Evaluation of the Effectiveness and Safety of Chitosan Derivatives as Adjuvants for Intranasal Vaccines

Abstract

Intranasal immunization is currently used to deliver live virus vaccines such as influenza. However, to develop an intranasal vaccine to deliver inactivated virus, a safe and effective adjuvant is necessary to enhance the mucosal immune response. Here, we demonstrate the effectiveness of a chitosan microparticle (1–20 μm, 50 kDa, degree of deacetylation=85%) and a cationized chitosan (1000 kDa, degree of deacetylation=85%) derived from natural crab shells as adjuvants for an intranasal vaccine candidate. We examined the effectiveness of chitosan derivatives as an adjuvant by co-administering them with ovalbumin (OVA) intranasally in BALB/c mice, polymeric Ig receptor knockout (pIgR-KO) mice, and cynomolgus monkeys (Macaca fascicularis). pIgR-KO mice were used to evaluate S-IgA production on the mucosal surface without nasal swab collection. Administration of OVA with chitosan microparticles or cationized chitosan induced a high OVA-specific IgA response in the serum of pIgR-KO mice and a high IgG response in the serum of BALB/c mice and cynomolgus monkeys. We also found that administration of chitosan derivatives did not have a detrimental effect on cynomolgus monkeys as determined by complete blood count, blood chemistries, and gross pathology results. These results suggest that chitosan derivatives are safe and effective mucosal adjuvants for intranasal vaccination.

Introduction

T he mucosal immune system is usually the first immunological barrier against infectious agents. The respiratory tract and vaginal mucosa are primary sites of infection and are the immunological compartments where the host immune system first comes into contact with a virus (8,19). Secretory IgA (S-IgA) antibodies are major effectors, providing a frontline of defense against viruses at the mucosal surface (19,23,26,27). Whereas most vaccines are given parenterally, and thus cannot stimulate mucosal immunity, mucosal immunizations stimulate both mucosal and systemic immunities, and antigen-specific mucosal immune responses can be expressed at all mucosal sites (5,8,9). Furthermore, the inoculation procedure is simple, reliable, and cheap, without the need for needles.

However, the mucosal immune response to protein antigen alone is weak, if present at all, thereby requiring a mucosal adjuvant that can sufficiently enhance mucosal antigen-specific IgA antibody responses for mucosal immunization (4,8,25,27). A study on influenza has shown that the mucosal immunity acquired by natural infection, primarily due to S-IgA in the respiratory tract, is more effective and cross-protective against viral infection than the systemic immunity induced by parenteral vaccines in humans and mice (26,27). In this regard, induction of S-IgA in the respiratory tract is advantageous for protection against unpredictable infections.

In developing intranasal (i.n.) vaccines, cholera toxin (CT) and Escherichia coli heat-labile toxin (LT) have been used as adjuvants to enhance mucosal immune responses. Although CT and LT are effective adjuvants, they have some side effects, including nasal discharge and Bell's palsy, in humans (18). Therefore, other adjuvants that are as effective as CT or LT but safer for human use have been developed for clinical application with i.n. vaccines. These include chitin microparticles, mutant CT, complement C3d, and synthetic double-stranded RNA (1,2,4,6,10,13,15,16,23,25). There are few mucosal vaccines currently available for human use, two of which are the oral polio vaccine and a nasal cold-adapted influenza virus vaccine, Flumist^®, both of which are live attenuated viral vaccines. The nasal enterotoxin-adjuvant inactivated influenza vaccine was introduced in the market, but withdrawn after a short time due to a serious adverse reaction, namely facial paresis (18). Therefore, further work is needed to develop adjuvants for mucosal vaccines for human use.

Over 25 years ago, chitin derivatives were found to be potent activators of macrophages and NK cells (20,21). Nishimura and colleagues used various chitin derivatives with antigen to measure adaptive immune response (22). Their studies indicated that deacetylated chitosan had adjuvant activity on antigen-specific serum antibody titers in mice and guinea pigs upon intraperitoneal (i.p.) injection (22).

