HUMAN IMMUNE RESPONSE TO POLYSACCHARIDE-PROTEIN CONJUGATE VACCINES

致病菌的表面多糖，包括荚膜多糖（CPS）或脂多糖（LPS），既可作为必要的毒力因子，也可作为保护性抗原。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，必须纯化：O-SP太小，不具有免疫原性（半抗原）。将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。同样，虽然IgM和IgG抗lps也引起了较小程度的升高。血清IgG抗lps水平等于或高于从志贺氏菌病恢复的新兵。再次注射flexneri 2a缀合物在新兵中诱导了增强反应。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。我们开发了两种方法来提高志贺氏菌结合物在小鼠体内的免疫原性。首先，基因灭活白喉棒状杆菌毒素（CRM9）是sonnei O-SP的优良载体。第二次用琥珀酸酐治疗rEPA，琥珀酸酐是一种无毒的温和的酰基化剂，可以将蛋白质的氨基转化为羧基，增加了S. flexneri 2a O- SP的免疫原性。这些改进的志贺氏菌偶联物的1期研究证实了它们的免疫原性，这些新产品将在以色列的3期试验中评估其临床疗效。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。艰难梭菌是使用抗生素后医院获得性腹泻的主要原因：腹泻是由两种外毒素介导的，a和b。毒素A被认为是主要的毒素，其极端形式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。rARU的溶解度提高了其溶解度，对其与抗血清的反应无明显影响。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。计划对这些旨在保护两种疾病的偶联物进行临床评估。-人体实验对象

Surface polysaccharides of pathogenic bacteria, including capsular polysaccharides (CPS) or lipopolysaccharides (LPS), may 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 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). Covalently binding 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 IgG anti-LPS were also induced. The levels of serum IgG anti-LPS were equal to or greater that those in recruits that recovered from shigellosis. Re-injection of S. flexneri 2a conjugate induced a booster response in the recruits. 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. First another carrier protein, a genetically-inactivated Corynebacterium diphtheriae toxin (CRM9) was a superior carrier for S. sonnei O-SP. Second 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 of these improved Shigella conjugates confirmed their immunogenicity and these new products will be evaluated for their clinical efficacy in a Phase 3 trial in Israel.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. In its extreme form. Toxin A, considered to be the major toxin, in its extreme form will cause pseudomembranous colitis. A genetically-derived toxin mutant (rARU) induces both antitoxin and protects animals from infection with C. difficile. The solubility of rARU improved its solubility and did not detectable affects its reaction with antiserum. 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. Clinical evaluation of these conjugates, designed to protect two diseases, is planned. - Human Subjects