Enhancement of the priming efficacy of DNA vaccines encoding dendritic cell-targeted antigens by synergistic toll-like receptor ligands

Abstract

Background: Targeting of protein antigens to dendritic cells (DC) via the DEC205 receptor enhances presentation of antigen-derived peptides on MHC-I and MHC-II molecules and, in the presence of costimulatory signals, antigen-specific immune responses. The immunogenicity and efficacy of DNA vaccination can also be enhanced by fusing the encoded antigen to single chain antibodies directed against DEC205. To further improve this strategy, we evaluated different toll-like receptor ligands (TLR) and CD40 ligands (CD40L) as adjuvants for DNA vaccines encoding a DEC205-single-chain antibody fused to the ovalbumin model antigen or HIV-1 Gag and assessed the priming efficacy of DNA in a DNA prime adenoviral vector boost immunization regimen.

Results: Mice were primed with the adjuvanted DEC-205 targeted DNA vaccines and boosted with adenoviral vectors encoding the same antigens. CD8+ T cell responses were determined after the adenoviral booster immunization, to determine how well the different DNA immunization regimens prime for the adenoviral boost. In the absence of adjuvants, targeting of DNA-encoded ovalbumin to DCs suppressed CD8+ T-cell responses after the adenoviral booster immunization. CD8+ T-cell responses to the DEC205 targeted DNA vaccines increased only slightly by adding either the TLR-9 ligand CpG, the TLR-3 ligand Poly I:C, or CD40 ligand expression plasmids. However, the combination of both TLR-ligands led to a strong enhancement of CD8+ T-cell responses compared to a non-targeted DNA vaccine. This finding was confirmed using HIV Gag as antigen.

Conclusion: Although DNA prime adenoviral vector boost immunizations belong to the strongest inducers of cytotoxic T cell responses in different animal models and humans, the CD8+ T cell responses can be further improved by targeting the DNA encoded antigen to DEC205 in the presence of synergistic TLR ligands CpG and Poly I:C.

Figure 1

CD8⁺ T cell responses after priming with a DNA vaccine encoding DEC-205 targeted ovalbumin and boosting with an adenoviral vector. Mice were immunized with the indicated plasmids prior to boosting with adenoviral vectors encoding ovalbumin (Ad-Ova) or GFP (Ad-GFP). Unless stated otherwise, a DNA dose of 50 μg was used. One week after the boost, the percentage of SIINFEKL/H-2K^b tetramer positive cells (A), IFN-γ and CD107a (B, C) or IFN-γ and IL-2 (D) double-positive cells after stimulation with the SIINFEKL peptide were determined. A, B) Mean percentages with SEM of three independent experiments with a total of eight mice/group are shown. Significant differences (Bonferroni Multiple Comparison test) between groups primed with different DNA vaccines and boosted with Ad-OVA are marked (* p < 0.05; ** p < 0.01; *** p < 0.001). C, D) Dose response curve for DEC-Ova priming. The percentage of IFN-γ and CD107a positive (C) and IFN-γ and IL-2 positive (D) cells of CD8⁺ lymphocytes is shown for individual mice.

Figure 2

CD8⁺ T-cell responses after priming with DEC-205 targeted DNA vaccines in the presence of costimulatory adjuvants and boosting with an adenoviral vector. Mice were primed with DEC-Ova or Con-Ova in the presence or absence of the indicated adjuvants (Adj.) prior to boosting with adenoviral vectors encoding ovalbumin (Ad-Ova) or GFP (Ad-GFP). The percentage of SIINFEKL/H-2K^b tetramer positive cells (A, C, E, F) and IFN-γ and CD107a double-positive cells after stimulation with the SIINFEKL peptide (B, D) were determined after the boost. Unless stated otherwise, DEC-Ova and Con-Ova were used at a dose of 50 μg. A, B, F) Fifty μg of plasmids encoding a soluble trimer (CD40L¹), a dimeric form (CD40L²) or a tetrameric form of that trimer (CD40L⁴) were co-administered with the DNA vaccine. C, D, F) CpG, Poly I:C or the combination of CpG and Poly I:C (CpI:C) were coinjected with the plasmids as indicated. E) CpI:C was added during DNA priming. Mean percentages with SEM of two independent experiments with a total of eight mice/group are shown (A-D). Mean percentages and SEM of one experiment with four (E) and six (F) mice per group are shown. For reason of clarity, the level of significance in the Bonferroni Multiple Comparison test is only indicated for selected group to group comparisons (* p < 0.05; ** p < 0.01; ***p < 0.001).

Figure 3

In vivo CTL activity after priming with adjuvanted DNA vaccine encoding ovalbumin and adenoviral vector booster immunization. Mice (n = 3) were primed either with DEC-Ova or Con-Ova in the presence of CpI:C and boosted with the Ad-Ova vector. One week after the boost, mice received a 1 to 1 mixture of SIINFEKL peptide-loaded and non-loaded syngenic donor splenocytes also differing in the intensity of the CFSE-labelling. One day after immunization, the percentage of SIINFEKL loaded donor cells (A) and tetramer ⁺ CD8⁺ cells (B) was determined. The levels of significance in the Bonferroni Multiple Comparison test are indicated (* p < 0.05; ** p < 0.01; ***p < 0.001).

Figure 4

HIV Gag-specific CD8⁺ T cell responses. Mice were primed with a DNA vaccine encoding DEC205-targeted HIV p41 Gag (DEC-p41) or control plasmids (Con-p41, mock) with or without CpI:C prior to boosting with an adenoviral vectors encoding HIV GagPol (Ad-Hgp^syn) or GFP (Ad-GFP). One week after the boost, the percentage of CD8⁺ cells also positive for IFN-γ and CD107a (A) or IFN-γ and IL2 (B) after stimulation with an immunodominant HIV Gag peptide were determined. Mean percentages with SEM of two independent experiments with a total of eight mice/group are shown. For reason of clarity, the level of significance in the Bonferroni Multiple Comparison test is only indicated for selected group to group comparisons (* p < 0.05; ** p < 0.01; ***p < 0.001).

Figure 5

HIV Gag-specific in vivo CTL activity. Mice (n = 4) were immunized as described in Figure 4. One week after the boost, mice received a 1 to 1 mixture of HIV Gag peptide-loaded and non-loaded syngenic donor splenocytes also differing in the intensity of the CFSE-labelling. One day after immunization, the percentage of HIV Gag peptide-loaded donor cells was determined. For reason of clarity, the level of significance in the Bonferroni Multiple Comparison test is only indicated for selected group to group comparisons (** p < 0.01).