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. 2019 Jun 21:10:1424.
doi: 10.3389/fimmu.2019.01424. eCollection 2019.

A Modified mRNA Vaccine Targeting Immunodominant NS Epitopes Protects Against Dengue Virus Infection in HLA Class I Transgenic Mice

Claude Roth  1   2 , Tineke Cantaert  3 , Chloé Colas  1   2 , Matthieu Prot  1   2 , Isabelle Casadémont  1   2 , Laurine Levillayer  1   2 , Jessie Thalmensi  4 , Pierre Langlade-Demoyen  4 , Christiane Gerke  5 , Kapil Bahl  6 , Giuseppe Ciaramella  7 , Etienne Simon-Loriere  1   2 , Anavaj Sakuntabhai  1   2
Affiliations

A Modified mRNA Vaccine Targeting Immunodominant NS Epitopes Protects Against Dengue Virus Infection in HLA Class I Transgenic Mice

Claude Roth et al. Front Immunol. .

Abstract

Dengue virus (DENV) induces strong T and B cell responses upon infection. Hence, it is difficult to determine the contribution of cell-mediated immunity alone in the long lasting protection against DENV infection and disease. Numerous CD4+ and CD8+ T cell epitopes have been identified, mainly in the non-structural proteins of DENV. Taking into account the immunogenicity and peptide sequence conservation among the different DENV serotypes, a minimal DENV antigen, called DENV1-NS, has been designed. This antigen is enriched in conserved and highly antigenic epitopes located in the NS3, NS4B, and NS5 regions of DENV1. To evaluate the ability of the DENV1-NS poly-epitope to express the antigenic peptides in the context of different HLA class I molecules, we established its in vivo immunogenicity by measuring, after DNA immunization and electroporation, the activation of DENV-specific CD8 T cells in transgenic mice expressing the human HLA-A*0201, -A*2402, -B*0702, and -B*3502 class I alleles. We then engineered a lipid nanoparticle (LNP) encapsulated modified mRNA vaccine encoding DENV1-NS and tested immunogenicity and protection in these human HLA class I transgenic mice, after transient blockade of the interferon (IFN) type I receptor. Significant protection was observed, after two injections of the mRNA vaccine. Collectively, these data strongly support the development of T cell-based vaccines targeting immunodominant T cell epitopes that generate potent virus-specific T cell responses conferring immunity against DENV infection.

Keywords: DNA vaccine; NS epitopes; T cells; chimeric vaccine; dengue virus (DENV); human HLA transgenic mice; vaccine.