In the present study, we demonstrate the effectiveness of chitosan derivatives as mucosal adjuvants in BALB/c and polymeric Ig receptor knockout (pIgR-KO) mice, and cynomolgus monkeys (Macaca fascicularis), through measurement of IgA and IgG after i.n. administration of OVA and chitosan derivatives. We also examined the safety of chitosan derivatives through complete blood counts, blood chemistries, and gross pathology in cynomolgus monkeys.

Materials and Methods

Mice

Female specific pathogen-free BALB/c mice were obtained from Shizuoka Agricultural Cooperative Association for Laboratory Animals, Hamamatsu, Japan. Mice were 6 weeks old at the time of immunization.

pIgR transgenic KO mice were generated as previously described (3,24). Heterozygous mice were generated by mating male chimeric mice and female BALB/c mice, and F₁ mice were backcrossed with BALB/c mice 8 times (N8). Some experiments were conducted using littermates of the N8 or N9 generation. Additional experiments were conducted by intercrossing pIgR-KO mice with homozygous mice from the N10 generation. To measure secretory IgA in sera, pIgR-KO mice were used in this study without sampling by a nasal swab.

Monkeys

Female cynomolgus monkeys (aged 5–10 years) obtained from Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Ibaraki, Japan, were used in this study.

All animal studies were carried out in accordance with the Guides for Animal Experiments performed at National Institute of Infectious Diseases (NIID) and were approved by the Animal Care and Use Committee of the NIID.

Ovalbumin

Two micrograms of ovalbumin (OVA) (5×crystallized, Seikagakusha Co. Ltd., Japan) was used for each mouse immunization. The product details did not contain endotoxin data of the OVA.

Preparation of chitosan derivatives and control adjuvants

Raw chitin from natural crab shells or chitosan derivatives (Dainichiseika Color and Chemicals Mfg. Co. Ltd., Tokyo, Japan) were modified chemically or mechanically to obtain Derivatives A-N listed below.

Derivative A: The chitosan (100 kDa, degree of deacetylation=100%) was refined and adjusted to 1% in HCl solution. The pH was adjusted to 6.1 with NaOH and filtered through with a 0.45 μm membrane filter.

Derivative B: The chitosan (100 kDa, degree of deacetylation=100%) was refined and adjusted to 1% in acetic acid solution. The pH was adjusted to 6.2 with NaOH, and the solution was filtered through with a 0.45 μm membrane filter.

Derivative C: The chitosan (50 kDa, degree of deacetylation=100%) was refined and adjusted to 1% in HCl solution. The pH was adjusted to 6.1 with NaOH, and the solution was filtered through a 0.45 μm membrane filter.

Derivative D: The chitosan (50 kDa, degree of deacetylation=100%) was refined and adjusted to 1% in acetic acid solution. The pH was adjusted to 6.2 with NaOH, and the solution was filtered through with a 0.45 μm membrane filter.

Derivative E: The chitosan (500 kDa, degree of deacetylation=67%) was refined and adjusted to 1% in HCl. The pH was adjusted to 5.8 with NaOH, and the solution was filtered through a 16 μm glass filter.

Derivative F: The chitosan (500 kDa, degree of deacetylation=67%) was refined and adjusted to 1% in acetic acid solution. The pH was adjusted to 5.8 with NaOH, and the solution was filtered through a 40 μm glass filter.

Derivative G: A 2.5% chitin solution was prepared by dissolving the chitin in HCl at 37°C for 30 min. The solution was then combined with cold (5°C) distilled water and precipitated overnight at 5°C.The precipitated sample was filtered through Grade No.2 filter paper (Toyo Roshi Kaisha, Japan). Colloidal chitin particles of 1–2 μm diameter were prepared by sonication and adjusted to a 1% suspension with distilled water.

Derivative H: A 6.7% chitin solution was prepared by dissolving the chitin in HCl at room temperature (RT) for 120 min. The solution was then combined with distilled water at RT. The solution was then centrifuged, and precipitated particles were washed with distilled water at pH 5.0. Colloidal chitin particles of approximately 500 nm were prepared by sonication and adjusted to a 1% suspension with distilled water.

