肺炎链球菌是一种革兰氏阳性菌，能够引起高死亡率的侵袭性肺炎疾病。荚膜多糖是肺炎链球菌的主要致病成分，早期曾以荚膜多糖作为疫苗预防肺炎链球菌的感染。然而，荚膜多糖是一种T细胞非依赖抗原，免疫持续时间短，不能诱导免疫记忆，不能为婴幼儿和儿童提供免疫保护作用。肺炎荚膜多糖与具有辅助性T细胞抗原表位的载体蛋白共价结合，这种多糖结合疫苗会极大改善多糖的免疫学性质，并能有效保护婴幼儿和儿童。例如，2010年于美国上市的13价肺炎结合疫苗显著降低了侵袭性肺炎链球菌疾病的发病率。然而，肺炎多糖结合疫苗的免疫保护作用有待进一步优化。研究表明多糖链的长度、糖蛋白比、化学结合策略等因素会影响结合疫苗的免疫学性质。有文献报道，适当的佐剂与多糖结合疫苗上的载体蛋白偶联会提高结合疫苗的免疫原性。此外，由于结合疫苗中存在载体蛋白诱导的表位抑制作用（CIES），即较高的载体蛋白特异性抗体滴度会抑制多糖特异性抗体的产生。因此，通过适当屏蔽载体蛋白的抗原表位降低CIES效应，是改善结合疫苗免疫学性质的有效策略之一。近期的研究结果表明，苯环作为连接桥可以改变PEG的构象，能更为有效地屏蔽葡激酶的抗原表位，从而改善葡激酶在体内的药学性质。本文分别以不带苯环、苯环、苯环-苯环为连接桥，将肺炎多糖与破伤风类毒素（一种载体蛋白，TT）进行共价结合。制备的多糖结合疫苗分别为PS-TT、PS-phe-TT和PS-2phe-TT。通过改变多糖结合疫苗的连接桥来提高疫苗的免疫原性。本文还对多糖结合疫苗的结构进行了鉴定，并检测了其免疫学性质。具体结果如下：利用化学偶联的方法，制备了PS-TT、PS-phe-TT和PS-2phe-TT三种结合疫苗。凝胶排阻层析色谱和1H NMR鉴定结果表明多糖能够有效的结合载体蛋白。动态光散射测定结果表明三种疫苗的水化半径分别为11.6±0.3 nm、10.9±0.4 nm和10.3±0.2 nm。 免疫学评价结果表明，苯环连接桥能降低TT特异性IgG的滴度，显著增加多糖特异性IgG的滴度和IL-5的分泌。这表明苯环连接桥能改变多糖构象，增强对TT抗原表位的屏蔽作用，降低结合疫苗的CIES效应；此外，PS-phe-TT能最大程度上保持多糖和载体蛋白抗原表位的完整性，能增强免疫记忆和Th2型细胞免疫应答。因此，PS-phe-TT有望成为预防效果更好的一种肺炎结合疫苗。 ;Streptococcus pneumonia is a serious Gram-positive pathogen and can lead to invasive pneumococcal diseases with high mortality rate. Capsular polysaccharide (PS) is the major virulence factor of S. pneumoniae. Previously, capsular PS was used for protection against pneumococcal diseases. However, capsular PS is a T-cell independent antigen that induces a poor immune response in infants and young children.In addition, the immune responses to PS are of short duration and cannot be boosted and the affinity does not mature. Thus, pneumococcal capsular PS has been conjugated with a carrier protein possessing T-helper cell epitopes to improve its immunogenicity, which effectively protects infants and young children against S. pneumonia. For example, a 13-valent pneumococcal conjugate vaccine (Pfizer Inc., USA) was licensed in 2010 and significantly reduced the incidence of invasive pneumococcal disease via the acquisition of PS-specific antibodies.However, the PS-specific immunogenicity of pneumococcal conjugate vaccines still needs to be improved. Several factors such as the length of PS chain, PS-to-protein ratio, and the conjugation chemistry are important to produce the PS-specific antibodies. Conjugation of the conjugate vaccines with adjuvant has also been used to stimulate the production of PS-specific antibodies. Beyond that, the carrier-induced epitopic suppression (CIES) mechanism has been observed in the study of conjugate vaccine. Through the CIES mechanism, the elicited high level carrier Protein specific antibodies can suppress the immune response to the capsular PS on the conjugate. Thus, the strategy to reduce the CIES effect of carrier proteins on the conjugate by introducing chemical linkers that moderately shield the epitope of the carrier protein is efficent. Recently, the hydrophobic phenyl linker has been found can induce a dense conformation of PEG by interaction with the hydrophilic PEG, which extensively shielded most domains adjacent to the antigen epitopes of SAK.Here, three conjugates (PS-TT, PS-phe-TT and PS-2phe-TT) were prepared to study whether the phenyl linker can reduce the CIES effect and improve the immunogenicity of the pneumococcal polysaccharide conjugates. The structural property and immunogenicity of the three pneumococcal polysaccharide conjugates were investigated. The details were as following:The three conjugates were purified by size exclusion chromatography and identified by 1H NMR, which indicated the successful conjugation of PS and TT. In addition, DLS analysis showed that the molecular radii of PS-TT, PS-phe-TT and PS-2phe-TT were 11.6±0.3 nm, 10.9±0.4 nm and 10.3±0.2 nm, respectively.As compared with the conjugate without the phenyl linker (PS-TT), the conjugate with the phenyl linker (PS-phe-TT) showed lower TT-specific IgG titers and significantly higher PS-specific IgG titers and higher IL-5 level. This indicated that the phenyl linker could alter the PS conformation and efficiently shield the TT epitopes. Thus, the PS-specific immunogenicity of the conjugate vaccine could be improved by decreasing the CIES effect of TT with the phenyl linker. In addition, the damage extent of PS epitopes and TT epitopes were minimized for PS-phe-TT. PS-phe-TT preferentially promoted a robust Th2-type immune response. In summary, PS-phe-TT is expected to act as an effective pneumococcal conjugate vaccine for protection against pneumococcal diseases.