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. 2015 Dec 29;10(12):e0145717.
doi: 10.1371/journal.pone.0145717. eCollection 2015.

The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

Hsin-Wei Chen  1   2 , Hui-Mei Hu  1 , Szu-Hsien Wu  1 , Chen-Yi Chiang  1 , Yu-Ju Hsiao  1 , Chia-Kai Wu  1 , Chun-Hsiang Hsieh  1 , Han-Hsuan Chung  1 , Pele Chong  1   2 , Chih-Hsiang Leng  1   2 , Chien-Hsiung Pan  1   2
Affiliations

The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

Hsin-Wei Chen et al. PLoS One. .

Abstract

Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3) is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4), we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost). A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4+ T-cell epitopes; one T-cell epitope located at E349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.

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Conflict of interest statement

Competing Interests: Dr. Hsin-Wei Chen, Dr. Pele Chong and Dr. Chih-Hsiang Leng are named on the patent entitled with “Consensus dengue virus envelope protein domain III polypeptides (cED III) and their methods of use.” (US patent 7,981,431) presented in this study. This does not alter our adherence to all PLOS ONE policies on sharing data and materials. All other authors have no conflict of interest.

Figures

Fig 1
Fig 1. Preparation of a tetravalent ED3-expressing DNA vaccine and a tetravalent recombinant ED3 protein vaccine.
(A) Schematic diagram of two ED3-expressing plasmids, pDV13-ED3 and pDV24-ED3, generated by cloning DNA fragments consisting of tandem repeats of ED3 from DENV-1 and 3, and DENV-2 and 4 into the pVax-1 vector between the Xho I and Apa I sites. The positions of the immunoglobulin light chain signal peptide (Ig) or three repeats of GGGGS linker (linker) are also indicated. (B) The presence of bivalent ED3 in the culture supernatants or cell lysates of 293Trex cells transfected with pVax-1, pDV13-ED3 or pDV24-ED3 for 24 h was detected with an ED3-specific monoclonal antibody. The expression of β-actin cellular protein in the cell lysate was used as a control. The recombinant ED3 proteins from DENV-1 to 4 were expressed in E. coli and purified with an affinity column. The final products were analyzed by SDS-PAGE (C) or Western blotting (D) with hyperimmune sera.
Fig 2
Fig 2. ED3-specific T-cell responses elicited by tetravalent dengue vaccines.
Groups of 6- to 8-week-old BALB/c female mice (n = 4) were immunized three times at 2-week intervals with pVax-1, pTDV-ED3 DNA vaccine (pTDV+pTDV), rTED3 protein vaccine (rTED3+rTED3) or prime-boost (pTDV+rTED3) as indicated in the top of figure. One week after last immunization, spleen cells were harvested for the detection of IFN-γ (A) or IL-4 (B) production specific to the ED3 of each serotype by ELISPOT assay. The results are presented with the mean and standard deviation (SD) of spot-forming cells (SFC) per million splenocytes. The significance shown in the graph was determined in comparison to the pVax-1 group, if there is no other indication. The typical results from one out of two independent experiments are presented.
Fig 3
Fig 3. ED3-specific antibody responses elicited by tetravalent dengue vaccines.
Groups of 6- to 8-week-old BALB/c female mice (n = 6) were immunized three times at 2-week intervals with pVax-1, pTDV-ED3, rTED3 or prime-boost as the same schedule in Fig 2. The results showing here come from the sera sample collected at week 6 after immunization. (A) The ED3-specific IgG titers assayed by ELISA is presented with the mean and SD. (B) The neutralizing antibody titers against four serotypes of DENV were determined by FRNT, and the endpoint titer leading to ≥50% reduction (FRNT50) is shown. Mann-Whitney t-tests were used for statistical analyses, and the significance compared to the pVax-1 group is shown, if nothing else is indicated. The data are representative of two independent experiments with similar results.
Fig 4
Fig 4. ED3-specific T-cell epitope mapping.
Mice (n = 4 per group) were immunized with either pDV13-ED3 or pDV24-ED3 and boosted with recombinant DENV-4 ED3 (rD4) with alum 2 weeks later. (A) Spleen cells were harvested 1 week after the boost, and T-cell epitopes were mapped using IFN-γ ELISPOT. The mean and SD of IFN-γ SFC per million cells responding to sixteen individual peptides for each serotype ED3 are shown. The significance shown in the graph was determined in comparison to other individual peptides. (B) Part of spleen cells were treated with anti-CD4 antibody conjugated with magnetic beads (MACs) for CD4+ cells depletion or were left untreated to analyze the CD4 dependence of specific IFN-γ response by ELISPOT. The mean and SD of IFN-γ SFC per million cells with or without CD4-depletion are indicated. (C) The amino acid sequences of ED3 peptide number 10 (E349-363) and 4 (E313-327) from DENV-1 to 4 are listed.
Fig 5
Fig 5. The time-course changes in CD4+ T-cell epitope-specific responses after tetravalent dengue vaccination.
Groups of 6- to 8-week-old BALB/c female mice (n = 4) were immunized three times at 2-week intervals with pTDV-ED3, rTED3 or prime-boost as indicated in Fig 2. Spleen cells from two mice per group were harvested one week after either the 1st or 2nd boost for the detection of IFN-γ production by ELISPOT. The mean and SD of SFC per million splenocytes responding to stimulation with the indicated individual peptides are shown.
Fig 6
Fig 6. Protection, IFN-γ responses and antibody titers in tetravalent dengue vaccine-immunized mice after DENV-4 challenge.
Groups of 6- to 8-week-old BALB/c female mice (n = 6, except n = 4 for pVax-1) were immunized three times at 2-week intervals with pVax-1, pTDV-ED3, rTED3 or prime-boost as the same schedule in Fig 2. Four weeks after the last immunization, mice were challenged with the intraperitoneal injection of 5 x 107 DENV-4 infected K562 cells. (A) Plasma viremia titers from individual mice were determined by viremia assay and are represented as the mean and SD. The significance shown in the graph was determined in comparison to the pVax-1 group, if nothing else is indicated. (B) Spleen cells were harvested for the detection of the ED3-specific IFN-γ production by ELISPOT at 1 month post-challenge. (C and D) The sera collected at 1 month post-challenge were used for the detection of neutralizing titers in an FRNT assay (C) or ED3-specific IgG titers by ELISA (D).
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Grants and funding

This study was supported by grants 00A1-VCPP09 and 01A1-IVPP29 (CHP) from the National Health Research Institutes (www.nhri.org.tw) and NSC99-2320-B-400-004-MY3 (CHP) from the Ministry of Science and Technology, Taiwan (www.most.gov.tw). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.