Derivative I: A 6.7% chitin solution was prepared by dissolving the chitin in HCl at RT for 120 min. The solution was then combined with distilled water and centrifuged and precipitated particles were washed with distilled water at pH 5.0. Colloidal chitin particles of approximately 500 nm diameter were prepared by coagulation and adjusted to a 1% suspension with distilled water.

Derivative J: Chitin microparticles of 1–20 μm diameter were obtained by crushing the chitin with a machine. The chitin was distributed by sonication and adjusted to a 1% suspension with distilled water.

Derivative K: The chitosan (100 kDa, degree of deacetylation=72%) was refined and adjusted to 1% in an acetic acid solution. The pH was adjusted to 6.3 with NaOH, and the solution was filtered through a 0.45 μm membrane filter.

Derivative L: Chitosan microparticles of 1–20 μm were obtained by the dry crushing of chitosan (50 kDa degree of deacetylation=85%). The obtained chitosan particles were distributed by sonication and adjusted to 0.5% to 5% solutions, L-0.5, L-1, L-2, L-3, and L-5 with distilled water.

Derivative M: Chitosan microparticles of approximately 4 μm diameter were obtained by H₂SO₄ hydrolysis of the chitosan (1500 kDa, degree of deacetylation=90%). The chitosan microparticles were distributed by sonication and adjusted to 1% and 2% suspensions, M-1 and M-2, with distilled water.

Derivative N: A chitosan derivative (cationized chitosan) was obtained by introducing glycidyl trimethylammonium chloride into the chitosan (1000 kDa, degree of deacetylation=85%). The cationized chitosan was dissolved with citric acid and adjusted to 0.125%–2% solution, N-0.125, N-0.25, N-0.5, N-1, and N-2, and then filtered through a 0.45 μm membrane filter.

WAKO-500: This chitosan was obtained commercially (Wako Pure Chemical Ltd., Tokyo, Japan) and dissolved in acetic acid to obtain 0.1% and 1% solution WAKO-500-0.1 and WAKO-500-1.

Cholera toxin: Cholera toxin B subunit (CTB) containing a trace amount of holotoxin (CTB*) was prepared by adding 0.1% holotoxin to CTB (Sigma, St. Louis, MO).

Immunization

Five mice in each experimental group were anesthetized with diethyl ether and primarily immunized i.n. by dropping 5–7 μL of PBS containing OVA (2 μg) and a chitosan derivative (100 μg) or CTB* (2 μg) into each nostril. Immunization was repeated 3 weeks after the initial administration. Additional immunization (booster) was conducted at different time points in some experiments. Some inoculums included twice the dose of OVA, as indicated. Dose-dependent adjuvanticity was evaluated for some chitosan derivatives by using a range of 6.25–250 μg.

Cynomolgus monkeys were anesthetized with ketamine and primarily immunized i.n. by dropping 500 μL of PBS containing either OVA (300 μg) alone (n=1), OVA (300 μg) and 1 mg of chitosan microparticles (derivative L) (n=2), or OVA (300 μg) and 1 mg of cationized chitosan (derivative N) (n=2). Immunization was repeated at 3 and 21 weeks after initial administration. Serum, vaginal wash, urine, and fecal samples were collected to detect the specific antibodies against OVA. The following parameters were monitored: white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, white blood cell:small cell ratio, white blood cell:large cell ratio, white blood cell:small cell count, white blood cell:large cell count, red blood cell distribution width, platelet distribution width, mean platelet volume, platelet:large cell ratio, total protein, albumin, albumin/globulin ratio, blood urea nitrogen, glucose, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, lactate dehydrogenase, alkaliphosphatase, C-reactive protein, creatinine, sodium chloride, potassium chloride, and r-GTP.

