Introduction Exosomes are nanograde membrane-bound vesicles secreted from most cell types through the fusion of multivesicular bodies with plasma membranes. Some of these exosomes are well defined, and are known to have immunomodulatory properties as well as play critical roles in intercellular communications. In this study, we characterized the exosomes derived from Toxoplasma gondii and their functions in aspect of immune responses. Methods T. gondii exosomes were isolated and identified using electron microscopy, nanoparticle tracking analysis, and Western blotting. The viability of macrophage RAW264.7 cells affected by exosomes was evaluated using a Cell Counting Kit (CCK-8). Then the uptake of T. gondii exosomes by RAW264.7 cells was detected by labeling with fluorescent dye PKH67. After exosomes stimulation, in vitro the production of interleukin (IL)-12, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10 in RAW264.7 cells were investigated using enzyme-linked immunosorbent assay (ELISA). In immunized BALB/c mice, the antibodies, cytokines as well as the percentage of CD4+ and CD8+ T cells were determined using ELISA and flow cytometric analysis. Protective efficacy was evaluated by challenging intraperitoneally with tachyzoites of T. gondii . Results We successfully isolated and characterized the exosomes derived from T. gondii . Functionally, the viability of macrophage RAW264.7 cells was significantly affected by exosomes at a high concentration (160 μg/mL). The production of IL-12, TNF-α and IFN-γ in macrophage cells were increased, and the level of IL-10 was decreased. Furthermore, BALB/c mice immunized with T. gondii exosomes showed both humoral and cellular immune responses and also exhibited a prolonged survival time. Conclusion T. gondii exosomes could modulate macrophage activation in vitro and trigger humoral and cellular immune responses and partial protection against acute parasite infection in mice, which suggested that exosomes may serve as a potential candidate against toxoplasmosis.

Toxoplasma gondii is an intracellular parasite that causes severe neurologic and ocular disease in immune-compromised and congenitally infected individuals. There is no vaccine protective against human toxoplasmosis. Herein, immunization of L d mice with HF10 (HPGSVNEFDF) with palmitic acid moieties or a monophosphoryl lipid A derivative elicited potent IFN-γ production from L drestricted CD8 + T cells in vitro and protected mice. CD8 + T cell peptide epitopes from T. gondii dense granule proteins GRA 3, 6, 7, and Sag 1, immunogenic in humans for HLA-A02 + , HLA-A03 + , and HLA-B07 + cells were identified. Since peptide repertoire presented by MHC class I molecules to CD8 + T cells is shaped by endoplasmic reticulum-associated aminopeptidase (ERAAP), polymorphisms in the human ERAAP gene ERAP1 were studied and associate with susceptibility to human congenital toxoplasmosis (p<0.05). These results have important implications for vaccine development.

BackgroundT follicular helper (TFH) cells are a special subpopulation of T helper cells and can regulate humoral immune responses. This study examined whether the frequency of CD4+CXCR5+ TFH cells could be associated with active immunity in chronic hepatitis B (CHB) patients.Methodology and FindingsThe frequencies of peripheral blood CD4+CXCR5+ TFH cells, inducible T cell costimulator (ICOS), and/or programmed death 1 (PD-1) positive CD4+CXCR5+ TFH cells in immune-active (IA), immune-tolerant (IT) CHB, and healthy controls (HC) were characterized by flow cytometry analysis. The effect of adevofir dipivoxil treatment on the frequency of CD4+CXCR5+ TFH cells, the concentrations of serum IL-2, IFN-γ, TNF-α, IL-4, IL-6, IL-10, IL-21, ALT, AST, HBsAg, HBsAb, HBeAg, HBeAb and HBV loads in IA patients were determined. The potential association of the frequency of CD4+CXCR5+ TFH cells with clinical measures was analyzed. In addition, the frequency of splenic and liver CD4+CXCR5+ TFH cells in HBV-transgenic mice was examined. We found that the frequency of CD4+CXCR5+ TFH cells in IA patients was significantly higher than that of IT patients and HC, and the percentages of CD4+CXCR5+ TFH in IA patients were positively correlated with AST. Furthermore, the percentages of ICOS+, PD-1+, and ICOS+PD-1+ in CD4+CXCR5+ TFH cells in CHB patients were significantly higher than that of HC. Treatment with adefovir dipivoxil reduced the frequency of CD4+CXCR5+ TFH, PD-1+CD4+CXCR5+ TFH cells and the concentrations of HBsAg and HBeAg, but increased the concentrations of HBsAb, HBeAb, IL-2 and IFN-γ in IA patients. Moreover, the frequency of splenic and liver CD4+CXCR5+ TFH cells in HBV-transgenic mice was higher than that of wild-type controls.ConclusionsThese data indicate that CD4+CXCR5+ TFH cells may participate in the HBV-related immune responses and that high frequency of CD4+CXCR5+ TFH cells may be a biomarker for the evaluation of active immune stage of CHB patients.

BackgroundToxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-γ production by human HLA-A03 supertype peripheral blood CD8+ T cells from seropositive but not seronegative persons.ResultsHerein, when these peptides were administered with the universal CD4+T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with Pam2Cys added, we found we successfully created preparations that induced IFN-γ and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam2Cys is an effective way to elicit IFN-γ producing CD8+ splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1224-232); AMLTAFFLR (GRA6164-172); RSFKDLLKK (GRA7134-142); STFWPCLLR (SAG2C13-21); SSAYVFSVK(SPA250-258); and AVVSLLRLLK(SPA89-98). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite, luciferin, this novel, HLA transgenic mouse model, and imaging with a Xenogen camera.ConclusionsToxoplasma gondii peptides elicit HLA-A03 restricted, IFN-γ producing, CD8+ T cells in humans and mice. These peptides administered with adjuvants reduce parasite burden in HLA-A*1101 transgenic mice. This work provides a foundation for immunosense based vaccines. It also defines novel adjuvants for newly identified peptides for vaccines to prevent toxoplasmosis in those with HLA-A03 supertype alleles.