SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts

Abstract\ud \ud \ud \ud Background\ud \ud Toxoplasma gondii is an intracellular parasite that causes relevant clinical disease in humans and animals. Several studies have been performed in order to understand the interactions between proteins of the parasite and host cells. SAG2A is a 22 kDa protein that is mainly found in the surface of tachyzoites. In the present work, our aim was to correlate the predicted three-dimensional structure of this protein with the immune system of infected hosts.\ud \ud \ud \ud Methods\ud To accomplish our goals, we performed in silico analysis of the amino acid sequence of SAG2A, correlating the predictions with in vitro stimulation of antigen presenting cells and serological assays.\ud \ud \ud \ud Results\ud Structure modeling predicts that SAG2A protein possesses an unfolded C-terminal end, which varies its conformation within distinct strain types of T. gondii. This structure within the protein shelters a known B-cell immunodominant epitope, which presents low identity with its closest phyllogenetically related protein, an orthologue predicted in Neospora caninum. In agreement with the in silico observations, sera of known T. gondii infected mice and goats recognized recombinant SAG2A, whereas no serological cross-reactivity was observed with samples from N. caninum animals. Additionally, the C-terminal end of the protein was able to down-modulate pro-inflammatory responses of activated macrophages and dendritic cells.\ud \ud \ud \ud Conclusions\ud Altogether, we demonstrate herein that recombinant SAG2A protein from T. gondii is immunologically relevant in the host-parasite interface and may be targeted in therapeutic and diagnostic procedures designed against the infection.The authors thank Marley Dantas Barbosa and Zilda Mendonça da Silva Rodrigues for technical assistance. This work was supported by Brazilian funding agencies CNPq, CAPES and FAPEMIG.The authors thank Marley Dantas Barbosa and Zilda Mendonça da Silva Rodrigues for technical assistance. This work was supported by Brazilian funding agencies CNPq, CAPES and FAPEMIG

Toxoplasma gondii-Derived Synthetic Peptides Containing B- and T-Cell Epitopes from GRA2 Protein Are Able to Enhance Mice Survival in a Model of Experimental Toxoplasmosis

Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2) is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN), as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b), mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-alpha and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii

Production, characterization and applications for Toxoplasma gondii-specific polyclonal chicken egg yolk immunoglobulins.

Toxoplasma gondii may cause abortions, ocular and neurological disorders in warm-blood hosts. Immunized mammals are a wide source of hyperimmune sera used in different approaches, including diagnosis and the study of host-parasite interactions. Unfortunately, mammalian antibodies present limitations for its production, such as the necessity for animal bleeding, low yield, interference with rheumatoid factor, complement activation and affinity to Fc mammalian receptors. IgY antibodies avoid those limitations; therefore they could be an alternative to be applied in T. gondii model.In this study we immunized hens with soluble tachyzoite antigens of T. gondii (STAg) and purified egg yolk antibodies (IgY) by an inexpensive and simple method, with high yield and purity degree. IgY anti-STAg antibodies presented high avidity and were able to recognize a broad range of parasite antigens, although some marked differences were observed in reactivity profile between antibodies produced in immunized hens and mice. Interestingly, IgY antibodies against Neospora caninum and Eimeria spp. did not react to STAg. We also show that IgY antibodies were suitable to detect T. gondii forms in paraffin-embedded sections and culture cell monolayers.Due to its cost-effectiveness, high production yield and varied range of possible applications, polyclonal IgY antibodies are useful tools for studies involving T. gondii

Immunocytochemistry for <i>T. gondii</i> using polyclonal IgY anti-STAg.

<p>HeLa cells infected with tachyzoites were fixed, permeabilized and incubated with IgY anti-STAg and rabbit IgG anti-IgY conjugated to fluorescein isothiocyanate (FITC). In fluorescence photomicrographs, or merged images with phase contrast. tachyzoites were detected into cell cytoplasm around the nucleus (blue, DAPI) with strong staining (arrows).</p

Purification of egg yolk antibodies.

<p>(<b>A</b>) Separation of the water soluble fraction (S1) from a lipid-rich precipitate (P1), after the incubation of crude egg yolk with acid water (pH 5.0–5.2); S1 precipitation by salting-out (19% Na₂SO₄) produced an enriched IgY pellet (P2) and a supernatant with contaminants (S2). (<b>B</b>): Purity degree of IgY samples determined by SDS-PAGE (8%), demonstrating that salting-out protocol produced high purity IgY antibodies. (<b>C</b>) Size-exclusion chromatography of the P2 fraction, with peak of IgY between 13th and 19th fractions, where (<b>D</b>) the 14th fraction presented the highest degree of purity. (<b>E</b>): IgY enrichment conferred by Slot-blot assay, IgY was detected until the concentration of 0.01 µg of protein (box). Bovine sera albumin (BSA) was used as negative control.</p

Immunohistochemistry for <i>T. gondii</i> detection using polyclonal IgY anti-STAg antibodies.

<p>Paraffin-embedded brain sections of mice chronically infected with ME-49 strain were incubated with IgY anti-STAg and rabbit IgG anti-IgY conjugated to fluorescein isothiocyanate (FITC). (<b>A</b> and <b>B</b>), segmented parasitophorus vacuoles were detected into host cell cytoplasm around the DAPI-stained nucleus (blue). (<b>C</b> and <b>D</b>), IgY anti-STAg antibodies strongly recognized antigens on outer walls of tissue cysts and free tachyzoites (<b>C</b>, arrow).</p

Recognition profile of STAg proteins using polyclonal antibodies raised in chicken and mice.

<p>(<b>A</b>) Differential reactivity to <i>Toxoplasma gondii</i> soluble antigens (STAg) of egg IgY antibodies and mouse serum IgG from animals immunized by intramuscular (i.m.) and subcutaneous (s.c.) routes initially was assessed by 1D immunoblotts (WB1D). Antigens with approximately (∼) 30 kDa (blue box) were evenly recognized by all antibodies tested, which was not observed in antigens with ∼40 kDa (green box) and ∼50 kDa (red box), which were mainly recognized by chicken and mouse antibodies, respectively. The same recognition pattern against STAg proteins was noted in two-dimensional immunoblotts (WB2D), probed with chicken IgY (<b>B</b>) or mouse IgG (<b>C</b>). (<b>D</b>) IgY antibodies obtained from eggs of chickens immunized with <i>T. gondii</i>, as well as IgY against <i>Neospora caninum</i> and <i>Eimeria</i> spp., were assayed against STAg-immobilized nitrocellulose membranes in order to check possible antibody cross-reactivity.</p

Production kinetics and avidity of IgY anti-STAg antibodies.

<p>Indirect ELISA showing (<b>A</b>) kinetics of chicken IgY and (<b>B</b>) IgY avidity maturation with 6M urea-treated (•) or untreated (○) immunocomplex. Black arrows indicate the three immunization doses. Samples with ELISA Index (EI) equal or superior to 1.2 were considered positive. (<b>C</b>) 1D-immunoblot presenting recognition kinetics (from 14 to 49 days p.i.) of STAg proteins by IgY and its respective avidity maturation, evaluated by 6M urea treatment of immunocomplexes.</p