Laboratório de Tecnologia Bacteriana, Fundação Oswaldo Cruz, Bio-ManguinhosRio de Janeiro, Brazil.
Laboratório de Química Biológica de Microrganismos, Instituto de Microbiologia Paulo de Goes, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, Brazil.
Glycoconj J. 2021 Oct;38(5):539-549. doi: 10.1007/s10719-021-10016-w. Epub 2021 Sep 13.
Recent changes in the epidemiology of meningococcal have been reported and meningococcal group W (MenW) has become the third most prevalent group isolated in Brazil in the last 10 years. In this study we have developed a conjugate vaccine for MenW using a modified reductive amination conjugation method through a covalent linkage between periodate-oxidized MenW non-O-acetylated polysaccharide and hydrazide-activated monomeric tetanus toxoid. Process control of bulks was done by physicochemical analysis including polysaccharide and protein quantification, high performance liquid chromatography - size exclusion chromatography, capillary electrophoresis, and hydrogen nuclear magnetic resonance. Conjugate bulks were best produced with concentration of polysaccharide twice as high as protein, at room temperature, and pH approximately 6.0. A scaled-up bulk (100 mg scale) was formulated and inoculated intramuscularly in mice in a dose-response study (0.1, 0.5, 1.0 and 10.0 µg of polysaccharide/dose). The immunogenicity of conjugate bulks was determined by serum bactericidal assay and ELISA assays of serum from immunized mice. ELISA and SBA titers revealed high titers of IgG and demonstrated the functionality of the antibodies produced in all doses studied 15 days after the third dose. However, significant differences were observed among them by ELISA. In conclusion, this study established the best conditions to produce MenW conjugate bulks and showed the efficacy of the obtained conjugate bulk in induce a good immune response in mice. Further experiments will need to be done to scale up the conjugation reaction and then allow the use of this conjugate in clinical trials.
最近报道了脑膜炎奈瑟菌流行病学的变化，脑膜炎奈瑟菌 W 群（MenW）已成为过去 10 年巴西分离的第三大常见菌群。在这项研究中，我们使用改良的还原胺化偶联方法，通过过碘酸钠氧化的 MenW 非-O-乙酰化多糖与酰肼活化的单体破伤风类毒素之间的共价键，开发了一种用于 MenW 的结合疫苗。通过包括多糖和蛋白质定量、高效液相色谱-尺寸排阻色谱、毛细管电泳和氢核磁共振在内的物理化学分析对批量进行过程控制。在多糖浓度是蛋白质浓度两倍、室温以及 pH 值约为 6.0 的条件下，可最佳生产结合物批量。进行了大规模批量（100mg 规模）的配方，并在剂量反应研究中（多糖剂量为 0.1、0.5、1.0 和 10.0μg）通过肌肉内接种在小鼠中进行接种。通过血清杀菌测定和免疫小鼠血清的 ELISA 测定来确定结合物批量的免疫原性。ELISA 和 SBA 滴度显示出 IgG 的高滴度，并证明了在所有研究剂量下 15 天后产生的抗体的功能。然而，通过 ELISA 观察到它们之间存在显著差异。总之，本研究确定了生产 MenW 结合物批量的最佳条件，并表明获得的结合物批量在诱导小鼠产生良好免疫应答方面的功效。需要进一步的实验来扩大偶联反应，然后允许在临床试验中使用这种结合物。
