Human Immune Response To Polysaccharide-protein Conjugat

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S.flexneri 2a的O-SP与细菌类毒素结合。两种缀合物都是安全的，并且在成人、4-7岁和1-4岁儿童中诱导对同源LPS的IgG抗体有统计学意义且长期升高。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结合菌在所有年龄组均能引起增强反应，而sonnei结合菌仅在1-4岁。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显著相关性。研究人员开发了两种方法来提高志贺菌结合物在小鼠体内的免疫原性：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。在一项针对1-4岁儿童的第二阶段研究中，弗氏胞杆菌2a结合物在92%的儿童中引起了4倍的上升，而索内胞杆菌结合物在85%的儿童中引起了4倍的上升。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。

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.