Ho Mei M, Bolgiano Barbara, Martino Angela, Kairo Satnam K, Corbel Michael J
Division of Bacteriology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire, UK.
Hum Vaccin. 2006 May-Jun;2(3):89-98. doi: 10.4161/hv.2755. Epub 2006 May 23.
A bivalent, unadjuvanted conjugate vaccine composed of Staphylococcus aureus capsular polysaccharides type 5 and 8 (T5 and T8 PS) conjugated to a novel carrier protein, the mutant nontoxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA), has been the subject of recent clinical trials. A program of preclinical laboratory evaluation was carried out in support of the clinical trials conducted by the National Vaccine Evaluation Consortium. This involved physical chemical characterization and limited assessment of toxicity and immunogenicity. The carrier protein showed good stability and its conformation was essentially maintained when conjugated. The T5- and T8-rEPA conjugates were of a size range (1-3 x 10(6) g/ mol) consistent with polysaccharide conjugates. Fluorescence spectroscopy and molecular sizing showed good batch-to-batch consistency. Although all batches of final fill preparations elicited positive immune responses in the mouse model with three schedule doses of 0.25 microg of each T5/8 conjugate per dose, the mouse serum IgG response to T8 PS varied from batch to batch. Storage temperature at 37 degrees C or below or with repetitive temperature fluctuations did not significantly affect the IgG responses to T5 or T8 PS. Storage at 56 degrees C, however, diminished the mouse serum IgG response to T5 PS. The conformation of the conjugated protein and size of the conjugates correlated well with mouse immunogenicity in the thermal stability samples; significant unfolding of the protein and downshifts in molecular size of the conjugate were only observed when stored at 56 degrees C. The relatively high stability of the novel carrier protein when conjugated to large polysaccharides makes this an attractive candidate carrier protein for other conjugate vaccines. When assayed for serum IgG concentration, the bivalent T5/ 8 conjugate was found to evoke an IgG response well over the threshold value of 10 microg/ ml anti-T5 and -T8 IgG established for the ELISA immunogenicity assay.
一种由5型和8型金黄色葡萄球菌荚膜多糖（T5和T8 PS）与新型载体蛋白——突变型无毒重组铜绿假单胞菌外毒素A（rEPA）偶联而成的二价、无佐剂结合疫苗，是近期临床试验的研究对象。为支持国家疫苗评估联盟进行的临床试验，开展了一项临床前实验室评估计划。这包括物理化学特性表征以及对毒性和免疫原性的有限评估。载体蛋白显示出良好的稳定性，偶联后其构象基本保持。T5 - rEPA和T8 - rEPA偶联物的大小范围（1 - 3×10(6) g/ mol）与多糖偶联物一致。荧光光谱和分子大小分析显示批次间一致性良好。尽管所有最终灌装制剂批次在小鼠模型中，通过每剂0.25微克的每种T5/8偶联物分三个剂量方案给药后均引发了阳性免疫反应，但小鼠血清IgG对T8 PS的反应因批次而异。在37摄氏度或以下储存或反复温度波动并不会显著影响对T5或T8 PS的IgG反应。然而，在56摄氏度储存会降低小鼠血清对T5 PS的IgG反应。在热稳定性样品中，偶联蛋白的构象和偶联物的大小与小鼠免疫原性密切相关；仅在56摄氏度储存时才观察到蛋白的显著解折叠和偶联物分子大小的下移。当与大多糖偶联时，这种新型载体蛋白相对较高的稳定性使其成为其他结合疫苗有吸引力的候选载体蛋白。在检测血清IgG浓度时，发现二价T5/8偶联物引发的IgG反应远超过ELISA免疫原性测定中确定的10微克/毫升抗T5和 - T8 IgG的阈值。
