Unveiling a Shield of Hope: A Novel Multiepitope-Based Immunogen for Cross-Serotype Cellular Defense against Dengue Virus

Abstract

Dengue virus (DENV) infection continues to be a public health challenge, lacking a specific cure. Vaccination remains the primary strategy against dengue; however, existing live-attenuated vaccines display variable efficacy across four serotypes, influenced by host serostatus and age, and predominantly inducing humoral responses. To address this limitation, this study investigates a multiepitope-based immunogen designed to induce robust cellular immunity across all DENV serotypes. The chimeric immunogen integrates H-2^d specific MHC-I binding T-cell epitopes derived from conserved domains within the DENV envelope protein. Immuno-informatics analyses supported its stability, non-allergenic nature, and strong MHC-I binding affinity as an antigen. To assess the immunogenicity of the multiepitope, it was expressed in murine bone-marrow-derived dendritic cells (BMDCs) that were used to prime mice. In this experimental model, simultaneous exposure to T-cell epitopes from all four DENV serotypes initiated distinct IFNγ-CD8 T-cell responses for different serotypes. These results supported the potential of the multiepitope construct as a vaccine candidate. While the optimization of the immunogen design remains a continuous pursuit, this proof-of-concept study provides a starting point for evaluating its protective efficacy against dengue infection in vivo. Moreover, our results support the development of a multiepitope vaccine that could trigger a pan-serotype anti-dengue CD8 response.

Keywords: MHC I-binding epitopes; cellular immunity; cross-serotype protection; dengue virus; immunization; memory T cells; multiepitope vaccine; vaccine candidate.

Figure 2

(A) The produced H2DVE sequence that comprises the Ig-κ signal peptide (purple), the XCL1 sequence (blue), the polyepitope sequence (cyan) with AAY spacers (green), a c-myc epitope (orange), and a polyhistidine tag (red); it is adorned with secondary structure elements extracted from the AlphaFold model and colored according to the pLDDT confidence score (blue for very high, cyan for high, yellow for low, orange for very low). Figure prepared with ESPript [42]. (B) The Cα trace of the Alphafold model of H2DVE with the three-stranded β-sheet/α-helix chemokine domain of XCL1 and the β-solenoid polyepitope domain. (C) The high confidence AlphaFold model of the polyepitope domain with IIF spacers displayed in atom mode. (D) The very low confidence model of the polyepitope domain with PPF spacers. All structures are rendered with ChimeraX [43]. The blue, cyan, yellow and orange color information in (B–D) represents pLDDT confidence score as depicted within the figure.

Figure 2

(A) The produced H2DVE sequence that comprises the Ig-κ signal peptide (purple), the XCL1 sequence (blue), the polyepitope sequence (cyan) with AAY spacers (green), a c-myc epitope (orange), and a polyhistidine tag (red); it is adorned with secondary structure elements extracted from the AlphaFold model and colored according to the pLDDT confidence score (blue for very high, cyan for high, yellow for low, orange for very low). Figure prepared with ESPript [42]. (B) The Cα trace of the Alphafold model of H2DVE with the three-stranded β-sheet/α-helix chemokine domain of XCL1 and the β-solenoid polyepitope domain. (C) The high confidence AlphaFold model of the polyepitope domain with IIF spacers displayed in atom mode. (D) The very low confidence model of the polyepitope domain with PPF spacers. All structures are rendered with ChimeraX [43]. The blue, cyan, yellow and orange color information in (B–D) represents pLDDT confidence score as depicted within the figure.

