实验海洋生物学重点实验室知识库

免疫佐剂是指可以增强抗原特异性免疫应答的物质，添加到疫苗中可延缓抗原的降解，提高免疫强度，减少抗原使用量；或增强抗原提呈细胞对抗原的处理和提呈能力；刺激淋巴细胞增殖分化；促进细胞释放细胞因子等，因而在疫苗中具有广阔的市场前景。理想的免疫佐剂能够在较少用量下刺激机体产生高效免疫应答，且自身无毒副作用，便于生产和使用。现有的兽用佐剂因不易代谢或易引起不良反应等缺点而越来越不满足市场的要求，因而亟需开发新型无毒副作用的佐剂。壳聚糖为具有一定免疫活性的天然多糖，生物相容性好、无毒且资源丰富，但由于在水中的不溶性限制了其应用，而其衍生物如壳聚糖季铵盐、壳聚糖硫酸酯具有良好的水溶性。因此，本文制备了不同分子量的α、β型壳聚糖及其水溶性衍生物——壳聚糖硫酸酯和壳聚糖季铵盐，并采用红外光谱、核磁共振一维、二维波谱、高效液相色谱等分析手段对其理化性质进行全面解析，确定了其取代位置；通过免疫调节实验筛选出免疫效果好的衍生物，并将衍生物制备成纳米粒，开发具有免疫刺激和抗原递送作用的新型免疫佐剂。主要研究结果如下：

1）成功制备了不同分子量的α、β-壳聚糖、壳聚糖硫酸酯和壳聚糖季铵盐衍生物。并以RAW 264.7细胞为模型进行体外细胞试验及免疫机制研究。首先通过细胞的一氧化氮产生量和细胞毒性的测试，发现不同分子量的α壳聚糖免疫效果优于β壳聚糖，壳聚糖硫酸酯和季铵盐衍生物的免疫效果优于低分子量的壳聚糖，而壳聚糖季铵盐衍生物的免疫效果最佳；采用荧光定量PCR、流式细胞术及蛋白印迹等技术，对三类样品的免疫机制研究表明：100 μg/mL的壳聚糖硫酸酯可以激活RAW 264.7细胞中的PI3K/Akt通路，并促进核转录因子NF-κB、AP-1进入核内，从而增加TNF-α等细胞因子的表达。100 μg/mL的壳寡糖可以激活RAW 264.7细胞中的MAPK及PI3K/Akt通路，并促进核转录因子NF-κB进入核内，从而增加IL-6等细胞因子的表达。而50 μg/mL的壳聚糖季铵盐即可激活RAW 264.7细胞中的MAPK, JAK/STAT 和 PI3K/Akt通路，并促进核转录因子NF-κB和AP-1 进入核内，从而增加IL-6等细胞因子的表达。

2）新城疫是由新城疫病毒感染导致的一种禽类传染病，自1926年首次发现以来给养禽业造成了巨大损失，因而受到了重视，目前主要通过接种疫苗预防该病。纳米载药系统能够延长药物释放时间，达到缓释效果，作为佐剂可以使抗原缓慢释放从而具有降低抗原使用量等优点。本研究以新城疫病毒为抗原，将壳聚糖季铵盐和壳聚糖作为佐剂，采用离子交联法制备了载新城疫病毒壳聚糖季铵盐/壳聚糖纳米粒，得到的纳米粒平均粒径为320±0.84 nm，多分散系数为0.36±0.02，zeta电位为+18.3±0.5 mV，包封率和载药量分别为37.4±2.69%和16.16±1.84%。通过扫描电镜观察发现纳米粒呈类球形，且表面光滑，无明显聚集现象，且粒径与粒度仪测定结果相近，进一步证明了纳米粒制备成功。体外细胞实验和体内动物实验证明纳米粒无明显细胞毒性，生物安全性好。使用载新城疫病毒纳米粒免疫无特定病原体的鸡后发现，载新城疫病毒壳聚糖季铵盐/壳聚糖纳米粒组的体液免疫水平稍低于白油佐剂组，但其细胞免疫水平显著优于白油组，且均可以使鸡对新城疫病毒的保护率达到100%。

综上所述, 壳寡糖、壳聚糖硫酸酯及壳聚糖季铵盐能够激活RAW 264.7细胞的信号通路，调节免疫应答。壳聚糖季铵盐/壳聚糖纳米粒作为新城疫疫苗的佐剂能够显著增强鸡的体液免疫和细胞免疫，保护免疫鸡不被新城疫病毒感染，且为壳聚糖季铵盐/壳聚糖纳米粒用作新型疫苗佐剂的研制提供依据。

