CSFV And PCV2 Infection And The Blocking Of Porcine PD-1 / PD-L1 Pathway

Immune inhibitory receptors have a key role in regulatory of adaptive immunity. They include programmed death-1(PD-1), cytotoxic T lymphocyte-associated antigen-4(CTLA-4), lymphocyte activation gene-3(LAG-3) and so on that deliver immune suppression and tolerance signals. During many chronic viral infection, overexpression of PD-1, CTLA-4 or LAG-3 can activate immune negative regulatory signal pathway resulted in virus-specific T cell exhaustion and immunosuppression through inhibiton of the lymphocyte proliferation and anti-virus cytokines secretion and acceleration of lymphocyte apoptosis. However, blockade of interaction inhibitory receptors with corresponding ligands can restore exhausted T cell function. Classical swine fever (CSF) and Postweaning Multisystemic Wasting Syndrome (PMWS) are the two important contagious diseases for swine. At present, although immunopathogenesis has been studied widely from pathogens and host for CSF and PMWS, research from immunoregulatroy of effector cells have been less. It has not yet been reported that the relationship between immune inhibitory receptors and immunophathogenesis of CSF and PMWS. Thus, this study explores the transcriptional level of inhibitory receptors during CSF and PMWS infection and immune effect changes. Polypeptides that blockade of PD-1/PD-L1 pathway are identificated. It further reveals the immunophathogenesis of CSF and PMWS. It provides the theoretical foundation for the study of immunomodulatory drugs and adjuvant.The activation of PD-1/PD-Ls pathway is the mechanism of immune dysfunction for many viral diseases. This part mainly studies the transcriptional level of porcine PD-1 and its ligands PD-L1 and PD-L2 during pig infected with CSFV. Piglets with 45 days of age and CSFV antibody negative were inoculated with CSFV Shimen strain. Experimental groups (10 pigs) and control groups (5 pigs) were inoculated intramuscularly with 105×medium tissue culture infective dose (TCID50) of CSFV Shimen strain or 0.5 mL PBS per pig, respectively. Five milliliter heparinized venous blood samples form the jugular vein were obtained at 0,3,7,10,14, and 21 days post infection, respectively. Peripheral Blood Mononuclear Cells (PBMCs) were isolated form fresh-heparinized venous blood by density gradient centrifugation. The mRNA expression of PD-1 and its ligands PD-L1 and PD-L2 in PBMC were evaluated by quantitative polymerase chain reaction (qPCR) during artificial CSFV infection. Simultaneously, expression of IL-2 and IL-10 mRNA were detected. The T cell proliferation and CSFV load in plasma were also measured. Results showed that the expression of PD-1 and its ligands mRNA were significantly increased (p<0.01) in PBMC from 3 to 7 days post infection (dpi). Meanwhile the level of IL-10 was up-regulated (p<0.01). The IL-2 mRNA was not obviously changed but it is significantly increased from 14 dpi. The T cell proliferation was notably decreased at 7 dpi. The CSFV load was also increased in plasma. Overall, our results suggest that the expression of PD-1 and its ligands were up-regulated and probably correlated with immune inhibition during acute CSFV infection.Two groups of pigs were selected from a conventional farm in the Henan province of China. The naturally occurring PMWS group infected with PCV2 included nine 45-day-old pigs with approximately 1 week of progressive weight loss, anorexia, pale skin, hair disheveled, and respiratory disorder. The control group tested negative for PCV2 infection and included six 45-day-old healthy pigs without any previous clinical signs of PMWS. Several viral nucleic acids including porcine circovirus 2 type (PCV2), Porcine Reproductive And Respiratory Syndrome Virus (PRRSV), porcine parvovirus (PPV), pseudorabies virus (PRV), and CSFV in plasma or lung and spleen were detected by PCR. The mRNA expression of immunomodulatory molecule (PD-1, PD-L1, PD-L2, PTEN, CTLA-4, and LAG-3) and cytokines (IL-10, IL-2, and IFN-γ) was determined by qPCR. Proliferation of PBMCs was also assessed by flow cytometry utilizing cellular labeling dilutions for detection. The results showed that specific bands of 381 bp product for PCV2 were found to be amplified in the plasma, lung, and spleen of PMWS-affected pigs, whereas no bands for PPV, PRV, PRRSV, and CSFV were observed in any of the samples. These data confirm our physiological observations of the PMWS-affected and uninfected groups. The mRNA levels of PD-L1 (p<0.01), PD-L2 (p<0.05), and PTEN (p<0.01) were remarkably increased in the PBMCs of diseased pigs compared to healthy pigs, whereas no change was observed for PD-1, CTLA-4, LAG-3, and Foxp3 expression. Cytokine IL-10 mRNA levels were significantly elevated (p<0.01), while IL-2 and IFN-c mRNA levels tended to be only slightly increased in the PBMCs of affected pigs compared to healthy controls. The proliferation of PBMCs was also decreased in diseased pigs. These data suggest that overexpression of PD-L1 and PD-L2 mRNA is one mechanism by which immunosupression of PMWS pigs occurs, supporting a new therapeutic strategy focused on PD-Ls for pigs suffering from PMWS.A series of polypeptides locate in extracellular domain of porcine PD-1 and PD-L1 protein were designed and synthesized by bioinformatics software and technique according to the three dimensional structure of PD-1 and PD-L1. We mainly Focuses on the combination epitope peptide with target protein and effect on immune function recovery of PBMCs by blockade of porcine PD-1/PD-L1 pathway. PBMCs were isolated from pigs suffering PMWS. PBMCs were labeled with Carboxy Fluoroscein Succinimidyl Ester (CFSE) and co-cultured with peptides and concanavalin A (con A) for 3 days. The proliferation of PBMCs was evaluated by flow cytometry. Peptides of promoting PBMCs proliferation were conjugated with FITC. We detected that whether Conjugated peptides combine with porcine recombinant PD-L1 protein or PBMCs. The anti-CSFV antibodies level in mouse serum was examined after Kunming mice were inoculated with peptides and inactivated CSFV. The resulted showed that peptides PD-15 can significantly accelerate porcine PBMCs proliferation. Interestingly, peptide PD-15 can also combine with porcine recombinant PD-L1 protein and PBMCs. And the level of anti-CSFV antibodies was elevated by peptide PD-15 stimulation. These data suggest that the peptide PD-15 can up-regulated the lymphocyte function by blockade of porcine PD-1/PD-L1 pathway. It provides a foundation for novel immunomodulatory adjuvant.