In situ engineering of the lymph node microenvironment via intranodal injection of adjuvant-releasing polymer particles

Abstract

Recent studies have demonstrated a simple, potentially universal strategy to enhance vaccine potency, via intralymph node (i.LN) injection. To date, intranodal immunization studies have focused on the delivery of unadjuvanted vaccines (e.g., naked DNA, peptide, or protein). We hypothesized that combining i.LN vaccination with controlled release biomaterials permitting sustained dosing of molecular adjuvants to the local tissue microenvironment would further enhance this promising vaccination strategy. To test this idea, we encapsulated the Toll-like receptor-3 ligand poly(inosinic:cytidylic acid) (polyIC) in biodegradable poly(lactide-co-glycolide) microparticles (MPs) designed to remain extracellular and release polyIC in the LN over several days. Intranodal injection of MPs increased persistence of polyIC in LNs compared to the same dose of soluble polyIC or polyIC formulated in nanoparticles, leading to increased accumulation of Toll-like receptor agonist in LN-resident antigen presenting cells and more enduring dendritic cell activation. Intralymph node injection of ovalbumin mixed with polyIC-releasing MPs enhanced the humoral response and expanded ovalbumin-specific T cells to frequencies as high as 18% among all CD8(+) cells following a single injection (8.2-fold greater than the same vaccine given i.m.), a response that could not be matched by antigen mixed with polyIC-loaded nanoparticles or a 10-fold greater dose of soluble polyIC. Thus, i.LN immunization with slow release-formulated adjuvants may be a broadly applicable strategy to enhance therapeutic or prophylactic vaccines.

Fig. 1.

Intranodal injection of polymer particles. (A) Intralymph node injection of adjuvant-loaded particles following s.c. injection of tracer to mark draining LNs. (B) Tracer drainage to inguinal LNs 2 h after injection of 10 μg dye. Dashed lines indicate exterior (Upper) and internal (Lower) views of LNs after tracer convection from the injection site (arrow, Upper). (C) Whole-animal imaging of an untreated mouse and a mouse injected i.LN with fluorescent MPs. Shown are live animals (Upper) or excised LNs (Lower) 24 h after injection. (D) Histological analysis of LNs from C showing MPs (pink channel) dispersed within the LN parenchyma. (Scale bar: 50 μm.) (E) Histology of LNs 24 h after i.m. or i.LN injection of fluorescent MPs or NPs, showing particle fluorescence (pink) overlaid with T cells (CD3, green). (Scale bar: 50 μm.) (F) LNs excised 24 h after i.m. or i.LN particle injection were analyzed by flow cytometry to characterize uptake by LN APC populations.

Fig. 2.

Particle encapsulation increases the persistence of intranodally injected polyIC. (A) In vivo imaging 24 and 96 h after i.LN injection of 50 μg polyIC (soluble, NP-polyIC, or MP-polyIC). Particles (red color scale) and polyIC (blue color scale) were labeled with distinct fluorophores. (B and C) Quantification of whole-animal NP/MP fluorescence (B) and polyIC fluorescence (C) 24 and 96 h after i.LN injection. Shown is mean radiant efficiency ± SEM (n = 4) determined from equivalent regions of interest (*, p < 0.05; **, p < 0.001). Data are from one representative of three independent experiments.

Fig. 3.

Intranodal MP-polyIC mediates sustained polyIC exposure and uptake by LN-resident APCs. Mice received i.LN injections of 50 μg soluble polyIC, MP-polyIC, or empty MPs. (A) Histological images of LNs 24 or 96 h after injection showing T cells (CD3, green), polyIC (red), and MPs (pink). (Scale bar: 50 μm.) (B and C) Flow cytometry analysis of fluorescent polyIC uptake by LN-resident APCs 24 (B) or 96 h (C) after injection. Uptake levels are reported as polyIC mean fluorescent intensity (MFI) ± SEM among dendritic cells, CD11c⁺; macrophages (MACs), F4/80⁺; and B cells, B220⁺. (*, p < 0.05; **, p < 0.01; and ***, p < 0.001 vs. soluble polyIC). Data (n = 4) are from one representative of two independent experiments.

Fig. 4.

i.LN injection of MP-polyIC induces enduring DC activation. (A) Flow cytometry analysis of surface markers on CD11c⁺ DCs from LNs of naïve mice or mice receiving i.LN injections of 50 μg of soluble OVA antigen mixed with either empty MPs, 50 μg soluble polyIC, empty MPs mixed with 50 μg polyIC, or MP-polyIC encapsulating 50 μg polyIC, 24 or 96 h after injection. (B) Quantification of number of MHCII^hi DCs for each immunization condition at 24 or 96 h. Shown are mean frequencies ± SEM measured per LN (*, p < 0.05; **, p < 0.01). Data (n = 5) are from one representative of two independent experiments.

Fig. 5.

Enhanced vaccine responses following i.LN immunization with MP-polyIC. (A) Mice were immunized with 50 μg OVA and 50 μg polyIC (soluble polyIC alone, NP-polyIC, or MP-polyIC), administered i.m. or i.LN. T-cell responses were analyzed by tetramer staining on day 7. Shown are mean frequencies of tetramer⁺ T cells. Data (n = 6) is representative of one of three independent experiments. (B–D) Mice were immunized i.LN with 50 μg of soluble OVA antigen and either empty MPs, soluble polyIC, empty MPs mixed with polyIC just prior to injection, MP-polyIC, or NP-polyIC. (B) Shown are mean tetramer⁺ CD8⁺ T-cell frequencies ± SEM from one (n = 4) representative of three independent experiments. (C and D) Seven days after immunization with 50 μg polyIC, peripheral blood mononuclear cells were restimulated ex vivo with SIINFEKL peptide, fixed, and stained to detect intracellular IFN-γ/TNF-α. Shown are flow cytometry analyses (C) and quantification of frequencies (D) of CD8⁺ T cells positive for IFN-γ, TNF-α. (E) Mice were immunized with soluble polyIC or MP-polyIC as in A, and OVA-specific IgG serum titers were measured by ELISA. Shown are mean titers ± SEM from onee (n = 4) representative of two independent experiments. (F and G) Mice were immunized i.LN with soluble polyIC or MP-polyIC as in A and, 6 wk after vaccination, antigen-specific CD8⁺ T-cell frequencies (F) and cytokine production (G) were measured in T cells isolated from blood or spleen. Data are mean ± SEM and are representative of one (n = 4) of two independent experiments. For all panels, *, p < 0.05; **, p < 0.01; ***, p < 0.001.