Figure 1

Schematic representation of the immunogen design and expression cassette subcloned in the pSecTag2B plasmid (referred to as ‘pH2DVE’ in the text). (A) The recombinant immunogen comprises of a dendritic cell-specific XCL1 ligand (yellow) at the N-terminus, merged with flexible and rigid linkers to sub-cassettes of MHC Class I binding epitopes from the DENV E protein (the primary antigen of the construct). The selected epitopes specific to the four dengue serotypes are represented by colored boxes (DENV1 in purple, DENV2 in green, DENV3 in light yellow, and DENV4 in grey), each connected by flanking AAY linker/spacer peptides. The pH2DVE cassette was designed using SnapGene Viewer software (v7.0). (B) The horizontal vector map illustrates the positioning of the primary antigen at the HindIII and XhoI restriction sites within the pH2DVE expression plasmid. (Note: H2DVE includes Ig-kappa light chain, XCL1, polyepitope antigen and 6XHis tag) (C) Description of the antigen source, MHC subclass and haplotype, length of each epitope, and the entire construct (aa: amino acids) is presented. Notably, the construct length mentioned here includes the entire open reading frame (ORF) from pH2DVE.

Figure 3

Western blots depicting the expression of H2DVE recombinant protein in a mammalian system (HEK293 cell line). (A) Time course expression profile using unpurified whole lysate (10⁶ cells) of pH2DVE transfected cells (odd numbered lanes) and mock-transfected (pSecTag2B, empty vector control) cells (even numbered lanes). The blot images were aligned with the 56 kDa bands for clarity and consistency. (B) Purified proteins from the whole lysate of mock-transfected control (lane 1) and pH2DVE transfected cells (lane 2). The recombinant proteins were tagged with poly-histidine tag at the C-terminal and were purified using Ni-NTA IMAC spin columns. All the blots were imaged using ChemiDoc system (Bio-Rad) and ImageLab software (v5) with automatic image exposure.

Figure 4

Jess SimpleWestern Immunoblot showing the H2DVE expression profile in pH2DVE transfected BMDCs (lane 1 to 3) and non-transfected control (lane 4 and 5). The immunoblot images were analyzed using ‘Compass for SimpleWestern’ software (v6.3).

Figure 5

CB6F1 mice were immunized twice with pH2DVE-transfected BMDCs at a two-weeks interval and H2DVE-specific CD8 T cell response was evaluated one week after second immunization and compared to naive mice (A). The percentage of total CD8⁺ (B) and CD44⁺ cells within CD8 T cells (C) were measured by flow cytometry. The number of CD44⁺ CD8⁺ T cells were determined (D). Total splenocytes were stimulated with DENV specific peptide pools Pool20, DENV1, DENV2, DENV3 and DENV4 (100 nM) for 6 hours and the expression of IFNγ was measured by flow cytometry (E). Total splenocytes were expanded in the presence of Pool20 peptides (100 nM) and IL-2 (11 ng/mL) or IL-2 alone for 8 days. The production of IFNγ was followed over time in the supernatants by ELISA (F). Following expansion, cells were restimulated as described in (E) and IFNγ production was measured by flow cytometry (G). One represented experiment out of two (N = 3–5/group). The statistical significance of differences was determined by Mann-Whitney test (* p < 0.05).

Figure 5

CB6F1 mice were immunized twice with pH2DVE-transfected BMDCs at a two-weeks interval and H2DVE-specific CD8 T cell response was evaluated one week after second immunization and compared to naive mice (A). The percentage of total CD8⁺ (B) and CD44⁺ cells within CD8 T cells (C) were measured by flow cytometry. The number of CD44⁺ CD8⁺ T cells were determined (D). Total splenocytes were stimulated with DENV specific peptide pools Pool20, DENV1, DENV2, DENV3 and DENV4 (100 nM) for 6 hours and the expression of IFNγ was measured by flow cytometry (E). Total splenocytes were expanded in the presence of Pool20 peptides (100 nM) and IL-2 (11 ng/mL) or IL-2 alone for 8 days. The production of IFNγ was followed over time in the supernatants by ELISA (F). Following expansion, cells were restimulated as described in (E) and IFNγ production was measured by flow cytometry (G). One represented experiment out of two (N = 3–5/group). The statistical significance of differences was determined by Mann-Whitney test (* p < 0.05).