Measurement of anti-OVA antibodies

Sera were collected from mice under anesthesia with diethyl ether at various time points to measure the levels of anti-OVA antibodies. The levels of IgA and IgG against OVA were determined by ELISA. Briefly, ELISA was performed sequentially from the solid phase (EIA plates; Costar, Cambridge, MA; 3690 A/2 or SUMILON, ELISA Plate S MS-8496F, Tokyo, Japan) with a ladder of reagents as follows: first, OVA from chicken eggs (Seikagakusha, Tokyo, Japan); second, serum; third, either goat anti-mouse IgA (KPL Inc., Gaithersberg, MD) or goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) conjugated with biotin; fourth, streptavidin conjugated with alkaline phosphatase (Invitrogen, Grand Island, NY); and fifth, p-nitrophenylphosphate (KPL). The chromogen produced was determined by measuring the absorbance at 405 nm using an ELISA reader (Bio-Rad Laboratories, Inc, Hercules, CA). The binding kinetics of the standard OVA monoclonal IgG was compared with that of OVA IgG from immunized mice. Serum was also collected from cynomolgus monkeys at various time points, as indicated. Anti-OVA antibodies against IgA and IgG were analyzed by ELISA with anti-monkey IgA or IgG (Rockland Immunochemicals, Gilbertsville, PA), following the same procedure described for the mice above. Serum dilutions in this ELISA procedure, ×100 for Mouse IgG, ×80 for KO-mouse IgA, ×200 for Monkey IgG, and ×20 Monkey IgA were used.

Monoclonal anti-chicken egg albumin (8.1 mg/mL IgG1, clone OVA-14, Sigma) was used as a positive control. Strong positive reaction was observed with ×20,000 dilution. In this study, OD value was measured at the same time for the collected samples in order to compare the antibody uptake over the experimental period.

Electron microscopy

A scanning electron microscope (Model S-5200, Hitachi, Japan) was used to observe the structure of the chitosan microparticles. The sample was coated with osmium using a Neoc-ST osmium plasma coater (Meiwaforsis, Osaka, Japan). The size of the particles was measured using a scale bar.

Effect of intranasal treatment of adjuvants

Six-week-old BALB/c female mice were inoculated i.n. with 200 μg chitosan microparticles, 200 μg cationized chitosan, and 2 μg CTB* in 10 μL of PBS, and body weights were monitored for 10 days. PBS was used as a negative control.

Histopathological examination

Excised nasal tissues were fixed with 10% neutral-buffered formalin, and the tissues were then decalcified in EDTA solution. After fixation, the tissues were embedded in paraffin by conventional methods and stained with hematoxylin-eosin (H&E).

Statistical analysis

Standard error (SE) was calculated for OD values of antibody response measured by ELISA for all experiments.

Results

Antibody responses against OVA in BALB/c mice immunized intranasally with chitosan derivatives as adjuvants

BALB/c mice were immunized three times i.n. with 2 μg of OVA and 100 μg of the various chitosan derivatives, A to N, commercially available chitosan (WAKO-500-1), CT (positive control), or OVA alone (negative control). Sera samples were collected sequentially from the mice after the third immunization. The activities of anti-OVA IgG in the sera of the immunized mice are summarized in Fig. 1A–D. High activity levels of serum IgG were observed in mice treated with derivatives B, C, D, E, K, L, and WAKO-500-1. After booster immunization, OD values quickly escalated for some derivatives (Fig. 1B–D). There were no detectable levels of specific antibody in control mice immunized with OVA alone. Immunization and additional immunization schedule are indicated in figure legends.

FIG. 1.

Anti-OVA-specific IgG antibody response in BALB/c mice. BALB/c mice were immunized intranasally, then immunization was repeated at 3 weeks; additional immunization (indicated by the arrow) was also conducted. Sera collected at the indicated times from each group were assayed for specific IgG levels by ELISA. Data are indicated as an average of absorbance (Abs) for 5 mice. Pre-immunization sera from mice had no detectable specific IgG. Data represent the mean±SE. (A) Chitosan derivatives A–F, WAKO-500-1, and WAKO-500-0.1. (B) OVA (negative control), chitosan derivatives G–L, and cholera toxin (positive control). Additional immunization was done week 18. (C) Chitosan derivatives H, J, L, and H–W, J–W, L–W, which are twice the dose of H, J, and L, respectively, and OVA (negative control). Additional immunization was done week 18. (D) Chitosan derivatives M-1 (1%), M-2 (2%), and N (0.125%–2%), and OVA (negative control). Due to the long color developing process for ELISA, OD values of B and C are higher than the other values. Additional immunization was done in week 13.

