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. 2020 Mar 31:11:546.
doi: 10.3389/fimmu.2020.00546. eCollection 2020.

Immunodomination of Serotype-Specific CD4+ T-Cell Epitopes Contributed to the Biased Immune Responses Induced by a Tetravalent Measles-Vectored Dengue Vaccine

Tsung-Han Lin  1 , Hsin-Wei Chen  1   2   3 , Yu-Ju Hsiao  1 , Jia-Ying Yan  1 , Chen-Yi Chiang  1 , Mei-Yu Chen  1 , Hui-Mei Hu  1 , Szu-Hsien Wu  1 , Chien-Hsiung Pan  1   2   3
Affiliations

Immunodomination of Serotype-Specific CD4+ T-Cell Epitopes Contributed to the Biased Immune Responses Induced by a Tetravalent Measles-Vectored Dengue Vaccine

Tsung-Han Lin et al. Front Immunol. .

Abstract

Dengue is an emerging mosquito-borne disease, and the use of prophylactic vaccines is still limited. We previously developed a tetravalent dengue vaccine (rMV-TDV) by a recombinant measles virus (MV) vector expressing envelope protein domain III (ED3). In this study, we used dengue-susceptible AG129 mice to evaluate the protective and/or pathogenic immune responses induced by rMV-TDV. Consistent with the previous study, rMV-TDV-immunized mice developed a significant neutralizing antibody response against all serotypes of DENV, as well as a significant IFN-γ response biased to DENV-3, compared to the vector controls. We further demonstrated that this DENV-3-specific IFN-γ response was dominated by one CD4+ T-cell epitope located in E349-363. After DENV-2 challenge, rMV-TDV-immunized mice showed a significantly lower viremia and no inflammatory cytokine increase compared to the vector controls, which had an ~100 times higher viremia and a significant increase in IFN-γ and TNF-α. As a correlate of protection, a robust memory IFN-γ response specific to DENV-2 was boosted in rMV-TDV-immunized mice after challenge. This result suggested that pre-existing DENV-3-dominated T-cell responses did not cross-react, but a DENV-2-specific IFN-γ response, which was undetectable during immunization, was recalled. Interestingly, this recalled T-cell response recognized the epitope in the same position as the E349-363 but in the DENV-2 serotype. This result suggested that immunodomination occurred in the CD4+ T-cell epitopes between dengue serotypes after rMV-TDV vaccination and resulted in a DENV-3-dominated CD4+ T-cell response. Although the significant increase in IgG against both DENV-2 and -3 suggested that cross-reactive antibody responses were boosted, the increased neutralizing antibodies and IgG avidity still remained DENV-2 specific, consistent with the serotype-specific T cell response post challenge. Our data reveal that immunodomination caused a biased T-cell response to one of the dengue serotypes after tetravalent dengue vaccination and highlight the roles of cross-reactive T cells in dengue protection.

Keywords: AG129 mice; dengue; dengue vaccine; envelope protein; immunodominance; recombinant measles virus.

