ABSTRACT
The binding motif of BF2*15 major histocompatibility complex (MHC) class I was explored by analyzing the interaction between an infectious bronchitis virus octapeptide and BF2*15, and the cytotoxic T lymphocyte (CTL) epitope from the nucleoprotein (NP) of H5N1 virus was identified using experimental methods. Computational methods, including homology modeling, molecular dynamics simulation, and molecular docking analysis, were used. The recombinant plasmid pCAGGS-NP was constructed, and NP expression was confirmed by indirect immunofluorescence and Western blot in transfected 293T cells. Antibodies against NP in pCAGGS-NP-inoculated specific-pathogen-free chickens were detected by enzyme-linked immunosorbent assay (ELISA). Interferon γ (IFN-γ) mRNA was quantified, and IFN-γ production was evaluated using quantitative reverse transcription PCR and capture ELISA, respectively. CD8(+) T-lymphocyte proliferation was detected using flow cytometric analysis. The BF2*15 MHC class I binding motif "x-Arg/Lys-x-x-x-Arg/Lys" was explored. Quantification of chicken IFN-γ mRNA, evaluation of IFN-γ production, and measurement of CD8(+) T-lymphocyte proliferation confirmed that the peptide NP67-74 of H5N1 was the BF2*15 MHC-class-I-restricted CTL epitope.
Subject(s)
Epitopes, T-Lymphocyte/metabolism , HLA-B Antigens/metabolism , Influenza A Virus, H5N1 Subtype/immunology , RNA-Binding Proteins/immunology , T-Lymphocytes, Cytotoxic/immunology , Viral Core Proteins/immunology , Animals , Binding Sites , Cell Line , Chickens , Humans , Molecular Docking Simulation , Nucleocapsid Proteins , Protein BindingABSTRACT
This study aims to construct a 3D structure of the avian major histocompatibility complex (MHC)-ß2M complex through homology modelling technology, perform molecular docking of the predicted infectious bronchitis virus (IBV) S1 protein potential epitope peptide Sp6 (NQFYIKLT) and the avian MHC-ß2M complex, and demonstrate the interactive mechanism between Sp6 and MHC using molecular dynamical simulations. The peptide Sp6 and the non-related peptide NP89-97 (PKKTGGPIY) were used to stimulate in vitro recombinant plasmid (pCAGGS-S1) avian splenic lymphocytes. Flow cytometric results show that CD8(+) T lymphocytes reproduce stimulated by the Sp6 and the nonrelated peptide proliferate by 34.8% and 2.6%, respectively. Meanwhile, fluorescent quantitative PCR results show that the secretion of IFN-γ in avian splenic lymphocytes increases after Sp6 stimulation. These data suggest that Sp6 can induce the activated avian lymphocytes in vitro to produce CTL, which is the CTL epitope in IBV S1.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/metabolism , Histocompatibility Antigens Class I/metabolism , Infectious bronchitis virus/immunology , Molecular Docking Simulation , Spike Glycoprotein, Coronavirus/metabolism , Cell Proliferation , Epitopes, T-Lymphocyte/chemistry , Flow Cytometry , Histocompatibility Antigens Class I/chemistry , Interferon-gamma/biosynthesis , Protein Binding , Real-Time Polymerase Chain Reaction , Spike Glycoprotein, Coronavirus/chemistryABSTRACT
The virulent isolate SDZB0808 of QX-type infectious bronchitis virus (IBV) was continuously passaged in chicken embryos for 110 generations. The safety and immune efficacy of the 110th generation of IBVs (P110) were evaluated. Damage was not found in the appearance of the 3-day-old specific-pathogen-free (SPF) chicks immunized with 10(4.5) EID50 (median embryo infective dose) of P110 by intranasal and ocular administration. At 14 d after the vaccination with 10(4.5) EID50 of P110, all the 3-day-old SPF chicks were immune from the attack of the homologous virulent strain SDZB0808 and the heterologous virulent strain SDIB821/2012. The whole genome sequencing of SDZB0808 of different generations (P1-P110) indicated that the replicase 1a sequences of P60-P110 all lost a length of 30bp in the same region. Specific primers were designed according to the differences in the genomes of P1-P110. SYBR Green I real-time quantitative PCR was adopted to analyze the proportion of the viruses with 30bp deletion in P60, P100, and P110. Results showed that with the passage in chicken embryos, the proportion of the viruses with 30bp deletion gradually increased. Almost 100% of the viruses in the P110 had 30bp deletion in the replicase 1a sequence. Therefore, the attenuation of IBV's virulence may be the outcome of directional screening in the chicken embryos. This work confirmed the high safety and immune efficacy of P110 in SPF chickens. Thus, P110 can serve as an attenuated IBV vaccine candidate.
