Henriques Ana M, Fevereiro Miguel, Prazeres Duarte M F, Monteiro Gabriel A
Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Lisboa, Portugal.
Vet Immunol Immunopathol. 2007 Oct 15;119(3-4):222-32. doi: 10.1016/j.vetimm.2007.05.004. Epub 2007 Jun 3.
DNA vaccine candidates against Maedi-Visna virus (MVV) infection in ovines were developed as an alternative to conventional vaccines. Candidates were constructed by cloning genes encoding the MVV gag polyprotein and gag proteins p16 and p25 fused to a beta-galactosidase reporter in a plasmid backbone. Transfection of different ovine cells showed a higher protein expression with plasmid lacZp16, which was hence further optimised by (i) removing a putative inhibitory sequence via reduction of the AU-content in the p16 gene or by (ii) introducing a secretory signal (Sc) to promote antigen secretion and increase its presentation to APCs. Unexpectedly, plasmids constructed on the basis of the first strategy by mutagenesis of lacZp16 (lacZp16mut(24)), led to a reduction in the expression of the antigen/reporter fusion in cultured ovine cells. This indicates that the high AU content in MVV does not inhibit protein expression. However, mice primed with lacZp16mut(24) and boosted with MVV protein displayed higher humoral response when compared with control lacZp16. The addition of the Sc signal (Sc-p16) led to lower amounts of intracellular antigen/reporter fusion in transfected ovine cells, thus confirming secretion. These findings correlate with in vivo experiments, which showed that mice primed with Sc-p16 and boosted with MVV exhibited stronger antibody responses when compared with control mice primed with lacZp16 and boosted with MVV. Stronger humoral responses were recorded by immunising mice with (i) Sc-p16 and lacZp16mut(24) plasmids together or with (ii) one plasmid containing both the mutations and the Sc signal.
开发了针对绵羊梅迪 - 维斯纳病毒（MVV）感染的DNA候选疫苗，作为传统疫苗的替代方案。通过在质粒骨架中克隆编码MVV gag多蛋白以及与β - 半乳糖苷酶报告基因融合的gag蛋白p16和p25的基因来构建候选疫苗。不同绵羊细胞的转染结果显示，质粒lacZp16具有更高的蛋白表达，因此通过以下方式对其进行进一步优化：（i）通过降低p16基因中的AU含量去除一个假定的抑制序列，或（ii）引入分泌信号（Sc）以促进抗原分泌并增加其向抗原呈递细胞（APC）的呈递。出乎意料的是，基于第一种策略通过对lacZp16进行诱变构建的质粒（lacZp16mut(24)），导致培养的绵羊细胞中抗原/报告基因融合体的表达降低。这表明MVV中的高AU含量并不抑制蛋白表达。然而，用lacZp16mut(24)免疫的小鼠并用MVV蛋白加强免疫后，与对照lacZp16相比，显示出更高的体液反应。添加Sc信号（Sc - p16）导致转染的绵羊细胞中细胞内抗原/报告基因融合体的量减少，从而证实了分泌。这些发现与体内实验相关，体内实验表明，与用lacZp16免疫并用MVV加强免疫的对照小鼠相比，用Sc - p16免疫并用MVV加强免疫的小鼠表现出更强的抗体反应。通过用（i）Sc - p16和lacZp16mut(24)质粒一起免疫小鼠或用（ii）一种同时包含突变和Sc信号的质粒免疫小鼠，记录到了更强的体液反应。
