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Principles of transcutaneous immunization using cholera toxin as an adjuvant
Vaccine 17 (1999) S37±S43
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Principles of transcutaneous immunization using cholera toxin as an adjuvant Tanya Scharton-Kersten a,b,*, Gregory M. Glenn a,b, Russell Vassell a,b, Jian-mei Yu a,b, Deborah Walwender a,b, Carl R. Alving a a
Walter Reed Army Institute of Research, Department of Membrane Biochemistry, 14th and Dahlia Sts. NW, Building 40, Rm 3049, Washington, DC 20307, USA b IOMAI Corporation, Washington, DC 20037, USA
Abstract Transcutaneous immunization is a novel strategy for immunization employing topical application of antigen and adjuvant to the skin surface and resulting in detectable antigen/adjuvant speci®c IgG in plasma and mucosal secretions. In this study we show that transcutaneous immunization with cholera toxin (CT) as an adjuvant can be used in several inbred mouse strains with varying H-2 major histocompatibility complex genes (C57BL/6 (H-2b), BALB/c (H-2d), and C3H (H-2k)). Although the primary anti-CT antibody responses re¯ected previously described MHC restriction patterns for this protein, the dierences were overcome after two booster immunizations. Potent antibody responses against hen egg lysozyme and/or diphtheria toxoid were observed using CT as adjuvant. We also demonstrate that the unshaved dorsal or ventral surface of the ear can be eectively used for transcutaneous immunization and that gentle swabbing with alcohol increases the magnitude of the host immune response. Together these data further our understanding of the principles governing this new platform technology and support its integration into novel and existing human vaccine strategies. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Strategies which avoid the use of needles are desirable for human and animal vaccines as such techniques greatly reduce barriers to immunization including the need for vaccine administration by trained personnel, incidence of needle related reactions and needle-borne diseases, and the physical and mental discomfort associated with needles themselves [1,2]. At present several oral and intranasal vaccines are under development although the rapid degradation of antigens in the gastro-intestinal tract, but the possibility of host pathology induced by antigens or adjuvants in nasal and bronchial tissues is a potential limitation to the broad implementation of these approaches. Thus,
* Corresponding author. Tel.: +1-202-782-3137; fax: +1-202-7821890. E-mail address: [email protected] (T. Scharton-Kersten)
the search continues for practical, alternative strategies to introduce vaccines into the host [1,3]. We have recently described a method for introducing antigen through the unbroken skin using bacterial ADP ribosylating agents as adjuvants [4]. It was observed that application of either Vibrio cholerae-derived cholera toxin (CT) or the related protein, heat labile enterotoxin (LT) from Escherichia coli to mouse skin for 2 h results in a potent (titers r103) anti-CT or LT IgG response in the sera within 2 weeks of the immunization and that the response is boosted with subsequent immunizations [5]. Application of CT with other proteins including tetanus toxoid, diphtheria toxoid, and bovine serum albumin also results in a systemic IgG response against the coadministered antigen [4,5] indicating that this method has broad utility for the induction of host immunity through the skin. The immune response induced using CT as an immunogen or adjuvant for TCI appears to be physiologically relevant since vaccinated mice have been shown to be
0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 2 3 3 - 9
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T. Scharton-Kersten et al. / Vaccine 17 (1999) S37±S43
