (IELs) in this process. Methods: We analyzed the histologic and immunologic consequences of adoptive transfer of antigen-primed IELs into susceptible mice treated with anti–TGF-β before oral challenge with T. gondii cysts. An in vitro coculture of enterocytes and IELs assessed the production of chemokines and cytokines in the presence of anti–TGF-β. Results: Antigen-primed IELs prevent acute ileitis in susceptible mice that is reversed with anti–TGF-β. Resistant mice (CBA/J) develop ileitis after treatment with anti–TGF-β. Antigen-primed IELs can induce systemic immunosuppression as measured by depressed IFN-γ production. In vitro, primed IELs reduce the production of inflammatory chemokines by infected enterocytes and IFN-γ by splenocytes. Conclusions: Regulation of the ileal inflammatory process resulting from T. gondii is dependent on TGF-β–producing IELs. The IELs are an essential component in gut homeostasis after oral infection with this parasite. GASTROENTEROLOGY 2001;120:914-924

The rhoptry 2 protein (Rop2) is an interesting protein of Toxoplasma gondii that is involved in the parasite invasion of host cell, it has three T-cell epitopes and high antigenic value. However, the expression of Rop2 as a recombinant protein in Escherichia coli is not an easy task, showing low levels of expression or degradation and solubility problems. Using a recombinant Rop2(196-561) fused to 6 histidine residues, we showed high levels of expression in bacteria growing in terrific broth. rRop2(196-561) was purified mainly as a soluble product and in high concentrations (approx 1 mg/mL) under native conditions (40 mM imidazol in phosphate buffer). However, after a cycle of freezing-thawing rRop2(196-561) became insoluble. When glycerol was added to 26%, immediately after purification, the protein stayed soluble after cycles of freezing-thawing. Finally, it was demonstrated that under these conditions soluble rRop2(196-561) keeps its diagnostic value in contrast with the insoluble protein.

A cDNA clone (Tgzy85d11.r1) obtained from the Toxoplasma Expressed Sequence Tag project was chosen due to its homology with proteins of the heat shock 90 family. The cDNA encodes 137 amino acids of the C-terminal portion of the Toxoplasma Hsp90 protein (TgHsp90). Serum samples obtained from orally infected BALB/c and C57BL/6 mice showed reactivity against a recombinant TgHsp90 (rTgHsp90) after 8 weeks postinfection. Isotype analysis showed an anti-rTgHsp90 IgG2a/IgG3 response in infected BALB/c and anti-rTgHsp90 IgG1/IgG2a/IgG2b response in infected C57BL/6 mice. Serum samples from individuals chronically and putative acutely infected with T. gondii showed a similar anti-rTgHsp90 IgG response. Our work identifies TgHsp90 as a novel parasite antigen that seems to elicit a higher relation of anti-TgHsp90/anti-T. gondii IgGs during chronic infection in comparison with the acute stage.

A novel Toxoplasma gondii interspersed repeat element (TgIRE), present in most of the tachyzoite chromosomes, was characterised. Two regions on the TgIRE sequence showed high identity to two different T. gondii expressed sequence tag cDNAs of unknown function, which seems to be TgIRE pseudogenes. Two set of primers were designed, 2-2' and 2-3, that amplify products of 1.02 and 0.62 kb, respectively. T. gondii DNA from RH and Me49 strains was amplified with TgIRE 2-2' primers, and the respective 1.02 kb products were digested with several endonucleases. Different fragment patterns by gel electrophoresis were found only with MboI. Sensitivity analysis revealed that the set 2-3 was more sensitive than 2-2', detecting by gel visualisation the amount of DNA equivalent to 1 and 10 parasites, respectively.

The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds. In human medicine, T. gondii can cause complications in pregnant women and immunodeficient individuals, while in veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock. Thus, the development of an effective anti-Toxoplasma vaccine would be of great value. In this study, we analysed the expression of T. gondii GRA4 antigen by chloroplast transformation (chlGRA4) in tobacco plants and evaluated the humoral and cellular responses and the grade of protection after oral administration of chlGRA4 in a murine model. The Western blot analysis revealed a specific 34-kDa band mainly present in the insoluble fractions. The chlGRA4 accumulation levels were approximately 6 μg/g of fresh weight (equivalent to 0.2% of total protein). Oral immunization with chlGRA4 resulted in a decrease of 59% in the brain cyst load of mice compared to control mice. ChlGRA4 immunization elicited both a mucosal immune response characterized by the production of specific IgA, and IFN-γ, IL-4 and IL-10 secretion by mesenteric lymph node cells, and a systemic response in terms of GRA4-specific serum antibodies and secretion of IFN-γ, IL-4 and IL-10 by splenocytes. Our results indicate that oral administration of chlGRA4 promotes the elicitation of both mucosal and systemic balanced Th1/Th2 responses that control Toxoplasma infection, reducing parasite loads., National Research Council (CONICET, Argentina), This work was supported by PIP 168/2010 of the National Research Council (CONICET, Argentina), PICT 691/2007 of the National Agency for Promotion of Science and Technology (ANPCyT, Argentina) and by Grant BIO2005-00155 (Ministerio de Educación y Ciencia, España)., National Agency for Promotion of Science and Technology (ANPCyT, Argentina), Ministerio de Educación y Ciencia, España

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) have been characterized as Th1-promoting immunopotentiators, an adjuvant activity desirable for vaccination against intracellular parasites like Toxoplasma gondii. In an attempt to find new antigen–adjuvant combinations that enhance the immunogenicity of antigen candidates for toxoplasma vaccines, we analyzed the extent of protection in mice immunized with ROP2 and GRA4 recombinant proteins when co-administered with CpG-ODN. Both GRA4 + CpG-ODN and ROP2 + CpG-ODN formulations were shown to induce a strong humoral Th1-biased response characterized by a high IgG2a to IgG1 antibody ratio. Both vaccination regimens led to increased secretion of IFN-γ and IL-10, and negligible amounts of IL-4, upon specific re-stimulation of spleen cells from these groups of mice. After a non-lethal challenge with tissue cysts of a moderately virulent strain, only the brains from mice vaccinated with ROP2 or GRA4 in combination with CpG-ODN showed a significant reduction (63% and 62%, respectively) in their parasite load compared to the controls. The rate of protection obtained with GRA4 + ROP2 + CpG-ODN resulted equivalent (66%) to those achieved with the single antigens plus CpG-ODN. Taken together, these results indicate that CpG-ODN is an important candidate adjuvant for use in potential multicomponent anti-T. gondii vaccines for animals and humans.

