Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

Abstract

Nucleic acid vaccines have grown in importance over the past several years, with the development of new approaches remaining a focus. We describe a lipid nanoparticle-formulated DNA (DNA-LNP) formulation which induces robust innate and adaptive immunity with similar serological potency to mRNA-LNPs and adjuvanted protein. Using an influenza hemagglutinin (HA)-encoding construct, we show that priming with our HA DNA-LNP demonstrated stimulator of interferon genes (STING)-dependent upregulation and activation of migratory dendritic cell (DC) subpopulations. HA DNA-LNP induced superior antigen-specific CD8⁺ T cell responses relative to mRNA-LNPs or adjuvanted protein, with memory responses persisting beyond one year. In rabbits immunized with HA DNA-LNP, we observed immune responses comparable or superior to mRNA-LNPs at the same dose. In an additional model, a SARS-CoV-2 spike-encoding DNA-LNP elicited protective efficacy comparable to spike mRNA-LNPs. Our study identifies a platform-specific priming mechanism for DNA-LNPs divergent from mRNA-LNPs or adjuvanted protein, suggesting avenues for this approach in prophylactic and therapeutic vaccine development.

Keywords: DNA-LNP; T cell; adjuvanted protein; antibody; lipid nanoparticle; mRNA; plasmid DNA; vaccine.

Graphical abstract

Figure 1

Initial immune characterization of HA DNA-LNP formulations and immunogenicity (A) Schematic of DNA-LNP N/P ratios and immunization regimen. (B–D) Biophysical characterization of DNA-LNPs at different N/P ratios. (B) Particle size; (C) polydispersity index (PDI); (D) zeta potential. (E) Representative fluorescence-activated cell sorting (FACS) plots of GC B cells. (F) Bar plots quantifying frequency of GC B cells. (G) Frequency of CA09 HA-specific GC B cells. (H) Frequency of activated Tfh cells. (I) IFNγ ELISpot of splenocytes. (J) Representative TEM images of HA DNA-LNP (top) and HA mRNA-LNP (bottom). Scale bar 100 nm (K and L) Fold change cytokine induction in DLNs at 4 h (K) and 24 h (L) after immunization quantified using Luminex. (M and N) ELISpot assay measuring IFNα (M) and IFNγ (N) 20 h after stimulation of splenocytes ex vivo with DNA-LNP, plasmid DNA, or DNA-LNP in the presence of chemical inhibitors to the indicated DNA sensors. (O) Schematic of relevant pathways implicated in DNA-LNP sensing. Dots represent individual animals; n = 8–9 (E–H), n = 5 (I, K, and L), or n = 3–4 animals per group (L and M); data pooled or representative from two independent experiments (E–H, M, and N) or from one independent experiment (I–L). Plots show mean with SD (B–D and I) or geometric mean with geometric SD (F–H and K–N). Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups (F–H, K, and L) or compared to DNA-LNP control (M and N). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 2

Early immune profiling of innate and adaptive populations reveals STING-dependent activation status after HA DNA-LNP immunization (A) Schematic of immunization regimen. (B–G) Frequency of total mDCs (B), CD11b⁺ mDCs (C), CD103⁺ mDCs (D), pDCs (E), neutrophils (F), and monocytes (G) in the iliac DLN. (H) Representative histograms of CD86 expression on total mDC and mDC subpopulations. (I) Quantification of CD86 expression. (J) Schematic of immunization regimen. (K–P) Frequency of total mDCs (K), CD11b⁺ mDCs (L), CD103⁺ mDCs (M), pDCs (N), neutrophils (O), and monocytes (P) in the iliac DLN. (Q) Representative histograms of CD86 expression on total mDC and mDC subpopulations. (R) Quantification of CD86 expression. Dots represent individual animals; for (A–I), n = 9–10 animals per group; for (J–R), n = 3–6 animals per group. Data are representative of two independent experiments. Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 3

Single-cell transcriptomics elucidates pro-activation and migration signature among innate immune subsets after priming with HA DNA-LNP (A) UMAP plot of innate immune subsets. (B) UMAP plot of clusters colored by sample. (C–G) Differentially expressed genes upregulated after immunization with DNA-LNP relative to naive, mRNA-LNP, and protein in adjuvant immunization in NK cells (C), neutrophils (D), monocytes (E), pDCs (F), and cDCs (G). Data represent one independent experiment of 10 pooled mouse popliteal LNs per group.

