INDEX
COMPARISON OF SYSTEMIC AND MUCOSAL RESPONSES OF MICE TO ALUMINIUM-ADSORBED DIPHTHERIA TOXOID BETWEEN INTRANASAL ADMINISTRATION AND SUBCUTANEOUS INJECTION

MASANORI ISAKA1), YOKO YASUDA1), TOORU TANIGUCHI1), SATOSHI KOZUKA 2), KEIKO MATANO1), JUN-ICHI MAEYAMA3), KAZUNORI MOROKUMA4), KUNIO OHKUMA4), NORIHISA GOTO3), AND KUNIO TOCHIKUBO1)

1)Department of Microbiology, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-8601, Japan.
2)Department of Nutrition, Faculty of Wellness, Chukyo Women's University, Obu 474-0011, Aichi, Japan.
3)Department of Safety Research on Biologics, National Institute of Infectious Diseases, Gakuen, Musashimurayama, Tokyo 208-0011, Japan. 4)Human Vaccine Production Department, The Chemo-Sero-Therapeutic Research Institute Okubo, Kumamoto 860-8568, Japan

Running title: Intranasal immunization of aDT
Key words: Aluminium compound, Diphtheria toxoid, Intranasal immunization, Mucosal adjuvant
Address for Correspondence: Masanori Isaka, Department of Microbiology , Nagoya City University School of Medicine, Mizuho-ku,Nagoya 467-8601,Japan TEL: +81-52-853-8166, FAX: +81-52-853-4451, E-mail: isaka@med.nagoya-cu.ac.jp

SUMMARY
We investigated the effect of aluminium compound as an adjuvant on systemic and mucosal immune responses of mice vaccinated intranasally and subcutaneously with aluminium-adsorbed diphtheria toxoid (aDT). Intranasal immunization with aDT induced high levels of DT-specific serum IgG and IgA antibody titers and lower levels of DT-specific serum IgE antibody responses in comparison with subcutaneous injection. High or moderate levels of mucosal DT-specific IgA antibody responses were observed in the lung, the nasal cavity, the small intestine, the fecal extract and the vagina. On the other hand, subcutaneous inoculation of aDT elicited high levels of serum DT-specific IgG antibody titers but hardly induced mucosal IgA antibody responses to DT at all mucosal sites examined. These results suggest that aluminium compound may act as a mucosal adjuvant and intranasal administration of aDT seems to be better than its subcutaneous injection with respect to additional protection at the mucosal sites, decreasing IgE mediated allergic reactions, and simpler inoculation procedure.

INTRODUCTION
Most currently available vaccines for humans against bacterial and viral infections contain aluminium compounds as an adjuvant and are usually given by parenteral administration. Parenteral immunization of these vaccines induces systemic immune responses but brings about little or no increase in mucosal immune responses. However, many bacterial and viral pathogens invade their hosts via the large surface area of mucosal membranes, and therefore, the development of vaccines effective in inducing protective immune responses at the mucosal surfaces is desirable. Recently, cholera toxin (CT) produced by Vibrio cholerae (1), the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (2,3,4), genetically detoxified mutants of CT and LT (5) and B subunits of CT and LT (CTB, LTB) (6,7) have been used as effective mucosal adjuvants in animal models. In addition, it has been reported that pertussis toxin, which is use of the major protective antigens of Bordetella pertussis, and its genetically detoxified derivative (8), and synthetic oligodeoxynucleotides containing CpG motifs (9,10) are potent mucosal adjuvants to unrelated antigens.
We have found that intranasal immunization with aluminium-adsorbed tetanus toxoid (aTT) without recombinant CTB (rCTB) induces high levels of tetanus toxoid (TT)-specific serum IgG antibody titers and high or moderate levels of mucosal TT-specific IgA antibody responses, and protects mice against subcutaneous tetanus toxin challenge (6). In this study, to ensure the mucosal immunoadjuvanticity of aluminium compounds, we examined systemic and mucosal immune responses of mice to intranasal immunization with aluminium-adsorbed diphtheria toxoid (aDT) now in use as a current vaccine compared with subcutaneous injection of aDT.