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Figures

Figure 1
Figure 1
Analysis of target regions for T cells and sequence conservation of non-structural proteins of DENV. Analyses of the magnitude of T cell responses, in SFC/106 cells (A), from Weiskopf et al. (24), and in SFC/106 cells (B), from Rivino et al. (28). Intra-serotypes and inter-serotypes sequence conservation are represented for DENV1 (yellow line), DENV2 (blue line), DENV3 (purple line), DENV4 (green line), and for all DENV serotypes (black lines), respectively.
Figure 2
Figure 2
Quantification of the T cell responses in HLA-A*0201, -A*2402, -B*0702, and -B*3501 transgenic mice by ELISpot assay. (A) Three independent experiments were performed, in which a total of 10 and 4 HLA-A*0201 transgenic mice received the DENV1-NS construct and the control plasmid (CT), respectively, 9 and 4 HLA-A*2402 transgenic mice received the DENV1-NS construct and the control plasmid, respectively, 10 and 5 HLA-B*0702 transgenic mice received the DENV1-NS construct and the control plasmid, respectively, and 8 and 3 HLA-B*3501 transgenic mice received the DENV1-NS construct and the control plasmid, respectively. All the animals were immunized by intradermic injection (100 μg DENV1-NS or control plasmid) followed by in vivo electroporation. Two immunizations were performed at 3-week interval, and spleen cells were tested for IFN-γ secretion by ELISpot 10 days after the second injection. Individual mice were tested in parallel with different peptides at 2 μg/ml and with concanavalin A (ConA) at 5 μg/ml, final concentration. Lines represent mean and SEM. Differences between mice immunized with the DENV1-NS construct and the control plasmid were evaluated using non-parametric Mann-Whitney U-test (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Schematic representation of the T cell epitopes from the DENV1-NS poly-epitope, which induce a significant T cell response in HLA transgenic mice. NS3.1, NS3.2, NS4B, and NS5 represent the 4 antigenic regions selected in the DENV1-NS poly-epitope.
Figure 3
Figure 3
Quantification of the T cell responses in HLA-B*3501 transgenic mice by intracellular cytokine staining and ELISpot assay. Two groups of 4 HLA-B*3501 transgenic mice received an intramuscular inoculation of 10 or 2 μg of the LNP-based mRNA encoding DENV1-NS and 1 group of 2 HLA-B*3501 transgenic received 10 μg of the LNP-based irrelevant mRNA as a negative control. Two immunizations were performed at 3-week interval and spleen cells were tested 8 days after the second immunization for IFN-γ and TNF-α secretion by intracellular staining, after 6 h in vitro stimulation with peptides, in the presence of Golgi stop or by ELISpot assay after 20 h stimulation with peptides. (A) Flow cytometry analyses of gated CD3+, CD8+, for IFN-γ and TNF-α secretion. Plots show CD8 T cell responses from one representative animal against p49, p50, and p51 derived from DENV1 sequence (upper panels) or from their DENV2 variants (lower panels) after immunization with 10 μg of the LNP-based mRNA encoding DENV1-NS. (B) Comparison of the T cell responses analyzed by intracellular cytokine staining (upper panels) and by ELISpot assays (lower panels). Closed circles: peptides derived from DENV1. Open circles: peptide variant derived from DENV2. Open squares: peptide variant from DENV3. Open triangles: peptide variant from DENV4.
Figure 4
Figure 4
Effect of transient blockade of type I IFN signals on the expansion and contraction phases of antigen-specific T cells. (A) Three groups of 3 HLA-A*02:01 transgenic mice and 3 groups of 3 HLA-A*24:02 transgenic mice received 2 intramuscular inoculations of 10 μg of the LNP-based mRNA encoding DENV1-NS at 4-week interval. One group of mice received in addition 1 intraperitoneal inoculation of 2 mg anti-IFNAR antibody at day 27, just before the boost. Spleen cells were tested by ELISpot assay for IFN-γ secretion at days 27 and 36, after in vitro stimulation with the p30 peptide from DENV1 or its peptide variant from DENV4 for HLA-A*0201 transgenic mice or the p32 peptide from DENV1 or its peptide variant from DENV2.1 for HLA-A*2402 transgenic mice. Mean and SEM are shown. Differences in the ELISpot responses between 2 groups were evaluated using unpaired t-test (ns, non-significant, *p < 0.05, **p < 0.01). (B) Three groups of 3 HLA-A*0201 transgenic mice and 3 groups of 3 HLA-A*2402 transgenic mice received 2 intramuscular inoculation of 10 μg of the LNP-based mRNA encoding DENV1-NS at 3-week interval. One group received in addition 1 intraperitoneal inoculation of 2 mg anti-IFNAR antibody at day 55. Spleen cells were tested by ELISpot assay for IFN-γ secretion at days 27, 36, or 56. Mean and SEM are shown. Differences in the ELISpot responses between 2 groups were evaluated using unpaired t-test (ns, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001).
Figure 5
Figure 5
Immune protection induced by immunization with the LNP-based mRNA encoding DENV1-NS. (A) Two groups of 6 HLA-A*0201 transgenic mice were immunized with the LNP-based mRNA encoding DENV1-NS or control mRNA, (B) Five and six HLA-A*2402 transgenic mice received the LNP-based mRNA encoding DENV1-NS and control mRNA, respectively, (C) Nine and ten HLA-B*3501 transgenic mice received the LNP-based mRNA encoding DENV1-NS and control mRNA, respectively. Mice were immunized by intramuscular injection of 10 μg LNP-based mRNA encoding DENV1-NS or control mRNA, at day 0 and at day 28. One day prior challenge with the virus, mice received 1 intraperitoneal inoculation of 2 mg anti-IFNAR antibody (MAR1-5A3) and they were challenged at day 56 by retro-orbital injection of 106 pfu DENV1 (KDH0026A strain). Quantification of virus replication in plasma samples was performed by qRT-PCR at days 1, 2, and 3 after the challenge. LLOQ: Lower limit of quantitative detection. Lines represent mean and SEM. Differences between mice immunized with the DENV1-NS vaccine and the CT vaccine were evaluated using non-parametric Mann-Whitney U-test (*p < 0.05, **p < 0.01, ***p < 0.001).
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