protected from a lethal intranasal challenge with CT and, in the case of animals immunized against tetanus, from a lethal challenge with tetanus toxin [5,6]. The adjuvant properties of CT and LT are most widely applied to oral and intranasal uses [7], however, the toxicity of the proteins has restricted their widespread use and led to development of strategies using non-toxic (and often less immunogenic) mutants and subunits [8±11]. In contrast, application of intact CT and LT to unbroken skin does not induce the systemic toxicities that are associated with their use via oral, nasal, or parenteral routes [4]. To better understand the potential applications and mechanism of TCI we have evaluated the immunological rules which in¯uence the success of this method. In this study we demonstrate that CT can function as a transcutaneous adjuvant in MHC-disparate mouse strains including lipopolysaccharide (LPS) responsive and non-responsive animals. The ability to induce immune responses in animals which are traditionally considered low-responders to CT (C3H) suggests that the TCI method has potential for broad use in outbred populations. Furthermore, we describe optimal methods for use of the ear, rather than the back, as a site for TCI. The latter studies, using the ear method which does not involve shaving of the skin surface, argue against a role for inadvertent abrasion or penetration in the TCI process. Finally, the ability to introduce antigen through the ear may assist in the discovery of the mechanisms involved in TCI as study of skin immune function is routinely conducted using mouse ear tissue. 2. Materials and methods 2.1. Immunization and antigen Cholera toxin and diphtheria toxoid (DT) were obtained from LIST Biologicals (Cambell, CA) and bovine serum albumin (BSA) and hen egg lysozyme (HEL) from Sigma (St. Louis, MO). BALB/c and C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and C3H/HeN and C3H/HeJ animals from Harlan Sprague-Dawley, Inc. (Frederick, MD). Mice (6±10 weeks of age) were maintained in pathogen-free conditions and fed rodent chow and water ad libitum. 2.1.1. Back immunization Mice were shaved on the dorsum with a No. 40 clipper and rested for 48 h. Groups of three to ®fteen mice were anaesthetized in the hind thigh intramuscularly with 110 mg/kg ketamine mixed with 11 mg/kg xylazine during the immunization procedure, to prevent grooming. The skin was wetted with 100 ml of
immunizing solution [CT (100 mg), CT/BSA (100/200 mg), DT (100 mg), CT/DT (100/100 mg), HEL (100 mg), or CT/HEL (100/100 mg)] placed on the shaved skin over a 2 cm2 area for 2 h. The mice were then extensively washed with approximately 1 l of lukewarm tap water, patted dry and washed again. No adverse eects from either the shaving, anesthesia, immunization, or washing procedures were observed. Neither erythema nor induration was seen at the immunization site for up to 72 h after the antigen exposure.
2.1.2. Ear immunization Mice were anaesthetized as above, with ketamine/ xylazine cocktail. As indicated antigen solution was either placed on the dorsal (outer) or ventral (inner) surface of the ear. For dorsal immunizations, the ear was treated by gently rubbing the outer skin surface with a cotton tipped applicator containing either 70% isopropanol or sterile water. After 5 min the excess water was blotted from water-treated ears and antigen [CT (50 mg), CT+BSA (50/100 mg) or BSA (50 mg)] in 50 ml phosphate buered saline (PBS; Biowhittaker, Walkersville, MD) was painted onto the prepared skin of all ears using a pipetter. At 2.5 h, the ears were rinsed and blotted dry twice. Mice were boosted in a similar fashion 4 and 8 weeks later. For ventral vaccinations, the ear was treated by gently rubbing the ear with a cotton tipped applicator wetted with sterile water. After 5 min, excess water was blotted from the skin and antigen (CT+HEL (100/100 mg)) in 25 ml PBS was painted onto the prepared skin using a pipetter. After 2 h the ears were rinsed and blotted dry twice. Mice were boosted 5 and 8 weeks later.
2.2. Antibody assay Antibody levels against CT, DT, BSA, and HEL were determined using ELISA. Immulon-2 polystyrene plates (Dynatech Laboratories, Chantilly, VA) were coated with 0.1 mg/well of antigen in saline, incubated at room temperature overnight, blocked with 0.5% casein-Tween-20, washed, serial dilutions of serum applied, and the plates incubated for 2 h at room temperature. Speci®c IgG (H+L) antibody was detected using HRP-linked goat anti-mouse IgG (H+L) (Biorad, Richmond VA) and revealed after 30 min using 2,2 '-azino-di (3-ethyl benzthiazolone) sulphonic acid substrate (ABTS; Kirkegaard and Perry, Gaithersburg, MD) and the reaction stopped using 1% SDS. The plates were read at 405 nm. Results are reported in ELISA Units which are de®ned as the inverse dilution of the sera that yields an OD of 1.0 at 405 nm.
