Toll-like receptor 8 agonist nanoparticles mimic immunomodulating effects of the live BCG vaccine and enhance neonatal innate and adaptive immune responses

Abstract

Background: Newborns display distinct immune responses, leaving them vulnerable to infections and impairing immunization. Targeting newborn dendritic cells (DCs), which integrate vaccine signals into adaptive immune responses, might enable development of age-specific vaccine formulations to overcome suboptimal immunization.

Objective: Small-molecule imidazoquinoline Toll-like receptor (TLR) 8 agonists robustly activate newborn DCs but can result in reactogenicity when delivered in soluble form. We used rational engineering and age- and species-specific modeling to construct and characterize polymer nanocarriers encapsulating a TLR8 agonist, allowing direct intracellular release after selective uptake by DCs.

Methods: Chemically similar but morphologically distinct nanocarriers comprised of amphiphilic block copolymers were engineered for targeted uptake by murine DCs in vivo, and a range of TLR8 agonist-encapsulating polymersome formulations were then synthesized. Novel 96-well in vitro assays using neonatal human monocyte-derived DCs and humanized TLR8 mouse bone marrow-derived DCs enabled benchmarking of the TLR8 agonist-encapsulating polymersome formulations against conventional adjuvants and licensed vaccines, including live attenuated BCG vaccine. Immunogenicity of the TLR8 agonist adjuvanted antigen 85B (Ag85B)/peptide 25-loaded BCG-mimicking nanoparticle formulation was evaluated in vivo by using humanized TLR8 neonatal mice.

Results: Although alum-adjuvanted vaccines induced modest costimulatory molecule expression, limited T_H-polarizing cytokine production, and significant cell death, BCG induced a robust adult-like maturation profile of neonatal DCs. Remarkably, TLR8 agonist polymersomes induced not only newborn DC maturation profiles similar to those induced by BCG but also stronger IL-12p70 production. On subcutaneous injection to neonatal mice, the TLR8 agonist-adjuvanted Ag85B peptide 25 formulation was comparable with BCG in inducing Ag85B-specific CD4⁺ T-cell numbers.

Conclusion: TLR8 agonist-encapsulating polymersomes hold substantial potential for early-life immunization against intracellular pathogens. Overall, our study represents a novel approach for rational design of early-life vaccines.

Keywords: BCG; Newborn; Toll-like receptor 8; dendritic cells; nanoparticle; polymersome; vaccine.

Graphical abstract

Fig 1

Polymersomes preferentially associate with DCs in vivo. A, Cryogenic transmission electron microscopy revealed the self-assembly of approximately 50-nm filomicelles (F; left, PEG₄₅-bl-PPS₄₄), approximately 120-nm vesicular polymersomes (PS; middle, PEG₁₇-bl-PPS₃₀), or approximately 30-nm spherical micelles (M; right, PEG₄₄-bl-PPS₂₉). B, Flow cytometric analysis of single-cell suspensions from mouse lymph nodes and spleens was conducted 24 hours after subcutaneous injection of fluorescent nanocarriers into the footpad (n = 5-8). A heat map demonstrates percentages of key phagocyte populations that associated with PEG-bl-PPS filomicelles (F), vesicular polymersomes (PS), and spherical micelles (M). Amine-functionalized PEG₁₇-bl-PPS₃₀ block copolymers were conjugated to fluorescent Dy647-N-hydroxysuccinimide and assembled in the presence of block copolymers engineered to generate filamentous, vesicular, or spherical morphologies. See also Fig E1.

Fig 2

Synthesis, loading, and characterization of CL075-PS. A,Left, Size exclusion chromatography with Sepharose CL-6B resin and a PBS mobile phase indicated there was no free CL075 present after purification of the CL075-loaded polymersomes. The green line indicates a strong peak for the polymersomes, which elute very early, without a second peak for CL075 (dashed blue line). Data are plotted against blank unloaded polymersomes, demonstrating the location of the polymer (purple line). Right, CL075 dissolved readily in the solvent dimethylformamide, as did the PEG-PPS block copolymer used to assemble polymersomes. CL075-PS was lyophilized so that both the polymer and adjuvant were completely dissolved in the dimethylformamide. Both free (dashed blue line) and loaded CL075 (green line) eluted at approximately 14.48 minutes. B, Graphic depiction of the CL075-PS nanoparticles. See also Figs E2-E4.