MATERIAL AND METHODS
2.1. Animals, immunogens, immunization and sample collection
All procedures performed on animals were conducted according to the Guideline for the Care and Use of Laboratory Animals of the Nagoya City University Medical School under protocols approved by the Institutional Animal Care and Use Committee at the Nagoya City University Medical School. Female BALB/c mice (SLC, Shizuoka, Japan) aged 7 weeks were used in this study. Each group consisted of five mice. Aluminium-adsorbed diphtheria toxoid (aDT) containing 25 Lf units ml-1 and aluminium-non-adsorbed diphtheria toxoid (nDT) containing 200 Lf units ml-1 (67 mg protein nitrogen ml-1, purity; 2,985 Lf mg-1 protein nitrogen) were provided by the Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan); nDT was used for an enzyme-linked immunosorbent assay (ELISA). Mice were immunized in two ways: (i) mice were intranasally immunized with 30ml of solution containing 3 Lf aDT under light ether anesthesia, and (ii) subcutaneously immunized with 100ml of solution containing 3 Lf aDT. Both intranasal and subcutaneous immunizations were performed on days 0, 14, 21 and 28, and mice were sacrificed on day 35. Collection of lung and nasal cavity lavages, small and large intestinal washes, saliva and vagina secretions, blood and fecal extracts was carried out as described before (6,11). These samples were stored at -20 until use for ELISA.

2.2. Measurement of antibody levels by ELISA
DT-specific IgG , IgG subclass and IgA antibody titers in serum were measured using ELISA as described previously (11). The measurement of mucosal IgA antibody titers in washing samples of lung, nasal cavity and small and large intestines, saliva and vaginal secretions were also carried out as previously described (11) except for the use of biotinylated goat anti-mouse IgA antibody as the second antibody and streptavidin-b-galactosidase. Briefly, after serial dilutions, samples were added to the wells coated with DT (1mg ml-1), incubated overnight at 4 and washed with PBS containing 0.05% Tween 20 (PBS-Tween). Fifty ml of a 2000-fold dilution of biotinylated goat anti-mouse IgA antibody (Southern Biotechnology, Birmingham, AL) in Tris-buffered saline, pH 7.5 (TBS) containing 0.1% bovine serum albumin (BSA) were added to each well and incubated for 2 h at room temperature. After five washings with PBS-Tween, 50 ml of a 1000-fold dilution of b-galactosidase-conjugated streptavidin (Southern Biotechnology) in TBS containing 0.1% BSA were added to each well as the detection enzyme, incubated for 1 h at room temperature and washed five times with PBS-Tween. The color was developed for 1 h at room temperature using 50ml each of 1 mg ml-1 o-nitrophenyl-b-D-galactopyranoside (Southern Biotechnology) in 10 mM Tris buffer, pH 7.5, containing 12 mM NaCl and 5 mM MgCl2. The reaction was stopped by adding 150 mM EDTA and read at 405 nm on an automatic microplate reader (Bio-Rad Laboratories, Richmond, CA). The mean and standard deviation (SD) of values at 450 and 405 nm were calculated with sera and each washing sample of five nonimmunized mice. Antibody-positive cut-off values were set as the mean + threefold SD of negative control. ELISA anti-DT antibody titers were expressed as the highest endpoint dilution of each sample giving the positive reaction and shown as geometric mean (GM) SD.

2.3 Measurement of antigen-specific mouse IgE by a fluorometric capture ELISA
DT-specific IgE antibody was detected by IgE caputer ELISA as described by Sakaguchi et al (12) except the use of 5-[5-(N-succinimidyloxycarbonyl) pentylamido] hexyl D-biotinamide (Dojindo Laboratories, Kumamoto, Japan) in place of N-hydroxysuccinimidobiotin to conjugate DT with biotin.

2.4 Statistics
Analysis of antibody titers was performed on logarithmically transformed data, and the GM and SD were calculated. Mann-Whitney's U-test was used to compare mean values of different groups with serum and mucosal anti-DT antibody titers. Statistical significance was designated as p<0.01 or p<0.05.

