Schematic representation of tandem EDIII constructs.

<p>The amino acid sequence covering EDIII (298aa∼400aa) from each serotype was arranged sequentially, and (Gly₄Ser)₃ sequence was used to connect EDIII regions. Thioredoxin (Trx) was fused at the N-terminal to increase solubility and the C-terminal His-Patch was included for purificaiton through Ni-NTA agarose.</p

Neutralizing antibody responses in mice immunized with MixBiEDIII.

<p>Neutralization titers of MixBiEDIII immune sera at two weeks after the last immunization were measured by PRNT₅₀. The neutralizing antibodies against four DENV serotypes were significantly induced by MixBiEDIII compared with control group. Dotted line represents limits of detection.</p

Characterization of the tandem bivalent recombinant EDIII antigens.

<p>ELISA of purified EDIII proteins was performed using corresponding monoclonal antibody against different serotype of DENV. Trx protein was control. The cut-off for the ELISA is shown by a dotted line.</p

Protection of anti-MixBiEDIII sera against all four serotypes DENV.

<p>The suckling mice were injected intracranially (IC) with the sera-virus mixture, and mortality was monitored for 21 days. Data represent the mean±SD.</p>a<p>P<0.05 for MixBiEDIII sera group vs. control sera group, by Log-Rank analysis.</p

IgG antibody responses in mice immunized with MixBiEDIII.

<p>The total lgG titers of immune sera from mice immunized with MixBiEDIII at two weeks after the last immunization were measured by ELISA. The lgG titers of immune sera against four DENV serotypes from mice immunized with MixBiEDIII were significant higher than control group. Dotted line represents limits of detection.</p

Protection of anti-MixBiEDIII sera against all four DENV serotypes in suckling mice.

<p>Sera from mice immunized with MixBiEDIII were pre-incubated with different serotype DENV and inoculated into suckling mouse (1-day-old) brain intracranially (IC), and mortality was recorder. (*) Survival rate of mice inoculated with virus plus sera from MixBiEDIII group was statistically significant compared with control group using Fisher’s exact test.</p