A TLR7/8 Agonist-Including DOEPC-Based Cationic Liposome Formulation Mediates Its Adjuvanticity Through the Sustained Recruitment of Highly Activated Monocytes in a Type I IFN-Independent but NF-κB-Dependent Manner

Abstract

Novel adjuvants, such as Toll-like receptors (TLRs) agonists, are needed for the development of new formulations able to circumvent limitations of current vaccines. Among TLRs, TLR7/8 agonists represent promising candidates, as they are well described to enhance antigen-specific antibody responses and skew immunity toward T helper (T_H) 1 responses. We find here that the incorporation of the synthetic TLR7/8 ligand 3M-052 in a cationic DOEPC-based liposome formulation shifts immunity toward T_H1 responses and elicits strong and long-lasting germinal center and follicular T helper cell responses in adult mice. This reflects the prolonged recruitment of innate cells toward the site of immunization and homing of activated antigen-loaded monocytes and monocyte-derived dendritic cells toward draining lymph nodes. We further show that this adjuvanticity is independent of type I IFN but NF-κB-dependent. Overall, our data identify TLR7/8 agonists incorporated in liposomes as promising and effective adjuvants to enhance T_H1 and germinal center responses.

Keywords: TLR7/8 agonist; adjuvants for vaccine; follicular T helper cells; germinal centers; liposome.

Figure 1

Liposomal-based vaccines elicit strong T_FH and germinal center (GC) B cell responses, which are further increased by TLR7/8 ligands. (A, B) Naïve C57BL/6 mice were immunized once intramuscularly (i.m.) in both hind legs and euthanized at days 6, 10, 17 and 28 p.i. Serum levels of gB-specific (A) IgG and (B) IgG1 and IgG2c were assessed by ELISA. Results are expressed as mean ± SEM obtained from three independent experiments with n = 3–4 mice per group each. (C) Mice were immunized i.m. with gB/PBS, gB/SPA06 and gB/SPA10 and boosted at day 21. Spleens were harvested at day 35, and IL-5 and IFN-γ secretion was quantified in supernatants of splenocytes restimulated or not with gB for 72h. The mean cytokine level by gB-stimulated cells after subtraction of medium condition ± SEM are given for n = 5 mice per group. One out of 2 independent experiment is shown. (D–G) Inguinal and para-aortic draining lymph nodes were harvested at indicated time-points after a single immunization with gB/PBS, gB/SPA06 or gB/SPA10, and analyzed by flow cytometry for the presence of GC and T_FH cells. Representative contour and zebra plots, respectively, show (D) the GL7⁺Fas⁺ GC B cell population gated on B220⁺ and (F) the CXCR5⁺PD1⁺ T_FH cell population gated on CD4⁺ T cells at days 10 and 28 p.i. Numbers in plots represent the frequency of the gated population. The mean frequency and number of (E) GC B cells and (G) T_FH cells ± SEM from three independent experiments with n = 3–4 mice per group are given. ^‡ p < 0.05 gB/PBS vs gB/SPA06; ^# p < 0.05 gB/PBS vs gB/SPA10; *p < 0.05 gB/SPA06 vs gB/SPA10.

Figure 2

SPA10 induces sustained innate cell recruitment at the site of immunization. Naïve C57BL/6 mice were immunized i.m. in both hind legs with fluorescently labeled gB-Alexa633 and SPA06-DiO or SPA10-DiO or PBS as control. Muscles were collected after 2 h, 24 h, and 5 days and individually analyzed by flow cytometry after enzymatic digestion. (A) Representative dot plots display the frequency of gB-, SPA06-, or SPA10-loaded cells within lymphocyte population after doublets exclusion. (B) Graphs show the mean number per muscle of gB⁺Adj^- (top graph) or gB⁺Adj⁺ (bottom graph) cells within lymphocytes ± SEM. (C) Histograms show the mean frequency of several innate cell types identified by specific markers among gB⁺SPA06⁺ and gB⁺SPA10⁺ cell populations in muscles 24 h and 5 days post immunization (monocytes:CD11c^- CD11b⁺ Ly6G^- Ly6C^high; PMNs: CD11c^- CD11b⁺ Ly6G⁺ Ly6C^int; B cells: CD11c^- CD11b^- B220⁺; cDCs: CD11c⁺ CD11b^+/- Ly6C^-; Mo-DCs: CD11c⁺ CD11b⁺ Ly6C⁺) ± SEM. (D) The kinetics of different total cell population recruitment are shown. Mean cell number ± SEM for each population is given. All results are obtained from three independent experiments with n = 2–3 mice per group each. ^# p < 0.05 for gB/PBS vs gB/SPA10; *p < 0.05 gB/SPA06 vs gB/SPA10.