Antibody responses against OVA in pIgR-KO mice immunized intranasally with chitosan derivatives as adjuvants

The effect of chitosan derivatives as an adjuvant of i.n. immunization with OVA was investigated in pIgR-KO mice. The mice were immunized three times i.n. with 2 μg of OVA and100 μg of the various chitosan derivatives, A to N, commercially available chitosan (WAKO-500-1), CT (positive control), or OVA alone (negative control). Sera samples were collected sequentially from the mice after the third immunization. The activity of anti-OVA IgA in the serum of the immunized mice is summarized in Fig. 2C and Fig. 3A–C. Relatively high activity levels of serum IgA were observed in mice treated with the derivatives B, C, N, and L. After booster immunization, OD values escalated for B, L–W, and N. There were no detectable levels of any specific antibody in control mice immunized with non-adjuvanted OVA.

FIG. 2.

Dose-dependent antibody response against chitosan derivatives in BALB/c mice and pIgR-KO mice. BALB/c mice and pIgR-KO mice were immunized intranasally, then immunization was repeated at 3 weeks; additional immunization (indicated by the arrow) was also conducted. Sera samples were collected at the indicated times from each group and were assayed for specific IgG and IgA by ELISA. Data are indicated as an average of absorbance (Abs) for 5 mice. Data represent the mean±SE. (A) IgG response in BALB/c mice administered derivative L (0.5%–5%) and OVA (negative control). Additional immunization was done week 13. (B) IgG response in pIgR-KO mice administered derivative N (0.125%–2.0%), OVA (negative control), and cholera toxin (positive control). (C) IgA response in pIgR-KO mice administered derivative N (0.125%–2.0%), OVA (negative control), and cholera toxin (positive control). Additional immunization was done week 13.

FIG. 3.

Anti-OVA-specific IgG antibody response in pIgR-KO mice. pIgR-KO mice were immunized intranasally, then immunization was repeated at 3 weeks; additional immunization (indicated by the arrow) was also conducted. Sera samples were collected at the indicated times from each group and were assayed for specific IgGs by ELISA. Data are indicated as the average of absorbance (Abs) of 5 mice. Data represent the mean±SE. (A) Chitosan derivatives A–F and WAKO-500-1. (B) Chitosan derivatives G–L. Additional immunization was done week 18. (C) Chitosan derivatives H, J, and L, and H–W, J–W, and L–W, which are twice the dose of H, J, and L, and each derivative. Additional immunization was done week 18.

Dose-dependent increase in antibody response with chitosan derivatives

Based upon the data obtained from the BALB/c mice experiments, derivative L, a chitosan microparticle, and derivative N, a cationized chitosan, were evaluated for their dose-dependent adjuvanticity in BALB/c mice and/or in pIgR-KO mice, respectively, with the same immunization protocol previously described in this study. As shown in Figure 2A and B, dose-dependent antibody uptake of IgG antibody was clearly demonstrated with both derivatives L and N. However, a 5% solution of derivative L, a chitosan microparticle, showed a similar antibody uptake to a 1%–3% solution of the same derivative. Dose-dependent antibody uptake was also observed for 0.125%–2% solutions of cationized chitosan. IgA response was also evaluated for cationized chitosan (Fig. 2C). Although the level of antibody response was lower than that for cholera toxin, after booster immunization, a dose-dependent antibody response was observed for cationized chitosan. There were no detectable levels of specific antibody in control mice immunized with OVA alone.

