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

Supplementary Figures

Supplemental Figure S1. Effects of epitope-grafting on the serospecificity of 3ED3. ELISA(OD450nm) of anti-3ED3, anti-3ED3epi1and anti-4ED3 sera against wildtype 3ED3 (A), 4ED3 (B),3ED3epi1(C) and 4ED3epi1(D) are shown. Both anti-3ED3 and anti-3ED3epi1sera recognized wildtype 3ED3, but anti-4ED3 sera did not cross-recognize 3ED3. Similarly, only anti-4ED3 sera recognized 4ED3, indicating that wildtype ED3s were serospecific, and epitope-grafting onto 3ED3 from 4ED3 did neither not much affect 3ED3 serospecificity nor transfer 4ED3 serospecificity onto 3ED3. Grafting of putative epitope 1 onto 3ED3 reduced its detection by anti-3ED3 sera. Similarly,anti-epitope-grafted 3ED3 sera had reduced recognition of wildtype 3ED3. However,sero-cross-recognition of 4ED3epi1by anti-3ED3 sera improved following epitope-grafting onto 4ED3 from 3ED3. These observations indicated that epitope-grafting from 3ED3 onto 4ED3 could transfer serospecificity of 3ED3 onto 4ED3. Legends are shown within the panels.

Supplemental Figure S2. Effects of epitope-grafting on serospecificity of 4ED3. ELISA(OD450nm) of anti-4ED3, anti-4ED3epi1and anti-3ED3 sera against wildtype 3ED3 (A), 4ED3 (B),3ED3epi1(C) and 4ED3epi1(D) are shown. Epitope-grafting from 4ED3 onto 3ED3 did not improve recognition of 3ED3 and serospecific recognition of 4ED3 by anti-4ED3epi1sera. Similarly,anti-4ED3 sera did not recognize 3ED3epi1as well. On the other hand, anti-3ED3 and anti-4ED3epi1sera showed similar recognition of 3ED3, 4ED3epi1, in addition to recognizing 4ED3. These observations indicated that 4ED3epi1possesses serospecificity of 3ED3, in addition to maintaining serospecificity of 4ED3 (Figure 3-4) and clearly suggest that 4ED3epi1is a chimeric ED3 possessing serospecificity of both 3ED3 and 4ED3 onto a common 4ED3 scaffold. Legends are shown within the panels.

Supplemental Figure S3. Chimeric ED3 conferring serospecificity of both 3ED3 and 4ED3.(A) Serospecificity and sero-cross-recognition of wildtype 3ED3s and their epitope-grafted variants by anti-3ED3, anti-3ED3epi1, anti-4ED3 and anti-4ED3epi1sera. 4ED3epi1maintained serospecificity of both 3ED3 and 4ED3 and could be recognized by both anti-3ED3 and anti-4ED3 sera. (B) Comparative view of ELISA OD450nmof anti-ED3 sera against 3ED3, 4ED3, and 4ED3epi1. Anti-4ED3epi1had almost the same OD450nmagainst 3ED3, 4ED3, and 4ED3epi1,indicating that 4ED3epi1is a chimeric ED3 possessing serospecificity of both 3ED3 and 4ED3,both in generating cross-reactive anti-ED3 antibodies and detecting both anti-3ED3 and anti-4ED3 sera. Legends are shown within the panels. Averaged OD450nmwith standard deviations are shown.Superimposed structures of 3ED3 and 4ED3 (C) and 3ED3 and 4ED3epi1(D) (dark gray: 3ED3;light gray: 4ED3). Grafted epitope residues are indicated in ball & stick model. The overall structure of modeled 4ED3epi1is very similar to that of 3ED3.

Supplemental Figure 4. Effects of epitope-grafting on the structures of 3ED3 and 4ED3. (A)CD-spectra of 3ED3 and its epitope-grafted variants and (B) CD-spectra of 4ED3 and its epitope-grafted variants. CD-spectra were measured under immunization condition (at 0.2mg/mL in PBS) and the secondary structure contents in 3ED3 and 4ED3 variants estimated using bestsel [39]are shown in panel C, and D, respectively. Epitope-grafting on 3ED3 resulted a considerable change in the CD spectrum, but not in the case of 4ED3. Legends are shown within the panels.