Early T-cell responses to dengue virus epitopes in Vietnamese adults with secondary dengue virus infections

Abstract

T-cell responses to dengue viruses may be important in both protective immunity and pathogenesis. This study of 48 Vietnamese adults with secondary dengue virus infections defined the breadth and magnitude of peripheral T-cell responses to 260 overlapping peptide antigens derived from a dengue virus serotype 2 (DV2) isolate. Forty-seven different peptides evoked significant gamma interferon enzyme-linked immunospot (ELISPOT) assay responses in 39 patients; of these, 34 peptides contained potentially novel T-cell epitopes. NS3 and particularly NS3200-324 were important T-cell targets. The breadth and magnitude of ELISPOT responses to DV2 peptides were independent of the infecting dengue virus serotype, suggesting that cross-reactive T cells dominate the acute response during secondary infection. Acute ELISPOT responses were weakly correlated with the extent of hemoconcentration in individual patients but not with the nadir of thrombocytopenia or overall clinical disease grade. NS3556-564 and Env414-422 were identified as novel HLA-A*24 and B*07-restricted CD8+ T-cell epitopes, respectively. Acute T-cell responses to natural variants of Env414-422 and NS3556-564 were largely cross-reactive and peaked during disease convalescence. The results highlight the importance of NS3 and cross-reactive T cells during acute secondary infection but suggest that the overall breadth and magnitude of the T-cell response is not significantly related to clinical disease grade.

FIG. 1.

T-cell responses by viral antigen. Shown is the frequency with which individual antigenic peptides in the (A) capsid, (B) preM/M, (C) Env, and (D) NS3 viral antigens were recognized by PBMC collected on study days 5 to 14 from 48 patients with secondary dengue. Shown within each viral protein are recognized structural features and (in parentheses) their amino acid locations with respect to the start of the protein. The relative position of the overlapping peptide (OVL) in each antigen that evoked a response is indicated on the x axis. (A) CV, capsid virion; CI, intracellularly anchored capsid.

FIG. 2.

Breadth and magnitude of the ELISPOT response to DV2 peptides in patients infected with a DV2 virus at different times postpresentation. The number of individual peptide antigens that evoked significant responses (>50 SFU/million PBMC) in PBMC samples collected from four prospectively recruited patients infected with DV2 was determined at different times postinfection. Shown are the number of peptides recognized in each patient at different times postdiagnosis.

FIG. 3.

Recognition by CD8⁺ T cells of truncated peptide variants of RMAILGDTAWDFGSL (Env_411-425). Shown are the frequencies of IFN-γ SFU elicited by truncated Env_411-425 peptides in IFN-γ ELISPOT assays against PBMC from BC503 after enrichment for CD8⁺ T cells.

FIG. 4.

Kinetics and cross-reactivity of responses to Env_414-422. The data depict the number of IFN-γ SFU detected in PBMC enriched for CD8⁺ T cells from six HLA-B*07-positive patients with secondary dengue virus infections. PBMC were incubated with peptides corresponding to DV1-3Env_414-422 (A) and DV4Env_414-422 (B). Patients BC385, BC389, BC400, BC395, and BC504 were DHF grade II. Patient BC414 was DHF grade III. Patients BC389, BC395, and BC385 were infected with DV2. Patient BC504 was infected with DV4. Virus was not detected in patient BC400 or BC414. Peptide titration experiments suggested that T cells from patients BC414 (C) and BC504(D) preferentially recognized the DV1 to DV3 Env_414-422 variant at high peptide concentrations but were cross-reactive for the variant peptides DV1 to DV3 Env_414-422 and DV4 Env_414-422 at peptide concentrations of <1 μM.

FIG. 5.

NS3_555-564 is a CD8⁺ T-cell epitope. (A) In IFN-γ ELISPOT assays, PBMC (study day 3) from subject BC313 responded to the DV2 NS3 peptide VAAEGINYADRRWCF (NS3_550-564) in a CD8-dependent fashion. (B) The truncated peptide INYADRRWCF (NS3_550-564) was the minimum peptide that evoked the strongest ELISPOT response at the lowest peptide concentration.

FIG. 6.

NS3_555-564 is recognized only in the context of HLA-A*24. PBMC (study day 5) from subject BC313 (A*24, A*26, B38, B15, Cw7, and Cw3) were cocultured with peptide-pulsed BCLs that were HLA matched (A*24 only) or mismatched to BC313 as described in Materials and Methods. After coculture, the frequency of CD3⁺ CD8⁺ T cells that expressed the early activation marker CD69⁺ and IFN-γ (upper right) was determined in combinations of PBMC and peptide-pulsed BCL targets. (A) Presentation of DV2 NS3_555-564 by targets matched to effectors at A*26 and B38, B15, or Cw7 (data not shown) did not elicit functional responses. (B) Activated (CD69⁺), IFN-γ-producing CD8⁺ T cells were only observed when DV2NS3_555-564 was presented to PBMC in an HLA-A*24-matched BCL. Functional CD8 T-cell responses were not observed when the truncated peptide DV2NS3_557-564 (YADRRWCF) (C and D) or no peptide (E and F) was presented by HLA-A*24-matched or mismatched BCLs.

FIG. 7.

Kinetics of T-cell responses to NS3_555-564. Shown are the kinetics and magnitude of IFN-γ ELISPOT responses in samples from four patients with secondary dengue virus infection to each of the recognized peptide variants of NS3_555-564. PBMC from each patient were stimulated with DV1NS3_555-564 (FQYSDRRWCF) (A), DV2NS3_555-564 (INYADRRWCF) (B), DV3NS3_555-564 (IKYTDRKWCF) (C), and DV4NS3_555-564 (ISYKDREWCF) (D). Subject BC311 was infected with DV4, and subject BC398 was infected with DV2.

FIG. 8.

T-cell responses to NS3_555-564 variants during acute secondary dengue virus infection. (A) Peptide titration curves in IFN-γ ELISPOT assays for PBMC collected in the acute phase of infection (study day 5) from patient BC311 (infected with DV4) and stimulated with DV4NS3_555-564 (ISYKDREWCF) or DV2NS3_555-564 (INYADRRWCF). (B) Peptide titration curves in IFN-γ ELISPOT assays for PBMC collected in the acute phase of infection (study day 5) from patient BC398 (infected with DV2) and stimulated with DV2NS3_555-564 (INYADRRWCF) and DV1NS3_555-564 (FQYSDRRWCF).