Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design. © 2013 Nascimento et al

ELISPOT data showing T-cell activation of HLA-A02 positive in dengue IgG negative individuals using dendritic cells pulsed with NS3_399–407 epitope on homologous region of DENV2, DENV3 and DENV4 viruses.

<p>ELISPOT data showing T-cell activation of HLA-A02 positive in dengue IgG negative individuals using dendritic cells pulsed with NS3_399–407 epitope on homologous region of DENV2, DENV3 and DENV4 viruses.</p

Inter-serotype variants and corresponding incidence among reported sequences in NCBI Entrez database, with binding affinity data to HLA-A*0201 molecule.

<p>The underlined sequences show the amino acid changes in relation to DENV3 sequence. ND: non-determined.</p

T-cell responses triggered by HLA-A*0201 and HLA-B*0702 epitopes in subjects with history of DENV3 infection.

<p>CD4 depleted PBMCs were cultured for a week in presence of peptide pool containing either A*0201 or B*0702 epitopes. The cells were then harvested, washed and ELISPOT assay for IFN-γ detection was performed. (A) and (B) represent the T-cell responses of two different HLA-A*02 positive subjects, whereas (C) and (D) represent T-cell responses of two different HLA-B*07 positive individuals.</p

DENV3 polyprotein schema showing the localization of potential T-cell epitopes identified in this study as well as the pan-dengue conserved regions (CR) and the list of DENV3 T-cell epitopes reported in the literature.

<p>DENV3 polyprotein schema showing the localization of potential T-cell epitopes identified in this study as well as the pan-dengue conserved regions (CR) and the list of DENV3 T-cell epitopes reported in the literature.</p

List of DENV3 epitopes along with HLA affinity (if known) described in the literature and comparison with the immunogenicity and binding affinity of the peptides tested in this study.

<p>The sequences in bold represent the epitopes previously identified within the peptides used in this study. ND: non-determined.</p

Identification of DENV3 HLA class I (A02, B07 and A24) T-cell epitopes using HLA transgenic mice, epitope prediction and binding assays.

<p>Identification of DENV3 HLA class I (A02, B07 and A24) T-cell epitopes using HLA transgenic mice, epitope prediction and binding assays.</p

Immunogenicity and binding affinity capability of peptides containing non-conserved and pan-dengue conserved sequences.

<p>The percentage of peptides containing non-conserved and pan-dengue conserved sequences was calculated based on their ability to (A) induce T-cell responses in HLA class II transgenic mice (DR2, DR3 and DR4); or (B) to bind to 14 different HLA molecules.</p

ELISPOT data showing T-cell activation of HLA-B07 positive in dengue IgG negative individuals using dendritic cells pulsed with DENV3 NS5_389–398 epitope.

<p>ELISPOT data showing T-cell activation of HLA-B07 positive in dengue IgG negative individuals using dendritic cells pulsed with DENV3 NS5_389–398 epitope.</p

List of the most conserved DENV3 peptides for which immunogenicity in HLA-DR02, -DR03 and -DR04 transgenic mice was supported by binding affinity analysis to the respective HLA.

<p>A representative ELISPOT data is shown along with the minimum peptide concentration that activated memory T-cells (functional avidity) is included. In addition, IC50 (nM) for HLA-DR02, -DR03 and -DR04 as well as the number of additional potential HLA binders (as many as 11) are also shown. Binding affinity is characterized by IC50 below 1000 nM (in bold).</p><p>Pan-dengue conserved sequences are shown underlined.</p