Antibody responses against OVA in cynomolgus monkeys immunized intranasally with chitosan derivatives as adjuvants

The effect of chitosan derivatives as an adjuvant of i.n. immunization with OVA was investigated in cynomolgus monkeys. The monkeys were immunized three times i.n. with 300 μg of OVA with chitosan derivatives, L and N, both of which demonstrated good adjuvanticity in our mouse experiments. Derivatives were used at a concentration of 1 mg/animal for immunization and were boosted 21 weeks after initial administration. Sera samples were collected sequentially from the monkeys after the third immunization. As shown in Figure 4A and B, IgG and IgA antibodies against OVA were clearly detected after the booster immunization. One monkey immunized with chitosan microparticles (derivative L-2.(1)) showed a low response compared with the control monkey. Anti-OVA IgG responses occurred faster in the groups immunized with chitosan microparticles and cationized chitosan after the second immunization. These results suggest that i.n. administration of OVA with chitosan microparticles or cationized chitosan as an adjuvant induces specific antibodies as seen in mice. No antibodies were detected in the urine, fecal, and vaginal wash (data not shown).

FIG. 4.

Anti-OVA-specific IgG and IgA antibody responses in cynomolgus monkeys. Cynomolgus monkeys were immunized intranasally, then immunization was repeated at 3 weeks; additional immunization (indicated by the arrow) was also conducted. Sera samples were collected at the indicated times from each monkey and assayed for specific IgGs and IgAs by ELISA. (A) IgG response for derivatives L and N, and OVA (negative control). Additional immunization was done week 20. (B) IgA response for derivatives L and N, and OVA (negative control). Additional immunization was done week 20.

The safety of chitosan microparticles and cationized chitosan was monitored by biochemical examination of sequential blood samples. There were no significant changes in complete blood counts or blood chemistries compared with the control monkey (OVA alone). These data were always within the normal range of age-matched cynomolgus monkeys. There were also no significant changes in the body weight of monkeys in any of the groups treated with chitosan microparticles or cationized chitosan as compared with the negative control (data not shown).

Upon necropsy, no gross pathological changes were observed in the heart, lung, stomach, spleen, liver, or kidney in the i.n. immunization groups or in the control group. These results suggest that i.n. administration of chitosan microparticles are not harmful to cynomolgus monkeys (data not shown).

Morphological observations of adjuvant candidates

Scanning electron micrograph images of chitosan microparticles show that the particles were rough and spherical in appearance. The arithmetic mean diameter of chitosan derivative L was 4 μm (Fig. 5).

FIG. 5.

Scanning electron micrograph images of chitosan derivative L (5000x). The sample was coated with osmium with a model Neoc-ST osmium plasma coater (Meiwaforsis, Osaka, Japan). The size of the particles was measured using a scale bar.

Safety of intranasal administration of the adjuvant

To examine the safety of chitosan derivatives in intranasal administration, 200 μg of chitosan microparticles, 200 μg of cationized chitosan, and CTB* were intranasally administered to mice daily for 10 days. The body weight of the mice was not significantly changed (Fig. 6). Histopathological examination revealed that the nasal areas of the mice administered with chitosan microparticles had no pathological changes, similar to those of the PBS-treated mice. On the other hand, mucus exudation with inflammatory cells was identified in the nasal areas of the mice treated with CTB* and cationized chitosan (Fig. 6).

FIG. 6.

(A) Body weight of mice after intranasal administration of chitosan derivative L (200 μg/day), chitosan derivative N (200 μg/day), and CTB*(2 μg/day) for 10 days. Each point represents the relative ratio to initial body weight (mean±SE) of mice on each day. (B) Histpathological findings of the mice intranasally administered with 200 μg of chitosan derivative L, 200 μg of chitosan derivative N, 2 μg of CTB*, and PBS daily for 10 days (×100; H&E). Black arrows indicate the mucus exudation with inflammatory cells.

