Transcutaneous pollinosis immunotherapy using a solid‐in‐oil nanodispersion system carrying T cell epitope peptide and R848

Abstract Antigen‐specific immunotherapy is the only curative approach for the treatment of allergic diseases such as Japanese cedar pollinosis. Immunotherapy using a T cell epitope vaccine in combination with the adjuvant R848 is of particular interest as a safe and effective approach to treat allergic diseases. Herein, we propose a simple and easy to handle vaccine administration method using the original solid‐in‐oil (S/O) nanodispersion system that permeates through the skin. The S/O nanodispersion system is composed of nanoparticles of hydrophilic molecules surrounded with hydrophobic surfactants that are dispersed in an oil vehicle. The system has potential to carry and deliver both hydrophilic and hydrophobic bioactives. Hydrophilic T cell epitope peptide was efficiently delivered through mouse skin using the S/O nanodispersion system and lowered antigen‐specific IgE levels in pollinosis model mice. Addition of the hydrophobic adju1vant R848 significantly lowered the antibody secretion and shifted the Th1/Th2‐balance toward Th1‐type immunity in the model mice, showing the potential to alleviate Japanese cedar pollinosis.


| I N T R O D U C T I O N
Vaccines have contributed to the decrease in the rate of infectious diseases since their introduction more than two centuries ago. 1 Recent research has revealed vaccines are useful treatments for a number of immune-related diseases including autoimmune diseases, cancers, and allergies. [2][3][4] Allergic diseases have been conventionally classified into four types (type I, II, III, and IV) according to Gell and Coombs,5 although many exceptions have recently been found that do not fit these clasifications. 6,7 Type I (immediate-type) allergy, such as pollinosis, cat allergy, house dust mite allergy, or allergic asthma, is caused by activation of type 2 helper T (Th2) cells and induction of IgE antibodies from B cells. The representative symptoms of type I allergy are rhinitis, conjunctivitis, pruritus, asthma, and lowered blood pressure. These allergic reactions are triggered by the binding of antigen molecules to IgE on mast cells, and the symptoms appear within 5-15 min from the time of contact with the antigens. Type I allergy holds risks of severe symptoms such as anaphylaxis 8 and the only curative treatments of this type of allergy are immunotherapies using whole antigen molecules, antigen derivatives, or T cell epitopes. 9,10 T cell epitopes are short specific regions of the antigen molecules (usually 10-20 amino acids) that are recognized by mammalian T Abbreviations: 5-CF, 5-carboxyfluorescein; DLS, dynamic light scattering; IPM, isopropyl myristate; PBS, phosphate buffered saline; Rho-DOPE, Rhodamine-dioleyl phosphatidylethanolamine; s.c. injection, subcutaneous injection; S/O nanodispersion, solid-in-oil nanodispersion; Th1 cell, type 1 helper T cell; Th2 cell, type 2 helper T cell; TLR, toll like receptor. cells. 11 Researches have recently suggested that several mechanisms are involved in peptide immunotherapy using T cell epitopes, including activation of regulatory T cells, induction of T cell anergy and deletion of allergen-specific Th2 cells. 12 Type 1 helper T (Th1) cell-dominant immunity is observed in the sera from patients that have been treated with T cell epitopes. Stimulation of IgG antibody secretions were also observed. 13 Serious adverse events are a problem using whole antigen molecules in immunotherapy. T cell epitopes do not bind to IgE and the likelihood of serious adverse events are expected to be low during T cell epitopetherapy. Several recent reports demonstrated that a conjugated peptide of seven T cell epitopes derived from Japanese cedar (Cryptomeria japonica) pollen allergen (Cry j 1 and 2) (7Crp) has potential to alleviate allergic symptoms in murine models. 14,15 Japanese cedar pollinosis is becoming a severe problem in Japan. One quarter of Japanese population is assumed to suffer from the pollinosis according to a survey in 2010 and its prevalence is thought to be increasing. 16,17 Therefore, development of an effective therapy is a high priority.
Conventional immunotherapy of type I allergy requires lengthy treatments from several months to years of vaccine inoculations administered by subcutaneous (s.c.) injection or by the sublingual mucosal route. The pain associated with injections lowers patient compliance using the s.c. route. Similarly, adverse events resulting from sublingual immunotherapy (i.e., local swelling, itchiness, and gastrointestinal inflammation) possibly raise the therapy discontinuation rate. Administration of vaccines via a patch is advantageous because of the ease of handling and painless application. 18 Vaccines delivered by the transcutaneous route encounter abundant immunerelated cells in the skin, [19][20][21] bypassing metabolic pathways. 22 The outermost layer of the skin is hydrophobic and functions as a barrier to prevent intrusion of extraneous molecules and organisms, making passive diffusion permeability of peptides and proteins quite low. 23,24 To address this issue, we have previously used transcutaneous administration of an arginine-modified 7Crp, 7CrpR, by a unique solid-in-oil (S/O) nanodispersion drug carrier system. 25 The S/O nanodispersion was prepared by lyophilizing a water-in-oil (W/O) emulsions consists of 7CrpR in water and a surfactant in vaporous organic solvents to give a solid paste that was redispersed in an oil vehicle. Dispersing the nanosized solid peptides in an oil vehicle ensured they permeated efficiently across the hydrophobic layer, 26,27 although the therapeutic effect of 7CrpR was unsatisfactory. In this study, we introduce a hydrophobic adjuvant, R848 (Resiquimod, Fig-ure 1), to the 7CrpR S/O nanodispersion system. R848 is a toll like receptor (TLR) 7/8 agonist that acts as an immune response modifier, which shifts the Th1/Th2 immune balance toward Th1-dominant immunity, and is known to alleviate allergic symptoms. 28 2 | E X P ER I M EN T A L

