Vaccine Division, Center for Genetic Engineering and Biotechnology (CIGB), Avenue 31, P.O. Box 6162, Havana 6, 10 600, Cuba.
Arch Virol. 2013 Jan;158(1):225-30. doi: 10.1007/s00705-012-1471-z. Epub 2012 Sep 18.
It was previously reported that DIIIC-2 (a fusion protein composed of domain III of the envelope protein and the capsid protein from dengue 2 virus), as an aggregate antigen from a partially purified preparation, induced a functional protective immune response against dengue 2 virus in the mouse encephalitis model. In the present work, a purification procedure was developed for DIIIC-2, and soluble and aggregated fractions of the purified protein were characterized and evaluated in mice. The purification process rendered a protein preparation of 91 % purity, and the remaining 9 % consisted of fragments and aggregates of the same recombinant protein. After the in vitro aggregation process, upon addition of oligodeoxynucleotides, 80 % of the protein formed aggregates, whereas 20 % remained as soluble protein. An immunological evaluation revealed the proper immunogenicity of the aggregated purified protein in terms of induction of antiviral and neutralizing antibodies, cell-mediated immunity and protection upon dengue 2 virus challenge in the mouse encephalitis model. Based on these results, we can assert that the purified protein DIIIC-2 is functional and could be used for further scalable steps and preclinical studies in non-human primates.
先前有报道称，DIIIC-2（一种由登革热 2 型病毒包膜蛋白的结构域 III 和衣壳蛋白组成的融合蛋白）作为一种从部分纯化制剂中提取的聚集抗原，在小鼠脑炎模型中诱导了针对登革热 2 型病毒的功能性保护免疫反应。在本研究中，开发了一种用于 DIIIC-2 的纯化程序，并对纯化蛋白的可溶性和聚集性部分在小鼠中进行了表征和评估。该纯化过程使蛋白质制剂的纯度达到 91%，其余 9%由相同重组蛋白的片段和聚集物组成。体外聚集过程后，加入寡脱氧核苷酸时，80%的蛋白形成聚集物，而 20%仍为可溶性蛋白。免疫学评估显示，聚集的纯化蛋白具有适当的免疫原性，可诱导抗病毒和中和抗体、细胞介导免疫，并在小鼠脑炎模型中预防登革热 2 型病毒的攻击。基于这些结果，我们可以断言，纯化的 DIIIC-2 蛋白是功能性的，可以用于进一步的规模化步骤和非人类灵长类动物的临床前研究。
