Cpg Oligodeoxynucleotide Is an Effective Adjuvant for Transcutaneous Immunization

Int J Oral-Med Sci 6(2):91-96,2007 Original Article

CpG Oligodeoxynucleotide is an Effective Adjuvant for Transcutaneous Immunization

ChenLu Liu,Tomomi Hashizume,Tomoko Kurita-Ochiai,and Masafumi Yamamoto

Department of Microbiology and Immunology,Nihon University School of Dentistry at Matsudo,Matsudo,Chiba

271-8587,Japan Tianjin Stomatology Hospital,Tianjin,China

Correspondence to: Masafumi Yamamoto Antigen delivery systems have been designed to facilitate the develop-

E-mail: yamamoto.masafumi＠ ment of vaccines that induce both mucosal and systemic immune nihon-u.ac.jp responses. Transcutaneous immunization (TCI) is a new method of

vaccination that can induce both mucosal and systemic immunities. However,because most protein antigens are rather weak immunogens

when given transcutaneously,the development of effective adjuvants is

of central importance for TCI.In this study,we assessed the potential

for the use of oligodeoxynucleotides containing cytosine-phosphate- guanosine motifs(CpG),as an adjuvant for transcutaneous immunization.When female C57BL/6 mice were immunized with tetanus toxoid (TT) by direct application to shaved skin,a TT-specific serum IgG

antibody response was induced; however,no response was induced in

feces.When TT was given together with CpG oligonucleotide(ODN) as an adjuvant, a higher TT-specific serum IgG and IgA antibody

response was induced, compared to TT alone.Furthermore,a TT- specific IgA antibody response was detected in the fecal extract of

mice immunized with CpG ODN. Dose response studies established

that 500μg of CpG induced the highest fecal anti-TT antibody

response.These results suggest that transcutaneous administration of

Keywords: CpG as an adjuvant is effective for the induction of mucosal and transcutaneous immunization, ad- systemic antigen-specific antibody responses and that CpG could be juvant,IgA antibody used as an adjuvant for transcutanous immunization.

cular or intradermal injection),inducing a systemic

The mucosal surface is the primary site of entry but not a mucosal immune response (4).Mucosal for most pathogens. Gastrointestinal, respiratory, delivery systems have been designed to facilitate the and urogenital tracts expose a large area to development of vaccines that induce both mucosal exogenous agents, including potentially harmful and systemic immune responses.At present,oral and microorganisms.Therefore,the induction of an effec- intranasal vaccines are under development,although tive immune response in the mucosal surface is the rapid degradation of antigens in the gastro-intes- important to prevent the invasion of pathogens.Im- tinal tract and the possibility of host pathology in- munoprophylaxis by the administration of vaccines duced by antigens or adjuvants in nasal and bron- represents a feasible tool for giving targeted immune chial tissues are potential limitations to the broad protection. Effective protection against pathogens implementation of these approaches (5,6).Further- requires both a mucosal response, including secre- more,oral administration of antigens may lead to tory IgA (S-IgA)antibodies as a first line of defense antigen unresponsiveness (7, 8). Thus, the search at the site of entry (1, 2),and a systemic immune continues for practical,effective strategies to intro- response to neutralize pathogens that have penetrat- duce vaccines (5,6).Transcutaneous immunization ed this barrier(3).However,most vaccines developed (TCI)is a novel delivery route for vaccination,using to date are delivered parenterally(i.e.by intramus- the topical application of vaccine antigens on intact Int J Oral-Med Sci 6(2):91-96,2007 bare skin. It has been shown that TCI elicits both Vaccines systemic and mucosal immune responses (9-12). Tetanus toxoid (TT) was kindly provided by Dr. Furthermore,transcutaneous vaccines have several Chikateru Nozaki (The Chemo-sero-Therapeutic advantages over traditional vaccines,including ease Research Institute, Kumamoto, Japan). Synthetic of administration,reduced risk of disease transmis- oligodeoxynucleotides containing CpG motifs (CpG sion,and the elimination of both needles and the need ODN) (5’-TCCATGACGTTCCTGACGTT-3’)were for specially trained healthcare specialists to admin- purchased from Coley Pharmaceutical Group (Wel- ister vaccines.However,the induction of significant lesley,MA,USA). secretory immunity by transcutaneous vaccinations has proven difficult and requires the use of adjuvants Immunization