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Figures

Figure 1
Figure 1
AG-hCD46 transgenic mice were more susceptible to the recombinant MV vector. AG-hCD46 transgenic and AG129 mice were intraperitoneally inoculated with 1 × 106 pfu of rMV-EGFP, and total RNA from different tissues or peripheral blood cells was isolated at day 9 (A) or day 15 (B) to determine MV RNA by quantitative RT-PCR. The MV RNA copies were normalized to GAPDH RNA (1 × 106 copies) and are presented as the mean ± SD of two mice. A 2-way ANOVA was used for statistical analysis (***p < 0.001).
Figure 2
Figure 2
The long-lasting MV- and DENV-specific antibody responses induced by rMV-TDV immunization. Groups of AG-hCD46 mice (n = 5–6) were infected with 2 × 105 pfu of recombinant tetravalent dengue vaccine (rMV-TDV) or vector control (rMV-EGFP) by ip injection and boosted 4 weeks later, after which sera were collected every 4 weeks until 20 weeks after immunization. (A–D) The specific IgG titers to DENV-1 to 4 were determined by recombinant ED3-based ELISA. (E) MV-specific IgG titers were measured by ELISA. (F) Neutralizing antibody titers to the 4 serotypes of DENV were assayed by FRNT. The results are shown as the mean ± SD, and the significance (*p < 0.05; **p < 0.01; ***p < 0.001) was analyzed by 2-way ANOVA and Student's t-test for the ELISA and NT assay, respectively.
Figure 3
Figure 3
The MV- and DENV-specific memory T-cell responses induced by the MV-vectored dengue vaccine. Groups of eight AG-hCD46 mice were infected with 2 × 105 pfu of rMV-TDV or rMV-EGFP by ip injection and boosted 4 weeks later. Fresh splenocytes from immunized mice (n = 2) were isolated at weeks 1, 5, and 20 to measure T-cell responses. MV- or DENV-1 to 4 ED3-specific IFN-γ (A) and IL-4 (B) responses after stimulation with ED3 peptide mixtures for each serotype (D1 to D4) or inactivated MV from cell lysate (MV) were assayed by ELISPOT. For the CD8-dependent T-cell response, rMV-TDV-immunized mice (n = 2) were sacrificed at week 6, and either CD8-depleted or non-depleted splenocytes were used for the IFN-γ ELISPOT assay (C). The results represent the mean ± SD of the numbers of spot-forming cells (SFC) per million splenocytes. The Mann–Whitney t-test was used for statistical analyses except for the CD8 depletion assay (2-way ANOVA), and the significance (*p < 0.05; **p < 0.01; ***p < 0.001) is indicated.
Figure 4
Figure 4
The immunization of rMV-TDV protected mice from DENV-2 challenge. Groups of AG-hCD46 transgenic mice (n = 4–6) were immunized with 2 × 105 pfu of rMV-EGFP or rMV-TDV by ip injection and boosted 4 weeks later as indicated at the top of the figure. Eight weeks after immunization, immunized AG-hCD46 mice were challenged with 1.5 × 107 ffu of DENV-2 (strain 16681) by ip injection. The viral loads (A) or cytokine (B–F) gene expression in peripheral blood cells were measured by quantitative RT-PCR with normalization to GAPDH expression. The results are shown as the mean ± SD, and the significance (*p < 0.05; ***p < 0.001) was analyzed by 2-way ANOVA, except for viremia duration, which was analyzed by 1-way ANOVA, compared to day 0.
Figure 5
Figure 5
The subdominant DENV-2-specific T-cell responses were recalled after challenge. All immunized AG-hCD46 mice were sacrificed 12 days after DENV-2 challenge for the T-cell responses determined by ELISPOT. IFN-γ (A) or IL-4 (B) responses and IFN-γ responses specific to MV- or DENV-1 to −4 ED3 mixed peptides, IFN-γ responses specific to individual peptides containing T-cell epitopes (C) or DENV specific IFN-γ responses with (CD4−) or without CD4 T-cell depletion (D) were detected, and the results are presented as the mean ± SD of SFC per million spleen cells. The significance (*p < 0.05; ***p < 0.001) is shown.
Figure 6
Figure 6
Changes in antibody responses after DENV-2 challenge. Serum samples from immunized AG-hCD46 mice 8 weeks after immunization (pre) or 12 days post challenge (post) were used for the assay. Either ED3- (A) or virion- (B) specific IgG titers were detected by ELISA. The neutralizing antibody titers (C) and ED3-specific IgG avidity (D) were measured by FRNT and avidity assays, respectively. The results are presented as the mean ± SD, and the significance (*p < 0.05; **p < 0.01; ***p < 0.001) was analyzed by 2-way ANOVA, except for IgG avidity, which was analyzed by the Mann–Whitney test.
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