Polysaccharide/Oligosaccharide-protein conjugates

多糖/寡糖-蛋白质缀合物

• 批准号: 7333982
• 负责人: RACHEL SCHNEERSON
• 金额: --
• 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/7333982
• 关键词: Polysaccharide; Oligosaccharide; protein; conjugates

SHIGELLAE: 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 CPS or of O-SP to medically-useful proteins to form conjugates both increases their immunogenicity and confers T-cell dependence to these saccharides making them suitable vaccines for infants and children. The O-SP of Shigella sonnei bound to recombinant non toxic P. aeruginosa exoprotein A (rEPA) 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 is in progress. 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. Modification of O-SP by glycosylation or acetylation is important for its antigenic specificity and immunogenicity. The degree of O-acetylation and localization of the O-acetyl groups and glucose substitution of S. flexneri 2a O-SP were investigated. The structure of the repeat unit was assigned. CROSS REACTING POLYSACCHARIDES: H. influenzae types b (Hib)and a (Hia), B. pumilus: Hia, sharing a D-1,5-ribitol phosphate in its repeat unit with Hib, was a (distant) second most common H. influenzae isolate from patients, causing up to 10% of disease in some populations. Its importance has risen with the successful control of Hib disease. To provide a potential vaccine for the prevention of Hia disease, its CPS was isolated and conjugated to a recombinant S. aureus enterotoxin C1 (rSEC). Injected sc as a saline solution into 5-6 week old mice, the conjugate induced CPS antibodies with bactericidal activity. The cell wall polysaccharide (PS) of B. pumilus Sh 18 was reported to cross react with Hib CPS, presumably due to its D-ribitol phosphate, a common component of bacilli and staphylococcal cell wall PS. Anti Hib CPS was also shown to precipitate with the cell wall PS of L. plantarium and S. aureus. The cell wall PS of Bacillus pumilus Sh18, was isolated and its structure investigated using GC-MS, NMR, fast atom bombardment and several sugar degrading techniques. It was shown to be composed of polyribitolphosphate (poly Rib-P), polyribitolphosphate substituted at C2 with N-acetyl glucosamine and polyglycerolphosphate (Gly-P). We failed to separate these components.. In addition to anti Hib this preparation bound to anti S. epidermidis, known to contain poly (gly-P) in its teichoic acid. The Sh18 PS was conjugated to carrier proteins and its immunogenicity evaluated in general purpose mice. Conjugate-induced antibodies reacted with the homologous and with several cross-reacting polysaccharides. Poly (Rib-P) was synthesized; an octamer and a dodecamer with terminal keto groups were conjugated to aminooxylated BSA or tetanus toxoid. MALDI-TOF showed 10-18 saccharide chains incorporated per mole of BSA. The immunogenicity of these conjugates is being studied. GROUP B N. MENINGITIDIS AND ESCHERICHIA COLI K1: Group B meningococcus (GBM) and Escherichia coli k1 meningitis continue to cause serious and difficult to treat diseases and there is no licensed vaccine for their prevention. The polysaccharide, alpha (2-8) N-acetylneuraminic acid (PSA), is the capsular polysaccharide (CPS) of GBM and of E. coli K1 and a component of mammalian glycopeptides. Because it is a "self antigen", vaccines