Conjugate-induced Polysaccharide Antibodies

革兰氏阴性肠道病原菌的表面多糖，以被膜或脂多糖的形式存在，既是重要的毒力因子，又是保护性抗原。这些多糖通过与载体蛋白结合而增强了免疫原性。在伤寒发病率较高的越南，成人和儿童的连续临床研究表明，与重组铜绿假单胞菌外蛋白A(REPA)结合的胶囊多糖(Vi)可引起对伤寒的高应答。在第三阶段的试验中，大约12,000名2-5岁的儿童注射了结合疫苗，没有表现出明显的副作用。监测48个月后，Vi-REPA有效率为89%。Pacebo组儿童在2.5年后接种结合疫苗。甲型副伤寒沙门氏菌是东南亚第二常见的肠道热病因，结合疫苗被发现对成人、青少年和2-4岁的儿童是安全和免疫原性的。在中国东南部，甲型副伤寒病例已超过斑疹伤寒。计划进行第三阶段临床试验。大肠杆菌O157是一种新出现的病原体，会导致幼儿溶血性尿毒症综合征。O157 O特异性多糖-REPA结合物在成年志愿者中的第一阶段研究证明了其安全性和免疫原性。大肠埃希菌O157结合物的II期研究，招募了52名2-5岁的儿童。给儿童注射一两次疫苗表明疫苗是安全的。初步免疫原性数据显示，儿童在第一次注射后6周，抗-LPSIg G升高了10倍以上，反应一致。无毒志贺毒素I和II是从突变的E.ColiO157中提纯出来的，并将与O特异的多糖偶联制成双价疫苗。O157大肠杆菌的主要宿主是牛。LPS-蛋白结合物在牛体内具有免疫原性，一项攻击性研究表明，血清抗LPSIg G水平与E.ColiO157在直肠的定植水平呈负相关。霍乱弧菌O1和O139是霍乱感染的主要血清型。O139的包膜多糖与O1的脂多糖合成的偶联物能诱导小鼠产生杀弧菌抗体。稻叶和小川两种血清型的脱毒脂多糖已被用作结合疫苗的基础。在动物实验中，结合物诱导的抗体水平高于单独使用内毒素的水平。将制备一种化学合成的小川O-特异性多糖，并将其偶联到蛋白质载体上。比较用天然纯化和化学合成的O-特异多糖制备的霍乱疫苗的免疫原性。这些结合物的临床试验正在计划中。在发达国家最常见的沙门氏菌感染之一是鼠伤寒沙门氏菌。耐药菌株在疾病分离株中很常见。我们比较了最常见和耐药菌株的O-特异性多糖结构。Abequose上的O-乙酰基被发现是一个主要的抗原位点和免疫显性决定簇。O-乙酰阳性菌株与O-乙酰阴性菌株的抗体反应较差。因此，疫苗制备菌株的选择将取决于流行病学调查和细菌学鉴定。空肠弯曲杆菌感染是美国和世界各地最常见的肠道感染之一。在美国最常见的类型是类型2。弯曲杆菌是微嗜氧性的，在液体介质中发酵一直很困难。对不同的培养基和发酵条件进行了研究，观察到在液体培养基中的适度生长。化学分析表明，该表面多糖中含有酮基，不含可可酸。从大小和染色性质来看，该多糖是一种低聚脂多糖。

Surface polysaccharides of Gram-negative enteric pathogens, in the form of capsule or lipopolysaccharide, are both essential virulence factors and protective antigens. The immunogenicity of these polysaccharides were enhanced by binding to carrier proteins. Sequential clinical studies in adults and in children in Vietnam, an area with a high attack rate of typhoid, showed that the capsular polysaccharide (Vi) conjugates bound to the recombinant Pseudomonas aeruginosa exoprotein A (rEPA) elicited high responses against typhi. In a Phase III trial, About 12,000 2-5 years old children injected with the conjugate vaccine showed no significant side reaction. The efficacy of Vi-rEPA was 89% after 48 months surveillance. Chidren in the pacebo group were given the conjugate vaccine 2.5 years later. Salmonella paratyphi A, the second most common cause of enteric fever in Southeast Asia, conjugate vaccine was found to be safe and immunogenic in adults, teenagers and then 2-4 year old children. In southeast China, cases of paratyphi A has exceeded those of typhi. A phase III clinical trial is planned. Escherichia coli O157, an emerging pathogen, causes hemolytic uremic syndrome in young children. Phase 1 study of E. coli O157 O-specific polysaccharide-rEPA conjugate demonstrated safety and immunogenicity in adult volunteers. The phase II study of E. coli O157 conjugate, 52 children 2-5 years old children were recruited. Children injected once or twice showed that the vaccine was safe. Preliminary immunogenicity data showed children responded uniformly with greater than 10 fold rise of anti-LPS IgG 6 weeks after the first injection. Non-toxic shiga toxin I and II are purified from mutant E. coli O157 and will be conjugated with O-specific polysaccharide for a bivalent vaccine. The major reservoir of E.coli O157 is cattle. LPS-protein conjugate showed to be immunogenic in cattle and a challenge study showed an inversed correlation between the serum anti-LPS IgG and the level of colonization of E. coli O157 at rectal. Vibrio cholera O1 and O139 are the major sero types in cholera infections. Conjugates synthesized with capsular polysaccharide of O139 and O-specific polysaccharide of LPS of O1 elicited vibriocidal antibodies in mice. Detoxified LPS from both Inaba and Ogawa serotypes have been used as a base for conjugate vaccine. In animal study, the conjugates elicited higher antibody level than LPS alone. A chemical synthesized O-specific polysaccharide for Ogawa will be prepared and conjugated to protein carrier. The immunogenicity of cholera vaccine prepared with naturally purified or chemically synthesized O-specific polysaccharide will be compared. Clinical trials of these conjugates are planned. One of the most common Salmonella infection in developed country is Salmonella typhimurium. Antibiotic resistant strains have been common in disease isolates. We have compared the O-specific polysaccharide structure of the most common and antibiotic resistant strains. The O-acetyl group on the abequose is found to be a major antigenic site and an immunodominant determinant. Strains that are O-acetyl positive reacted with antibodies from O-acetyl negative strains poorly. Selection of strains for vaccine preparation will therefore rely on the epidemiology survey and bacteriology identification. Campylobacter jejuli infection is one of the most common enteric infection in the US and around the world. The most common type in US is type 2. Campylobacter is microaerophilic and fermentation in liquid media has been difficult. Various media and fermentation conditions was studied and moderate growth in liquid media were observed. Chemical analysis showed that the surface polysaccharide contains keto and does not contain colominic acid. From the size and the staining properties, we concluded that the polysaccharide is an oligo LPS.