T. Scharton-Kersten et al. / Vaccine 17 (1999) S37±S43
2.3. T cell proliferation C3H/HeJ mice were immunized with CT or CT/DT on the back at 0, 4, and 8 weeks as described above. Tissues were removed 19 weeks after the primary immunization spleens, and draining lymph node (LN; inguinal) and single cell suspensions prepared using a manual tissue grinder. Red blood cells were lysed with ACK lysis buer (Biowhittaker, Walkerville, MD). Spleen and LN cells from four mice were pooled and cultured at 4 105 cells per well in 96-well plates for 5 days at 378C, 5% CO2 in the presence or absence of 10 mg/ml of DT. Concanavalin A (ConA) at 5 mg/ml was used as a positive control. Culture media contained RPMI 1640 (BioWhittaker), 10% Fetal calf serum (GibcoBRL, Grand Island, NY), penicillin (10 U/ml; BioWhittaker), streptomycin (100 mg/ml; BioWhittaker), L-glutamine (2 mM; Sigma), Hepes (10 mM; BioWhittaker) and 2-mercaptoethanol (50 mM; BioRad, Hercules, CA). [3 H]-thymidine (1 mCi/ well) was added to the cultures during the last 20 h of the 5 day culture period. Thymidine incorporation was assessed by harvesting cellular DNA onto glass-®ber ®lters followed by liquid scintillation counting.
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C57BL/6 or BALB/c mice suggested that the former mice are superior responders to transcutaneously administered CT as determined by both the plasma anti-CT IgG titer and the number of responding animals. To formally test this concept we evaluated the immune response to CT administered by the transcutaneous route in BALB/c, C57BL/6, C3H/HeN and C3H/HeJ mice. Animals were exposed to 100 mg of CT on the back at 0 weeks and boosted 4 and 8 weeks later. Serum was collected 2, 8 and 12 weeks after the primary immunization. As shown in Fig. 1, the plasma IgG response to CT was distinct among the strains at the 2 and 8 week time points with the geometric mean plasma IgG response in C57BL/6> BALB/c r C3H. Although the hierarchy of responders persisted for 8 weeks, the degree of dierence was most apparent 2 weeks after the primary immunization, when the geometric mean titer in the C57BL/6 mice was more than 1 log of that observed in the other strains, and the number of C57BL/6 responders (12 of 12) was greater than that in BALB/c (13 of 15), C3H/HeJ (6 of 12) or C3H/HeN (1 of 9) animals. One hypothesis which might explain the dierences in the immune response following primary and booster
2.4. CD4+ T cell puri®cation CD4+ T cells were isolated from pooled spleen cells from the CT/DT immunization group (see above) using a CD4+ T cell selection column as per the manufacturer's instructions (R and D systems, Minneapolis, MN). Brie¯y, cells eluted from the column (CD4+) were cultured in 96-well plates at 1 105 cells per well in the presence or absence of 3 105 irradiated (3000 RAD) feeder cells from naive mice. Proliferation assays were conducted as described in detail above. 2.5. Statistical analysis Unless otherwise indicated, the data shown are the geometric means of values from individual animals. Comparisons between antibody titers in groups were performed using an unpaired, two-tailed Student's ttest and p-values <0.05 regarded as signi®cant. 3. Results and discussion 3.1. Transcutaneous delivery of cholera toxin as antigen in C57BL/6, BALB/c and C3H mouse strains Over the course of developing the transcutaneous immunization (TCI) method we have evaluated the immunogen and adjuvant activities of CT in several inbred mouse strains. Numerous studies employing
Fig. 1. Serum anti-cholera toxin titers in C57BL/6, BALB/c and C3H mice following TCI with 100 mg cholera toxin on the back at 0 and 4 weeks. Serum was collected at 0, 2 and 8 weeks after the primary immunization and analyzed for CT speci®c IgG by ELISA as described in the Materials and Methods. Results shown are the IgG titers in individual animals (open circles) and the geometric mean for the group (solid bars) in ELISA Units. ELISA units are de®ned as the inverse dilution at which the OD is 1.0 at 405 nm. Statistical signi®cance between C57BL/6 values and other mouse strains are indicated.