Figure 3

SPA10 induces sustained influx of Ag-loaded cells into the draining lymph nodes (dLNs). (A–D) naïve C57BL/6 mice were immunized i.m. in both hind legs with fluorescently labeled gB-Alexa633 and SPA06-DiO or SPA10-DiO or PBS as control. Draining LNs from each mouse side (inguinal and para-aortic) were collected after 2 h, 24 h, and 5 days and separately analyzed by flow cytometry after enzymatic digestion. (A) Representative dot plots display the frequency of gB-, SPA06-, or SPA10-loaded cells within lymphocyte population after doublets exclusion. (B) The mean number per two dLNs (para-aortic + inguinal) of gB⁺Adj^- (top graph) or gB⁺Adj⁺ (bottom graph) cells within lymphocytes ± SEM is shown. (C) Histograms show the mean frequency of various innate cell types among gB⁺SPA06⁺ and gB⁺SPA10⁺ cell populations (defined as in Figure 2) 24 h and 5 days post immunization ± SEM. (D) Graphs on the top represent the kinetics of innate cell recruitment with the mean cell number ± SEM for each population. Graphs on the bottom show the mean cell number of gB⁺Adj⁺ cells for each sub-population ± SEM. All results are obtained from two independent experiments with n = 4 dLNs per group each (2 mice). (E) Draining LNs from each mouse side (inguinal + para-aortic) were separately treated by enzymatic digestion 24h post i.m. immunization with gB/PBS, gB/SPA06-DiO or gB/SPA10-DiO in both hind legs and CD80 and CD86 expression was assessed on Adj^- and Adj⁺ monocytes, Mo-DCs and cDCs (identified as in Figure 2 ) by flow cytometry. The fold-change of geometrical mean fluorescence intensity over PBS (dotted line) is shown ± SEM for n = 10 dLNs per group (5 mice). ^‡ p < 0.05 for gB/PBS vs gB/SPA06; ^# p < 0.05 for gB/PBS vs gB/SPA10; *p < 0.05 gB/SPA06 vs gB/SPA10.

Figure 4

SPA10 induces a potent type I IFN response in adult mice. Naïve C57BL/6 mice were immunized (A, C, D) i.m. or (B) intraperitoneally with gB/SPA06, gB/SPA10 or gB/PBS as control. The mRNA expression levels of (A) IFN-α, (C) TLR7, MyD88, IRF7 and (D) IRF9, STAT1, and STAT2 were assessed by semi-quantitative real-time PCR in whole dLNs and muscles after 12 h and normalized to three constitutively expressed control genes. Results are represented as arbitrary units ± SEM for n ≥ 3 mice per group. One of two independent experiments is shown. (B) IFN-α was measured by ELISA in cell-free supernatants of peritoneal lavages of immunized mice performed 12 h post i.p. immunization. The mean IFN-α concentration ± SEM from two independent experiments with n ≥ 3 mice per group is given. *p < 0.05.

Figure 5

SPA10 immune responses are type I IFN-independent and NF-κB-dependent. Naïve WT C57BL/6 (dashed lines) and (A, B) IFNAR^-/- or (C, D) NF-κB^-/- (plain lines) mice were immunized twice i.m. with gB/SPA06 or gB/SPA10 in the hind leg 21 days apart. (A, C) Mice were bled at days 10, 20, and 35 and serum levels of gB-specific IgG, IgG1 and IgG2c were assessed by ELISA. Data are represented as mean ± SEM gB-specific Ab titers for n=5 mice per group. (B, D) Spleens were harvested at day 35, and IL-5 and IFN-γ secretion was quantified in supernatants of splenocytes restimulated or not with gB for 72h. The mean cytokine level by gB-stimulated cells after subtraction of medium condition ± SEM are given for n = 4–5 mice per group. One out of two independent experiment is shown. ^‡ p < 0.05 WT gB/SPA06 vs KO gB/SPA06; ^# p < 0.05 WT gB/SPA10 vs KO gB/SPA10; *p < 0.05.