(1,13,14).Recent studies have shown that cholera Each mouse was anaesthetized with ketamine toxin(CT)is an effective adjuvant for TCI in animal before immunization.Fur was shaved from a section models(9).However,its toxicity prevents its use in of the upper back with care taken not to break the humans. In this regard, genetically detoxified skin. The skin was swabbed with 70% ethanol. A mutants of CT have been developed using site-direct- wound plaster soaked with 150μl phosphate-buffer- ed mutagenesis,which in animal models appear to be ed saline(PBS)containing 100μg TT with 0μg,100 nontoxic which retaining adjuvanticity (4).Despite μg, 200μg, or 500μg of CpG ODN was applied on this progress,there is still a need for novel safe and days 0,7,and 14. effective adjuvants for TCI. Bacterial DNA containing cytosine-phosphate- Detection of antigen-specific antibody responses guanosine(CpG)motifs with unmethylated cytokine, Serum and fecal samples were collected 2 days as well as synthetic oligodeoxynucleotides contain- before immunization as control samples and 6 days ing immunostimulatory CpG motifs(CpG ODN),has after the final immunization and examined for TT- a direct immunostimulatory effect in vitro due to the specific antibody responses (16,17).Antibody titers presence of CpG motifs within a particular base in serum and fecal extracts were determined by context (4).Furthermore,it appears that the rapid enzyme-linked immunosorbent assay(ELISA).Brief- immune activation in response to CpG DNA may ly,the plates were coated with TT (5μg/ml) and evolve as one component of the innate immune blocked with PBS containing 1% bovine serum albu- defense mechanisms that recognize structural pat- min.The starting dilution of serum was 1: 2and terns specific to microbial molecules(4,15).Thus,in fecal samples were 1: 2by concentration. After 4 this study,we assessed the potential of CpG ODN as hours incubation at room temperature, the plates an adjuvant for TCI to induce both mucosal and were washed and peroxidase-labeled goat anti- systemic antibody responses. mouseγorαheavy chain-specific antibodies(South- ern Biotechnology Associates, Birmingham, AL, USA) was added to the appropriate wells. Finally, Animals 2,2’-azino-bis (3-ethylbenzene-thiazoline-6-sulfonic

Female C57BL/6 mice purchased from Sankyo acid)with HO(Moss Inc.,Pasadena,MD,USA)was

Laboratories were maintained in a pathogen-free added for color development. Endpoint titers were experimental facility at Nihon University School of expressed as the reciprocal logof the last dilution

Dentistry at Matusdo. All mice were given sterile that gave an optical density at 415 nm of 0.1 greater water,food,and bed dressing.All experiments were than nonimmunized control samples after 15 minutes performed using 8- to 12-week old mice that were of incubation. randomly assigned in groups of 3 or 4. Int J Oral-Med Sci 6(2):91-96,2007

Statistical analysis Toll-like receptor 9 expressed by B cells and plas-

Results were expressed as the mean ± standard macytoid dendritic cells(18).The interaction of Toll deviation. Statistical significance (p＜0.05) was -like receptor 9 with CpG motifs triggers an immune determined by unpaired Student’t test. cascade,resulting in improved antigen uptake and

presentation by antigen-presenting cells,antibodies, and chemokines,as well as cytokine secretion by B

Induction of TT -specific serum antibody responses by cells, NK cells, dendritic cells, and monocytes (18, transcutaneous administration of TT and CpG 19). Synthetic CpG ODN mimics the immunos-

Effective protection against pathogens requires timulatory activity of bacterial DNA. This activity both a mucosal immune response and a systemic enables CpG ODN to act as an immune adjuvant, immune response.Transcutaneous immunization has accelerating and boosting antigen-specific immune several advantages over traditional vaccines and responses.This might be due to the close physical other mucosal vaccines(2).In this study,to evaluate contact between the CpG ODN and the immunogen the ability of TCI with TT to induce serum antibody (18, 19).Thus, in this study, we assessed whether responses, a group of mice were transcutaneously transcutaneous administration of CpG ODN would immunized with TT.When the TT-specific antibody act as an adjuvant in the induction of TT-specific response was analyzed by ELISA, a significant IgG antibody responses.Mice were transcutaneously im- antibody response was found to have been induced. munized with TT in the presence of different concen- However, the responses were relatively low (Fig. trations of CpG ODN. TCI with TT plus CpG ODN

1A).Furthermore,an IgA response was not detected induced a significantly higher TT-specific IgG anti- (Fig.1B).These results suggest that TCI with TT body response compared to TT alone.All doses of alone may not be an effective vaccine for the induc- CpG ODN tested in this study (100-500μg) gave tion of protective immunity against tetanus toxin. comparable adjuvant activity and induced serum IgG