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exposures to CT by the transcutaneous route is the eects of the H-2 MHC genes of the strains tested; C3H (H-2k), C57BL/6 (H-2b) and BALB/c (H-2d). Our ®nding of a hierarchy among these strains is consistent with a series of experiments by Elson et al. which clearly show that the plasma IgG response to CT following oral or parenteral administration is under the genetic control of the I-A region of the H-2 major histocompatibility complex [12±14]. Thus, inbred mice with H-2b or H-2q haplotypes were high responders, strains with the H-2s and H-2k low responders and those with the H-2d haplotype intermediate responders. Further study in H-2 congenics on similar backgrounds (i.e., B10 (H-2b) vs B10.BR (H-2k) vs B10.D2 (H-2d) is necessary to formally test the in¯uence of MHC genes on the transcutaneous method. The rise in anti-CT titers in C3H strains following the second immunization suggested that the inferior response in these animals is transient. Accordingly, the animals were boosted a second time and, after the third immunization, the resulting plasma anti-CT levels were comparable to the other strains (Fig. 2A). Thus the quantitative dierence in antigenicity of CT administered by TCI is not absolute and may be overcome by additional exposure to CT. Oral administration of CT to lipopolysaccharide (LPS) congenic mice (C57BL/10 SnJ (lps+) and ScN (lpsÿ)) has been shown to result in a higher plasma
anti-CT IgG titer in the endotoxin responsive strain suggesting a role for the LPS genes in controlling this response [14]. In contrast, our studies with LPS congenics on the C3H background (HeJ (lpsÿ), and HeN(lps+)) failed to reveal an in¯uence of the LPS genes on CT IgG levels following TCI (Fig. 1). The cause of the discrepancy between these studies is unclear and may re¯ect true dierences in the genetic control of oral and TCI procedures or relate to background genes in the C3H and C57BL/10 mouse strains employed. 3.2. Transcutaneous adjuvant activity of CT for diphtheria toxoid and hen egg lysozyme in C57BL/6, C3H and BALB/c mice To assess the adjuvant activity of CT delivered by the transcutaneous route, mice were exposed to adjuvant (CT), antigen (DT or HEL), or adjuvant + antigen at 0, 4 and 8 weeks. After 4 weeks of the ®nal immunization the animals were bled and the plasma anti-CT, DT and HEL titers measured by ELISA. Neither DT nor HEL were able to induce immune responses to themselves after application to the skin (Fig. 2B and C). As stated above, all three mouse strains raised similar anti-adjuvant (CT) responses after three exposures to this protein. However, the antigen (DT and HEL) speci®c IgG titers showed con-
Fig. 2. Serum anti-CT, DT, and HEL titers in C57BL/6, BALB/c and C3H mice following TCI with CT and/or antigen on the back at 0, 4 and 8 weeks. Serum was collected 0 and 12 weeks after the primary immunization and analyzed for CT speci®c IgG by ELISA. Results shown are the IgG titers in individual animals (open circles) and the geometric mean for the group (bars) in ELISA units.
T. Scharton-Kersten et al. / Vaccine 17 (1999) S37±S43
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Table 1 Proliferative response to DT in C3H micea Immunization proteins
CT CT/DT CT CT DT
Source of cells
spleen spleen CD4+APC APC LN LN
In vitro stimulation media (cpm)
(cpm)
1486 236 43 3700 6061
2766 9512 46 4407 55056
DT
SI
(cpm)
1.9 40.3 1.1 1.2 9.1
27725 20247 176 23425 27696
ConA
SI 18.7 85.8 4.1 6.3 4.6
a
Mice were immunized with CT (adjuvant) or CT (adjuvant) and DT (antigen) at 0.4, and 8 weeks as described in the Materials and Methods. Spleen and lymph node cells were collected nineteen weeks after the primary immunization. Data shown are the proliferation values (cpm) obtained from cultures of pooled cells stimulated with media alone DT (10 mg/ml) or the lectin concanavalin A (5 mg/ml). The stimulation index (SI) was calculated with the formula:cpm (experimental)/cpm (media).
