A Novel Orf Virus D1701-VrV-Based Dengue Virus (DENV) Vaccine Candidate Expressing HLA-Specific T Cell Epitopes: A Proof-of-Concept Study

Abstract

Although dengue virus (DENV) affects almost half of the world's population there are neither preventive treatments nor any long-lasting and protective vaccines available at this time. The complexity of the protective immune response to DENV is still not fully understood. The most advanced vaccine candidates focus specifically on humoral immune responses and the production of virus-neutralizing antibodies. However, results from several recent studies have revealed the protective role of T cells in the immune response to DENV. Hence, in this study, we generated a novel and potent DENV vaccine candidate based on an Orf virus (ORFV, genus Parapoxvirus) vector platform engineered to encode five highly conserved or cross-reactive DENV human leukocyte antigen (HLA)-A*02- or HLA-B*07-restricted epitopes as minigenes (ORFV-DENV). We showed that ORFV-DENV facilitates the in vitro priming of CD8⁺ T cells from healthy blood donors based on responses to each of the encoded immunogenic peptides. Moreover, we demonstrated that peripheral blood mononuclear cells isolated from clinically confirmed DENV-positive donors stimulated with ORFV-DENV generate cytotoxic T cell responses to at least three of the expressed DENV peptides. Finally, we showed that ORFV-DENV could activate CD8⁺ T cells isolated from donors who had recovered from Zika virus (ZIKV) infection. ZIKV belongs to the same virus family (Flaviviridae) and has epitope sequences that are homologous to those of DENV. We found that highly conserved HLA-B*07-restricted ZIKV and DENV epitopes induced functional CD8⁺ T cell responses in PBMCs isolated from confirmed ZIKV-positive donors. In summary, this proof-of-concept study characterizes a promising new ORFV D1701-VrV-based DENV vaccine candidate that induces broad and functional epitope-specific CD8⁺ T cell responses.

Keywords: CD8+ T cells; HLA class I; ORFV; T cell epitope; dengue virus; immune response; parapoxvirus; vaccine; viral vector.

Figure 1

Selection of DENV-derived epitopes based on a query of the Immune Epitope Database (IEDB) of the NIAID. Five DENV-derived HLA-A*02- or HL-B*07-restricted epitopes were selected according to the magnitude of cross-reactive functional CD8⁺ T cell responses reported and/or conservation among the various DENV serotypes.

Figure 2

(A) Genomic map of ORFV-DENV depicting the arrangement of the minigenes encoding HLA-A*02- (red boxes) and HLA-B*07- (blue boxes) restricted DENV-derived epitopes controlled by the authentic early promoter Pvegf. The GFP gene used as a selection marker for virus purification is expressed under the control of the synthetic eP2 promoter. The 5′ and 3′ sequences homologous to the vegf-e locus of ORFV flanked the minigenes and the GFP sequence, enabling direct homologous recombination with the virus backbone. * DNA fragments affected by the genomic deletions (B) The genetic homogeneity of novel recombinants was verified by PCR targeting the vegf-e locus and resulted in the predicted amplicon size for ORFV-DENV (1640 bp). (C) DENV-specific PCR revealed the predicted amplicon sizes for ORFV-DENV (277 bp). Transfer plasmid pV-DENV-2-GFP was used as the positive control. DNA isolated from the parental ORFV V12-Cherry and non-infected cells served as the negative control.

Figure 3

ORFV-DENV induces CD8⁺ T cells against several DENV-derived epitopes within the same individual in vitro. The responses of each donor are identified with different symbols and colors, as shown. Each symbol corresponds to a biological replicate exhibiting a positive response out of thirty (Donors 78, 27, and 25) or twenty (Donor 31) replicates. Effective priming was verified by (A) HLA tetramer staining and/or (B) ICS. Data for only four donors are shown. Cells from a fifth donor (data included in Table 2 only) showed a cytokine response to a pool of HLA-A*02-restricted DENV and control peptides; the specificity of the observed responses against individual peptides was not determined due to rapid cell death. CD8⁺ T cells from a sixth donor were not primed against any of DENV-derived or control epitopes (data not included). N/D, not determined (negative).

Figure 4

Restimulation of CD8⁺ T cells from six confirmed DENV-positive donors with ORFV-DENV. The responses of each donor are identified with a different symbol and color, as shown. Each symbol corresponds to a biological replicate exhibiting a positive response. Specific CD8⁺ T cell activation was verified by (A) HLA tetramer staining and/or (B) ICS. PBMCs from donor DENV-015 showed no specific CD8⁺ T cell responses against any of DENV-derived or control epitopes after restimulation (data not shown).

Figure 5

Cross-reactive CD8⁺ T cell responses in PBMCs from confirmed ZIKV-positive donor DENV-021 after in vitro restimulation with ORFV-DENV expressing DENV-derived peptide epitopes. Frequencies and functionality of epitope-specific CD8⁺ T cells were determined by (A) HLA tetramer staining, (B) ICS, and (C) specific lysis after 20 h of co-incubation with peptide-pulsed target cells at an effector-to-target ratio of 0.1:1. The percentage of target cell lysis corrected for spontaneous background lysis is shown. A synthetic peptide was used as a control.