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Identification of conserved and HLA promiscuous DENV3 T-cell epitopes.

Nascimento EJ, Mailliard RB, Khan AM, Sidney J, Sette A, Guzman N, Paulaitis M, de Melo AB, Cordeiro MT, Gil LV, Lemonnier F, Rinaldo C, August JT, Marques ET - PLoS Negl Trop Dis (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Disease and Microbiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America ; Center for Vaccine Research, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
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.

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T-cell responses triggered by HLA-A*0201 and HLA-B*0702 epitopes in subjects with history of DENV3 infection.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.
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pntd-0002497-g003: T-cell responses triggered by HLA-A*0201 and HLA-B*0702 epitopes in subjects with history of DENV3 infection.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.

Mentions: TgM and binding affinity assays are useful tools for determination of HLA affinity of an epitope. However, experimental validation of the T-cell epitopes in humans is necessary for accurate interpretation of results. Hence, a subset of the T-cell epitopes, identified herein by use of TgM with HLA affinity confirmed by binding assays, was selected for immunogenicity study in humans by use of PBMC collected from subjects immune to DENV3 (Supplementary Tables S1 and S2). In addition, DR2 epitopes were selected based on their functional avidity, defined as the ability to activate CD4 T-cells at concentrations below 0.1 µg/mL and high affinity to HLA-DR2 molecule (IC50 below 20 nM). This additional criterion was applied in order to reduce the number of DR2 peptides to test and also to select only the peptides with increased likelihood to induce T-cell responses. PBMCs from A02 (n = 2), B07 (n = 2) and DR2 (n = 3) positive subjects were either CD4 depleted (A02 and B07) or CD8 depleted (DR2) and cultured for a week with a peptide pool containing the HLA-matched epitopes, followed by analysis of T-cell activation by use of ELISPOT to detect IFN-γ secretion. Among the A02 positive subjects tested, consistent response against peptide NS3399–407 was observed in all subjects, whereas only one of the two responded against the peptide NS5318–326 (Figure 3B). No T-cell response was detected against the peptides Env106–114 and NS5325–333 (Figure 3A & 3B). All subjects analyzed for B07 epitopes elicited T-cell response against the peptide NS5389–398 (Figure 3C & 3D). However, T-cell response against Env226–234 and NS3593–601 was observed only in one of the two subjects analyzed (Figure 3C). The peptides Env126–140 and NS185–99 reproducibly activated memory T-cells on all the subjects analyzed for DR2 epitopes (Figure 4). On the other hand, the peptides Env231–245, NS169–83 and NS3357–371 elicited T-cell response in one of the volunteers while the remaining peptides did not elicit any memory T-cell response (Figure 4). Therefore, majority of the HLA class I and II T-cell epitopes analyzed activated memory T-cell response in at least one individual that had experienced DENV3 infection in the past. This suggests that the epitopes are naturally processed and presented to T-cells during DENV3 infection.


Identification of conserved and HLA promiscuous DENV3 T-cell epitopes.

Nascimento EJ, Mailliard RB, Khan AM, Sidney J, Sette A, Guzman N, Paulaitis M, de Melo AB, Cordeiro MT, Gil LV, Lemonnier F, Rinaldo C, August JT, Marques ET - PLoS Negl Trop Dis (2013)

T-cell responses triggered by HLA-A*0201 and HLA-B*0702 epitopes in subjects with history of DENV3 infection.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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3794980&req=5

pntd-0002497-g003: T-cell responses triggered by HLA-A*0201 and HLA-B*0702 epitopes in subjects with history of DENV3 infection.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.
Mentions: TgM and binding affinity assays are useful tools for determination of HLA affinity of an epitope. However, experimental validation of the T-cell epitopes in humans is necessary for accurate interpretation of results. Hence, a subset of the T-cell epitopes, identified herein by use of TgM with HLA affinity confirmed by binding assays, was selected for immunogenicity study in humans by use of PBMC collected from subjects immune to DENV3 (Supplementary Tables S1 and S2). In addition, DR2 epitopes were selected based on their functional avidity, defined as the ability to activate CD4 T-cells at concentrations below 0.1 µg/mL and high affinity to HLA-DR2 molecule (IC50 below 20 nM). This additional criterion was applied in order to reduce the number of DR2 peptides to test and also to select only the peptides with increased likelihood to induce T-cell responses. PBMCs from A02 (n = 2), B07 (n = 2) and DR2 (n = 3) positive subjects were either CD4 depleted (A02 and B07) or CD8 depleted (DR2) and cultured for a week with a peptide pool containing the HLA-matched epitopes, followed by analysis of T-cell activation by use of ELISPOT to detect IFN-γ secretion. Among the A02 positive subjects tested, consistent response against peptide NS3399–407 was observed in all subjects, whereas only one of the two responded against the peptide NS5318–326 (Figure 3B). No T-cell response was detected against the peptides Env106–114 and NS5325–333 (Figure 3A & 3B). All subjects analyzed for B07 epitopes elicited T-cell response against the peptide NS5389–398 (Figure 3C & 3D). However, T-cell response against Env226–234 and NS3593–601 was observed only in one of the two subjects analyzed (Figure 3C). The peptides Env126–140 and NS185–99 reproducibly activated memory T-cells on all the subjects analyzed for DR2 epitopes (Figure 4). On the other hand, the peptides Env231–245, NS169–83 and NS3357–371 elicited T-cell response in one of the volunteers while the remaining peptides did not elicit any memory T-cell response (Figure 4). Therefore, majority of the HLA class I and II T-cell epitopes analyzed activated memory T-cell response in at least one individual that had experienced DENV3 infection in the past. This suggests that the epitopes are naturally processed and presented to T-cells during DENV3 infection.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Disease and Microbiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America ; Center for Vaccine Research, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus