Xu Peng, Kelly Meagan, Vann Willie F, Qadri Firdausi, Ryan Edward T, Kováč Pavol
NIDDK, LBC, Section on Carbohydrates, National Institutes of Health (NIH), Bethesda, MD, 20892-0815, USA.
Department of Immunology and Infectious Diseases, Massachusetts General Hospital, 55 Fruit Street, Boston, MA, 02114, USA.
Chembiochem. 2017 Apr 18;18(8):799-815. doi: 10.1002/cbic.201600699. Epub 2017 Mar 23.
By using O-SP-core (O-SPcNH ) polysaccharide, isolated from Vibrio cholera O1 lipopolysaccharide (LPS) and related synthetic substances, a detailed study of factors that affect conjugation of bacterial polysaccharides to protein carriers through squaric acid chemistry to form conjugate vaccines has been carried out. Several previously unrecognized processes that take place during the squarate labeling of the O-SPcNH and subsequent conjugation of the formed squarate (O-SPcNH-SqOMe) have been identified. The efficiency of conjugation at pH 8.5, 9.0, and 9.5 to bovine serum albumin (BSA) and to the recombinant tetanus toxin fragment C (rTT-Hc) has been determined. The study led to a protocol for more efficient labeling of O-SPcNH antigen with the methyl squarate group, to yield a higher-quality, more potent squarate conjugation reagent. Its use resulted in about twofold increases in conjugation efficiency (from 23-26 % on BSA to 51 % on BSA and 55 % on rTT-Hc). The spent conjugation reagent could be recovered and regenerated by treatment with MeI in the absence of additional base. The immunological properties of the experimental vaccine made from the regenerated conjugation reagent were comparable with those of the immunogen made from the parent O-SPcNH-SqOMe.
通过使用从霍乱弧菌O1脂多糖（LPS）中分离得到的O - SP - 核心（O - SPcNH ）多糖及相关合成物质，开展了一项关于影响细菌多糖通过方酸化学与蛋白质载体结合以形成结合疫苗的因素的详细研究。已确定在O - SPcNH的方酸标记及随后形成的方酸（O - SPcNH - SqOMe）的结合过程中发生的几个先前未被认识的过程。已测定在pH 8.5、9.0和9.5条件下与牛血清白蛋白（BSA）及重组破伤风毒素片段C（rTT - Hc）结合的效率。该研究得出了一种用方酸甲酯基团更有效地标记O - SPcNH抗原的方案，以产生更高质量、更强效的方酸结合试剂。其使用使结合效率提高了约两倍（从与BSA结合时的23 - 26％提高到与BSA结合时的51％以及与rTT - Hc结合时的55％）。用过的结合试剂可在不添加额外碱的情况下用MeI处理进行回收和再生。由再生结合试剂制成的实验疫苗的免疫特性与由母体O - SPcNH - SqOMe制成的免疫原的免疫特性相当。
