Vanesa R. Sánchez

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.

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.

Regulatory cells induced by acute toxoplasmosis prevent the development of allergic lung inflammation.

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.

Toxoplasma gondii infection modulate systemic allergic immune response in BALB/c mice

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.

Elevating CLIC4 in Multiple Cell Types Reveals a TGF-β Dependent Induction of a Dominant Negative Smad7 Splice Variant

CLIC4 (Chloride intracellular channel 4) belongs to a family of putative intracellular chloride channel proteins expressed ubiquitously in multiple tissues. CLIC4 is predominantly soluble and traffics between the cytoplasm and nucleus and participates in cell cycle control and differentiation. Transforming growth factor beta (TGF-β) elevates CLIC4, which enhances TGF-β signaling through CLIC4 mediated stabilization of phospho-Smad2/3. CLIC4 is essential for TGF-β induced conversion of fibroblasts to myofibroblasts and expression of matrix proteins, signaling via the p38MAPK pathway. Therefore, regulation of TGF-β signaling is a major mechanism by which CLIC4 modifies normal growth and differentiation. We now report that elevated CLIC4 alters Smad7 function, a feedback inhibitor of the TGF-β pathway. Overexpression of CLIC4 in keratinocytes, mouse embryonic fibroblasts and other mouse and human cell types increases the expression of Smad7Δ, a novel truncated form of Smad7. The alternatively spliced Smad7Δ variant is missing 94bp in exon 4 of Smad 7 and is conserved between mouse and human cells. The deletion is predicted to lack the TGF-β signaling inhibitory MH2 domain of Smad7. Treatment with exogenous TGF-β1 also enhances expression of Smad7Δ that is amplified in the presence of CLIC4. While Smad7 expression inhibits TGF-β signaling, exogenously expressed Smad7Δ does not inhibit TGF-β signaling as determined by TGF-β dependent proliferation, reporter assays and phosphorylation of Smad proteins. Instead, exogenous Smad7Δ acts as a dominant negative inhibitor of Smad7, thus increasing TGF-β signaling. This discovery adds another dimension to the myriad ways by which CLIC4 modifies TGF-β signaling.

Toxoplasma gondii serine-protease inhibitor-1: A new adjuvant candidate for asthma therapy

Serine-proteases are important players in the pathogenesis of asthma, promoting inflammation and tissue remodeling. It’s also known that many serine protease inhibitors display immunomodulatory properties. TgPI-1 is a Toxoplasma gondii protein that exhibits broad spectrum inhibitory activity against serine proteases. In view of the increased prevalence of atopic disorders and the need to develop new treatment strategies we sought to investigate the potential of TgPI-1 for treating respiratory allergies. For this purpose, we developed a therapeutic experimental model. BALB/c mice were rendered allergic by intraperitoneal ovalbumin-alum sensitization and airway-challenged. Once the asthmatic phenotype was achieved, mice were intranasally treated with rTgPI-1 alone or with a mixture of rTgPI-1 and ovalbumin (OVA). A week later mice were given a secondary aerosol challenge. Treatment with rTgPI-1 alone or co-administered with OVA diminished bronchoalveolar eosinophilia, mucus production and peribronchial lung infiltration. This effect was accompanied by a lung resistance reduction of 26.3% and 50.3% respectively. Both treatments resulted in the production of lower levels of IL-4, IL-5, IFN-γ and regulatory IL-10 by thoracic lymph node cells stimulated with OVA. Interestingly, significant decreases in OVA specific IgE and T cell proliferation, and increases in FoxP3+ T cells at local and systemic levels were only detected when the inhibitor was administered along with OVA. These results show that both rTgPI-1 treatments reduced asthma hallmarks. However, co-administration of the inhibitor with the allergen was more effective. Hence, rTgPI-1 emerges as a novel adjuvant candidate for asthma treatment.

Genotyping of Toxoplasma gondii: The advantages of variable number tandem repeats within coding regions

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 (<20), and the absence of introns within the repeat region. The selected panel of eight molecular markers was analyzed in PRU and RH strains. As a first step, the variability of the sequence size allowed us to differentiate PRU from ME49 (two type II strains) and RH from GT1 (two type I strains). Additionally, amplification products from PRU and RH strains were sequenced to study intra-lineage variability. Aside from size polymorphisms in the amplification products we were able to identify sequence variability in polymorphic markers.