Systematic mutational analysis of epitope-grafted ED3's immunogenicity reveals a DENV3-DENV4 bi-serospecific ED3 mutant

Dengue viruses are classified into four serotypes (DENV1[~]4), and the severe forms of dengue disease, the dengue hemorrhagic fever and shock syndrome, are caused by sero-cross-reacting antibodies. However, the residue determinants of the serospecificity and sero-cross-reactivity are yet to be identified. Here, we report an epitope grafting mutational analysis of the serospecificity and cross-serospecificity of the envelope protein domain 3 (ED3; 107 residues, [~]11.6kDa), which contains two major putative epitopes of DENVs. To this end, we constructed ED3 from DENV3 (3ED3) and DENV4 (4ED3), and six epitope-grafted variants, where we transferred epitope 1 (L304I, K305D, V309M, and S310A) and/or epitope 2 (D383N, K384S, K387T, and N389H) of 4ED3 onto 3ED3 and vice versa. Mice immunization using 3ED3 and 4ED3 generated serotype-specific antisera, as expected. Similarly, most epitope-grafted ED3s produced antisera serospecific to the template ED3 with little or no cross-recognition of ED3 of the serotype from which the epitopes were taken. This result indicated that a mere grafting of the epitope was not sufficient to transfer serospecificity, contrary to our expectations. However, one epitope grafted ED3 mutant, where epitope 1 of 3ED3 was grafted onto 4ED3 (4ED3epi1), generated antisera that was serospecific to both 4ED3 and 3ED3. The 4ED3epi1 is thus a chimeric ED3 that produces antisera possessing serospecificity to both 3ED3 and 4ED3. The 4ED3epi1 provides a unique tool for analyzing serospecificity and cross-reactivity in dengue, and we hope it will serve as a template for trivalent and eventually tetravalent antisera.