Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors
Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors
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DOI：
10.1128/cvi.00075-15
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发表时间：
2015-07-01
影响因子：
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通讯作者：
Frothingham, Richard
中科院分区：
文献类型：
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作者：
Hart, Bryan E.;Asrican, Rose;Frothingham, Richard
结核疫苗株牛分支杆菌卡介苗(BCG)良好的安全性使其成为异源表达临床相关病原体抗原的有吸引力的载体。然而，成功构建具有一致插入表达的重组卡介苗菌株在稳定性方面遇到了挑战。在这里，我们描述了一种利用可选择的亮氨酸营养缺陷性互补来开发稳定表达慢病毒抗原人类免疫缺陷病毒(HIV)gp120和猴免疫缺陷病毒(SIV)Gag的重组卡介苗大批量的方法。疫苗稳定性的成功建立源于严格的质量控制标准，这些标准不仅筛选高度稳定的补充BCG Delta leuCD转化子，而且彻底表征后期生产质量。这些参数包括正确大小的抗原的一致生产，序列纯质粒DNA的保留，冻融恢复，CFU计数，以及细胞聚集体的评估。重要的是，这些质量保证程序表明了疫苗的总体稳定性，可以预测在随后的体外和体内传代中抗原的成功表达，并与诱导小鼠模型的免疫反应有关。本研究生产了一种质量可控的表达HIV gp120的BCG Delta leuCD疫苗，该疫苗在体外扩增10(24)倍并在小鼠体内生长60天后保持稳定的全长表达。第二批疫苗在体外表达了全长SIV Gag，体外扩增了10(68)倍，并在接种的小鼠中诱导了强大的抗原特异性T细胞群。大量、定义明确的重组BCG Delta leuCD批次的生产可以确保用于免疫原性和保护研究的疫苗材料不会受到不稳定或新生长生产批次之间的差异的负面影响。
The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guerin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG Delta leuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG Delta leuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG Delta leuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.