Photon Factory Activity Report 2014 #32 (2015) B

BL-5A, BL-17A/2013G216

Analysis of Sero-Specific Immune Responses using Epitope Grafted Dengue ED3 Mutants
Manjiri R. Kulkarnia, Nobutaka Numotob, Nobutoshi Itob and Yutaka Kurodaa,*a

Department of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology,
Tokyo 184-8588, Japan and bDepartment of Structural Biology, Medical Research Institute, Tokyo
Medical and Dental University, Tokyo 113-8510, Japan
1 Introduction
Dengue fever is a re-emerging tropical disease and its
severe form is caused by cross-reactivity between its four
serotypes. The third domain of the viral envelope protein(ED3) contains the two major putative epitopes(E1 andE2) and is a highly suitable model protein for examining
the molecular determinants of a virus’ sero-specificity.
Here, we aim at characterizing the sero-specificity andcross-reactivity of the immune response against DEN3 andDEN4 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.
Further, we report the structure of a DEN4 ED3 variant,where E2 was grafted from DEN3 (referred as
DEN4_E2DEN, hereafter).

the epitopes in the counter serotype's ED3 by those against
which the serum was raised was weak in absolute terms,
the relative reactivity pattern was approximately in linewith our specificity-transfer hypothesis (Fig 1). Finally,
we solved the crystal structure of a DEN4 ED3 variant,where E2 was grafted from DEN3 ED3. The structure ofDEN4_E2DEN3 was essentially identical to that of the wild
type DEN4 ED3 (RMSD: 0.33 Å) despite E2’s residues
being substituted from DEN3. Detailed examination of theelectrostatic surfaces (Fig.2) nevertheless provided someinsights into the sero-specificity and cross-reactivity of
DEN ED3s that were unnoticed from sequence
information only [2].

2 Experiments
Synthetic genes encoding the ED3 sequences were cloned
into a pET15b vector (Novagen) as reported previously [1].
The proteins were over-expressed in E. coli, purified byreverse phase HPLC, and lyophilized. 3-4 week old SwissAlbino mice were immunized by weekly injection, andafter the final injection, the sera were collected from thesupernatant of blood sampled from heart. Interaction
strengths were monitored by ELISA using purified ED3.
Finally, X-ray diffraction data were recorded from single
crystals at the Photon Factory, and DEN4_E2DEN3‘sstructure was determined by molecular replacement.
3 Results and Discussion
We prepared six ED3 variants, in which the surfaceexposed epitope residues from DEN3 ED3 were switchedto those of DEN4 ED3 and vice versa. We raised sera
against DEN3 ED3 and DEN4 ED3 and analyzed theirreactivity against the grafted ED3 variants. Both anti-seraexhibited high sero-specificity with little cross reactivity totheir counter serotype. Reactivity against epitope-grafted
mutants indicated that E2 in DEN3 ED3 and E1 in DEN4
ED3 play a comparatively major role in determining serospecificity. Although the reactivity recovered by replacing

Fig 1: ELISA of anti-DEN4
sera. DEN4 are shown withback and DEN3 are in graybars. The variant’s identities
are: a) DEN3 ED3, b) DEN3
_E1DEN4, c) DEN3_E2DEN4, d)
DEN3_E1E2DEN4, e) DEN4
ED3, f) DEN4_E1DEN3, g)
DEN4_E2DEN3 and h) DEN4
_E1E2DEN3, where superscripts indicate the epitope’s
serotype.

Fig 2: Electrostatic potential
surface of DEN4 and its
epitope-graftedmutants.
The epitope regions areshown in wire models andencircled.

References[1] Elahi M et al Biochim Biophys Acta. Proteins andProteomics ,1844(3), 585-92 (2014)[2] Kulkarni M et al, Biochim Biophys Acta. Proteins andProteomics in press (2015). The coordinates ofDEN4_E2DEN3 are deposited in the Protein Data Bank(PDB: 4x42).
*E-mail YK: ykuroda@cc.tuat.ac.jp