Results
3.1. Serum DT-specific antibody responses to intranasally and subcutaneously administered aDT
Intranasal administration of aDT showed high levels of DT-specific serum IgG antibody titers in all mice and high or moderate levels of DT-specific serum IgA antibody titers (Fig. 1). Subcutaneous injection of aDT also elicited high levels of DT-specific serum IgG antibody responses in all mice but DT-specific serum IgA antibody responses were very low (Fig. 1). The major subclass of serum IgG antibody responses to aDT was IgG1 antibodies followed by IgG2b antibodies with intranasal immunization and there were equally high levels of serum DT-specific IgG1 and IgG2b antibodies with subcutaneous injection (Fig. 2). DT-specific serum IgE antibody responses were observed in both administration methods but intranasal immunization showed much lower DT-specific IgE responses than subcutaneous injection (Fig. 3).

3.2. Mucosal DT-specific immune responses to intranasally and subcutaneously administered aDT
We next examined mucosal immune responses to intranasal administration of aDT and subcutaneous immunization of aDT. Intranasal immunization of aDT showed high levels of IgA antibody responses to DT in the lungs of all mice and high or moderate levels of DT-specific IgA antibody responses in the vaginal secretions of all mice (Fig. 4). Moderate levels of mucosal DT-specific IgA responses were observed in the small intestines of all mice, in the fecal extracts of four mice and in the nasal cavities of two mice (Fig. 4). No anti-DT IgA antibody was detected in the saliva of all mice (Fig. 4). On the other hands, subcutaneous injection of aDT did not induce mucosal DT-specific antibodies at all mucosal sites examined (Fig. 4). From these results, the mucosal adjuvanticity of aluminium present in aDT was confirmed.

Discussion
Subcutaneous injection of aDT elicited high levels of DT-specific serum IgG antibody titers but hardly induced mucosal IgA antibody responses to DT at all mucosal sites, whereas intranasal administration of aDT resulted in high titers of serum DT-specific IgG antibody responses and high or moderate titers of mucosal DT-specific IgA antibody responses (Figs. 1 and 4). As shown in our previous study, intranasal administration of nDT alone could not induce systemic and mucosal antibody responses to DT at all (11). These results are almost consistent with those obtained from intranasal immunization with aTT or nTT alone (6) and suggest strong mucosal adjuvanticity of the aluminium compound.
It is generally considered that antitoxin titer of 0.1 international units (IU) ml-1 is the smallest level necessary to protect man from a challenge of diphtheria toxin. When mice intranasally immunized with 5 Lf nDT plus rCTB showed high levels of DT-specific serum IgG antibody titers, they gave sufficiently high antitoxin titers greater than 0.1 IU ml-1 (11). In this experiment, both groups of mice administered intranasally and subcutaneously showed serum antitoxin titers higher than 0.1 IU ml-1 in all mice (data not shown).
The major serum IgG subclass responding to DT by intranasal immunization of aDT was IgG1 followed by IgG2b (Fig. 2) in the same manner as serum IgG subclass responses to DT by that of nDT plus rCTB (11), indicating a Th2-biased response. Intranasal administration of aDT induced high or moderate levels of mucosal DT-specific IgA antibodies especially in the lung, the small intestine and the vagina (Fig. 4), whereas that of nDT with rCTB did at all mucosal sites examined (11).
Corynebacterium diphtheriae is mainly remaining in the superficial layers of the respiratory mucosa, vagina on rare occasions, where it can induce a inflammatory reaction by the action of diphtheria toxin in the local tissue. Heart and peripheral nerves are damaged by diphtheria toxin which penetrates through the mucosal membrane into general circulation. Production of high titers of DT-specific IgA antibody at the lung and vagina and IgG in the serum may be useful to prevent diphtheria toxin from inflammatory reaction at the mucosal sites and in the blood, respectively .
DT-specific serum IgE antibody responses were observed in both intranasal and subcutaneous routes but were much higher in all mice inoculated subcutaneously compared with those administered intranasally (Fig. 3), coinciding with the idea that lymphoid tissue stimulated by the subcutaneous route is under different control of immune responses from the nasopharyngeal lymphoid tissue stimulated by intranasal administration (13). In addition to route of administration, types of adjuvants are also important for serum IgE antibody response, because higher levels of TT-specific serum IgE antibody were induced in mice vaccinated intranasally or subcutaneously with aTT alone in comparison with intranasal or subcutaneous inoculation of aluminium-non-adsorbed TT (nTT) and rCTB which showed no or slight level of serum TT-specific IgE antibody response (14). Moreover, there is a report that both eosinophilopoiesis and IgE production increase with aluminium adjuvant and only eosinophilopoiesis occurs with Freund's complete adjuvant (15).
In addition to high IgE production, aluminium adjuvants have been reported to occasionally bring about subcutaneous nodules, granulomatous inflammation, and sterile abscesses after their intramuscular injection (16-20). And intranasal administration of aluminium compound occasionally induces irregular arrangement in the epithelium of mice (21). On the other hand, rCTB produced by Gram-positive bacterium Bacillus brevis acts as a mucosal adjuvant (6,11,22) and elicits no toxic effects to macrophages, no vascular permeability-increasing effects and no local histopathological reactions in the nasal cavity, the small-intestinal loop or the muscle given rCTB (21).
Our study showed that induction of mucosal DT-specific IgA antibody responses occurred in the intranasal route but not in the subcutaneous route and, on the contrary, higher levels of serum DT-specific IgE antibody titers were induced by the subcutaneous route than by the intranasal route. Mucosal gd T cells may play an important role in the regulation of serum IgE and mucosal IgA immune responses because the level of IgA in fecal extracts is low in mutant mouse lacking gd T cells (23, 24); the mucosal immune system of the mutant mice contains lower numbers of IgA antibody producing cells than these of control mice (23, 24), and gd T cells down-regulate primary IgE responses in rats and mice to inhaled soluble protein antigen (25, 26). An increase in serum IgA antibody responses to aDT given intranasally may be related to that in mucosal IgA antibody responses (Figs. 1 and 4).
Results from this study suggest that aluminium compound may act as an adjuvant for mucosal immunization strategies and intranasal administration of aDT seems to be better than its subcutaneous injection with respect to additional protection at the mucosal sites, decreasing IgE mediated allergic reactions, and simpler inoculation procedure.

