Human Immune Response To Polysaccharide-protein Conjugat

病原菌表面多糖，包括衣壳多糖(CPS)和脂多糖(LPS)，既是重要的毒力因子，又是保护性抗原。CPS的年龄相关和T细胞非依赖性免疫原性限制了其作为疫苗的使用，特别是在婴幼儿中。脂多糖毒性太大，不能用药。因此，它们的O-特异性多糖(O-SP)具有CPS的毒力促进和保护作用，必须进行纯化：O-SP太小而不能产生免疫原性(半抗原)。CPS或O-SP的共价结合到医学上有用的蛋白质上形成偶联物，既增加了它们的免疫原性，又使T细胞对这些糖类产生依赖。宋内氏志贺氏菌和福氏2a志贺氏菌的O-SP与细菌类毒素结合。这两种结合物都是安全的，并在成人、4-7岁和1-4岁儿童中诱导了具有统计学意义的同源内毒素抗体的长期升高。类似地，尽管IgM和IgA抗内毒素的升高幅度较小，但也是如此。再次注射福氏2a链球菌结合物在所有年龄组都能引起增强反应，而宋内氏链球菌结合物仅在1-4岁时才能产生增强反应。3期试验表明，一次注射宋内氏链球菌O-SP，与无毒的重组铜绿假单胞菌外蛋白A(REPA)结合，可保护新兵免受这种病原体的爆发。重要的是，血清中抗内毒素抗体的水平与结合物的疗效有显著的相关性。研究了两种提高志贺氏菌结合物免疫原性的方法：基因灭活的白喉棒状杆菌毒素(CRM9)是宋内氏志贺氏菌O-SP的优良载体；琥珀酸酐处理REPA可提高福氏2a志贺菌O-SP的免疫原性。这些志贺氏菌结合物在成人中的第一阶段研究证实了它们的安全性和免疫原性；改善的免疫原性不如在小鼠身上明显。在一项在1-4岁进行的第二阶段研究中，福氏2a志贺氏菌结合物使92%的儿童发病率增加了4倍，宋内氏志贺氏菌结合物的发病率增加了85%。改良的福氏2a志贺氏菌和原始宋内氏志贺氏菌结合物的3期研究正在准备中。在兰州疫苗研究所和河南省医学中心中国的合作下，这两种结合物的临床试验正在计划中。 为了研究交叉反应和同源CPS同时使用是否比单独使用同源CPS更有优势，用常规方法分离了短小芽孢杆菌SH18与b型流感嗜血杆菌CPS交叉反应的胞壁多糖，并用GC-MS、核磁共振、快原子轰击和几种糖降解技术对其结构进行了研究。它被证明是由多聚核糖醇磷酸结合到聚甘油磷酸上，并可能通过聚甘油磷酸结合到细胞壁的胞壁酸。这两种多元醇在C2位被N-乙酰氨基葡萄糖部分取代，除了与抗Hib发生交叉反应外，还与抗表皮葡萄球菌发生交叉反应。将该多糖与载体蛋白偶联，并在GP小鼠体内检测其免疫原性。结合物诱导的抗体与同源和几种交叉反应的多糖反应。 A组脑膜炎奈瑟菌引起地方性和流行性脑膜炎，特别是在非洲的脑膜炎地带。一种有效和可用的CPS疫苗没有得到充分利用。为了进一步提高其免疫原性，将其与牛血清白蛋白偶联。与单独使用CPS相反，它在小鼠体内具有免疫原性，在第二次和第三次注射后有增强反应。交叉反应多糖的偶联物E.coliK93和B.puilus SH17不能诱导抗人类抗体。一个CPS。 伯氏疏螺旋体是莱姆病的病原体，是一种通过受感染的硬蜱叮咬传播的螺旋体。目前有一种针对这种疾病的蛋白质疫苗，但在12岁以下无效。脂多糖曾在其他螺旋体中被描述过，但它在伯氏杆菌中的存在一直存在争议。到目前为止，我们还无法确认它的存在。对脂多糖的搜索揭示了一种独特的糖脂，它由C16、C18脂肪酸组成，可能是甘油和半乳糖作为碳水化合物部分。有证据表明，这种糖脂是表面暴露的，注射在完全弗氏佐剂中，它会诱导特异性抗体。这种糖脂在各种制剂中的免疫原性和抗体的生物学效应正在调查中。 炭疽杆菌是人类致命感染的潜在原因，它有两个基本的毒力因子，如果没有这两个因子，它就不会对人类产生致病作用。这些因素是：1.炭疽毒素；2.胶囊。该毒素由致死因子、水肿素和保护性抗原3种多肽组成，每种多肽本身均无毒。PA是与哺乳动物细胞结合的毒素部分。它必须有一个20 KDa的多肽被水解，暴露出一个Lf或EF可能结合的位置，从而产生酶修饰的毒素 哺乳动物细胞胞浆中的底物。该胶囊由聚D-γ-谷氨酸组成，无免疫原性，其保护作用尚不清楚。获得许可的疫苗是安全和保护的，但也有局限性，证明有理由开发改进的疫苗。 从发酵罐中培养的无囊化菌株中分离到一株重组PA。几种经甲醛处理和明矾吸附材料的配方在小鼠身上被发现具有免疫原性。临床批次正在准备中。该胶囊是从一种无毒菌株中分离出来的 目前正在研究将其与载体蛋白偶联的方法。

Surface polysaccharides of pathogenic bacteria, including capsular polysaccharides (CPS) or lipopolysaccharides (LPS),serve both as essential virulence factors and as protective antigens. The age-related and T-cell independent immunogenicity of CPS limit their use as vaccines especially in infants and young children. LPS are too toxic to be administered. Accordingly, their O-specific polysaccharide (O-SP), that share the virulence promoting and protectiveness of CPS, must be purified: O-SP are too small to be immunogenic (haptens). Covalent binding of CPS or of O-SP to medically-useful proteins to form conjugates both increases their immunogenicity and confers T-cell dependence to these saccharides. The O-SP of Shigella sonnei and of S.flexneri 2a were bound to bacterial toxoids. Both conjugates were safe and induced statistically significant and long-lived rises of IgG antibodies to the homologous LPS in adults, 4-7 and 1-4 year-olds. Similar, though lesser rises of IgM and IgA anti-LPS were also induced. Re-injection of S. flexneri 2a conjugate induced a booster response in all age groups,of the S. sonnei conjugate only in the 1-4 year old. A Phase 3 trial showed that one injection of S. sonnei O-SP, bound to a non-toxic recombinant Pseudomonas aeruginosa exoprotein A (rEPA) protected army recruits against outbreaks with this pathogen. Importantly, there was a significant correlation between the levels of serum IgG anti-LPS and the efficacy of the conjugate. Two methods were developed that increased the immunogenicity of the Shigella conjugates in mice: a genetically-inactivated Corynebacterium diphtheriae toxin (CRM9) was a superior carrier for S. sonnei O-SP and treatment of rEPA with succinic anhydride, a non-toxic mild akylating agent that converts amino groups of proteins to carboxyls, increased the immunogenicity of S. flexneri 2a O-SP. A phase 1 study in adults of these Shigella conjugates