designed to induce PSA immune responses have been considered as a potential cause of immunopathology. Antibodies to PSA bind to human tissues in-vitro but have not been shown to induce pathology in-vivo. The incidence, severity, and nature of systemic infection caused by GBM are similar to those caused by the closely related group C meningococci (GCM). As shown for other polysaccharides, there is an age-related acquisition of serum PSA antibodies in humans: most human neonates and adults have IgG anti-PSA. Purified PSA is not immunogenic but as a component of bacteria or bound to proteins as a conjugate this CPS induces antibodies of the 3 major isotypes with its IgM and IgG components being protective in in-vitro and in-vivo models. Despite its similarity to mammalian glycoproteins, there are no data indicating an association of IgG anti-PSA with immunopathology in experimental animals or in humans. A follow up of ~50,000 person years of individuals with a past history of meningococcal infection, in Denmark and in Iceland, found no increased association with autoimmune disease in patients with GBM disease compared to those with group C meningococcal disease. We propose that clinical trials of PSA conjugate vaccines, shown to be immunogenic and protective in animals, be considered. BORRELIA BURGDORFERI, a spirochete transmitted through the bite of infected Ixodes ticks, is the etiologic agent of Lyme disease. A protein vaccine against it is not effective below the age of 12 years, and has been taken off the market recently. LPS has been described in other spirochetes but its presence in B. burgdorferi has been debated. We have not been able to confirm its presence. The search for LPS revealed 2 unique glycolipids: cholesteryl 6-O-acyl-beta-D-galactopyranoside (BbGL I) and 1,2-di-O-acyl-3-O-alpha-D-galactopyranosyl-sn-glycerol (BbGL II) There is evidence that they are surface exposed. Injected in various formulations into mice BbGL I induced specific antibodies, in order of induced levels: CFA, PBS, DMSO, and squalene. Investigation of the effect of BbGL-I and II upon human PMNs and monocytes showed increased levels of IFN-gamma, IL-4 and TNF-alpha secreted in their presence. The cholesteryl palmitoyl !Vgalactopyranoside was synthesized with an aldehydo group at the palmitoyl end which was used to bind to aminooxylated BSA. An average of 18 glycolipid groups were incorporated per BSA molecule. The immunogenicity of these conjugates is being studied in mice. BORDETELLAE: Pertussis, whooping cough in infants, is caused by B. pertussis. B. parapertussis causes pertussis in humans, though of a milder form and lower frequency than B. pertussis. B. bronchiseptica causes respiratory infection in animals, rarely in humans. B. parapertussis and B. bronchiseptica do not produce pertussis toxin. The Lipid A and core regions of the three Bordetella species are similar but only B. parapertussis and B. bronchiseptica posses an O-SP, the structure of which was reported but the linkages to the core PS were not identified. We analyzed the carbohydrate structures of the LPS from several strains of Bordetella bronchiseptica and Bordetella parapertussis, using different chemical degradation methods, NMR spectroscopy and mass spectrometry, and identified a novel pentasaccharide that links the O-chain to the core in all LPS studied. Additionally, whereas the O-chain of these bacteria was reported to be composed of a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-??