Protein and Polysaccharide Conjugate Vaccines to enteric diseases

革兰氏阴性菌表面多糖，即衣壳或脂多糖，是重要的毒力因子和保护性抗原。通过与载体蛋白的共价结合，提高了多糖的免疫原性。要用作疫苗，LPS必须解毒，其O-特异性多糖(O-SP)必须分离并与蛋白质结合。细菌毒素或类毒素和病毒衣壳蛋白可能是保护性抗原，也可能是载体蛋白。 伤寒沙门氏菌衣壳多糖(Vi)是一种获得许可的疫苗，但对5岁以下儿童的疗效有限。为给幼儿提供疫苗，将Vi与重组铜绿假单胞菌外源蛋白A(REPA)偶联。在注射Vi-REPA的2-5岁儿童中，47个月时的有效率为89%。对301名越南婴儿在2、4、6、12个月常规免疫的同时接种Vi-REPA，评价其安全性和免疫原性。对照组接受Hib-TT+DTP或DTP治疗。两组均未发生严重不良事件。Vi-REPA组免疫后GM-Ig G抗Vi水平显著高于对照组。各组间血清中抗DT抗体水平无明显差异。对TT和PT的抗体水平正在进行检测。 果胶是由多聚半乳糖醛酸组成的，结构上与Vi的主干相同。果胶可以在C2和C3位进行O-乙酰化(OacPec)，以模拟Vi的抗原性。OacPec-REPA在25名健康成年志愿者中一次性注射的I期研究表明它是安全的。注射6周后，60%的志愿者血清中的抗Vi抗体水平升高了4倍。抗果胶免疫球蛋白抗体未见升高。 甲型副伤寒沙门氏菌(SPA)是发展中国家肠热病的第二大常见原因，通过摄入受感染者污染的食物或饮料传播。SPA慢性携带者的存在尚未得到证实。我们与广西疾病预防控制中心合作，调查了一所寄宿中学的一次大规模疫情。一名慢性携带者，最有可能是暴发的源头，被确定为具有异常高水平的血清免疫球蛋白抗体和直肠拭子阳性。使用同样的方法，启动了一项扩大的调查。 O1霍乱弧菌仍然是印度次大陆和非洲的一个主要健康问题。现场研究表明，血清杀灭弧菌活性是针对其内毒素的。在一期试验中，O-SP偶联产生具有杀灭弧菌活性的抗脂多糖抗体。为了提高免疫原性，设计了一种结合方案，该方案利用非双功能试剂对载体蛋白和O-SP进行衍生化。我们合成了一个霍乱弧菌O-SP六聚体的结合物，改进后的O-SP结合物比第一阶段研究中使用的结合物具有更高的杀弧活性。 轮状病毒是全球婴儿腹泻的最常见原因。两种轮状病毒疫苗获得许可，一种是减毒活疫苗，另一种是重组疫苗，均为口服疫苗。我们正在设计一种基于衣壳蛋白的非肠道疫苗。重组衣壳蛋白具有截短的C或N端，并在大肠杆菌中表达。注射了重组蛋白的小鼠产生了中和抗体。重组蛋白与多糖的偶联提高了其溶解度

Surface polysaccharides of Gram-negative pathogens, capsules or lipopolysaccharides (LPS), are essential virulence factors and protective antigens. The immunogenicity of polysaccharides is enhanced by covalent binding to carrier proteins. To serve as vaccines, LPSs must be detoxified and their O-specific polysaccharides (O-SP) isolated and conjugated to proteins. Bacterial toxins or toxoids and viral capsid proteins may be protective antigens and may serve as carrier proteins. The capsular polysaccharide of Salmonella typhi (Vi) is a licensed vaccine but with limited efficacy in children less than 5 years old. To provide a vaccine for younger children, Vi was conjugated to recombinant exoprotein A of Pseudomonas aeruginosa (rEPA). An efficacy of 89% at 47 months was shown in 2-to-5-year olds injected with Vi-rEPA. Safety and immunogenicity of Vi-rEPA were evaluated in 301 Vietnamese infants administered concurrently with their routine immunizations at 2, 4, 6 and 12 months. Controls received Hib-TT +DTP or DTP. No serious adverse events occurred in any group. The post immunization GM IgG anti-Vi level in the Vi-rEPA group was significantly higher than in the control groups. There was no difference in the level of IgG anti-DT among the groups. Antibody levels to TT and PT are being assayed. Fruit pectin is composed of polygalacturonic acid, structurally identical to the backbone of Vi. Pectin can be O-acetylated chemically at the C2 and C3 positions (OacPec) to mimic Vi antigenically. A phase I study of OacPec-rEPA injected once into 25 healthy adult volunteers showed it to be safe. Six weeks after injection, a > 4 fold rise in IgG anti-Vi was found in 60% of the volunteers. No rise in IgG anti-pectin was found. Salmonella paratyphi A (SPA) is the second most common cause of enteric fever in developing countries transmited is through ingestion of food or drink contaminated by infected persons. The presence of chronic carriers of SPA is not established. In collaboration with the Guangxi Center for Disease Control and Prevention, we investigated a large outbreak in a residential middle school. A chronic carrier, most likely the source of the outbreak, was identified by having an exceptionally high level of serum IgG anti-LPS and a positive rectal swab. An expanded survey, using the same methods, was initiated. Vibrio cholerae O1 remains a major health problem in the Indian subcontinent and in Africa. Field studies showed that serum vibriocidal activity is directed toward its LPS. In a phase 1 trial, O-SP conjugates elicited IgG anti-LPS with vibriocidal activity. To enhance immunogenicity, a conjugation scheme was devised employing derivatization of both the carrier protein and the O-SP with heterobifunctional reagents. Conjugates of a V. cholerae O-SP hexamer we synthetized and the improved O-SP conjugates induced higher vibriocidal activities than the conjugate used in the Phase I study. Rotavirus is the most common cause of infantile diarrhea worldwide. Two rotavirus vaccines are licensed, a live attenuated and a reassortant, both orally administered. We are designing a parenteral vaccine based on capsid proteins. Recombinant capsid proteins with truncated C or N termini were expressed in E. coli. Mice injected with the recombinant proteins developed neutralizing antibodies. Conjugation of the recombinant proteins to polysaccharide increased their solubility