Lockyer Kay, Gao Fang, Derrick Jeremy P, Bolgiano Barbara
Division of Bacteriology, National Institute of Biological Standards and Control, Medicines and Healthcare Products Regulatory Agency, South Mimms, Potters Bar EN6 3QG, UK.
Faculty of Life Sciences, The University of Manchester, Manchester, UK.
Vaccine. 2015 Mar 10;33(11):1345-52. doi: 10.1016/j.vaccine.2015.01.046. Epub 2015 Jan 29.
An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8×10(6) g/mol to larger than 20×10(6) g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines.
对九种已获许可的多糖 - 破伤风类毒素（TT）结合疫苗中的结构 - 抗体识别关系进行了分析。所使用的结合物组包括用于预防由b型流感嗜血杆菌、A、C、W和Y群脑膜炎奈瑟菌以及18C血清型肺炎链球菌引起疾病的疫苗成分。构象和结构分析包括采用多角度光散射的尺寸排阻色谱法来确定大小，以及采用内源荧光光谱法和荧光猝灭法来评估蛋白质折叠和色氨酸残基的暴露情况。一种捕获ELISA使用四种大鼠单克隆抗体来检测结合物中TT表位的识别情况：两种定位于重链（HC）结构域，其中两种依赖于全类毒素构象。这些结合物的平均分子量范围很广，从1.8×10⁶ g/mol到大于20×10⁶ g/mol。发现该结合物组折叠良好，并且没有聚集TT的典型光谱特征。在分子量和表位识别之间发现了部分相关性。分子大小不一定会阻碍对HC结构域或整个类毒素上的表位的识别。在色氨酸侧链的可及性和多糖负载量之间也发现了相关性，这也表明较高水平的结合多糖不一定会干扰类毒素的可及性。定位于HC结构域的载体蛋白色氨酸侧链和表位可及性存在不同水平；尽管这些结合物是独立生产的，但它们与糖类类型有关。这些发现扩展了我们对TT结合疫苗中载体蛋白识别分子基础的理解。