Discussion

The present study results clearly demonstrate that chitosan microparticles and cationized chitosan are effective as mucosal adjuvants when administered i.n. with the antigen OVA. It has been reported that effective immunization strategies to protect against virus infection involve the induction of mucosal immune responses at the nasal mucosal epithelium, the initial target of viral infections. To provide effective protection against viral infection at the mucosa, bacterial toxin-derived adjuvants such as CT have been administered in combination with vaccines. To reduce the toxicity of bacterial toxin-derived adjuvants, mutant toxins or low doses of CTB* have been applied in experimental animal models (27). However, the use of these adjuvants in human vaccination strategies is still somewhat problematic. Thus, there is a need for an effective and safe adjuvant for i.n. vaccination in humans. This report presents new adjuvant candidates for i.n. vaccination without the use of bacterial toxins or derivatives. A major objective of i.n. vaccine development is to design an adjuvant that can provide both effective immune activity and that is safe for clinical application in humans. Chitosan microparticles and cationized chitosan are adjuvants derived from a natural, non-microbial source that induce anti-OVA antibodies when administered i.n. with OVA using a three-dose immunization protocol. Nasal immunization resulted in not only an increase in mucosal S-IgA but also a high level of anti-OVA IgG in the serum. These data showed that chitosan derivatives enhanced humoral immunity.

This study also showed that in the pIgR-KO mice, the blockade of transepithelial transport of dIgA by pIgR resulted in IgA accumulation in the serum. In the pIgR-KO mice immunized with the chitosan derivatives, a higher concentration of the OVA-reactive IgA in the serum indicated the blockade of the pIgR-mediated transcytosis of pIgA.

In this study, IgG1 antibody were dominant (Th-2 type) when OVA was administrated i.n. with chitosan microparticles and cationized chitosan (data not shown). Also when recombinant HIV-1 env protein was administrated i.n. with cationized chitosan, the env specific antibody was detected by a particle agglutination assay (PA genedia HIV-1/-2 Fujirebio Inc. Tokyo Japan) (data not shown).

The i.n. route of vaccination is advantageous for protection against viral infection due to the induction of S-IgA in the mucosal epithelium, which elicits a more effective cross-protective immunity compared with that induced by serum IgG in mouse influenza studies (11,12,14). The mechanism of the adjuvant effect of chitosan derivatives remains unclear. However, chitosan microparticles that are 1–20 μm in diameter may be the targets of phagocytosis by macrophages and dendritic cells. Prophylactic agents, including vaccines, must be proven sufficiently safe for clinical use.

Recent research has suggested that immune cell recruitment is the main mechanism underlying adjuvant actions in general, and that aluminum salts induce this recruitment via inflammation at the injected site (28). We also attempted to use the chitosan derivative for i.p administration, but we required a dose of more than 1000 μg to achieve the same OD values as those in i.n administration (data not shown).

Since the nasal cavity and the forebrain communicate directly via the olfactory nerve, the safety of an i.n. vaccine with adjuvant for the central nervous system should be evaluated. The safety of chitosan in the mouse and human system has already been demonstrated by Zaharoff et al. (30).

Moreover, the use of an inactivated virus vaccine with a safe adjuvant and not a live virus is very important with regard to safety, because vaccines are recommended for high-risk populations such as infants and the elderly. Therefore, a chitosan adjuvant is a good candidate for an effective and safe mucosal adjuvant when administered i.n. with a vaccine for use in humans. Cellular immunity against antigens and chitosan derivatives used in this study should be evaluated as a next step before these derivatives are tested in a human clinical trial, to determine whether such a nasal vaccine would be effective in humans.

Footnotes

Acknowledgments

We are grateful to Dr. S. Tamura, Dr. T. Ichinohe, and Dr. J. Maeyama (National Institute of Infectious Diseases, Japan) for assistance with the discussion, and Mr. M. Suzuki, Mr. T. Ohba, Mr. H. Abe, Mr. Y. Yamashita, Ms. E. Sakurada, Ms. N. Nishikawa, and Mr. N. Abe for technical assistance. This work was supported in part by grants from the Ministry of Health, Labor, and Welfare.

Author Disclosure Statement

No competing financial interests exist.

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