Identification of Continuous Human B-Cell Epitopes in the Envelope Glycoprotein of Dengue Virus Type 3 (DENV-3)

BACKGROUND:Dengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes. METHODOLOGY/PRINCIPAL FINDINGS:Synthetic peptides were used to identify B-cell epitopes in the envelope (E) glycoprotein of dengue virus type 3 (DENV-3). Eleven linear, immunodominant epitopes distributed in five regions at amino acid (aa) positions: 51-65, 71-90, 131-170, 196-210 and 246-260 were identified by employing an enzyme- linked immunosorbent assay (ELISA), using a pool of human sera from dengue type 3 infected individuals. Peptides 11 (aa51-65), 27 and 28 (aa131-150) also reacted with dengue 1 (DENV-1) and dengue 2 (DENV-2) patient sera as analyzed through the ROC curves generated for each peptide by ELISA and might have serotype specific diagnostic potential. Mice immunized against each one of the five immunogenic regions showed epitopes 51-65, 131-170, 196-210 and 246-260 elicited the highest antibody response and epitopes131-170, 196-210 and 246-260, elicited IFN-gamma production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively. CONCLUSIONS/SIGNIFICANCE:Our study identified several useful immunodominant IgG-specific epitopes on the envelope of DENV-3. They are important tools for understanding the mechanisms involved in antibody dependent enhancement and immunity. If proven protective and safe, in conjunction with others well-documented epitopes, they might be included into a candidate epitope-based vaccine

T-cell response through ELISPOT assay for IFN-γ elicited in Balb/c mice immunized with the envelope epitopes identified in human.

<p>The T cell response was assessed using total splenocytes harvested from immunized mice as well as from CD8+ T cell enriched splenocytes (CD4+T cell-depleted) or CD4+ T enriched splenocytes (CD8+ T cell -depleted). In asterisk are the groups considered positive in all mice analyzed based upon the following criteria: spot forming cells (SFC) less 2 standard deviation (SD) was greater than the average of the background plus 2 SD; and the average of the experimental peptides was greater than 10 SFCs per 10⁶ cells. The result shown is the average and standard deviation from two different mice.</p

Performance of E protein peptides to discriminate dengue infections: DENV-3 and dengue for other serotypes infections.

<p>Sensibility (Sens.), specificity (Spec.), cut-off (CO) and area under curve (AUC) generated for each one of the eleven peptides in the ROC curve. Confidence interval (CI) and <i>p value</i> are shown.</p

Total IgG production in Balb/c mice elicited by envelope epitopes.

<p>(A) 51–65, (B) 71–90, (C) 131–170, (D) 196–210 and (E) 246–260 identified in naturally dengue-infected humans. Balb/c mice were immunized twice with different epitopes and the humoral immune response (pooled sera, n = 2 per group) was assessed through ELISA after priming and boosting in different serum dilutions. NC, negative control.</p

ROC curves generated by Medcalc Software, 8.2 version.

<p>(A) Peptides 15, 16, 28, 29, 30, 31, 32, 40, 50 and its power to discriminate between DENV-3 infections and non dengue. (B) Peptides 11, 27, 28 and its power to differentiate DENV-3 infection from dengue infection of other serotypes.</p

Immunoreactive DENV- 3 E-glycoprotein peptides.

<p><b>ELISA</b>; enzyme linked immunosorbent assay.</p><p><b>OD</b>; optical densities.</p

Performance of E protein peptides to discriminate dengue infections: DENV-3 infections and non-dengue.

<p>Sensibility (Sens.), specificity (Spec.), cut-off (CO) and area under curve (AUC) generated for each one of the eleven peptides in the ROC curve. Confidence interval (CI) and <i>p value</i> are shown.</p

Top view of the homology model of the DENV-3seq E-glycoprotein dimer and location of the eleven mapped peptides.

<p>Peptides are represented in colorful space-filling: peptide 11 in green, peptides 15 and 16 in orange, peptides 27, 28, 29, 30, 31 e 32 in blue, 40 in red and 50 in violet. For illustrative reasons one monomer is represented as space-filling and the other as ribbons.</p