Caley I J, Betts M R, Davis N L, Swanstrom R, Frelinger J A, Johnston R E
Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill 27599, USA.
Vaccine. 1999 Aug 6;17(23-24):3124-35. doi: 10.1016/s0264-410x(99)00142-5.
A live virus vaccine vector has been constructed from a molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE). High levels of foreign protein expression are regulated by an additional copy of the 26 S viral subgenomic RNA promoter. The position of this additional promoter and foreign gene in the VEE genome was predicted to have a major influence on expression level of the heterologous protein. Two sites in the genome were tested to determine the optimal site for expression of the matrix/capsid (MA/CA) coding region of human immunodeficiency virus (HIV-1). One vector contained the additional promoter and the MA/CA genes immediately downstream of the VEE E1 gene at the 3' end of the genome. In the second vector, the additional promoter was introduced immediately upstream from the authentic 26 S subgenomic promoter. Significantly higher levels of MA/CA were expressed from the downstream vector compared to the upstream vector. However, the stability of expression for both vectors was similar following passage in baby hamster kidney cells (BHK) cells. In BALB/c mice, the two vectors elicited similar levels of cellular immune responses to MA/CA as determined by bulk cytotoxic T-lymphocyte assays and precursor frequency analysis, but the humoral response induced by the downstream vector was significantly stronger. At 11 months post boosting with the downstream vector, serum antibody levels against HIV MA/CA were undiminished, and MA/CA specific CTLp were detectable in all mice tested. These findings suggest that VEE vectors can be optimized to elicit strong, balanced and long-lived immune responses to foreign viral proteins.
一种活病毒疫苗载体是由分子克隆的委内瑞拉马脑炎病毒（VEE）减毒株构建而成。高水平的外源蛋白表达受26S病毒亚基因组RNA启动子的额外拷贝调控。该额外启动子和外源基因在VEE基因组中的位置预计会对异源蛋白的表达水平产生重大影响。对基因组中的两个位点进行了测试，以确定人免疫缺陷病毒（HIV-1）基质/衣壳（MA/CA）编码区表达的最佳位点。一种载体在基因组3'端的VEE E1基因下游紧邻位置含有额外启动子和MA/CA基因。在第二种载体中，额外启动子被引入到天然26S亚基因组启动子的紧邻上游。与上游载体相比，下游载体表达的MA/CA水平显著更高。然而，两种载体在幼仓鼠肾细胞（BHK）中传代后的表达稳定性相似。在BALB/c小鼠中，通过大量细胞毒性T淋巴细胞测定和前体频率分析确定，两种载体引发的针对MA/CA的细胞免疫反应水平相似，但下游载体诱导的体液反应明显更强。用下游载体加强免疫11个月后，针对HIV MA/CA的血清抗体水平未降低，并且在所有测试小鼠中均可检测到MA/CA特异性CTLp。这些发现表明，VEE载体可以进行优化，以引发对外源病毒蛋白的强烈、平衡且持久的免疫反应。
