Mesoporous Silicon Particles Favor the Induction of Long-Lived Humoral Responses in Mice to a Peptide-Based Vaccine

Abstract

Vaccinology faces the challenge of developing improved immunization approaches that are able to induce long-term immunity with the desired Th profile according to the pathology. In this context, new vehicles for efficient antigen delivery that exert adjuvant effects play a critical role in addressing this goal. Herein, mesoporous silicon particles (PSiP) were assessed as carriers for a peptide-based vaccine targeting the receptor for advanced glycation end products (RAGE), which is a relevant receptor in Alzheimer´s disease and other diseases. A RAGE peptide was adsorbed onto PSiP (PSiP vaccine) and administered to BALB/c mice, leading to immune responses that were similar in magnitude to those induced by the soluble peptide. However, the response induced by PSiP lasted for a significantly longer period when compared with the behavior of the group immunized with the peptide alone. Therefore, PSiP are proposed as carriers to enhance immune memory, which is critical in vaccination. This study opens interesting perspectives related to the application of PSiP in vaccinology.

Keywords: adjuvant; humoral response; peptide vaccine; receptor for advanced glycation end products; vaccine delivery vehicle.

Figure 1

Morphology characterization of mesoporous silicon particles (PSiP). (a) SEM top view of PSiP showing irregular discoid structures; (b) Size distribution of PSiP. The statistical analysis of the SEM image displays an average particle size of 3 ± 1 μm; (c) Front view of a porous layer; (d) Pore size distribution of PSiP. The statistical analysis of the SEM image indicates an average pore size of 40 μm.

Figure 2

Schematic representation of the adsorption of RAGEp onto the PSiP surface and pores in the particle structure. The PSiP are porous irregular discoids with a size distribution of 3 ± 1 µm, a thickness of ~400 nm, and a pore size of 40 nm. The anionic PSiP surface interacts with the cationic RAGEp molecules through electrostatic and/or ion dipole forces.

Figure 3

Titration curve for PSiP (•) and PSiP-RAGEp (▲). Titrations were performed in deionized water at different pH values. The selected titrants were HCl and NaOH.

Figure 4

Sequence of RAGEp. The receptor for advanced glycation end products peptide (RAGEp) is a synthetic peptide comprising the amino acids 23–54 from the human RAGE, at the extracellular region. Red indicates COOH residues with negative charge (COO⁻) at a physiological pH, and blue stands for NH₂ residues with positive charge (NH₃⁺) at a physiological pH. Green specifies hydrophobic uncharged residues, and black represents other residues.

Figure 5

RAGEp adsorption onto PSiP. Adsorption experiments were carried out using 100 μg of PSiP and different initial RAGEp amounts in μg: (a) 25, (b) 40, and (c) 60. A final volume of 250 μL was attained with PBS solution (pH = 7.4). All of the suspensions were stirred at 6 °C during the experiment. The concentration of soluble RAGEp was determined by measuring absorbance at λ_max = 199 nm. All of the experiments were conducted by triplicate, and the %SD were as follows: (a) 1.7–3.0%, (b) 0.1–2.5%, and (c) 2.0–3.0. (d) Linear concentration-dependent RAGEp adsorption. Data obtained after 4 h of experiment in the described conditions, r² = 0.78. (e) Percentage of RAGEp adsorbed onto the PSiP surface (%_RAGEp/PSiP) with respect to the initial RAGEp amount (m_0,RAGEp). Data are the average values of three experiments.

Figure 6

Infrared spectra of (a) PSiP, (b) RAGEp, and (c) RAGEp/PSiP conjugates. IR analyses were carried out after four hours of the adsorption experiment at the described conditions. PSiP and RAGE–PSiP were centrifuged, and pellets were washed twice with phosphate-buffered saline solution (PBS) before Fourier transform infrared (FT-IR) analyses. Chemical groups are assigned below relevant bands.

Figure 7

Long-lasting immune responses in BALB/c mice immunized with RAGEp/PSiP. Mice received four subcutaneous doses of one of the following treatments: PBS as negative control, RAGEp alone, PSiP, RAGEp/PSiP, or RAGEp/complete Freund’s adjuvant (CFA)/incomplete Freund’s adjuvant (IFA) as positive control. Serum samples were obtained at day 0 (before the first immunization) and at days 7 (a) and 52 (b) after the fourth immunization. Antibody levels were determined by ELISA, and data are presented as the average optical density (O.D.) (1:20 dilution) at either day 7 or 52 min average O.D. at day 0. Statistical differences (p < 0.05) versus the group treated with PBS are indicated by an asterisk (*), while statistical differences versus the group treated with RAGEp alone are indicated by double asterisk (**).