confirmed their safety and immunogenicity; the improved immunogenicity was less marked than in mice. In a phase 2 study in 1-4 years old the S.flexneri 2a conjugate induced a 4-fold rise in 92% of the children, the S.sonnei conjugate in 85%. A phase 3 study of the modified S.flexneri 2a and the original S. sonnei conjugates are in preparation. In collaboration with the Lanzhou Vaccine Institute and Provincial Medical Center in Henan, China, a clinical trial of these two conjugates is being planned. To investigate if concurrent administration of a cross-reacting along with a homologous CPS has an advantage over the use of the homologous CPS alone, the cell wall polysaccharide (PS) of Bacillus pumilus, SH18, reported to cross react with the CPS of haemophilus influenzae type b (Hib), was isolated by conventional methods and it's structure investigated using GC-MS,NMR, fast atom bombardment and several sugar degrading techniques. It was shown to be composed of polyribitolphosphate bound to polyglycerolphosphate and likely through the latter to the muramic acid of the cell wall. Both polyols are partially substituted at C2 with N-acetylglucosamine.Besides with the anti Hib it cross reacted with anti Staphylococcus epidermidis. The polysaccharide was conjugated to carrier proteins and it's immunogenicity evaluated in GP mice. Conjugate-induced antibodies reacted with the homologous and several cross-reacting polysaccharides. Neisseria meningitidis group A causes endemic and epidemic meningitis, notably in the meningitis belt of Africa. A CPS vaccine , effective and available, is underutilized. To further improve it's immunogenicity it was conjugated to BSA . Contrary to the CPS alone it was immunogenic in mice, with booster responses after 2nd and 3rd injections. Conjugates of the cross reactive polysaccharides, e.coli K93 and B.pumilus SH17,did not induce anti Men. A CPS. Borrelia burgdorferi, a spirochete transmitted though the bite of infected Ixodes ticks, is the etiologic agent of Lyme disease. A protein vaccine against it is available but is not effective below the age of 12 years. LPS has been described in other spirochetes but it's presence in B. burgdorferi has been debated. So far we have not been able to confirm it's presence. The search for LPS revealed a unique glycolipid composed of C16, C18fatty acids possibly glycerol and galactose as the carbohydrate moiety. There is evidence that this glycolipid is surface exposed Injected in complete Freund's adjuvant it induced specific antibodies. The immunogenicity of this glycolipid in various formulations and the biological effect of the antibodies are being ingestigated. Bacillus anthracis, a potential cause of lethal human infection, has 2 essential virulence factors without either of which it is not pathogenic for humans. these factors are: 1.anthrax toxin, 2.a capsule. The toxin is composed of 3 peptides: Lethal Factor, Edema Factor and Protective Antigen, each by itself non toxic. PA is the toxin part that binds to mammalian cells. It has to have a 20 KDa peptide hydrolyzed off exposing a site to which LF or EF may bind rendering toxins that enzymatically modify substrates in mammalian cell cytosol. The capsule is composed of poly-D-gamma-glutamic acid.It is non-immunogenic and it's protective effect unknown. The licensed vaccine is safe and protective but has limitations that justify development of improved vaccines. A recombinant PA was isolated from an unencapsulated strain grown in a fermenter. Several formulations with formaldehyde treated and alum adsorbed materials were found to be immunogenic in mice. Clinical lots are being prepared. The capsule has been isolated from a non toxic strain and methods to conjugate it to carrier proteins are being investigated.