Immune adjuvants are substances which promote the potency of antigen-specific immune responses. When added to vaccines, they can delay antigen degradation, enhance immune intensity and reduce the amount of antigen. Furthermore, adjuvants may enhance the ability of antigen presenting cells to process and present antigens, stimulate lymphocyte proliferation and differentiation, or promote the release of cytokines. Therefore, adjuvants have a broad market prospect in vaccines. An ideal immune adjuvant is supposed to produce an efficient immune response at a low dosage without side effects, and is easy to be produced and be applied. The existing veterinary adjuvants are increasingly unable to meet market requirements due to disadvantages such as difficulty in metabolism along with serious adverse reactions, thus it is urgent to develop new adjuvants with no toxic side effects. Chitosan is a natural polysaccharide with certain immunological activity, it is biocompatible, non-toxic and rich in resources, but its low solubility in neutral environment limits its application, while its derivatives such as quaternized chitosan and sulfated chitosan showed better water solubility. Therefore, in this article, we prepared α- and β-chitosan with different molecular weights and the water-soluble derivatives – sulfated chitosan and quaternized chitosan. Then the infrared spectrum, nuclear magnetic resonance (NMR) one-dimensional and two-dimensional spectrum, high performance liquid chromatography (HPLC) were determined to characterize the derivatives. Finally, the immunostimulatory effects of the derivatives were compared and the screened derivative with highest effect were used to prepare antigen-loaded nanoparticles to develop new types of adjuvant with immune stimulation and antigen delivery function. The main results are as follows:

1) α- and β-chitosan, chitosan sulfates and quaternized chitosan with different molecular weights were successfully prepared. RAW 264.7 cells were used as a model for in vitro to study the immune mechanisms of the derivatives. Firstly, through the test of nitric oxide production and cytotoxicity of cells, it was found that the immune effect of α-chitosan is better than that of β-chitosan, the immunological effects of chitosan sulfate and quaternized chitosan are better than low molecular weight chitosan, and α-quaternized chitosan showed the best activity. Quantitative RT-PCR, flow cytometry and western blot were used to study the immune mechanisms of three kinds of samples. The results showed that 100 μg/mL chitosan sulfate could activate the PI3K-Akt pathway in RAW 264.7 cells and promote the translocation of NF-κB and AP-1 into the nucleus, thereby increasing the expression of cytokines such as TNF-α. 100 μg/mL chitooligosaccharide can activate the MAPK and PI3K/Akt pathways in RAW 264.7 cells, and promote the entry of NF-κB into the nucleus, thereby increasing the expression of IL-6 and other cytokines. The quaternized chitosan at 50 μg/mL can activate the MAPK, JAK/STAT and PI3K/Akt pathways in RAW 264.7 cells, and promote the the translocation of NF-κB and AP-1 into the nucleus, thereby increasing the expression of IL-6 and other cytokines.

2) Newcastle disease (ND) is an avian infectious disease caused by Newcastle disease virus (NDV) infection. ND has caused great losses to the poultry industry since it was first discovered in 1926. Therefore, it has attracted much attention and is now mainly prevented by vaccination. Nano-drug delivery system can prolong the drug release time and achieve a sustained release effect. As an adjuvant, it can release antigens slowly and thus reduce the usage amount of antigen. In this study, NDV was used as the antigen, and NDV-loaded nanoparticles were prepared by ion-crosslinking method using quaternized chitosan and chitosan as adjuvants. The average particle size of the nanoparticles was 320±0.84 nm, the polydispersity index was 0.36±0.02, the zeta potential was +18.3±0.5 mV, and the encapsulation efficiency and drug loading were 37.4±2.69% and 16.16±1.84%, respectively. Scanning electron microscopy (SEM) results showed that the nanoparticles were almost spherical, with smooth surface and no obvious aggregation, and the particle size was similar to that of the particle size meter, which further proved the successful preparation of nanoparticles. In vitro MTT assay and in vivo animal experiments showed that the nanoparticles had no obvious cytotoxicity. Then the NDV-loaded nanoparticles were used to immunize chickens. It was found that the humoral immunity level of the nanoparticle immunized group was slightly lower than that of the oil emulsion vaccine group, but its cellular immunity level was significantly higher than that of the white oil group, and the protection rate of nanoparticle immunized chickens could reach 100%.

In summary, chitosan, chitosan sulfate and quaternized chitosan can activate the signaling pathways of RAW 264.7 cells and regulate the immune response. Quaternized chitosan nanoparticles as adjuvants for Newcastle disease vaccine can significantly enhance humoral and cellular immunity of chickens, protect immune chickens from Newcastle disease virus infection, and provide a basis for the development of quaternized chitosan/chitosan as new vaccine adjuvants.