疫苗免疫是预防感染性疾病的主要手段，它能有效降低各类传染性疾病的发病率和死亡率。猪圆环病毒（Porcine circovirus type 2, PCV2）引起的多种疾病给养猪业造成了重大的经济损失，但现有的猪圆环病毒疫苗存在免疫效力较低、油乳佐剂不易代谢及易引起不良反应等缺点，亟待寻找新的开发策略以提高猪圆环病毒疫苗的免疫效力及安全性。壳寡糖（Chitosan oligosaccharide, COS）可与机体特定受体结合激活免疫系统，发挥免疫调节功能，已被证明具有疫苗佐剂活性。相较于简单的物理混合，将佐剂和抗原化学偶联成一个整体可更大程度地增强疫苗的免疫原性。本论文通过化学手段，用具有佐剂活性的壳寡糖修饰猪圆环病毒疫苗，并通过细胞和动物实验评价其对细胞免疫和体液免疫功能的影响，分析其潜在的作用机制。论文的主要研究内容如下： （1）优化反应条件，制备壳寡糖修饰猪圆环疫苗。通过凝胶色谱、圆二色光谱、动态光散射、内源性荧光色谱和核磁共振光谱分析对寡糖修饰疫苗的结构进行表征。动物实验表明，壳寡糖修饰疫苗可显著提高PCV2特异性抗体滴度，促进T、B淋巴细胞增殖，提高细胞因子分泌水平，并对动物注射部位和代谢脏器没有任何毒副作用。（2）在上述研究基础上，探讨了壳寡糖脱乙酰度与修饰疫苗免疫原性之间的关系。本章实验中，制备三种不同脱乙酰度的壳寡糖，分别修饰PCV2疫苗。动物实验结果表明，脱乙酰度与壳寡糖修饰疫苗免疫原性呈正相关，高脱乙酰度壳寡糖更易诱导IgG抗体分泌，提高Th1和Th2型细胞因子表达水平，增加脾脏中CD4+ T细胞和CD8+ T细胞比例。（3）为进一步增强PCV2疫苗的免疫原性，将壳寡糖通过载体蛋白与疫苗偶联，构建壳寡糖-载体蛋白-疫苗新型修饰疫苗系统（COS-OVA-PCV2），并对其免疫学活性进行评价。细胞实验表明，COS-OVA修饰疫苗能更显著地促进DC细胞表面共刺激分子的表达，促进DC细胞活化。动物实验表明，COS-OVA修饰疫苗更有利于PCV2特异性抗体的产生，促进Th1和Th2型细胞因子分泌，增强体液免疫应答和细胞免疫应答。以上结果表明，载体蛋白可进一步提升壳寡糖修饰疫苗的免疫原性。（4）以巨噬细胞RAW264.7为实验模型，通过对其细胞增殖、活化、细胞因子分泌、TLRs受体蛋白表达及吞噬功能等方面的研究，探索壳寡糖修饰疫苗的作用机制。结果表明，壳寡糖修饰疫苗可激活巨噬细胞上的TLR2/4受体，进而激活NF-κB转录因子，诱导细胞因子转录表达，然后释放NO等细胞因子促进细胞活化和增殖，并增强巨噬细胞对抗原的吞噬功能。综上所述，本论文证实壳寡糖修饰能显著增强PCV2疫苗的体液免疫和细胞免疫，并发现修饰疫苗通过TLR2/4受体和NF-κB途径激活巨噬细胞，进而调节免疫应答，该研究成果将为壳寡糖作为新型疫苗佐剂用于兽用及人用疫苗研制提供重要的理论依据。;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 immunogenicity 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 immunostimulatory 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 Th1/Th2 response, including the raised levels of PCV2-specific antibodies and increased production of Th1 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.