Conjugate-induced Polysaccharide Antibodies

革兰氏阴性肠道病原菌的表面多糖以荚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。这些多糖的免疫原性通过与载体蛋白结合而增强。在伤寒高发地区越南的成人和儿童中进行的连续临床研究表明，与重组铜绿假单胞菌胞外蛋白A（rEPA）结合的荚膜多糖（Vi）缀合物引起了对伤寒的高应答。在第三阶段试验中，约12，000名2 - 5岁儿童注射了结合疫苗，没有出现明显的副反应。48个月监测后，Vi-rEPA的有效率为89%。Pacebo组在2.5年后接种结合疫苗。在越南北方的2 - 5岁儿童中进行了一项剂量研究。结果显示存在剂量依赖性抗体应答。25微克剂量的反应最高，其次是12.5微克剂量。两个剂量之间没有统计学意义。然而，注射5微克缀合物多糖的儿童的Vi抗体水平显著低于接受较高剂量的儿童。甲型副伤寒沙门氏菌是东南亚第二大常见的肠道发热原因，结合疫苗被发现在成人、青少年和2 - 4岁儿童中安全且具有免疫原性。对沙门氏菌结合疫苗进行了重新审视。甲型副伤寒表明，偶联疫苗的O-乙酰基含量与偶联疫苗的免疫原性直接相关。在中国东南部，甲型副伤寒病例已超过伤寒病例。计划进行III期临床试验。大肠杆菌O157是一种新出现的病原体，可引起幼儿溶血性尿毒综合征。I期研究E.大肠杆菌O157 O-特异性多糖-rEPA结合物在成年志愿者中显示出安全性和免疫原性。Ⅱ期研究表明，E. coliO157偶联物，招募55名2 - 5岁儿童。儿童注射一次或两次表明疫苗是安全的。免疫原性数据显示，98%的儿童在首次注射后6个月抗LPS IgG升高4倍以上。在第一次注射后6周接受第二次剂量的儿童中没有加强反应。从突变株E. coli O157，并与O-特异性多糖缀合用于双价疫苗。大肠杆菌O157的主要宿主是牛。LPS-蛋白结合物在牛体内具有免疫原性，攻毒试验表明血清抗LPS IgG与E.大肠杆菌O157。O1和O139是霍乱的主要血清型。用O139的荚膜多糖和O1的LPS的O特异性多糖合成的缀合物在小鼠中引起杀弧菌抗体。来自Inaba和Ogawa血清型的脱脂LPS已用作缀合物疫苗的基础。在动物研究中，缀合物比单独的LPS引起更高的抗体水平。将制备用于Ogawa的化学合成的O特异性多糖并将其缀合至蛋白质载体。临床研究中将比较天然纯化和化学合成O-特异性多糖制备的霍乱疫苗的免疫原性。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。抗生素耐药菌株在疾病分离株中很常见。我们比较了最常见的抗生素耐药菌株的O-特异性多糖结构。发现阿倍糖上的O-乙酰基是主要的抗原位点和免疫显性决定簇。O-乙酰基阳性菌株与O-乙酰基阴性菌株的抗体反应较差。因此，疫苗制备菌株的选择将依赖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。美国最常见的类型是2型。弯曲杆菌是微需氧菌，在液体培养基中发酵一直是困难的。研究了不同的培养基和发酵条件，并成功地在液体培养基中生长。化学分析表明，表面多糖含有酮基，不含多粘菌素酸。从大小和染色性质，我们得出结论，多糖是一个寡脂多糖。用LPS或去乙酰化LPS制备的缀合物是免疫原性的，并且在补体存在下显示杀昆虫活性。

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. A dosage study in Northern Vietnam was conducted in children 2-5 years old. Results showed there was a dose dependent antibody response. The 25 microgram dose gave the highest response, followed by 12.5 microgram dose. There is no statistical significance between the two dosages. Children injected with 5 microgram of conjugate polysaccharide,however, had significantly lower Vi antibody levels than those received higher dosages. 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. A careful re-examination of the conjugate vaccines of S. paratyphi A indicated that the O-acetyl content of the conjugate vaccine is directly related to the immunogenicity of the conjugate vaccine. 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, 55 children 2-5 years old children were recruited. Children injected once or twice showed that the vaccine was safe. Immunogenicity data showed 98% children responded with greater than 4 fold rise of anti-LPS IgG 6 months after the first injection. There was no booster response in children receiving a second dose 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 in clinical studies. 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 mmedia has been difficult. Various media and fermentation conditions was studied and growth in liquid media were succeeded. Chemical analysis showed that the surface polysaccharide contains keto groups and does not contain colominic acid. From the size and the staining properties, we concluded that the polysaccharide is an oligo LPS. The conjugate prepared with LPS or deacetylated LPS were immunogenic and showed bactercidal activity in the presence of complement.