There is a link between increased allergy and a reduction of some infections in western countries. Epidemiological data also show that respiratory allergy is less frequent in people exposed to orofaecal and foodborne microbes such as Toxoplasma gondii. Infection with T. gondii induces a strong cell-mediated immunity with a highly polarized T helper type 1 (Th1) response in early stages of infection. Using a well-known murine model of allergic lung inflammation, we sought to investigate whether T. gondii infection could modulate the susceptibility to develop respiratory allergies. Both acute and chronic infection with T. gondii before allergic sensitization resulted in a diminished allergic inflammation, as shown by a decrease in bronchoalveolar lavage (BAL) eosinophilia, mononuclear and eosinophil cell infiltration around airways and vessels and goblet cell hyperplasia. Low allergen-specific immunoglobulin (Ig)E and IgG1 and high levels of allergen-specific IgG2a serum antibodies were detected. A decreased interleukin (IL)-4 and IL-5 production by lymph node cells was observed, while no antigen-specific interferon-γ increase was detected. Higher levels of the regulatory cytokine IL-10 were found in BAL from infected mice. These results show that both acute and chronic parasite infection substantially blocked development of airway inflammation in adult BALB/c mice. Our results support the hypothesis that T. gondii infection contributes to protection against allergy in humans. © 2008 British Society for Immunology. Fil: Fenoy, Ignacio Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Giovannoni, M.. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Batalla, Estela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Martín, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina Fil: Frank, Fernanda María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Piazzon, Margarita Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Leucemia Experimental; Argentina Fil: Goldman, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina

The value of T. gondii recombinant antigens rRop2, rGra4, rGra7 and rSAG1m (mature version) or rSAG1ct (C-terminal version) in differentiating recently acquired from chronic infections was determined by IgG-ELISA. The general highest sensitivity was observed with rRop2 whereas rSAG1m was not recognized by any of the serum samples, suggesting an incorrect folding. rGra4 and rGra7 showed significant higher sensitivity and absorbance values with serum samples from recently infected individuals compared to those with chronic infection. In contrast, rRop2 and rSAG1ct did not show differences in the reactivity pattern between both groups of serum samples.

The Toxoplasma gondii rhoptry protein Rop2 was expressed in Escherichia coli as a fusion protein containing 44 kDa of the 55-kDa mature Rop2, supplied with six histidyl residues at the N-terminal end (Rop2 196–561 ). Humoral response during Toxoplasma infection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2 196–561 as the antigen substrate. The analyzed sera were divided according to T. gondii -specific serological tests (IgG, IgA, or IgM indirect immunofluorescence and IgA or IgM immunosorbent agglutination assay) as group A (IgG + IgA − IgM − ; n = 35), group B (IgG + IgA + IgM + ; n = 21), group C (IgG + IgA + IgM − ; n = 5), and group D (IgG + IgA − IgM + ; n = 16). Twenty-six T. gondii -seronegative sera from individuals with other infections were also included (group E). Anti-Rop2 IgG antibodies were detected in 82.8% of group A sera and in 97.6% of the sera with acute-phase marker immunoglobulins (groups B, C, and D). The percentage of IgA antibody reactivity against Rop2 196–561 was 17.1% in group A, 50% in group D, and 80.8% in groups B and C. The percentage of IgM antibody reactivity was 0% in groups A and C and 62% in groups B and D. Sera from group E failed to show IgA, IgM, or IgG antibody reactivity. Since T. gondii Rop2 elicits a strong humoral response from an early stage of infection, it is suggested that recombinant Rop2 196–561 would be suitable for use in diagnostic systems, in combination with other T. gondii antigens, to detect specific IgG, IgA, and IgM antibodies.

Allergic asthma is an inflammatory disorder characterized by infiltration of the airway wall with inflammatory cells driven mostly by activation of Th2-lymphocytes, eosinophils and mast cells. There is a link between increased allergy and a reduction of some infections in Western countries. Epidemiological data also show that respiratory allergy is less frequent in people exposed to orofecal and foodborne microbes such as Toxoplasma gondii. We previously showed that both acute and chronic parasite T. gondii infection substantially blocked development of airway inflammation in adult BALB/c mice. Based on the high levels of IFN-γ along with the reduction of Th2 phenotype, we hypothesized that the protective effect might be related to the strong Th1 immune response elicited against the parasite. However, other mechanisms could also be implicated. The possibility that regulatory T cells inhibit allergic diseases has received growing support from both animal and human studies. Here we investigated the cellular mechanisms involved in T. gondii induced protection against allergy. Our results show for the first time that thoracic lymph node cells from mice sensitized during chronic T. gondii infection have suppressor activity. Suppression was detected both in vitro, on allergen specific T cell proliferation and in vivo, on allergic lung inflammation after adoptive transference from infected/sensitized mice to previously sensitized animals. This ability was found to be contact-independent and correlated with high levels of TGF-β and CD4(+)FoxP3(+) cells.