Zhao Chunxia, Crews Charles Jefferson, Derdeyn Cynthia A, Blackwell Jerry L
Emory Vaccine Center, Emory University, Atlanta, GA 30329, United States.
J Virol Methods. 2009 Sep;160(1-2):101-10. doi: 10.1016/j.jviromet.2009.04.028. Epub 2009 May 3.
Adenovirus (Ad) vectors have been developed as human immunodeficiency-1 (HIV-1) vaccine vectors because they consistently induce immune responses in preclinical animal models and human trials. Strong promoters and codon-optimization are often used to enhance vaccine-induced HIV-1 gene expression and immunogenicity. However, if the transgene is inherently cytotoxic in the cell line used to produce the vector, and is expressed at high levels, it is difficult to rescue a stable Ad HIV-1 vaccine vector. Therefore we hypothesized that generation of Ad vaccine vectors expressing cytotoxic genes, such as HIV-1 env, would be more efficient if expression of the transgene was down-regulated during Ad rescue. To test this hypothesis, a Lac repressor-operator system was applied to regulate expression of reporter luciferase and HIV-1 env transgenes during Ad rescue. The results demonstrate that during Ad rescue, constitutive expression of the Lac repressor in 293 cells reduced transgene expression levels to approximately 5% of that observed in the absence of regulation. Furthermore, Lac-regulation translated into more efficient Ad rescue compared to traditional 293 cells. Importantly, Ad vectors rescued with this system showed high levels of transgene expression when transduced into cells that lack the Lac repressor protein. The Lac-regulated system also facilitated the rescue of modified Ad vectors that have non-native receptor tropism. These tropism-modified Ad vectors infect a broader range of cell types than the unmodified Ad, which could increase their effectiveness as a vaccine vector. Overall, the Lac-regulated system described here (i) is backwards compatible with Ad vector methods that employ bacterial-mediated homologous recombination, (ii) is adaptable for the engineering of tropism-modified Ad vectors, and (iii) does not require co-expression of regulatory genes from the vector or the addition of exogenous chemicals to induce or repress transgene expression. This system therefore could facilitate the development of Ad-based vaccine candidates that otherwise would not be feasible to generate.
腺病毒（Ad）载体已被开发用作人类免疫缺陷病毒1型（HIV-1）疫苗载体，因为它们在临床前动物模型和人体试验中能持续诱导免疫反应。强启动子和密码子优化常被用于增强疫苗诱导的HIV-1基因表达和免疫原性。然而，如果转基因在用于生产载体的细胞系中具有内在细胞毒性且高水平表达，就很难拯救出稳定的Ad HIV-1疫苗载体。因此，我们推测，如果在Ad拯救过程中转基因表达被下调，那么表达细胞毒性基因（如HIV-1 env）的Ad疫苗载体的产生效率会更高。为了验证这一假设，应用了乳糖阻遏物-操纵子系统来在Ad拯救过程中调节报告荧光素酶和HIV-1 env转基因的表达。结果表明，在Ad拯救过程中，293细胞中乳糖阻遏物的组成型表达将转基因表达水平降低至无调控时观察到的约5%。此外，与传统293细胞相比，乳糖调节转化为更有效的Ad拯救。重要的是，用该系统拯救的Ad载体转导到缺乏乳糖阻遏蛋白的细胞中时显示出高水平的转基因表达。乳糖调节系统还促进了具有非天然受体嗜性的修饰Ad载体的拯救。这些嗜性修饰的Ad载体比未修饰的Ad感染更广泛的细胞类型，这可能会提高它们作为疫苗载体的有效性。总体而言，这里描述的乳糖调节系统（i）与采用细菌介导的同源重组的Ad载体方法向后兼容，（ii）适用于嗜性修饰的Ad载体工程，并且（iii）不需要从载体共表达调控基因或添加外源化学物质来诱导或抑制转基因表达。因此，该系统可以促进基于Ad的候选疫苗的开发，否则这些疫苗将无法产生。