Figure 4

HA DNA-LNP elicits potent antigen-specific CD8⁺ and CD4⁺ T cell responses (A) Schematic of immunization regimen. (B and C) Representative FACS plots (B) and frequency (C) of IFNγ⁺ effector CD8⁺ T cells. (D) CD107a⁺ effector CD8⁺ T cells. (E) TNF-α⁺ effector CD8⁺ T cells. (F) IFNγ⁺ effector CD4⁺ T cells. (G) TNF-α⁺ effector CD4⁺ T cells. (H) IL-2⁺ effector CD4⁺ T cells. (I–K) Frequency of effector CD8⁺ T cells expressing IFNγ (I), CD107a (J), or TNF-α (K) after dose de-escalation. Dots represent individual animals; for (C–H), n = 10 animals per group; for (I–K), n = 5 animals per group. Data pooled from two (C–H) or one (I–K) independent experiment(s). Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 5

HA DNA-LNP induces robust GC and serum responses Mice were immunized with HA DNA-LNP (2 μg), HA mRNA-LNP (2 μg), or adjuvanted HA protein (1 μg). GC responses were assessed in the DLNs 14 days post immunization and serum responses longitudinally. (A) Representative FACS plots of activated Tfh cells. (B and C) Bar plots show quantification of frequency (B) and numbers (C) of activated Tfh cells. (D) Representative FACS plots of total GC B cells. (E and F) Bar plots show quantification of frequency (E) and numbers (F) of total GC B cells. (G) Representative FACS plots of CA09 HA-specific GC B cells. (H and I) Bar plots show frequency (H) and numbers (I) of CA09 HA-specific GC B cells. (J) Area under the curve (AUC) of total A/California/04/2009 HA-specific serum IgG ELISA data. (K) Serum endpoint titers at week 8 to various H1N1 HAs. (L) HAI titers at week 8 to A/California/07/2009 X-179A. (M and N) AUC of serum binding antibodies to A/Guangdong-Maonan/SWL1536/2019 HA (M) and A/Victoria/4897/2022 HA (N). (O and P) HAI titers to A/Netherlands/602/2009 (O) and A/New York City/PV63249/2022 (P). Dots represent individual animals (B, C, E, F, H, I, K, and L); n = 9–10 animals per group; data pooled from two independent experiments. Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups (A–I) or active immunization groups (K). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 6

HA DNA-LNP induces potent memory responses in mice and rabbits (A) Schematic of mouse immunization regimen. (B) IFNγ-secreting cells in splenocytes by ELISpot. (C) IFNγ-secreting effector CD8⁺ T cells by flow cytometry. (D) CA09 HA-specific ASC responses in bone marrow by ELISpot. (E) Representative FACS plot of CA09 HA-specific MBCs. (F and G) Bar plots show frequency (F) and numbers (G) of CA09 HA-specific MBCs. (H) Schematic of rabbit immunization regimen. (I–K) IFNγ ELISpot on peripheral blood mononuclear cells (PBMCs) at day 42 (I), day 105 (J), and day 202 (K). (L) AUC of total A/California/04/2009 HA-specific serum IgG ELISA data. (M and N) HAI titers to A/Netherlands/602/2009 (M) and A/New York City/PV63249/2022 (N). Dots represent individual animals (C, D, F, and G); n = 9–10 animals per group (B–D, F, and G), n = 5 animals per group (I–N); data pooled from two independent experiments. Plots show mean with SD (B and I–K) or geometric mean with geometric SD (C, D, F, G, and L–N). Unpaired one-way or two-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. ANOVA was performed at the final time point for (L–N). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 7

SARS-CoV-2 spike DNA-LNP elicits serum antibody responses and is protective in lethal challenge (A) Schematic of immunization regimen and challenge. (B–E) Serum endpoint titers at week 8 after immunization to wild-type spike RBD (B), D614G full-length spike (C), B.1.617.2 (Delta) full-length spike (D), and BA.2 full-length spike (E). (F) Serum neutralization infective dose (ID)₅₀ against wild-type SARS-CoV-2 pseudovirus. (G and H) Weight loss (G) and survival (H) of mice challenged with 1 × 10⁵ plaque-forming unit (PFU) mouse-adapted SARS-CoV-2 with an 80% weight loss cutoff. (I) Clinical score representing clinical signs of morbidity 4 days after challenge. Dots represent individual animals; n = 5–8 animals per group; data combined from two independent studies (A–F). Plots show geometric mean with geometric SD. Non-parametric Mann-Whitney U test (B–F) or log rank (Mantel-Cox) test (H) was used to compare groups. ∗∗p < 0.01.