| Immune adjuvants are molecules,compounds or macromolecular complexes that can enhance the antigen response.Most adjuvants assist the antigen response through initiating the innate immune system.Active protection is mediated by the innate and adaptive immune systems.The innate immune system senses pathogen-associated molecular patterns(PAMPs)that are expressed by a wide variety of infectious microorganisms through pattern recognition receptors(PRR),which trigger the activation of anti pathogenic defenses and stimulate the adaptive immune response.Addition of natural or synthetic molecules can improve the immunogenicity of vaccines and regulate the type of immune response,this is important in modern vaccine research.The synthetic oligodeoxynucleotides(ODNs)containing CpG sequences,are recognized as a "danger" signal by the mammalian immune system.Therefore,CpG ODNs can be used as immune adjuvant to enhance the immunogenicity of the vaccine.We found that the sequence and structural characteristics of different CpG ODNs porformenced different immunogenicity,and this difference affected the immunoregulation effect.We designed and synthesized 11 CpG ODNs and simulated their 3D structural and analyzed their internal energy by Chem office software.Three kinds of pig cells(IPECJ2,D34/21,PBMCs)were used to explored transcriptional level changes including that TLRs,signal pathway molecules,cytokines(interleukin,interferon,tumor necrosis factor,etc.)and antigen-related molecules.CpG ODNs on the immunoregulation of cells treated in vitro.The ability of interferon production induced by different CpG ODNs cells and their antiviral effects were investigated.The main results list as follows:(1)Design and analysis of CpG ODNs motifs of swine: We analyzed different types with different motifs and functions of CpG ODNs in mice,humans and existing animals.11 types of CpG ODN motifs with potential immunoregulation were designed and synthesized by the selection of CpG motif,flanking,terminal sequence motif and phosphorothioate bond.(2)Immunoregulatory effect of CpG ODNs motif on porcine cell line:CpG ODNs mainly activate the following signaling pathways: TLR9-MyD88-NF-κB,TLR9-MyD88-MARK p38,TLR3-NF-κB,TLR3-IRF3;CpG ODNs promoted the expression of multiple interleukins and inflammatory factors(Th1 type cytokine IL-12,IFN-γ;Th2 cytokine IL-4,local immune factor IL-17,etc.),regulated cellular immune response types,balanced Th1 / Th2 responses;CpG ODNs enhanced antigen presentation of APC: CpG ODNs promoted 3D4 / 21 and PBMCs to expressed CD86 and MHC;CpG ODNs can promote PBMCs expression of IFN-γ except CpG3,CpG1,CpG6,CpG7,CpG8,CpG11 can be used as specific activation type I interferon sequence;Meanwhile the immunoregulatory effect of CpG ODNs is cell-dependent.(3)Antiviral effect of different CpG ODNs: The antiviral ability of CpG ODNs was studied by using porcine epidemic diarrhea virus(PEDV)and GFP-labeled vesicular stomatitis virus(VSV-GFP).CpG ODNs antiviral and induced IFNβ / α ability due to the type and structure of CpG ODNs.CpG ODNs showed an antiviral effect that was significantly better than that of non-CpG motifs(scrabled sequences).In this study,we found that CpG ODNs were able to activate TLR9 and / or TLR3 to initiate downstream immune responses through four signaling pathways and cause different Th types of immune responses by promoting the expression of different Th cytokines meanwhile they could enhance humoral immunity and mucosal immunity and promot APC antigen presentation capacity.All CpG ODNs are able to assist cells in resisting both PEDV and VSV infections but they showed differently in antiviral capacity.And this study selected oligonucleotide sequences containing CpGs suitable for activating different signaling pathways,modulating different immune types,and promoting different cytokine expression. |
