Since the successful use of vaccinia virus (VACV) in the immunization strategies to eliminate smallpox, research has been focused on the development of recombinant VACV strains expressing proteins from various pathogens. Attempts at decreasing the side effects associated with exposure to recombinant, wild-type viral strains have led to the development of attenuated viruses. Yet while these attenuated VACV’s have improved safety profiles compared to unmodified strains, their clinical use has been hindered due to efficacy issues in stimulating a host immune response. This deficiency has largely been attributed to decreased production of the target protein for immunization. Efforts to increase protein production from attenuated VACV strains has largely centered around modulation of viral factors, while manipulation of the translation of viral mRNAs has been largely unexplored. In this study we evaluate the use of translation enhancing element hTEE-658 to increase recombinant protein production in an attenuated VACV system. Optimization of the use of this motif is also attempted by combining it with strategies that have demonstrated effectiveness in previous research. We show that extension of the 5′ leader sequence containing hTEE-658 does not improve motif function, nor does the combination with other known translation enhancing elements. However, the sole use of hTEE-658 in an attenuated VACV system is shown to increase protein expression levels beyond those of a standard viral promoter when used with a wild-type virus. Taken together these results highlight the potential for hTEE-658 to improve the effectiveness of attenuated VACV vaccine candidates and give insights into the optimal sequence context for its use in vaccine design.

中文翻译：

---

优化翻译增强元件用于增加痘苗病毒系统中的蛋白质表达

自从在消除天花的免疫策略中成功使用痘苗病毒 (VACV) 以来，研究一直集中在开发表达来自各种病原体的蛋白质的重组 VACV 毒株上。减少与暴露于重组野生型病毒株相关的副作用的尝试导致了减毒病毒的发展。然而，虽然这些减毒的 VACV 与未修饰的菌株相比具有更高的安全性，但由于刺激宿主免疫反应的功效问题，它们的临床应用受到了阻碍。这种缺陷主要归因于免疫目标蛋白的产生减少。增加减毒 VACV 毒株蛋白质产量的努力主要集中在病毒因子的调节上，而操纵病毒 mRNA 的翻译在很大程度上尚未得到探索。在这项研究中，我们评估了使用翻译增强元件 hTEE-658 来增加减毒 VACV 系统中重组蛋白的产生。还尝试通过将其与先前研究中已证明有效的策略相结合来优化该基序的使用。我们表明，包含 hTEE-658 的 5' 前导序列的扩展不会改善基序功能，也不会与其他已知的翻译增强元件组合。然而，当与野生型病毒一起使用时，在减毒 VACV 系统中单独使用 hTEE-658 显示出的蛋白质表达水平高于标准病毒启动子的表达水平。