Preliminary Study Of T Cell Epitopes-based Vaccine Against Porcine Circovirus Type 2 In Mice

Porcine circovirus type 2(PCV2)is widely present in pigs and considered to be one of the main causes of immunosuppression in pigs.The inoculation of PCV2 vaccine has played a certain preventive effect,and these vaccines induce protection through humoral immunity.However,existing studies have shown that humoral immunity is not sufficient to eliminate PCV2 virus in infected pigs,and PCV2 virus is widely carried in vaccine-immunized pigs,suggesting that the complete protection of PCV2 may require the participation of cellular immunity.However,there is no in-depth study on the cellular immune response induced by PCV2 infection and the epitope recognized by activated T cells.BALB/c mice are widely used as a small animal model for PCV2 infection and vaccine evaluation.This project uses this model to analyze the cellular immune response induced by PCV2 infection and to screen mouse T cell epitopes on the PCV2 Cap protein.Mainly divided into two parts of work:1.Mice were infected with PCV2 TB16 strain.At different time points after infection,different tissues and organs were collected for detection of virus content by fluorescence quantitative PCR.Sera were collected to detect the antibody titer of PCV2 Cap protein by ELISA.Spleens and lymph nodes were collected to prepare single-cell suspension.The dynamic changes and activation status of NK cells and different T cell subsets were analyzed by multicolor flow cytometry.Results showed that virus replication in PCV2-infected mice peaked on day 7 and then began to decline but persisted in tissues up to 28 days after infection(by the end of the study),with the highest virus content in lung tissues.Antibody titers peaked 14 days after infection and remained at 28 days.These results showed that PC V2 successfully infected mice,as expected.Polychromatic flow analysis showed that NK cells were increased and activated in spleen and lymph nodes,while γδ T cells,CD4~+T cells and CD8~+T cells were decreased in PCV2 7 days after infection,showing a state of immunosuppression.On the 14th day,γδ T cells,CD4~+T cells,CD8~+T cells,effector memory T cells and central memory T cells increased significantly,and PD-1 was significantly upregulated.The number of effector memory T cells increased significantly on the 28th day after infection.Intracellular cytokine staining showed that CD4~+and CD8~+T cells secreted more IFN-y and TNF-α in mice stimulated by mitogen on the 28th day after infection.s2.Using the IEDB database bioinformatics online software,the mouse or pig T cell epitopes on the PCV2 Cap protein were predicted,and 14 peptides containing 9-15 amino acids were synthesized.T cells activated after PCV2 infection or immunization were used as effector cells,and IFN-γ ELISpot was used to screen and identify the ability of different polypeptides to activate mouse T cells to secrete IFN-γ.The results showed that T cells activated by PCV2 infection recognized peptide No.19(116-125 aa),No.25(93-107 aa),No.26(89-103 aa),No.27(144-158 aa)and No.28(179-193 aa);the epitopes recognized by T cells elicited activated by the inactivated PCV2 liposome vaccine were No.19,No.25 and No.28,while commercial PCV2 vaccine only recognize No.25(93-107 aa)on the 14th day after immunization,indicating that the vaccine mainly induces humoral immunity rather than cellular immunity.These results provide an important date and basis for the further identification of porcine T cell epitopes on the PCV2 Cap protein and the development of new PCV2 T cell vaccines.