mhc-binding-peptide and Disease-Models--Animal

Rhoptry proteins (ROPs) are involved in the cell invasion and parasitophorous vacuole (PV) formation and also vital for survival of Toxoplasma gondii (T. gondii) within host cells. ROP8 have a main role during the early phase of infection and can express in tachyzoite and bradyzoite forms. In the present study, we designed a novel multi-epitope DNA vaccine encoding the potential B and T-cell epitopes from ROP8 protein to evaluate the immunity and protective efficacy against acute T. gondii infection in BALB/c mice. For this purpose, several bioinformatics online servers were used. At first, the potential epitopes were selected for T and B cells using immune epitope database (IEDB) and BCPREDS online services. Then, the selected epitopes were fused together by SAPGTP linker. Finally, the physico-chemical features, secondary and tertiary structures, antigenicity, and allergenicity of the peptide were evaluated through different bioinformatics tools. Lastly, the multi-epitope peptide was successfully cloned into pcDNA3.1 expression vector. The DNA vaccine was subcutaneously injected into BALB/c mice and the immune responses of the vaccinated mice and controls were determined. The obtained results revealed that the multi-epitope ROP8 peptide has 183 amino acid residues with average molecular weight (MW) of 18.974 kDa. More than 98 % residues of the peptide were incorporated in favored and allowed regions of the Ramachandran plot. The antigenicity of multi-epitope peptide were estimated 0.8751 and 0.7649 by ANTIGENpro and VaxiJen servers, respectively. BALB/c mice immunized with DNA vaccine showed significantly increased the level of specific anti-T. gondii antibodies (P < 0.05), and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a TH1-type cellular immune response with production of IFN-γ and prolonged survival time, compared with the control groups (P < 0.05). The findings revealed that the multi-epitope ROP8 DNA vaccine induced strong humoral and cellular responses and prolonged the survival time in BALB/c mice, suggesting selection of potential epitopes may be a promising strategy for the design of multi-epitope-based vaccines.

Topics: Amino Acid Sequence; Animals; Antibodies, Protozoan; Antigens, Protozoan; B-Lymphocytes; Disease Models, Animal; Epitope Mapping; Epitopes; Female; H-2 Antigens; Immunization; Mice; Oligopeptides; Protozoan Vaccines; T-Lymphocytes; Toxoplasma; Toxoplasmosis; Vaccination; Vaccines, DNA

The identification and analysis of allergen-specific CD4+ T cells is critical for understanding how these cells contribute to atopic disease and how to subvert their behavior through immune therapy. The advent of fluorescently labeled soluble tetramers of peptide:MHCII complexes (pMHCII tetramers) has provided investigators with an invaluable means to achieve this goal. Although pMHCII tetramers were first developed over two decades ago, their widespread use has been limited by the technical difficulty of generating these reagents. However, the adoption of various technical innovations from several labs over time has contributed greatly to the increased success in tetramer generation today. Here, we describe a comprehensive protocol for generating pMHCII tetramers using as an example a Derp1:I-A

Topics: Allergens; Animals; CD4-Positive T-Lymphocytes; Cell Line; Disease Models, Animal; Epitopes; Histocompatibility Antigens Class II; Humans; Mice; Oligopeptides; Protein Multimerization; Pyroglyphidae; Respiratory Hypersensitivity