Trypanosoma cruzi, the aetiologic agent of Chagas disease, releases vesicles containing a wide range of surface molecules known to affect the host immunological responses and the cellular infectivity. Here, we compared the secretome of two distinct strains (Y and YuYu) of T. cruzi, which were previously shown to differentially modulate host innate and acquired immune responses. Tissue culture‐derived trypomastigotes of both strains secreted extracellular vesicles (EVs), as demonstrated by electron scanning microscopy. EVs were purified by exclusion chromatography or ultracentrifugation and quantitated using nanoparticle tracking analysis. Trypomastigotes from YuYu strain released higher number of EVs than those from Y strain, enriched with virulence factors trans‐sialidase (TS) and cruzipain. Proteomic analysis confirmed the increased abundance of proteins coded by the TS gene family, mucin‐like glycoproteins, and some typical exosomal proteins in the YuYu strain, which also showed considerable differences between purified EVs and vesicle‐free fraction as compared to the Y strain. To evaluate whether such differences were related to parasite infectivity, J774 macrophages and LLC‐MK2 kidney cells were preincubated with purified EVs from both strains and then infected with Y strain trypomastigotes. EVs released by YuYu strain caused a lower infection but higher intracellular proliferation in J774 macrophages than EVs from Y strain. In contrast, YuYu strain‐derived EVs caused higher infection of LLC‐MK2 cells than Y strain‐derived EVs. In conclusion, quantitative and qualitative differences in EVs and secreted proteins from different T. cruzi strains may correlate with infectivity/virulence during the host–parasite interaction.

Purpose. Vogt-Koyanagi-Harada disease (VKH), an autoimmune disease targeted against melanocytes, is associated with HLA-DRB1*0405. This study was undertaken to analyze T-cell recognition and the cytokine expression profile induced by melanocyte epitopes in HLA-DRB1*0405-positive and -negative patients with VKH uveitis. methods. Peripheral blood mononuclear cells (PBMC) proliferation and Th1 (IFN-γ) and Th2 (IL-4 and IL-5) cytokine production were analyzed in HLA-DRB1*0405-positive (n = 12) and -negative (n = 22) patients with VKH and HLA-DRB1*0405-positive (n = 9) and -negative (n = 8) control subjects in response to human melanoma cell line lysate (HMCLL) and 28 synthetic peptides derived from the human melanocyte differentiation proteins TYR, TRP1, TRP2, and Pmel17. The peptides were selected using the TEPITOPE algorithm, based on their predicted binding to HLA-DRB1*0405 and to the non–disease-related HLA-DRB1*15. results. HMCLL was recognized exclusively by the patients’ PBMC (44%) but not by those of the control subjects (P < 0.01). PBMC from patients with VKH recognized an increased breadth of melanocyte-derived peptides at lower peptide concentrations than in the control subjects (68% vs. 25%; P < 0.01, at 1 μM) and did not produce the Th2 cytokine IL-4 in response to disease-specific peptides (0% vs. 50%, P < 0.001). Five peptides were exclusively recognized in patients bearing HLA-DRB1*0405. Furthermore, HLA-DRB1*0405-bearing patients, but not those with HLA-DRB1*15, recognized an increased breadth of melanocyte epitopes in comparison to HLA-matched control subjects (60% vs. 28%; P < 0.05). conclusions. These data indicate that patients with VKH are sensitized to melanocyte epitopes and display a peptide-specific Th₁ cytokine response. In addition, the data indicate that patients bearing HLA-DRB1*0405 recognize a broader melanocyte-derived peptide repertoire, reinforcing the importance of this allele in susceptibility to the development of VKH disease.

One of the major drawbacks limiting the use of synthetic peptide vaccines in genetically distinct populations is the fact that different epitopes are recognized by T cells from individuals displaying distinct major histocompatibility complex molecules. Immunization of mice with peptide (181–195) from the immunodominant 43 kDa glycoprotein of Paracoccidioides brasiliensis (gp43), the causative agent of Paracoccidioidomycosis (PCM), conferred protection against infectious challenge by the fungus. To identify immunodominant and potentially protective human T-cell epitopes in gp43, we used the TEPITOPE algorithm to select peptide sequences that would most likely bind multiple HLA-DR molecules and tested their recognition by T cells from sensitized individuals. The 5 most promiscuous peptides were selected from the gp43 sequence and the actual promiscuity of HLA binding was assessed by direct binding assays to 9 prevalent HLA-DR molecules. Synthetic peptides were tested in proliferation assays with peripheral blood mononuclear cells (PBMC) from PCM patients after chemotherapy and healthy controls. PBMC from 14 of 19 patients recognized at least one of the promiscuous peptides, whereas none of the healthy controls recognized the gp43 promiscuous peptides. Peptide gp43(180–194) was recognized by 53% of patients, whereas the other promiscuous gp43 peptides were recognized by 32% to 47% of patients. The frequency of peptide binding and peptide recognition correlated with the promiscuity of HLA-DR binding, as determined by TEPITOPE analysis. In silico prediction of promiscuous epitopes led to the identification of naturally immunodominant epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous patient population exposed to P. brasiliensis. The combination of several such epitopes may increase the frequency of positive responses and allow the immunization of genetically distinct populations.

Antigen B (EgAgB) is an abundant lipoprotein released by the larva of the cestode Echinococcus granulosus into the host tissues. Its protein moiety belongs to the cestode-specific family known as hydrophobic ligand binding protein (HLBP), and is encoded by five gene subfamilies (EgAgB8/1-EgAgB8/5). The functions of EgAgB in parasite biology remain unclear. It may play a role in the parasite’s lipid metabolism since it carries host lipids that E. granulosus is unable to synthesise. On the other hand, there is evidence supporting immuno-modulating activities in EgAgB, particularly on innate immune cells. Both hypothetical functions might involve EgAgB interactions with monocytes and macrophages, which have not been formally analysed yet. EgAgB binding to monocytes and macrophages was studied by flow cytometry using inflammation-recruited peritoneal cells and the THP-1 cell line. Involvement of the protein and phospholipid moieties in EgAgB binding to cells was analysed employing lipid-free recombinant EgAgB subunits and phospholipase D treated-EgAgB (lacking the polar head of phospholipids). Competition binding assays with plasma lipoproteins and ligands for lipoprotein receptors were performed to gain information about the putative EgAgB receptor(s) in these cells. Arginase-I induction and PMA/LPS-triggered IL-1β, TNF-α and IL-10 secretion were examined to investigate the outcome of EgAgB binding on macrophage response. Monocytes and macrophages bound native EgAgB specifically; this binding was also found with lipid-free rEgAgB8/1 and rEgAgB8/3, but not rEgAgB8/2 subunits. EgAgB phospholipase D-treatment, but not the competition with phospholipid vesicles, caused a strong inhibition of EgAgB binding activity, suggesting an indirect contribution of phospholipids to EgAgB-cell interaction. Furthermore, competition binding assays indicated that this interaction may involve receptors with affinity for plasma lipoproteins. At functional level, the exposure of macrophages to EgAgB induced a very modest arginase-I response and inhibited PMA/LPS-mediated IL-1β and TNF-α secretion in an IL-10-independent manner. EgAgB and, particularly its predominant EgAgB8/1 apolipoprotein, are potential ligands for monocyte and macrophage receptors. These receptors may also be involved in plasma lipoprotein recognition and induce an anti-inflammatory phenotype in macrophages upon recognition of EgAgB.