title: Structural and biophysical analysis of sero-specific immune responses using epitope grafted Dengue ED3 mutants.
abstract: 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.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan.
authors.forename: Manjiri R
authors.initials: MR
authors.lastname: Kulkarni
authors.affiliation: 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.
authors.forename: Monirul M
authors.initials: MM
authors.lastname: Islam
authors.affiliation: Department of Structural Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Numoto
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan.
authors.forename: Montasir
authors.initials: M
authors.lastname: Elahi
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chittagong, Bangladesh.
authors.forename: Mamunur R
authors.initials: MR
authors.lastname: Mahib
authors.affiliation: Department of Structural Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan.
authors.forename: Nobutoshi
authors.initials: N
authors.lastname: Ito
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan. Electronic address: ykuroda@cc.tuat.ac.jp.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kuroda
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000276:immunology; D000818:Animals; D003429:Cross Reactions; D018360:Crystallography, X-Ray; D003716:Dengue Virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D007106:Immune Sera / Q000737:chemistry*; D007114:Immunization; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D012703:Serotyping; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D000596:Amino Acids; D000939:Epitopes; D007106:Immune Sera; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins
keywords: ELISA; Epitope residues; Immune response; Molecular modeling; Sero-specificity; X-ray crystallography