Unmethylated CpG motifs,which are often found in anti-TT antibody response (Fig.1A).Significantly, bacterial but not vertebral DNA,are recognized by administration of 500μg of CpG resulted in the

Fig.1. Induction of TT-specific antibody responses in serum by transcutaneous immunization with TT plus CpG ODN.Groups of female C57BL/6 mice were transcutaneously immunized with 100μg of TT combined with 0μg (control),100μg,200μg,or

500μg of CpG ODN on days 0,7,and 14.After the final immunization,serum samples were collected and assessed on day 21 for

TT-specific IgG (A)and IgA (B) antibody responses.The results are expressed as the mean ± standard deviation and are representative of three separate experiments containing three to four mice in each group/experiment.p＜0.05 compared with the control of mice immunized with TT alone. Int J Oral-Med Sci 6(2):91-96,2007 highest TT-specific IgA antibody response of sev- transcutaneously. eral doses tested (Fig.1B). These finding clearly The amounts of CpG as adjuvant used in this study indicate that CpG ODN is an effective adjuvant for are much higher than those used in nasal immuniza- induction of TT-specific antibody responses when tion in previous studies(4).In this regard,it has been given transcutaneously. known that nasally administered antigen interacts

directly with IgA-inductive sites, known as nasal- Antigen-specific mucosal IgA immune responses are associated lymphoreticular tissues, while trans- induced by the co-administration of CpG cutaneous immunization does not.Therefore,higher

It is important to note that transcutaneous admin- doses of vaccine are required for transcutaneous istration of CpG ODN as an adjuvant induced a immunization when compared with nasal immuniza- secretory IgA antibody response to co-administrated tion.However,previous studies have suggested that

TT in mucosal secretions.Thus,TCI with TT plus nasally administered vaccines accumulate in the

CpG ODN resulted in the induction of a significant olfactory nerves and epithelial regions (20,21).On

TT-specific IgA antibody response in fecal extracts the other hand,a recent study has demonstrated that

(Fig.2).Of all the dosages of CpG ODN tested, the transcutaneous immunization of human volunteers administration of 500μg of CpG produced the largest elicits antigen-specific antibody responses without mucosal IgA anti-TT antibody response.In contrast, significant side effects (22).Taken together, these

IgA antibodies were not induced in mice by trans- findings suggest that transcutaneous immunization cutaneous immunization with TT alone (Fig.2). represents a novel and completely independent

These results indicate that CpG is a potent adjuvant antigen delivery system that significantly reduces the for the induction of immune responses in both negative effects produced by nasal immunization. mucosal and systemic areas when administrated The improved adjuvant effect of CpG ODN has

been shown by linking of CpG ODN directly to the

antigen (23,24)and co-encapsulating CpG ODN in

liposome vesicles (25).These findings suggest that

optimal immune stimulation may occur when antigen

and adjuvant are presented to the immune system in

close spatial and temporal proximity.We are cur- rently investigating the mucosal adjuvant effect of

CpG ODN by transcutaneous immunization when the

CpG ODN is directly linked to antigen. In summary,our current study provides evidence

that transcutaneous administration of CpG ODN as

an adjuvant elicits TT-specific serum IgG and IgA

as well as mucosal IgA antibody responses. These

findings reveal a promising future for the application Fig.2. Detection of TT-specific antibody responses in feces of CpG as a potent adjuvant for transcutaneous by transcutaneous immunization with TT plus CpG ODN. Groups of female C57BL/6 mice were transcutaneously im- immunization against tetanus as well as a range of munized with 100μg of TT combined with 0μg (control),100 other infectious diseases in humans. μg, 200μg, or 500μg of CpG on days 0,7,and 14.After the final immunization,fecal samples were collected and assessed on day 21 for comparison with TT-specific IgA antibody responses.The results are expressed as the mean ± standard deviation and are representative of three separate experiments This work was supported by a Grant-in-Aid for / ＜ containing three to four mice in each group experiment.p Scientific Research(18592270,19791624 and 19390537) 0.05 compared with the control of mice immunized with TT alone. from the Japan Society for the Promotion of Science, Int J Oral-Med Sci 6(2):91-96,2007 an “Academic Frontier”Project for Private Univer- AM, Hansbro PM, Gockel CM,Beagley KW : Trans- cutaneous immunization with combined cholera toxin sities matching fund subsidy from the Ministry of and CpG adjuvant protects against Chlamydia mur- Education,Culture,Sports,Science and Technology, idarum genital tract infection.Infect Immun,72: 1019 2007-2011. -1028,2004. 13.Thapar MA, Parr EL, Parr MB : The effect of adjuvants on antibody titers in mouse vaginal fluid after

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