Hart Bryan E, Asrican Rose, Lim So-Yon, Sixsmith Jaimie D, Lukose Regy, Souther Sommer J R, Rayasam Swati D G, Saelens Joseph W, Chen Ching-Ju, Seay Sarah A, Berney-Meyer Linda, Magtanong Leslie, Vermeul Kim, Pajanirassa Priyadharshini, Jimenez Amanda E, Ng Tony W, Tobin David M, Porcelli Steven A, Larsen Michelle H, Schmitz Joern E, Haynes Barton F, Jacobs William R, Lee Sunhee, Frothingham Richard
Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
Clin Vaccine Immunol. 2015 Jul;22(7):726-41. doi: 10.1128/CVI.00075-15. Epub 2015 Apr 29.
The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (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 ΔleuCD 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 ΔleuCD 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 ΔleuCD 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.
结核疫苗菌株卡介苗（BCG）已确立的安全特性使其成为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。在此，我们描述了一种利用可选择的亮氨酸营养缺陷型互补来开发大量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的重组卡介苗保藏株的方法。疫苗稳定性的成功建立源于严格的质量控制标准，该标准不仅筛选高度稳定的互补卡介苗ΔleuCD转化体，还全面表征生产后的质量。这些参数包括正确大小抗原的一致生产、序列纯质粒DNA的保留、冻融回收率、CFU计数以及细胞聚集体评估。重要的是，这些质量保证程序表明了整体疫苗稳定性，可预测体外和体内后续传代中抗原的成功表达，并与小鼠模型中免疫反应的诱导相关。本研究产生了一种质量可控的表达HIV gp120的卡介苗ΔleuCD疫苗，该疫苗在体外10（24）倍扩增后以及在小鼠中生长60天后仍保持稳定的全长表达。另一批疫苗表达全长SIV Gag，在体外扩增>10（68）倍，并在接种疫苗的小鼠中诱导产生强效的抗原特异性T细胞群体。大量、明确的重组卡介苗ΔleuCD保藏株的生产可以让人相信，用于免疫原性和保护研究的疫苗材料不会受到不稳定性或新鲜生产批次之间差异的负面影响。
