Study On The Immunogenicity And Molecular Mechanism Of Porcine Circovirus Vaccine(PCV) Modified With Chitosan Oligosaccharides

Vaccination has been regarded as one of the most effective tools to control infectious diseases.It can effectively reduce the morbidity and mortality of various infectious diseases.Porcine circovirus type 2(PCV2)-associated diseases have led to great economic losses to the pig industry.However,the commercial vaccines fail to completely block the infection and transmission of PCV2.And the severe side effects,caused by traditional adjuvants such as oil-in water emulsions,also caused lots of safety concerns about their applications.Thus,a novel and safe adjuvant capable of eliciting both humoral and cellular immune responses is necessary to enhance the efficacy of PCV2 vaccines.As a potential adjuvant,chitosan oligosaccharides(COS)was confirmed to have versatile biological functions such as immunostimulating,anti-inflammation and anti infection.To ensure the strong immunogenicity of COS while avoiding its possible toxicity,we hypothesized that covalently linking COS to PCV2 vaccine may be an alternative to traditional vaccine application by physical mixing with adjuvants.In this study,we covalently linked COS to PCV2 vaccine,followed by the assessment of the enhanced immunogenicity of PCV2 modified by COS via in vivo and in vitro studies.Additionally,the mechanisms related to signal transduction pathways was also investigated.(1)In this part,the reaction conditions were optimized to prepare COS-modified PCV2 vaccine.The physicochemical characterizations were characterized by gel chromatography,circular dichroism,dynamic light scattering,intrinsic fluorescence and 1H-NMR Animal experiments showed that the modified vaccine significantly increased PCV2-specific antibody titer,promoted the proliferation of T and B lymphocytes,increased the secretion of cytokines,and had no toxic side effects at the injection site and in metabolic organs of animals.(2)Based on the above studies,the relationship between the degree of deacetylation of COS and the immuno genic ity of modified vaccines was investigated.In this part,three COSs with different degrees of deacetylation were prepared and coupled to PCV2 respectively.The results of animal experiment showed that the immuno stimulatory activity of COSs was positively correlated with their degrees of deacetylation.High-deacetylated COS more easily induced IgG antibodies secretion,increased the levels of Th1 and Th2 cytokine,and promoted the population of CD4+and CD8+ lymphocytes.(3)In this part,an effective adjuvant system was developed by covalent conjugation of COS via a carrier protein(OVA)to further increase the immunogenicity of PCV2 vaccine.The cellular experiments showed that COS-OVA-modified vaccine can significantly stimulated DCs to express higher co-stimulatory molecules and promoted DC cell activation to a larger extent.Animal experiments show that COS-OVA-modified PCV2 vaccine remarkably enhanced both humoral and cellular immunity against PCV2 by promoting the lymphocyte proliferation and initiating a mixed Thl/Th2 response,including the raised levels of PCV2-specific antibodies and increased production of Thl and Th2-type cytokines.These results indicate that modification by COS via a carrier protein might be a promising strategy to enhance the immunogenicity of vaccines.(4)To better understand the immunostumulationg mechanism of COS-modified PCV2 vaccine,a murine macrophage cell line(RAW264.7)was chosen as an experimental model.The results showed that the COS-modified PCV2 vaccine can activate the TLR2/4 receptor on macrophages,induce the NF-κB transcription factor,promote the cytokine secretions,and release NO and other cytokines to enhance macrophagic function,including cell proliferation,activation and phagocytosis.In summary,the present study demonstrated that COS modification can significantly enhance vaccine-specific humoral and cellular immunity.The mechanism of enhancing the adjuvant activity was also studied and explained.The present study will provide an important theoretical basis for the development of COS as vaccine adjuvant for veterinary or human vaccines.