Salmonella enterica serotype Typhimurium is a nontyphoidal and common foodborne pathogen that causes serious threat to humans. There is no licensed vaccine to prevent the nontyphoid bacterial infection caused by S. Typhimurium. To develop conjugate vaccines, the bacterial lipid-A free lipopolysaccharide (LFPS) is prepared as the immunogen and used to synthesize the LFPS–linker–protein conjugates 6a–9b. The designed bifunctional linkers 1–5 comprising either an o-phenylenediamine or amine moiety are specifically attached to the exposed 3-deoxy-D-manno-octulosonic acid (Kdo), an α-ketoacid saccharide of LFPS, via condensation reaction or decarboxylative amidation. In addition to bovine serum albumin and ovalbumin, the S. Typhimurium flagellin (FliC) is also used as a self-adjuvanting protein carrier. The synthesized conjugate vaccines are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fast performance liquid chromatography (FPLC), and their contents of polysaccharides and protein are determined by phenol–sulfuric acid assay and bicinchoninic acid assay, respectively. Enzyme-linked immunosorbent assay (ELISA) shows that immunization of mouse with the LFPS–linker–protein vaccines at a dosage of 2.5 μg is sufficient to elicit serum immunoglobulin G (IgG) specific to S. Typhimurium lipopolysaccharide (LPS). The straight-chain amide linkers in conjugates 7a–9b do not interfere with the desired immune response. Vaccines 7a and 7b derived from either unfractionated LFPS or the high-mass portion show equal efficacy in induction of IgG antibodies. The challenge experiments are performed by oral gavage of S. Typhimurium pathogen, and vaccine 7c having FliC as the self-adjuvanting protein carrier exhibits a high vaccine efficacy of 74% with 80% mice survival rate at day 28 post the pathogen challenge. This study demonstrates that lipid-A free lipopolysaccharide prepared from Gram-negative bacteria is an appropriate immunogen, in which the exposed Kdo is connected to bifunctional linkers to form conjugate vaccines. The decarboxylative amidation of Kdo is a novel and useful method to construct a relatively robust and low immunogenic straight-chain amide linkage. The vaccine efficacy is enhanced by using bacterial flagellin as the self-adjuvanting carrier protein.

中文翻译：

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利用脂质A游离脂多糖构建鼠伤寒沙门氏菌结合疫苗。

肠炎沙门氏菌血清型鼠伤寒沙门氏菌是一种非伤寒和常见的食源性病原体，会对人类造成严重威胁。没有可预防由伤寒沙门氏菌引起的非伤寒细菌感染的许可疫苗。要开发结合疫苗，需要制备细菌脂质A游离脂多糖（LFPS）作为免疫原，并用于合成LFPS-连接子-蛋白质结合物6a-9b。设计的包含邻苯二胺或胺部分的双功能接头1-5通过缩合反应或脱羧酰胺化作用专门连接到裸露的3-脱氧-D-甘露糖辛酸（Kdo），LFPS的α-酮酸糖上。除牛血清白蛋白和卵清蛋白外，鼠伤寒沙门氏菌鞭毛蛋白（FliC）也被用作自佐剂蛋白载体。合成的结合疫苗通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和快速液相色谱（FPLC）表征，多糖和蛋白质的含量分别通过苯酚-硫酸测定法和二辛可宁酸测定法测定。酶联免疫吸附试验（ELISA）表明，以2.5μg剂量的LFPS-接头-蛋白疫苗免疫小鼠足以引起鼠伤寒沙门氏菌脂多糖（LPS）特异的血清免疫球蛋白G（IgG）。缀合物7a-9b中的直链酰胺接头不会干扰所需的免疫反应。衍生自未分级的LFPS或高质量部分的疫苗7a和7b在诱导IgG抗体方面显示出相同的功效。通过口服强饲S进行攻击实验。鼠伤寒病原体和具有FliC作为自佐剂蛋白载体的疫苗7c在病原体攻击后第28天显示出74％的高疫苗效力和80％的小鼠存活率。这项研究表明，由革兰氏阴性细菌制备的不含脂质A的脂多糖是合适的免疫原，其中暴露的Kdo与双功能接头连接形成偶联疫苗。Kdo的脱羧酰胺化是一种新型且有用的方法，可用于构建相对健壮和低免疫原性的直链酰胺键。通过使用细菌鞭毛蛋白作为自佐剂载体蛋白可以增强疫苗效力。这项研究表明，由革兰氏阴性细菌制备的不含脂质A的脂多糖是合适的免疫原，其中暴露的Kdo与双功能接头连接形成偶联疫苗。Kdo的脱羧酰胺化是一种新型且有用的方法，可用于构建相对健壮和低免疫原性的直链酰胺键。通过使用细菌鞭毛蛋白作为自佐剂载体蛋白可以增强疫苗效力。这项研究表明，由革兰氏阴性细菌制备的不含脂质A的脂多糖是合适的免疫原，其中暴露的Kdo与双功能接头连接形成偶联疫苗。Kdo的脱羧酰胺化是一种新型且有用的方法，可用于构建相对健壮和低免疫原性的直链酰胺键。通过使用细菌鞭毛蛋白作为自佐剂载体蛋白可以增强疫苗效力。