Acknowledgements
The authors thank Dr. Roy H. Doi, Section of Molecular and Cellular Biology, University of California, Davis for his critical reading of the manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research(C)10670266 from the Ministry of Education, Science, Sports and Culture, Japan and was partially supported by a grant from the Japan Health Sciences Foundation (Research on Health Sciences focusing on Drug Innovation, No. 51138).

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FIGURE LEGENDS
Fig. 1. Serum DT-specific IgG and IgA antibody responses to aDT administered intranasally (IN) and injected subcutaneously (SC). DT specific IgG and IgA antibody in serum were measured by ELISA. Antibody-positive cut-off values were set as the mean + threefold SD of negative control . Anti-DT antibody titers were expressed as the highest endpoint dilution of each sample giving the positive reaction and shown as geometric mean (GM) SD. Each circle represents data for an individual animal. Statistically significant differences , * p<0.01
Fig. 2. Serum DT-specific IgG subclass responses to aDT administered intranasally (IN) and subcutaneously (SC). IgG subclass : () IgG1 ; () IgG2a ; ( IgG2b.
DT specific IgG subclass antibodies were measured by ELISA. Antibody titers were expressed as the highest endpoint dilution of each sample giving the positive reaction and shown as geometric mean (GM)SD. Symbols of circle ,square and triangular represent IgG1, IgG2a and IgG2b, respectively.
Fig. 3. Serum DT-specific IgE antibody responses to aDT administered intranasally (IN) and subcutaneously (SC). Antibody titers were expressed as the highest endpoint dilution of each sample giving the positive reaction and shown as geometric mean (GM)SD. Each circle represents data for an individual animal. Statistically significant differences , * p<0.01
Fig. 4. Mucosal DT-specific IgA antibody responses to aDT administered intranasally (IN) and subcutaneously (SC). Antibody titers were expressed as the highest endpoint dilution of each sample giving the positive reaction and shown as geometric mean (GM)SD. Each circle represents data for an individual animal. Statistically significant differences , * p<0.01 , **p<0.05

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