Protein and Polysaccharide Conjugate Vaccines to enteric diseases

革兰氏阴性病原体的表面多糖，即荚膜或脂多糖（LPS），是重要的毒力因子和保护性抗原。多糖的免疫原性通过与载体蛋白的共价结合而增强。为了用作疫苗，LPS必须被解毒，并且它们的O-特异性多糖（O-SP）被分离并与蛋白质缀合。细菌毒素或类毒素和病毒衣壳蛋白可以是保护性抗原并且可以充当载体蛋白。 伤寒沙门氏菌（Vi）的荚膜多糖是一种获得许可的疫苗，但对5岁以下儿童的疗效有限。为了给年幼的儿童提供疫苗，将Vi与铜绿假单胞菌的重组外蛋白A（rEPA）缀合。在注射Vi-rEPA的2至5岁儿童中，47个月时的有效性为89%。 Vi-rEPA的安全性和免疫原性在301名越南婴儿中进行了评价，这些婴儿在2、4、6和12个月时同时进行常规免疫接种。对照组接受Hib-TT + DTP或DTP。任何组均未发生严重不良事件。 Vi-rEPA组中免疫后GM IgG抗Vi水平显著高于对照组。各组间IgG抗DT水平无差异。 TT和PT的抗体水平正在检测中。 果实果胶由多聚半乳糖醛酸组成，其结构与Vi的主链相同。 果胶可以在C2和C3位置（OacPec）处被0-乙酰化以在抗原性上模拟Vi。 一项对25名健康成年志愿者注射一次OacPec-rEPA的I期研究表明它是安全的。 注射后6周，在60%的志愿者中发现IgG抗Vi升高> 4倍。 未发现IgG抗果胶升高。 甲型副伤寒沙门氏菌（SPA）是发展中国家肠道发热的第二大常见原因，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确定。 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大爆发。 一个慢性携带者，最有可能的爆发源，被确定为具有异常高水平的血清IgG抗LPS和阳性直肠拭子。 采用同样的方法进行了一次扩大调查。 霍乱弧菌O1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，血清杀弧菌活性针对其LPS。在1期试验中，O-SP缀合物引发具有杀弧菌活性的IgG抗LPS。为了增强免疫原性，设计了一种缀合方案，其采用用异双功能试剂衍生化载体蛋白和O-SP。 我们合成了霍乱弧菌O-SP六聚体的缀合物，并且改进的O-SP缀合物比I期研究中使用的缀合物诱导更高的杀弧菌活性。 轮状病毒是全球婴儿腹泻的最常见原因。有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，均口服给药。我们正在设计一种基于衣壳蛋白的非肠道疫苗。在E.杆菌 注射重组蛋白的小鼠产生了中和抗体。 重组蛋白与多糖的偶联增加了它们的溶解性

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