Shigellae

志贺氏菌属

• 批准号: 7734840
• 负责人: Rachel Schneerson
• 金额: $ 33.92万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734840
• 关键词: 2 year old; 4 year old; Adult; Antibodies; Antigens; Binding; Blinded; Caco-2 Cells; Carrier Proteins; Cells; Child; Condition; Coupling; Dependence; Developed Countries; Developing Countries; Diphtheria Toxin; Dose; Dysentery; Epithelial; Goals; Growth; Hela Cells; Human; Immune Sera; Immunoglobulin G; In Vitro; Incidence; Infant; Institution; Intestines; Length; Licensing; Lipopolysaccharides; Mass Spectrum Analysis; Military Personnel; Mus; O Antigens; Oligosaccharides; Organism; Oximes; Patients; Phase III Clinical Trials; Plesiomonas shigelloides; Polysaccharides; Prevention; Proteins; Pseudomonas aeruginosa; Publishing; Randomized; Rate; Reaction; Recombinants; Recruitment Activity; Refugees; Serum; Shigella; Shigella Infections; Shigella dysenteriae; Shigella flexneri; Shigella sonnei; Structure; Surface; T-Lymphocyte; Temperature; Tetanus Toxoid; Time; Vaccines; Virulence Factors; World Health Organization; age related; anti-IgG; immunogenicity; molecular mass; mortality; mutant; pathogen; pathogenic bacteria; vaccine development; young adult

Surface polysaccharides of pathogenic bacteria, including capsular polysaccharides (CPS) or the O-specific polysaccharide (O-SP) of lipopolysaccharides (LPS), serve both as essential virulence factors and as protective antigens. Covalent binding of these saccharides to medically-useful proteins to form conjugates both increases their immunogenicity and confers to them T-cell dependence making them suitable vaccines for infants and children. The O-SP of Shigella sonnei bound to recombinant non toxic P. aeruginosa exoprotein A (rEPA) had an efficacy of over 70% in young adults exposed to 6-14% attack rates. This conjugate and of S. flexneri 2a bound to the succinylated exoprotein A (rEPA-succ) were safe and induced IgG antibodies to the homologous LPS in 1-4 year-olds. A randomized, blinded, phase 3 study of these conjugates in 1-4 year-olds with each conjugate serving as a control for the other showed these vaccines to be safe. Immunogenicity and efficacy were age related with little efficacy in 1-2 years old, about 70% in 3-4 years old. Fold increases in antibody levels were, as before, similar to those of adults but the actual levels achieved were lower. The effect of these children's immune sera and IgG isolated from them upon Shigella invasion into epithelial intestinal cells was studied in vitro using Caco-2 and HeLa cells. The sera inhibited Shigella invasion in a type specific manner. Pretreatment of the sera or of Caco-2 cells with O-SP abrogated these effects in a type specific and dose dependent manner. To enhance the immunogenicity of these conjugates S. sonnei and S. flexneri 2a O-SPs were bound to additional carrier proteins: tetanus toxoid (TT) and recombinant protective antigen (rPA). Mice were injected twice, with the same or a different carrier the second time. For S. flexneri 2a the TT conjugate injected twice induced the highest (>4-fold) antibody levels. For S. sonnei, the TT conjugate followed by the rPA conjugate induced significantly higher levels than the other combinations. We have shown that synthetic oligosaccharides of S. dysenteriae type 1 O-SP bound by their reducing end to a carrier protein (sun configuration) induced significantly higher antibody levels than conjugates of the native O-SP bound to protein by multiple point attachments (lattice configuration). Synthesis of S. sonnei oligosaccharides was not successful. Therefore, the low molecular mass O-SP-core (O-SPC) fragments of the native O-SP were isolated and used to bind to proteins by oxime linkages between the terminal Kdo residues of the reducing end core and aminoxy linkers bound to BSA or to a non toxic diphtheria toxin mutant. The coupling reaction was done at a neutral pH, room temperature and in a short time. IgG anti S. sonnei LPS levels induced by these conjugates in young outbred mice were significantly higher than those induced by the full length O-SP conjugates. The O-SP of Plesiomonas shigelloides O17 is structurally identical to that of S. sonnei but the core structure of S. sonnei only is published. We investigated the core structure of P. shigelloides O17, including its linkage to the O-SP by NMR and mass spectroscopy. The structure was assigned.

病原菌的表面多糖，包括荚膜多糖（CPS）或脂多糖（LPS）的O-特异性多糖（O-SP），既是必需的毒力因子，也是保护性抗原。这些抗体与医学上有用的蛋白质共价结合以形成缀合物，既增加了它们的免疫原性，又赋予它们T细胞依赖性，使它们成为适合婴儿和儿童的疫苗。与重组无毒铜绿假单胞菌胞外蛋白A（rEPA）结合的宋内志贺菌O-SP在暴露于6-14%发病率的年轻成人中具有超过70%的疗效。这种共轭和S.与琥珀酰化外蛋白A（rEPA-succ）结合的福氏2a是安全的，并在1-4岁儿童中诱导针对同源LPS的IgG抗体。这些结合物在1-4岁儿童中进行的随机、盲法、3期研究表明，这些疫苗是安全的，每种结合物都作为另一种结合物的对照。免疫原性和有效性与年龄有关，1-2岁的有效性很小，3-4岁的有效性约为70%。 与以前一样，抗体水平的倍数增加与成人相似，但实际达到的水平较低。 利用Caco-2和HeLa细胞体外研究了这些儿童的免疫血清和从他们中分离的IgG对志贺氏菌侵入肠上皮细胞的影响。血清以类型特异性的方式抑制志贺菌的侵袭。用O-SP预处理血清或Caco-2细胞以类型特异性和剂量依赖性方式消除这些作用。 为了增强这些缀合物的免疫原性，S. sonnei和S.将弗氏2a O-SP结合到另外的载体蛋白：破伤风类毒素（TT）和重组保护性抗原（rPA）。小鼠注射两次，第二次注射相同或不同的载体。对于鼠伤寒沙门氏注射两次的TT缀合物诱导最高（>4倍）抗体水平。对于鼠伤寒沙门氏sonnei，TT缀合物接着rPA缀合物诱导比其它组合显著更高的水平。 我们已经证明，S。通过其还原端结合至载体蛋白的1型放线菌O-SP（太阳构型）比通过多点连接（晶格构型）结合至蛋白的天然O-SP的缀合物诱导显著更高的抗体水平。S. Sonnei寡糖没有成功。因此，分离天然O-SP的低分子量O-SP-核心（O-SPC）片段，并用于通过还原末端核心的末端Kdo残基与结合BSA或无毒白喉毒素突变体的氨氧基接头之间的肟键与蛋白质结合。偶联反应在中性pH、室温和短时间内完成。IgG抗S.在年轻远系杂交小鼠中由这些缀合物诱导的sonnei LPS水平显著高于由全长O-SP缀合物诱导的那些。 类志贺邻单胞菌O 17的O-SP结构与S. sonnei，但S.只有索尼出版。我们通过NMR和质谱研究了类志贺毕赤酵母O 17的核心结构，包括其与O-SP的连接。结构已分配。