siderable variation among the three mouse strains (Fig. 2B and C). BALB/c mice displayed potent titers against both HEL and DT and, in the case of HEL, the antibody response in mice immunized transcutaneously was comparable to that observed in animals given HEL in alum by the intramuscular route. C57BL/6 mice responded well to DT but failed to display detectable anti-HEL responses even after three immunizations. In contrast, C3H mice (HeN and HeJ) displayed high anti-HEL IgG responses and minimal anti-DT titers. The variable response to antigen among the strains appears to be in¯uenced by several factors. First, it is well established that mice bearing the H-2b haplotype (i.e., C57BL/6) fail to produce detectable antibody titers after injection of HEL as they do not bear appropriate MHC molecules to successfully present this antigen [15]. On this basis it was not unexpected that C57BL/6 mice immunized with CT and HEL by the transcutaneous route did not raise an anti-HEL antibody response. One implication of this result is that TCI using CT does not overcome established MHC restriction in the host and thus is unlikely to cause undesirable immune responses against endogenous skin antigens. Consistent with this result we failed to see a rise in titers against cholesterol, a common skin lipid in mice which were treated with CT at doses between 0 and 500 mg (Nabila Wassef, personal communication). C3H mice, on the other hand, did not exhibit substantial IgG titers against DT and the response to this antigen is not known to be restricted by particular MHC H-2 haplotypes [16]. One possible explanation for the de®cient DT response in these animals is that, the less potent immune response to the adjuvant (CT; Fig. 1) contributes to the diminished antigen speci®c anti-DT IgG response in these animals. Detectable anti-DT IgG titers were observed in sera from three of ®ve C3H mice and puri®ed splenic CD4+ T cells and
LN cells from the C3H/HeJ mice clearly showed antigen speci®c proliferative responses to DT (Table 1). Thus, while the antibody response was poor, the C3H mice clearly responded to the antigen. 3.3. Ability to immunize in the ear Immunization on the back has been routinely conducted after shaving of the skin surface to remove hair. The shaving procedure assists in the controlled delivery of the antigen and allows visual assessment of the applied solution. However, one concern with this method has been that small abrasions resulting from the shaving process itself may contribute to the potent systemic immune response observed, following TCI.
Fig. 3. Serum anti-CT titers of BALB/c mice immunized transcutaneously on the ear with 50 ug CT following pretreatment with water (A) or isopropanol (B). Mice were immunized at 0, 4 and 8 weeks and serum collected 0, 4 and 12 weeks after the primary immunization. Anti-CT IgG titers were analyzed by ELISA. Data shown are titers in individual mice (gray bars) and the geometric mean of the group (black bars) in ELISA units. Numeric values for the geometric mean of the group are shown above the black bars.
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HEL as antigen. Thus, the penetration and immunization of the host with topically applied antigens is not dependent upon shaving related insults to the stratum corneum.
Fig. 4. Serum antibody titers of individual BALB/c mice immunized transcutaneously with CT and BSA: (A) on the back following pretreatment by hydration or (B) on the ear following pretreatment with isopropanol. Mice were immunized at 0, 4 and 8 weeks. Serum was collected 10 weeks after the primary immunization and assayed for CT and BSA speci®c IgG by ELISA. Values shown are the prebleed (gray bars) and ten week (black bars) titers for individual mice in ELISA units. NSA = no serum available.
To exclude this possibility we have transcutaneously immunized mice on the ear which does not require shaving because of its naturally hairless surface. As shown in Figs. 3±5 we have successfully achieved TCI on the ear utilizing CT as adjuvant and both BSA and
Fig. 5. Serum antibody titers of individual C3H mice immunized transcutaneously with 100 ug CT and 100 ug HEL: (A) on the back or (B) on the ventral surface of the ear following simple water hydration. Mice were immunized at 0, 4 and 8 weeks. Serum was collected 12 weeks after the primary immunization and assayed for CT and HEL speci®c IgG by ELISA. Values shown are the prebleed (grey bars) and twelve week (black bars) titers for individual mice in ELISA units.
