Valentina Martin

Recombinant GRA4 or ROP2 Protein Combined with Alum or the gra4 Gene Provides Partial Protection in Chronic Murine Models of Toxoplasmosis

ABSTRACT 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.

Evaluation of Toxoplasma gondii recombinant proteins for the diagnosis of recently acquired toxoplasmosis by an immunoglobulin G analysis

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.

Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice

BackgroundCodon optimization and subcellular targeting were studied with the aim to increase the expression levels of the SAG178-322 antigen of Toxoplasma gondii in tobacco leaves. The expression of the tobacco-optimized and native versions of the SAG1 gene was explored by transient expression from the Agrobacterium tumefaciens binary expression vector, which allows targeting the recombinant protein to the endoplasmic reticulum (ER) and the apoplast. Finally, mice were subcutaneously and orally immunized with leaf extracts-SAG1 and the strategy of prime boost with rSAG1 expressed in Escherichia coli was used to optimize the oral immunization with leaf extracts-SAG1.ResultsLeaves agroinfiltrated with an unmodified SAG1 gene accumulated 5- to 10-fold more than leaves agroinfiltrated with a codon-optimized SAG1 gene. ER localization allowed the accumulation of higher levels of native SAG1. However, no significant differences were observed between the mRNA accumulations of the different versions of SAG1. Subcutaneous immunization with leaf extracts-SAG1 (SAG1) protected mice against an oral challenge with a non-lethal cyst dose, and this effect could be associated with the secretion of significant levels of IFN-γ. The protection was increased when mice were ID boosted with rSAG1 (SAG1+boost). This group elicited a significant Th1 humoral and cellular immune response characterized by high levels of IFN-γ. In an oral immunization assay, the SAG1+boost group showed a significantly lower brain cyst burden compared to the rest of the groups.ConclusionTransient agroinfiltration was useful for the expression of all of the recombinant proteins tested. Our results support the usefulness of endoplasmic reticulum signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The results showed that this plant-produced protein has potential for use as vaccine and provides a potential means for protecting humans and animals against toxoplasmosis.

Combination of CpG-oligodeoxynucleotides with recombinant ROP2 or GRA4 proteins induces protective immunity against Toxoplasma gondii infection

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 IgG(2a) to IgG(1) 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.

Toxoplasma gondii protease inhibitor-1 (TgPI-1) is a novel vaccine candidate 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.

A chloroplast‐derived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice

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.

High level of expression of the Toxoplasma gondii-recombinant rop2 protein in Escherichia coli as a soluble form for optimal use in diagnosis

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 Rop2196–561 fused to 6 histidine residues, we showed high levels of expression in bacteria growing in terrific broth. rRop2196–561 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 rRop2196–561 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 rRop2196–561 keeps its diagnostic value in contrast with the insoluble protein.

Toxoplasma gondii Infection Induces Suppression in a Mouse Model of Allergic Airway Inflammation

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.

Homologous prime-boost strategy with TgPI-1 improves the immune response and protects highly susceptible mice against chronic Toxoplasma gondii infection.

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.

Elevated serum levels of macrophage migration inhibitory factor are associated with progressive chronic cardiomyopathy in patients with Chagas disease.

Clinical symptoms of chronic Chagas disease occur in around 30% of the individuals infected with Trypanosoma cruzi and are characterized by heart inflammation and dysfunction. The pathogenesis of chronic chagasic cardiomyopathy (CCC) is not completely understood yet, partially because disease evolution depends on complex host-parasite interactions. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that promotes numerous pathophysiological processes. In the current study, we investigated the link between MIF and CCC progression. Immunohistochemical analysis demonstrated MIF overexpression in the hearts from chronically T. cruzi-infected mice, particularly those showing intense inflammatory infiltration. We also found that MIF exogenously added to parasite-infected murine macrophage cultures is capable of enhancing the production of TNF-α and reactive oxygen species, both with pathogenic roles in CCC. Thus, the integrated action of MIF and other cytokines and chemokines may account for leukocyte influx to the infected myocardium, accompanied by enhanced local production of multiple inflammatory mediators. We further examined by ELISA the level of MIF in the sera from chronic indeterminate and cardiomyopathic chagasic patients, and healthy subjects. CCC patients displayed significantly higher MIF concentrations than those recorded in asymptomatic T. cruzi-infected and uninfected individuals. Interestingly, increased MIF levels were associated with severe progressive Chagas heart disease, in correlation with elevated serum concentration of high sensitivity C-reactive protein and also with several echocardiographic indicators of left ventricular dysfunction, one of the hallmarks of CCC. Our present findings represent the first evidence that enhanced MIF production is associated with progressive cardiac impairment in chronic human infection with T. cruzi, strengthening the relationship between inflammatory response and parasite-driven pathology. These observations contribute to unravel the elements involved in the pathogenesis of CCC and may also be helpful for the design of novel therapies aimed to control long-term morbidity in chagasic patients.