Immune Response To Polysaccharide-protein Conjugate Vacc

病原菌的表面多糖，包括荚膜多糖（CPS）或脂多糖（LPS），既是必需的毒力因子，也是保护性抗原。CPS的年龄相关性和不依赖t细胞的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，不能给药。因此，它们的o特异性多糖（O-SP），共享CPS的毒力促进和保护作用，必须被纯化：O-SP太小而不具有免疫原性（半抗原）。CPS或O-SP与医学上有用的蛋白质共价结合形成偶联物，既增加了它们的免疫原性，又赋予t细胞对这些糖的依赖性。sonnei志贺氏菌和flexneri志贺氏菌2a的O-SP与细菌类毒素结合。在成人和4-7岁儿童中，这两种结合物都是安全的，并诱导IgG抗体对同源LPS具有统计学意义和长期的上升。类似地，虽然IgM和IgA抗lps也引起了较小的升高。再次注射flexneri 2a结合物在新兵和4-7岁的儿童中引起了增强反应。一项三期试验表明，注射与无毒重组铜绿假单胞菌外蛋白A （rEPA）结合的sonnei S. O-SP可以保护新兵免受这种病原体的爆发。重要的是，血清IgG抗lps水平与偶联物的疗效之间存在统计学意义上的相关性。研究人员开发了两种方法来提高志贺菌偶联物在小鼠体内的免疫原性：另一种载体蛋白，一种基因灭活的白喉棒杆菌毒素（CRM9）是sonnei S. O-SP的优良载体；用琥珀酸酐（一种无毒的轻度乙酰化剂，可将蛋白质的氨基转化为羧基）处理rEPA，增加了S. flexneri 2a O-SP的免疫原性。成人志贺氏菌偶联物的1期研究证实了它们的安全性和免疫原性；与小鼠相比，免疫原性的改善不明显。一项针对1-4岁儿童的2期研究显示，新的s.f flexneri 2a结合物的免疫原性得到改善，但s.s sonnei结合物的免疫原性较差。改良的S.flexneri 2a和原S. sonnei偶联物的3期研究正在准备中。与兰州疫苗研究所和中国河南省医学中心合作，正在计划对这两种结合物进行临床试验。为了研究与同源CPS交叉反应的同时给药是否比单独使用同源CPS有优势，用常规方法分离了与b型流感嗜血杆菌（Hib）的CPS交叉反应的杆状芽孢杆菌（Bacillus pumilus, SH18）的细胞壁多糖（PS）。采用GC-MS对其结构进行了分析。结果表明，它含有甘油、利比醇和2-乙酰氨基-2-脱氧葡萄糖，摩尔比为0.2:1.0:0.2，磷酸盐为17%。此外，它还能与抗Hib抗表皮葡萄球菌交叉反应。研究了该聚合物共轭物的制备方法。A群脑膜炎奈瑟菌引起地方性和流行性脑膜炎，特别是在非洲脑膜炎带。一种有效和可用的CPS疫苗没有得到充分利用。要进一步改进吗？与其他CPS的免疫原性一样，目前正在研究将其与载体蛋白结合的方法。研制了一种百日咳杆菌双突变体，产生一种基因灭活毒素，缺乏FHA合成。目前正努力增加这种百日咳b型菌株的生产，作为一种更容易纯化的百日咳毒素，用于单组分疫苗，并作为14型肺炎球菌CPS的载体蛋白。艰难梭菌是抗生素使用后医院获得性腹泻的主要原因：腹泻由a和b两种外毒素介导。a毒素被认为是主要毒素，在极端情况下会引起假膜性结肠炎。一种基因衍生的毒素突变体（rARU）既能诱导抗毒素，又能保护动物免受艰难梭菌感染。琥珀酰化提高了rARU的溶解度，对其抗原性没有明显影响。研制出了用于临床的突变毒素A的制备技术。将14型肺炎球菌、K1型大肠杆菌和2a型弗氏杆菌三种不同组成的多糖与琥珀酰化的rARU结合。所得的偶联物诱导了高水平的抗多糖和抗毒素。用于临床评价的毒素A缀合物的制备正在进行中。伯氏疏螺旋体是一种通过受感染的硬蜱叮咬传播的螺旋体，是莱姆病的病原体。目前有一种蛋白质疫苗可以预防这种疾病，但对12岁以下儿童无效。脂多糖在其他螺旋体中也有描述，但它在伯氏疏螺旋体中的存在一直存在争议。到目前为止，我们还不能证实它的存在。对脂多糖的研究发现了一种独特的糖脂，由甘油和半乳糖作为碳水化合物组成。有证据表明，这种糖脂是表面暴露的，注射在完全的弗氏佐剂中，它会诱导特异性抗体。

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 Shigella flexneri 2a were bound to bacterial toxoids. In adults and then in 4-7 year-olds, both conjugates were safe and induced statistically significant and long-lived rises of IgG antibodies to the homologous LPS. 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 the recruits and the 4-7 years 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 statistically-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: another carrier protein, 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. A phase 2 study in 1-4 years old showed an improved immunogenicity of the new S.flexneri 2a conjugate but lesser immunogenicity of the S.sonnei conjugate. 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. It was shown to contain glycerol, ribitol and 2-acetamido-2-deoxyglucose in a molar ratio of 0.2:1.0:0.2 and 17% phosphate. Besides with the anti Hib it cross reacted with anti Staphilococcus epidermidis. Methods to prepare a conjugate of this PS are investigated. 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, as was done for other CPS, methods of binding it to a carrier protein are being investigated. A double mutant of Bordetella pertussis, producing a genetically-inactivated toxin and deficient in FHA synthesis was developed. Effort is directed towards increasing production of this B. pertussis strain as a more easily purified pertussis toxin for a monocomponent vaccine and as a carrier protein for pneumococcal type 14 CPS. Clostridium difficile is a major cause of hospital-acquired diarrhea following antibiotic usage: the diarrhea is mediated by two exotoxins, A and B. Toxin A, considered to be the major toxin, in the extreme form will cause pseudomembranous colitis. A genetically-derived toxin mutant (rARU) induces both antitoxin and protects animals from infection with C. difficile. The succinylation of rARU improved its solubility and did not detectably affects its antigenicity. Techniques to prepare the mutant toxins A for clinical use have been worked out. Three polysaccharide of varying composition, pneumococcus type 14, Escherichia coli K1 and S. flexneri 2a were conjugated to succinylated rARU. The resultant conjugates induced high levels of both anti-polysaccharide and antitoxin. Preparation of toxin A conjugates for clinical evaluation is underway. 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 cosisting of 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.