3.3.1. Immunization on the dorsal ear surface The mechanism by which TCI permits induction of a systemic immune response against topically administered antigen remains to be de®ned. One hypothesis is that hydration of the skin allows penetration of antigen into the epidermis where Langerhans cells (LCs) subsequently pick up and convey antigen to the draining lymph node for presentation to T cells [4]. To test this hypothesis we have optimized TCI methods for the ear since it is a convenient source of LCs for study of their function. Our initial attempts to immunize on the ear utilized the dorsal (outer) surface. Although we achieved a detectable antibody response using this approach, the magnitude of the response was considerably less than that achieved with TCI on the back (Fig. 1 vs Fig. 3A). In this regard, the antibody titers against CT were dramatically improved when the area for immunization was gently wiped with isopropanol and allowed to dry, prior to application of the antigen (Fig. 3). This procedure is thought to dilapidate the aected skin and appeared to enhance penetration, as the immunizing solution was absorbed more rapidly as compared to our standard procedure which involves hydration with water prior to immunization. To evaluate the eectiveness of TCI on the ear, BALB/c mice were immunized with CT and BSA on the dorsal surface of the ear or on the back at 0, 4, and 8 weeks and the serum anti-adjuvant and antigen IgG titers determined 10 weeks after the primary immunization. The immune response to adjuvant (CT) and antigen (BSA) was comparable in both groups (Fig. 4). 3.3.2. Immunization on the ventral ear surface Although the dorsal ear method described achieved high titer antibodies, we were concerned that treatment of the skin with chemical agents can alter Langerhans cell function [17]. Thus, we have continued to search for alternate methods which require only minimal modi®cation of the skin surface. To this end we tested whether TCI could be used to introduce antigen through the inner (ventral) ear surface. C3H/HeN animals were immunized with CT and HEL on the back or on the ventral surface of the ear without alcohol swabbing at 0, 4 and 8 weeks and the serum anti-adjuvant and antigen IgG titers determined 12 weeks after the primary immunization. The immune response to the adjuvant (CT) and antigen (HEL) was comparable in both groups (Fig. 5). Thus, with minimal manipulation of the inner ear (i.e., water hydration) animals can be eectively vaccinated using TCI. Together the series of experiments on the ear pro-
T. Scharton-Kersten et al. / Vaccine 17 (1999) S37±S43
vide several key insights related to the TCI method. The observation that animals can be immunized on the ear indicates that the shaving procedure used for TCI on the back is not required for the introduction of antigens to the host through the skin. Moreover, experiments to evaluate the cellular mechanisms of TCI can now be designed, given the body of research available, regarding Langerhans cell morphology and function in the mouse ear and our ability to eectively transcutaneously immunize these animals via the ear. Finally, these data provide strong rationale for integration of TCI into domestic and production animal medicine where the ear is a desirable site for immunization. Acknowledgements We thank Elaine Morrison and Julie Weitz for technical assistance and Dr. Nabila Wassef for measuring the anti-cholesterol antibodies and critically evaluating the manuscript. The work presented was supported and performed under a Cooperative Research and Development Agreement between Walter Reed Army Institute of Research and Medical Technologies and Practice Patterns Institute, Washington DC. References [1] Katz SL. Future vaccines and a global perspective. Lancet 1997;13:1767±70. [2] Reis EC, Jacobson RM, Tarbell S, Weninger BG. Taking the sting out of shots: control of vaccination-associated pain and adverse reactions. Pediatr Ann 1998;27:375±86. [3] Liu MA. Vaccine developments. Nature Med 1998;4:515±9. [4] Glenn GM, Rao M, Matyas G, Alving CR. Skin immunization made possible by cholera toxin. Nature 1998;391:851.