-galacturonic acid, we discovered that it contains amidated uronic acids, the number of which varies among strains. HAEMOPHILUS DUCREYI: is the cause of chancroid, a sexually transmitted disease characterized by painful genital ulceration. Chancroid also enhances the spread of HIV infection. A major virulence factor and a potentially protective antigen of H. ducreyi is its lipooligosaccharide (LOS).

志贺氏菌：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)，既可作为必需毒力因子，又可作为保护性抗原。 CPS 或 O-SP 与医学上有用的蛋白质共价结合形成缀合物，既增加了它们的免疫原性，又赋予了 T 细胞对这些糖的依赖性，使其成为适合婴儿和儿童的疫苗。宋内志贺氏菌与重组无毒铜绿假单胞菌外蛋白 A (rEPA) 结合的 O-SP 和与琥珀酰化外蛋白 A (rEPA-succ) 结合的弗氏志贺氏菌 2a 的 O-SP 是安全的，可在 1-4 岁儿童中诱导针对同源 LPS 的 IgG 抗体。对 1-4 岁儿童进行的一项随机、盲法 3 期研究正在进行中，其中每种缀合物作为另一种缀合物的对照。 使用 Caco-2 和 HeLa 细胞在体外研究了这些儿童的免疫血清和从中分离的 IgG 对志贺氏菌侵入肠上皮细胞的影响。该血清以特定类型的方式抑制志贺氏菌入侵。用 O-SP 预处理血清或 Caco-2 细胞可以以类型特异性和剂量依赖性方式消除这些效应。 O-SP 的糖基化或乙酰化修饰对其抗原特异性和免疫原性很重要。研究了福氏链霉 2a O-SP 的 O-乙酰化程度、O-乙酰基的定位以及葡萄糖取代。指定了重复单元的结构。 交叉反应多糖：b 型流感嗜血杆菌 (Hib) 和 a 型 (Hia)、短小芽孢杆菌：Hia 与 Hib 重复单元中具有 D-1,5-核糖醇磷酸盐，是患者中​​第二常见的流感嗜血杆菌分离株，在某些人群中引起高达 10% 的疾病。随着 Hib 疾病的成功控制，其重要性也随之上升。为了提供预防希亚病的潜在疫苗，分离了其 CPS 并将其与重组金黄色葡萄球菌肠毒素 C1 (rSEC) 结合。将盐溶液皮下注射到 5-6 周龄的小鼠体内，结合物诱导产生具有杀菌活性的 CPS 抗体。 据报道，短小芽孢杆菌 Sh 18 的细胞壁多糖 (PS) 会与 Hib CPS 发生交叉反应，这可能是由于其 D-核糖醇磷酸盐（杆菌和葡萄球菌细胞壁 PS 的常见成分）所致。抗 Hib CPS 也被证明与植物乳杆菌和金黄色葡萄球菌的细胞壁 PS 一起沉淀。分离短小芽孢杆菌 Sh18 的细胞壁 PS，并使用 GC-MS、NMR、快原子轰击和多种糖降解技术研究其结构。它显示出由聚核糖醇磷酸酯(poly Rib-P)、在C2处被N-乙酰葡糖胺取代的聚核糖醇磷酸酯和聚甘油磷酸酯(Gly-P)组成。我们未能分离这些成分。除了抗 Hib 之外，该制剂还与抗表皮葡萄球菌结合，已知其磷壁酸中含有聚（甘氨酸）。 Sh18 PS 与载体蛋白缀合，并在通用小鼠中评估其免疫原性。缀合物诱导的抗体与同源物和几种交叉反应的多糖发生反应。合成了聚（Rib-P）；带有末端酮基的八聚体和十二聚体与氨氧基化 BSA 或破伤风类毒素缀合。 MALDI-TOF 显示每摩尔 BSA 掺入 10-18 个糖链。 正在研究这些缀合物的免疫原性。 B 组脑膜炎球菌和大肠杆菌 K1：B 组脑膜炎球菌 (GBM) 和大肠杆菌 k1 脑膜炎继续引起严重且难以治疗的疾病，并且没有获得许可的疫苗可用于预防。多糖 α (2-8) N-乙酰神经氨酸 (PSA) 是 GBM 和大肠杆菌 K1 的荚膜多糖 (CPS)，也是哺乳动物糖肽的组成部分。由于它是一种“自身抗原”，旨在诱导 PSA 免疫反应的疫苗已被认为是免疫病理学的潜在原因。 PSA 抗体在体外与人体组织结合，但尚未显示出会在体内诱发病理。 GBM 引起的全身感染的发生率、严重程度和性质与密切相关的 C 族脑膜炎球菌 (GCM) 引起的全身感染相似。正如其他多糖所示，人类血清 PSA 抗体的获得与年龄相关：大多数人类新生儿和成人都有 IgG 抗 PSA。纯化的 PSA 不具有免疫原性，但作为细菌的成分或作为缀合物与蛋白质结合，该 CPS 诱导 3 种主要同种型的抗体，其 IgM 和 IgG 成分在体外和体内模型中具有保护作用。尽管它与哺乳动物糖蛋白相似，但没有数据表明 IgG 抗 PSA 与实验动物或人类的免疫病理学相关。在丹麦和冰岛，对过去有脑膜炎球菌感染史的个体进行了约 50,000 人年的随访，发现与 C 组脑膜炎球菌病患者相比，GBM 病患者与自身免疫性疾病的相关性并未增加。我们建议考虑对已证明在动物中具有免疫原性和保护性的 PSA 结合疫苗进行临床试验。 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。针对该病的蛋白质疫苗对 12 岁以下儿童无效，最近已从市场上撤下。其他螺旋体中也有 LPS 的描述，但伯氏疏螺旋体中是否存在 LPS 仍存在争议。我们无法确认它的存在。 LPS 的搜索揭示了 2 种独特的糖脂：胆固醇 6-O-酰基-β-D-吡喃半乳糖苷 (BbGL I) 和 1,2-二-O-酰基-3-O-α-D-吡喃半乳糖基-sn-甘油 (BbGL II) 有证据表明它们是表面暴露的。以各种制剂注射到小鼠体内，BbGL I 诱导了特异性抗体，按照诱导水平的顺序：CFA、PBS、DMSO 和角鲨烯。 BbGL-I 和 II 对人 PMN 和单核细胞的影响的研究表明，在它们存在的情况下，分泌的 IFN-γ、IL-4 和 TNF-α 水平增加。合成了在棕榈酰末端具有醛基的胆固醇棕榈酰基半乳糖苷，该醛基用于与氨氧基化的BSA结合。每个 BSA 分子平均掺入 18 个糖脂基团。正在小鼠中研究这些缀合物的免疫原性。 博德特氏菌：百日咳（婴儿百日咳）是由百日咳博德特氏菌引起的。副百日咳博德特氏菌会引起人类百日咳，但其形式比百日咳博德特氏菌更温和且频率较低。支气管败血杆菌会引起动物呼吸道感染，但很少会引起人类呼吸道感染。副百日咳博德特氏菌和支气管败血博德特氏菌不产生百日咳毒素。三种博德特氏菌属的脂质 A 和核心区域相似，但只有副百日咳博德特氏菌和支气管败血博德特氏菌拥有 O-SP，其结构已被报道，但与核心 PS 的联系尚未确定。我们使用不同的化学降解方法、核磁共振波谱和质谱分析了支气管败血博德特氏菌和副百日咳博德特氏菌的几种菌株的脂多糖的碳水化合物结构，并在所有研究的脂多糖中鉴定出了一种将 O 链连接到核心的新型五糖。此外，据报道这些细菌的O链由1,4-连接的2,3-二乙酰氨基-2,3-二脱氧-β-半乳糖醛酸的均聚物组成，但我们发现它含有酰胺化糖醛酸，其数量因菌株而异。 杜克雷嗜血杆菌：是软下疳的病因，软下疳是一种以生殖器疼痛性溃疡为特征的性传播疾病。软下疳还会促进艾滋病毒感染的传播。杜克雷嗜血杆菌的主要毒力因子和潜在保护性抗原是其脂寡糖 (LOS)。