Robust vaccine formulation produced by assembling a hybrid coating of polyethyleneimine-silica

Abstract

Exploring formulations that can improve the thermostability and immunogenicity of vaccines holds great promise in advancing the efficacy of vaccination to combat infectious diseases. Inspired by biomineralized core-shell structures in nature, we suggest a polyethyleneimine (PEI)-silica-PEI hybrid coated vaccine formulation to improve both thermostability and immunogenicity. Through electrostatic adsorption, in situ silicification and capping treatment, a hybrid coating of silica and PEI was assembled around a vaccine to produce vaccine@PEI-silica structures. Both in vitro and in vivo experiments demonstrated that the thermostability and immunogenicity of the modified vaccine were significantly improved. The modified vaccine could be used efficiently after long-term exposure at room temperature, which would facilitate vaccine transport and storage without a cold chain. Furthermore, mechanistic studies revealed that the PEI-silica-PEI coating acted as a physiochemical anchor as well as a mobility-restricting hydration layer to stabilize the enclosed vaccine. This achievement demonstrates a biomimetic surface-modification-based strategy to confer desired properties on biological products.

Fig. 1. Polycationic molecule mediated in situ silicification of JEV. (A) Schematic illustration of the assembly of PEI–silica hybrid nanocoatings on the vaccine. (B) The sensitivity of JEV to pH-tuned silicification by adjusting solution pH to different acidities, with their remaining infectivity and silicification efficacies examined. (C) Zeta-potential of JEV, and JEV coated with PEI, PEI–SiO₂ and PEI–SiO₂–PEI sandwich layers. (D) The silicification efficacies of JEV at pH 7.0 with or without adding PEI as a nucleating agent. (E) TEM images of silicified vaccine JEV@PEI–SiO₂ without any staining treatment. (F and G) TEM images of negatively stained JEV@PEI–SiO₂, image (G) depicts silicified JEV that was almost totally encased by PEI–silica composites. (H) SEM images of JEV@PEI–SiO₂ nanoparticles, inset represents EDX analysis of JEV@PEI–SiO₂. (I) pH sensitivity of JEV, JEV@PEI and JEV@PEI–SiO₂. (*P < 0.05, **P < 0.01, n ≥ 3, data represented as means ± SDs).

Fig. 2. Biological activity and immunogenicity of JEV and the silicified vaccine JEV@PEI–SiO₂. (A) Plaque morphologies in BHK21 cells. (B) The indirect immunofluorescence assays (IFA) of JEV and JEV@PEI–SiO₂ in BHK21 cells at 36 h post-infection. (C) Growth curves of JEV and JEV–PEI–SiO₂ in BHK21 cells, and (D) Vero cells (M.O.I. = 0.1). (E) The levels of serum IgG antibody and (F) neutralization antibody in mice immunized by the same amount of JEV or JEV@PEI–SiO₂. (*P < 0.05, n ≥ 3, data represented as means ± SDs).

Fig. 3. Thermostabilities of native JEV, JEV@PEI and JEV@PEI–SiO₂ in liquid form. (A) Thermal-inactivation curves of JEV in native and JEV@PEI–SiO₂ formulations at 25 °C, or (B) 37 °C, or (C) 42 °C, with the thermal-inactivation curves of native JEV at 4 °C as a reference. (D) Thermal-inactivation curves of JEV, JEV@PEI, and JEV mixed with ex situ synthesized PEI–silica composites at 42 °C. (n ≥ 4, data represented as means ± SDs).

Fig. 4. Animal experiments with fresh or stored JEV and JEV@PEI–SiO₂. (A) The levels of elicited serum IgG antibody and (B) serum neutralization antibody in mice immunized by fresh or 18 day-stored (25 °C) JEV and JEV@PEI–SiO₂. (C) The frequencies of JEV-specific IFN-γ secreting splenocytes of mice 12 days post-immunization were determined by quantifying the numbers of spot-forming cells (SFCs) with an ELIspot assay.

Fig. 5. TGA-DSC analyses of freeze-dried powders of (A) native JEV and (B) JEV@PEI–SiO₂. The results revealed that almost all water molecules in native vaccines were deprived at 60 °C, whereas most water molecules in JEV@PEI–SiO₂ were not lost until the temperature reached 150 °C, indicating that the PEI–silica composite had an excellent water confining effect.