| Animals
Female BALB/c mice (7-week-old) were purchased from Kyudo (Saga, Japan) a week prior to experimentation, and housed at a controlled temperature (

| Preparation of S/O nanodispersions
The peptide 7CrpR (GIIAAYQNPASWKRRRSMKVTVAFNQFGPRRRDI FASKNFHLQKN RRRKLTSGKIASCLNRRRYGLVHVANNNYDPRRRSG KYEGGNIYTKKEAFNVERRRQFAKLTGFTLMG, underlined amino acids are arginine linkers) was produced in Escherichia coli and purified following our previously described method. 25

| Drug release test
The drug release test was performed using custom-fabricated Franztype diffusion cells with an effective diffusion area of 0.785 cm 2

S/O nanodispersions containing 5-CF-7crpR (1 mg/ml in IPM) and
Rho-DOPE (50 lg/ml in IPM) were prepared as previously described. 29 Mouse ear auricles were collected from ddY mice (7-week-old, female, Kyudo) and stored at 280 8C until use. Tissue papers impregnated with S/O nanodispersions (25 ll) were placed onto the dorsal skin of defrosted mouse auricles, tightly sealed in place with adhesive tape to model occlusive patches and incubated at 32 8C for 24 h. After removal of patches, the ear pieces were washed thoroughly with 99% ethanol followed by Milli-Q water, and placed onto glass slides. Fluorescence images were obtained with a confocal laser scanning microscope LSM700 (Carl Zeiss, Oberkochen, Germany), by excitation at 488 nm (5-CF) and at 555 nm (Rhodamine). A series of Z sectioning images was obtained at 5 lm intervals. The images showing 5-CF (green) and Rhodamine (red) were exported as separate jpeg files (8-bit RGB format, 512 pixels 3 512 pixels, each). The RGB pixel values of green and red images were converted to brightness values (G and R, respectively), using a software ImageJ without any image processing.

| Sensitization and immunotherapy
Mice were sensitized to Cj pollen according to our previous report. 25 The Cj pollen extract was dissolved in PBS (100 lg/ml). Cj pollen extract in PBS (100 ll) and Imject Alum (100 ll) were mixed for 30 min and administered to mice by s.c. injection once a week for 3 weeks. Six days after the final s.c. injection, histamine dihydrochlor-  IgE and Cry j 1-specific IgE levels were measured by ELISA using a kit Mouse IgE Ready-SET-Go! (eBioscience) as previously described. 25 A standard serum sample was obtained from a mouse subjected to s.c. injection of a mixture of Cj pollen extract and ImjectAlum once a week for 8 weeks. Each level of Cry j 1-specific IgE and IgG2a in undiluted standard serum was assigned the arbitrarily value of 10,000 relative units (RU)/ml. The results are expressed as the mean 6 standard deviation (n 5 6). A one-way analysis of variance followed by Tukey's test for multiple comparison was used to determine the significance of the data (* p < .05 and ** p < .01) using Prism6 software (Graph Pad Software, La Jolla, CA). c. injection ( Figure 5C). The ratio of Cry j 1-specific IgG2a to Cry j 1specific IgE levels increased by administration of 7CrpR and R848 with the S/O nanodispersion system ( Figure 5D).

