Kulkarni Manjiri R, Islam Monirul M, Numoto Nobutaka, Elahi Montasir, Mahib Mamunur R, Ito Nobutoshi, Kuroda Yutaka
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan.
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan; Department of Biochemistry and Molecular Biology, University of Chittagong, Bangladesh.
Biochim Biophys Acta. 2015 Oct;1854(10 Pt A):1438-43. doi: 10.1016/j.bbapap.2015.07.004. Epub 2015 Jul 6.
Dengue fever is a re-emerging tropical disease and its severe form is caused by cross-reactivity between its four serotypes (DEN1, DEN2, DEN3 and DEN4). The third domain of the viral envelope protein (ED3) contains the two major putative epitopes and is a highly suitable model protein for examining the molecular determinants of a virus' sero-specificity. Here we examine d the sero-specificity and cross-reactivity of the immune response against DEN3 and DEN4 ED3 using six epitope grafted ED3 variants where the surface-exposed epitope residues from DEN3 ED3 were switched to those of DEN4 ED3 and vice versa. We prepared anti-DEN3 and anti-DEN4 ED3 serum by immunizing Swiss albino mice and measured their reactivities against all six grafted mutants. As expected, both sera exhibited strong reactivity against its own serotype's ED3, and little cross-reactivity against their counterpart serotype's ED3s. E2 played a major role in the sero-specificity of anti-DEN3 serum, whereas E1 was important for DEN4 ED3's sero-specificity. Next, the reactivity patterns corroborated our working hypothesis that sero-specificity could be transferred by grafting the surface exposed epitope residues from one serotype to the other. To analyze the above results from a structural viewpoint, we determined the crystal structure of a DEN4 ED3 variant, where E2 was grafted from DEN3 ED3, at 2.78Å resolution and modeled the structures of the five remaining grafted variants by assuming that the overall backbone remained unchanged. The examination of the electrostatic and molecular surfaces of the variants suggested some further rationale for the sero-specificity of the immune responses.
登革热是一种再度出现的热带疾病，其严重形式是由四种血清型（DEN1、DEN2、DEN3和DEN4）之间的交叉反应引起的。病毒包膜蛋白的第三个结构域（ED3）包含两个主要的假定表位，是用于研究病毒血清特异性分子决定因素的高度合适的模型蛋白。在这里，我们使用六种表位嫁接的ED3变体研究了针对DEN3和DEN4 ED3的免疫反应的血清特异性和交叉反应性，其中将DEN3 ED3表面暴露的表位残基替换为DEN4 ED3的表位残基，反之亦然。我们通过免疫瑞士白化小鼠制备了抗DEN3和抗DEN4 ED3血清，并测量了它们对所有六种嫁接突变体的反应性。正如预期的那样，两种血清对自身血清型的ED3都表现出强烈的反应性，而对其对应血清型的ED3几乎没有交叉反应性。E2在抗DEN3血清的血清特异性中起主要作用，而E1对DEN4 ED3的血清特异性很重要。接下来，反应模式证实了我们的工作假设，即血清特异性可以通过将一种血清型的表面暴露表位残基嫁接到另一种血清型上来转移。为了从结构角度分析上述结果，我们确定了一种DEN4 ED3变体的晶体结构，其中E2是从DEN3 ED3嫁接而来的，分辨率为2.78Å，并通过假设整体骨架保持不变对其余五种嫁接变体的结构进行了建模。对变体的静电和分子表面的研究为免疫反应的血清特异性提供了一些进一步的理论依据。
