Fuerst T R, Stover C K, de la Cruz V F
Department of Molecular Genetics, MedImmune, Inc., Gaithersburg, Maryland 20878.
Biotechnol Ther. 1991;2(1-2):159-78.
Bacille Calmette-Guèrin (BCG), a live attenuated tubercle bacillus, is currently the most widely used vaccine in the world. Because of its unique characteristics, including low toxicity, adjuvant potential, and long-lasting immunity, BCG represents a novel vaccine vehicle with which to deliver protective antigens of multiple pathogens. We have developed episomal and integrative expression vectors employing regulatory sequences of major BCG heat shock proteins for stable maintenance and expression of foreign antigens in BCG vaccine strains (22). Shuttle plasmids capable of autonomous replication in Escherichia coli and BCG were constructed with a DNA cassette containing a minimal replicon derived from the Mycobacterium fortuitum plasmid pAL5000. Efficient and stable chromosomal integration of recombinant plasmids into BCG was achieved using a DNA segment containing the mycobacteriophage L5 attachment site and integrase coding sequence. Using the BCG hsp60 and hsp70 stress gene promoters, we were able to express Escherchia coli beta-galactosidase to levels in excess of 10% of total cell protein. The major antigens of HIV-1 gag, pol, and env were also stably expressed using our vector systems. The recombinant BCG elicited long-lasting humoral and cellular immune responses to these antigens in mice. Antibody responses to beta-galactosidase using as few as 200 colony-forming units were detected 6 weeks after immunization, and titers (1:30,000) were sustained for more than 10 weeks. Cellular immune responses, of both cytotoxic T cell (CTL) and helper T lymphocytes, were detected to beta-galactosidase. CTL responses were also induced to the HIV-1 envelope protein. Thus, we have demonstrated stable recombinant antigen expression, processing, and presentation using our recombinant BCG vector system. This live recombinant vector system shows promise as a universally applicable and safe vaccine vehicle for protection against various infectious diseases.
卡介苗（BCG）是一种减毒活结核杆菌，是目前世界上使用最广泛的疫苗。由于其具有低毒性、佐剂潜力和持久免疫力等独特特性，卡介苗是一种新型疫苗载体，可用于递送多种病原体的保护性抗原。我们利用卡介苗主要热休克蛋白的调控序列开发了游离型和整合型表达载体，以在卡介苗疫苗株中稳定维持和表达外源抗原（22）。构建了能够在大肠杆菌和卡介苗中自主复制的穿梭质粒，其DNA盒包含源自偶然分枝杆菌质粒pAL5000的最小复制子。使用包含分枝杆菌噬菌体L5附着位点和整合酶编码序列的DNA片段，实现了重组质粒高效稳定地整合到卡介苗染色体中。利用卡介苗hsp60和hsp70应激基因启动子，我们能够将大肠杆菌β-半乳糖苷酶表达至总细胞蛋白的10%以上。HIV-1 gag、pol和env的主要抗原也利用我们的载体系统稳定表达。重组卡介苗在小鼠中引发了对这些抗原的持久体液免疫和细胞免疫反应。免疫6周后，使用低至200个菌落形成单位即可检测到对β-半乳糖苷酶的抗体反应，且滴度（1:30,000）持续超过10周。检测到针对β-半乳糖苷酶的细胞毒性T细胞（CTL）和辅助性T淋巴细胞的细胞免疫反应。对HIV-1包膜蛋白也诱导了CTL反应。因此，我们已经证明了使用我们的重组卡介苗载体系统能够实现重组抗原的稳定表达、加工和呈递
