The present studies prove that conjugation of meningococcal lipooligosaccharides through their non-reducing terminus conserves their inner epitopes resulting in conjugates potent to induce a protective immune response. Four different oligosaccharides were obtained by specific degradations of the same L7 lipooligosaccharide (L7-LOS), and each was linked to tetanus toxoid by direct reductive amination. Two were truncated oligosaccharides with incomplete inner epitopes and were obtained by mild acid hydrolysis of lipooligosaccharide. The terminal galactose of one oligosaccharide was additionally enzymatically oxidized. These oligosaccharides were conjugated through a newly exposed terminal Kdo in reducing end or through oxidized galactose localized at non-reducing end of the core, respectively. The third was a full-length oligosaccharide obtained by O-deacylation of the L7-LOS and subsequent enzymatic removal of phosphate substituents from its lipid A moiety. The fourth one was also a full-length O-deacylated lipooligosaccharide, but treated with galactose oxidase. This allowed direct conjugation to tetanus toxoid through terminal 2-N-acyl-2-deoxy-D-glucopyranose or through oxidized galactose, respectively. Comparison of the immune performance of four conjugates in mice revealed, that while each was able to induce significant level of L7-LOS-specific IgG antibody, the conjugates made with the full-length saccharides were able to induce antibodies with increased bactericidal activity against homologous meningococci. Only full-length oligosaccharides were good inhibitors of the binding of L7-LOS to the bactericidal antiserum. Moreover, induction of the significant level of the L7-LOS-specific antibody by full-length lipooligosaccharide conjugated from non-reducing end, provided also the direct evidence that internal core epitopes are fully responsible for the immunorecognition and immunoreactivity.

中文翻译：

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脑膜炎球菌脂寡糖的结合通过其非还原性末端导致改善的诱导性保护性免疫反应。

本研究证明脑膜炎球菌脂寡糖通过其非还原性末端缀合可保留其内部表位，从而导致有效诱导保护性免疫应答的缀合物。通过对相同的L7脂寡糖（L7-LOS）进行特异性降解，获得了四种不同的寡糖，每种寡糖都通过直接还原胺化与破伤风类毒素连接。两个是具有不完整内部表位的截短寡糖，并且是通过脂寡糖的温和酸水解而获得的。一种寡糖的末端半乳糖另外被酶氧化。这些寡糖分别通过还原端新暴露的末端Kdo或位于核心非还原端的氧化半乳糖结合。第三是通过L7-LOS的O-脱酰作用并随后从其脂质A部分酶促除去磷酸取代基而获得的全长寡糖。第四个也是全长的O-去酰基化的脂寡糖，但是用半乳糖氧化酶处理。这允许分别通过末端2-N-酰基-2-脱氧-D-吡喃葡萄糖或通过氧化的半乳糖与破伤风类毒素直接缀合。比较四种偶联物在小鼠中的免疫性能，发现每种偶联物均能诱导显着水平的L7-LOS特异性IgG抗体，而用全长糖制备的偶联物却能够诱导对同源菌具有增强杀菌活性的抗体脑膜炎球菌。仅全长寡糖是L7-LOS与杀菌抗血清结合的良好抑制剂。此外，