Fig 3

The TLR8 agonist CL075 retains robust TNF-inducing activity toward human newborn and adult MoDCs when encapsulated in polymersome nanoparticles. Human newborn (A) and adult (B) MoDCs were cultured in 10% (vol/vol) autologous plasma and stimulated for 24 hours with fixed concentrations of monophosphoryl lipid A (MPLA) or LPS (100 ng/mL) or increasing concentrations of free CL075 (dashed blue line), CL075-PS (green line), or CL075:Gag-PS (red line; mean ± SEM, n = 8-14). For analyses of individual treatments (eg, control RPMI vs 10 μmol/L CL075), the unpaired Mann-Whitney test was applied at each concentration, and statistical significance was denoted as follows: *P < .05, **P < .01, and ***P < .001. For comparisons between newborn and adult TLR4 agonist–treated conditions (ie, newborn LPS vs adult LPS), significance was denoted as follows: +P < .05 and ++P < .01. See also Fig E6.

Fig 4

CL075-PS induces a robust MoDC activation profile similar to that of BCG and distinct from that of alum-adjuvanted subunit vaccines. A and B, Newborn (Fig 4, A) and adult (Fig 4, B) MoDCs were cultured in 10% (vol/vol) autologous plasma and stimulated for 24 hours with fixed concentrations of monophosphoryl lipid A (MPLA; 100 ng/mL), CL075, CL075-PS (both 0.1, 1, 5, and 10 μmol/L), alum (5, 50, and 500 μg/mL), BCG, PCV13, or HBV (each at 1:1000, 1:100, and 1:10 vol/vol). Surface expression of costimulatory molecules and HLA was determined by using flow cytometry and analyzed as fold change in mean fluorescent intensity (MFI) versus vehicle control (mean ± SEM, n = 5-8). C, Neither polymersomes (PS), CL075, nor CL075-PS induced cell death. Undetectable LDH levels are indicated by a circle with a diagonal line through it. D, PGE₂ production, as measured by means of ELISA, benchmarked to adjuvants and licensed pediatric vaccines (mean ± SEM, n = 6). See also Figs E7 and E8 and Table E1.

Fig 5

TLR8 agonist–encapsulating polymersomes match or exceed BCG's cytokine-inducing activity, dramatically enhancing human neonatal and adult MoDC IL-12p70 production. Newborn (A and C) and adult (B and D) MoDCs were cultured in 10% (vol/vol) autologous plasma and stimulated for 24 hours with increasing concentrations of CL075:Gag-PS (red; 1, 5, and 10 μmol/L), BCG (blue), PCV13 (purple), HBV (green; each at 1:1000, 1:100, and 1:10 vol/vol), or alum (5, 50, 500 and μg/mL; orange). Concentration-dependent cytokine production induction was measured in supernatants by using the multiplex assay. Data represent fold change over vehicle control (black; mean ± SEM, n = 6). For analyses of individual treatments (eg, control 10 μmol/L CL075:Gag-PS vs BCG 1:10 [vol/vol]), the unpaired Mann-Whitney test was applied at each concentration, and statistical significance was denoted as follows: **P < .01. For comparisons between overall groups (eg, CL075:Gag-PS vs BCG), significance was denoted as follows: ++P < .01. See also Figs E9 and E10.

Fig 6

CL075:Ag85Bp25-PS and BCG induce comparable Ag85B-specific CD4⁺ effector T cells in the spleens of neonatal huTLR8Tg mice. A, Experimental design: 7-day-old huTLR8Tg and matched littermate WT mice were immunized subcutaneously and killed 14 days later before harvesting spleens and staining splenocytes with a control versus Ag85B_280–294 MHCII tetramer and for surface marker expression. B, Representative flow cytometric plots showing Tet⁺ T cells as CD44⁺ Neg Tet⁺ or CD44⁺ Ag85B Tet⁺ cells gated on live CD4⁺ splenic lymphocytes of BCG- or CL075-Ag85Bp25-PS–vaccinated mice 2 weeks after immunization. C and D, Similar to BCG, CL075-Ag85Bp25-PS induced a significant increase in CD44⁺ Ag85B Tet⁺ cells compared with CD44⁺ Neg Tet⁺ cells. Results represent means ± SEMs (n = 14-15), with significance relative to unspecific control tetramer. E, Neonatal humanized TLR8 mice have greater numbers of splenic Ag85B-specific CD4⁺ T cells versus their WT littermates. The unpaired Mann-Whitney test was applied, and statistical significance was denoted as follows: **P < .01 and ***P < .001. NS, Not significant.

Fig E1

Fig E2

Fig E3

Fig E4

Fig E5

Fig E6

Fig E7

Fig E8

Fig E9

Fig E10

Fig E11

Fig E12

Fig E13