Dus Santos María J, Wigdorovitz Andrés, Trono Karina, Ríos Raúl D, Franzone Pascual M, Gil Felix, Moreno Javier, Carrillo Consuelo, Escribano José M, Borca Manuel V
Instituto de Virología, C.I.C.V., INTA-Castelar, CC77, Morón (1708), Pcia. De, Buenos Aires, Argentina.
Vaccine. 2002 Jan 15;20(7-8):1141-7. doi: 10.1016/s0264-410x(01)00434-0.
The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the recombinant antigens in the plant tissues, which reduces the possibilities of practical applications. Because the site of insertion of the transferred DNA into the cellular chromosomal DNA is at random, different levels of foreign protein expression in independent transformants is expected. Strategies to allow the evaluation of a high number of the transgenic individuals, usually an expensive and very time consuming process, would permit the selection of those plants presenting the highest levels of recombinant protein expression. Here, we present the development of an experimental immunogen based in the expression of a highly immunogenic epitope from foot and mouth disease virus (FMDV) fused to the glucuronidase (gus A) reporter gene, which allows selection of the transgenic plants by the ss-glucuronidase (ssGUS) enzymatic activity. We produced transgenic plants of alfalfa expressing the immunogenic site between amino acid residues 135-160 of structural protein VP1 (VP135-160), fused to the ssGUS protein. Plants expressing the highest levels of the immunogenic epitope VP135-160, analyzed by Western blot, were efficiently selected based on their levels of ssGUS enzymatic activity. The FMDV epitope expressed in plants was highly immunogenic in mice which developed, after immunization, a strong anti-FMDV antibody response against a synthetic peptide representing the region VP135-160, to native virus VP1, and to purified FMDV particles. Additionally, these mice were completely protected against experimental challenge with the virulent virus. To our knowledge, this constitutes the first report of a peptide-based vaccine produced in transgenic plants that induces a protective immune response when used in experimental hosts. Also, these results demonstrated the possibility of using a novel and simple methodology for obtaining transgenic plants expressing high levels of foreign immunogenic epitopes, which could be directly applied in the development of plant-based vaccines.
在实验性疫苗的研发中,转基因植物中抗原的表达已越来越多地被用作经典抗原表达方法的替代方案。然而,在大多数情况下,一个重要的限制是植物组织中重组抗原的浓度较低,这降低了实际应用的可能性。由于转入细胞染色体DNA的转移DNA的插入位点是随机的,因此预计独立转化体中外源蛋白的表达水平会有所不同。评估大量转基因个体的策略通常是一个昂贵且非常耗时的过程,通过这种策略可以选择那些重组蛋白表达水平最高的植物。在此,我们展示了一种基于口蹄疫病毒(FMDV)高免疫原性表位与葡糖醛酸酶(gus A)报告基因融合表达的实验性免疫原的开发,该方法允许通过ss-葡糖醛酸酶(ssGUS)的酶活性来选择转基因植物。我们培育了表达结构蛋白VP1氨基酸残基135 - 160之间免疫原性位点并与ssGUS蛋白融合的苜蓿转基因植物。通过蛋白质免疫印迹分析,基于其ssGUS酶活性水平有效地筛选出了表达最高水平免疫原性表位VP135 - 160的植物。植物中表达的FMDV表位在小鼠中具有高度免疫原性,免疫后小鼠针对代表VP135 - 160区域的合成肽、天然病毒VP1以及纯化的FMDV颗粒产生了强烈的抗FMDV抗体反应。此外,这些小鼠在实验性攻毒中得到了完全保护。据我们所知,这是关于转基因植物中生产的基于肽的疫苗在实验宿主中使用时诱导保护性免疫反应的首次报道。而且,这些结果证明了使用一种新颖且简单的方法来获得表达高水平外源免疫原性表位的转基因植物的可能性,该方法可直接应用于基于植物的疫苗开发。