Transcutaneous Immunization System Using a Hydrotropic Formulation Induces a Potent Antigen-Specific Antibody Response

<div><h3>Background</h3><p>Transcutaneous immunization (TCI) is a novel vaccination strategy, which is expected to have therapeutic applications. However, to develop effective TCI systems, a simple, non-invasive and safe transdermal formulation is required. This study developed a novel TCI system utilizing the co-administration of a liposoluble absorption enhancer, propylene glycol monocaprylate (PGMC) and hydrosoluble protein antigen without pretreatment of any typical adjuvants and disruption of the skin. Novel transdermal formulations were also prepared with sodium salicylate (NaSal) as a hydrotropic agent to improve the solubility of poorly water-soluble substances.</p> <h3>Methodology/Principal Findings</h3><p>The TCI system, which used a transdermal formulation containing hen lysozyme (HEL) and PGMC, solubilized with NaSal, resulted in a substantial HEL-specific antibody response in an HEL dose-dependent manner even in the absence of potent adjuvants, such as cholera toxin (CT). We also investigated whether NaSal activates antigen-presenting cells <em>in vitro</em> to clarify the mechanisms of antibody production by the hydrotropic formulation. NaSal enhanced the expression of MHC class II molecules and increased the production of IL-12 and TNF-α in dendritic cells, which were stimulated by lipopolysaccharide <em>in vitro</em>, indicating that NaSal had an effective adjuvant-like property. Moreover, the use of NaSal in the TCI system did not induce an HEL-specific, IgE-dependent anaphylactic reaction.</p> <h3>Conclusion/Significance</h3><p>Our TCI system using a hydrotropic formulation effectively and safely induced the intended immune response, and this system thus represents a new advantageous method that will result in improved TCI strategies.</p> </div

High levels of anti-HEL antibody production after TCI using the hydrotropic formulation solubilized with NaSal.

<p>(A) The transdermal formulations of HEL (1.0 mg/mouse) alone (▾) or together with (•) or without (▪) 5% PGMC as an absorption enhancer in the presence of 30% NaSal as a hydrotropic agent, or HEL (1.0 mg/mouse) in CT as an adjuvant (♦) were applied directly to the shaved abdominal skin of BALB/c mice for 3 h as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047980#s2" target="_blank">Materials and Methods</a>. Immunizations for each group of mice were performed in the same manner at 0, 2, and 4 weeks. Anti-HEL antibody titers in serum samples were determined every 2 weeks by ELISA. ^***, <i>p</i><0.0001, compared with HEL without adjuvant. (B) Effects of the hydrotropic agent for transdermal immunization was tested by replacing 30% NaSal with 58% NaBen in the formulation of HEL (1.0 mg/mouse) with (▪) or without (♦) 5% PGMC. ^***, <i>p</i><0.0001, compared with HEL without adjuvant. Arrowhead indicates transdermal immunization point. (C) Anti-HEL antibody titers in serum and skin were determined at 12 weeks after the primary immunization with the transdermal formulation of HEL (1.0 mg/mouse) and 5% PGMC in the presence of 30% NaSal or 58% NaBen. ^*, <i>p</i><0.05; ^***, <i>p</i><0.0001, compared with HEL and PGMC in the presence of NaBen. The data represent the mean and SD of six mice in each experimental group.</p

Transcutaneous immunization with NaSal did not induce an IgE-dependent anaphylactic reaction.

<p>The shaved abdominal skin of BALB/c mice was immunized with a transdermal hydrotropic formulation containing HEL (1.0 mg/mouse) and 5% PGMC solubilized in the presence of 30% NaSal for 3 h as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047980#s2" target="_blank">Methods</a>. Immunizations for each group of mice were performed in the same manner at 0, 2, and 4 weeks. At 6 weeks, mice were sensitized intraperitoneally with HEL (2500 µg/mouse) in FIA. After 9 days of sensitization, the mice were treated with HEL (5 µg/site) on the abdominal wall. Individual VPVs (vascular permeability values) are displayed with the median (bars) of 6–12 mice in each experimental group. ^**, <i>p</i><0.001, compared with the group sensitized with HEL.</p

Analysis of HEL-specific antibody subtypes.

<p>The shaved abdominal skin of BALB/c mice was immunized with a transdermal hydrotropic formulation containing HEL (1.0 mg/mouse) and 5% PGMC with 30% NaSal for 3 h. Immunizations for each group of mice were performed in the same manner at 0, 2, and 4 weeks. Serum samples collected from the mice every 2 weeks were assayed for HEL-specific antibody subtype (IgG, IgM, IgA) titers by ELISA. The data represent the mean and SD of six mice in each experimental group. ^**, <i>p</i><0.001, compared with the primary immunization at week 0. Arrowhead indicates transdermal immunization point.</p

NaSal enhanced the expression of MHC class II molecules and the secretion of cytokines in LPS-stimulated BMDCs.

<p>BMDCs were differentiated from bone marrow cells in mice by the addition of mouse granulocyte macrophage-colony stimulating factor (250 U/mL) and 2-mercaptoethanol (50 µM) for 10 days. BMDCs were stimulated with LPS (0.001 ng/mL) in the presence or absence of NaSal for 72 h. (A) Cell surface expression levels of MHC class II molecules were determined by FACS. Mean fluorescence intensity (MFI) is expressed as the mean and SD of three independent experiments. (B) The concentrations of IL-12 and TNF-α in the culture supernatants were determined by ELISA. The data represent the mean and SD of three independent experiments. ^*, <i>p</i><0.05; ^**, <i>p</i><0.001, compared with the control group with LPS treatment alone.</p

Evaluation of dose-dependent effects of HEL and NaSal on immune response after TCI.

<p>The shaved abdominal skin of BALB/c mice was immunized with the transdermal formulation containing the indicated dose of HEL from 0 to 2.0 mg and 5% PGMC in the presence of 30% NaSal (A) or HEL (1.0 mg/mouse) and 5% PGMC with the indicated dose of NaSal as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047980#s2" target="_blank">Materials and Methods</a> (B) for 3 h. Immunizations for each group of mice were performed in the same manner at 0, 2, and 4 weeks. Anti-HEL antibody titers in serum were determined at 10 weeks after the primary immunization. Data represent the mean and SD of six mice in each experimental group. ^**, <i>p</i><0.001, compared with the control group without HEL treatment. ^*, <i>p</i><0.05, compared with the control group without NaSal treatment.</p