| DISCUSSION
The peptide 7CrpR comprises seven human T cell determinants derived from two antigen molecules found in cedar pollen. 32 Tri-arginine linkers were used between each determinant of the 7CrpR, ensuring it dissolved in Milli-Q water but not in the organic solvent IPM without encapsulation in the S/O nanoparticles. We have previously reported that the in vitro skin permeation efficiency of 7CrpR increased fourfold by applying the S/O nanodispersion system compared with that of naked 7CrpR dissolved in PBS. 24 However, only a slight decrease of the IgE level was observed after administration of the peptide alone. In   FIG URE 5 Serum antibody responses after immunotherapy using 7CrpR with or without R848 in the pollinosis model mice. The solid-in-oil nanodispersion system was administered once a week for 3 weeks, and the serum levels of total IgE (A), Cry j 1-specific IgE (B) and Cry j 1specific IgG2a (C) antibodies were measured by ELISA. The ratios of Cry j 1-specific IgG2a to Cry j 1-specific IgE were calculated (D). The data represent the mean 6 SD (n 5 6). *p < .05 the present study, we introduced the adjuvant R848 to the S/O nanodispersion system. R848 is a small (Mw 314.38), imidazoquinoline compound, and is a known TLR 7/8 agonist that efficiently alleviates allergic symptoms by modulating the balance of immune responses in the body. 32 R848 was expected to permeate through the skin efficiently because of its size and moderate hydrophobicity. DLS analysis indicated that the inclusion of R848 to the S/O particles had negligible effect on particle sizes, regardless of whether it was added into the Type I allergies, such as pollinosis, display the distinctive symptom of enhanced production of IgE antibodies in sera, 34

and the serum total
IgE level is often used as a marker of possible allergy. An antigenspecific IgE level could be used to assess whether or not the patient has an allergy to the specific antigen. In our experiments, unprimed mice produced undetectable levels of total IgE in sera. The mice sensitized to the Cj pollen extract produced more than 20 lg/ml of total IgE. After the treatment of the pollinosis model mice with the S/O nanodispersions, the total IgE and the Cry j 1-specific IgE levels in the sera declined, even without R848. The decrease in IgE levels was caused by efficient delivery of 7CrpR to the viable epidermis beneath the stratum corneum using the S/O nanodispersion system. The total and antigen specific IgE levels significantly decreased with the addition of R848, indicating alleviation of allergy.
R848 is recognized by TLR7 and TLR8 in the humans, and by TLR7 in mice, and is known to activate immune cells via the MyD88dependent signaling pathway. 35 Several reports indicated that R848 had the ability to skew the Th1/Th2 balance to Th1-type immunity through the activation of Th1-related lymphocytes. 36,37 Secretion of IL-12, induction of IgG2a and reduction of IgE had been reported after administration of R848, [38][39][40] while T cell epitope vaccine itself also enhances the production of IgG subclass antibodies that are related to the Th1-type immune response. 12 Therefore, we examined the antibody responses in the sera of mice that had been subjected to S/O and R848out S/O nanodispersions. Increases in the IgG2a antibody levels were observed in all the sera collected from the mice treated by S/O patch or s.c. injection, regardless of the peptide administration method. The ratio of Cry j 1-specific IgG2a to Cry j 1-specific IgE levels indicated that the immune response was biased to Th1-type immunity in the mice subjected to 7CrpR. Moreover, the bias of the immune response to the Th1-type immunity was enhanced by the addition of R848. Overall antibody levels decreased in the presence of R848, indicating that the immune response itself might be inhibited by the transcutaneous administration of the adjuvant. These results indicated that both hydrophilic 7CrpR and hydrophobic R848 were efficiently delivered through the skin.

| C O NC LU S I O N S
We have demonstrated the potency of the S/O nanodispersion system for the transcutaneous immunotherapy of Japanese cedar pollinosis.
The peptide vaccine was delivered to the viable epidermis through the stratum corneum. The S/O nanodispersion system was able to carry both a hydrophilic peptide vaccine and a hydrophobic adjuvant simultaneously. Transcutaneously administered T cell epitope peptide in combination with the hydrophobic adjuvant R848 using the S/O nanodispersion system efficiently lowered antigen-specific IgE levels, and shifted the Th1/Th2 immune balance in the pollinosis model mice toward Th1-type immunity.