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[5] Glenn GM, Scharton-Kersten T, Vassell R, Matyas GR, Alving CR. Transcutaneous immunization using bacterial ADP-ribosylating exotoxins as antigens and adjuvants. Infection and Immunity 1999; 67:1100±1106. [6] Glenn GM, Scharton-Kersten T, Vassell R, Mallett CP, Hale TL, Alving CR. Transcutaneous immunization with cholera toxin protects mice against lethal mucosal toxin challenge. J Immunol 1998;161:3211±4. [7] Lycke N. The mechanism of cholera toxin adjuvanticity. Res Immunol 1997;148:504±20. [8] Marchetti M, Rossi M, Giannelli V, Giuliani MM, Pizza M, Censini S, Covacci A, Massari P, Pagliaccia C, Manetti R, Telford JL, Douce G, Dougan G, Rappuoli R, Ghiara P. Protection against Helicobactor pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant. Vaccine 1998;16:33±7. [9] Komase K, Tamura S, Matsuo K, Watanabe K, Hattori N, Odaka A, Suzuki Y, Kurata T, Aizawa C. Mutants of Escherichia coli heat-labile enterotoxin as an adjuvant for nasal in¯uenza vaccine. Vaccine 1998;16:248±54. [10] Rappuoli R, Pizza M. Novel molecular biology approaches to acellular vaccines. Biotech Ann 1996;2:391±408. [11] O' Hagan DT. Recent advances in vaccine adjuvants for systemic and mucosal administration. J Pharm Pharmacol 1997;49:1±10. [12] Elson CO, Ealding W. Genetic control of the murine immune response to cholera toxin. J Immunol 1985;135:930±2. [13] Elson CO, Ealding W. Ir gene control of the murine secretory IgA response to cholera toxin. Eur J Immunol 1987;17:425±8. [14] Elson CO. Cholera toxin as a mucosal adjuvant: eects of H-2 major histocompatibility complex and lps genes. Inf Immun 1992;60:2874±9. [15] Hill SW, Sercarz EE. Fine speci®city of the H-2 linked immune response gene for the gallinaceous lysozymes. Eur J Immunol 1975;5:317±24. [16] Kameyama S, Nagaoka F, Matuhasi T. Comparison of immune responses to diphtheria and tetanus toxoids of various mouse strains. Japan J Med Sci Biol 1989;42:83±99. [17] Katoh N, Hirano S, Kishimoto S, Yasuno H. Acute cutaneous barrier perturbation induces maturation of Langerhans' cells in hairless mice. Acta Dermato-Venereologica 1997;77:365±9.
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Principles of transcutaneous immunization using cholera toxin as an adjuvant Tanya Scharton-Kersten a,b,*, Gregory M. Glenn a,b, Russell Vassell a,b, Jian-mei Yu a,b, Deborah Walwender a,b, Carl R. Alving a a
Walter Reed Army Institute of Research, Department of Membrane Biochemistry, 14th and Dahlia Sts. NW, Building 40, Rm 3049, Washington, DC 20307, USA b IOMAI Corporation, Washington, DC 20037, USA
Abstract Transcutaneous immunization is a novel strategy for immunization employing topical application of antigen and adjuvant to the skin surface and resulting in detectable antigen/adjuvant speci®c IgG in plasma and mucosal secretions. In this study we show that transcutaneous immunization with cholera toxin (CT) as an adjuvant can be used in several inbred mouse strains with varying H-2 major histocompatibility complex genes (C57BL/6 (H-2b), BALB/c (H-2d), and C3H (H-2k)). Although the primary anti-CT antibody responses re¯ected previously described MHC restriction patterns for this protein, the dierences were overcome after two booster immunizations. Potent antibody responses against hen egg lysozyme and/or diphtheria toxoid were observed using CT as adjuvant. We also demonstrate that the unshaved dorsal or ventral surface of the ear can be eectively used for transcutaneous immunization and that gentle swabbing with alcohol increases the magnitude of the host immune response. Together these data further our understanding of the principles governing this new platform technology and support its integration into novel and existing human vaccine strategies. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Strategies which avoid the use of needles are desirable for human and animal vaccines as such techniques greatly reduce barriers to immunization including the need for vaccine administration by trained personnel, incidence of needle related reactions and needle-borne diseases, and the physical and mental discomfort associated with needles themselves [1,2]. At present several oral and intranasal vaccines are under development although the rapid degradation of antigens in the gastro-intestinal tract, but the possibility of host pathology induced by antigens or adjuvants in nasal and bronchial tissues is a potential limitation to the broad implementation of these approaches. Thus,
* Corresponding author. Tel.: +1-202-782-3137; fax: +1-202-7821890. E-mail address: [email protected] (T. Scharton-Kersten)
the search continues for practical, alternative strategies to introduce vaccines into the host [1,3]. We have recently described a method for introducing antigen through the unbroken skin using bacterial ADP ribosylating agents as adjuvants [4]. It was observed that application of either Vibrio cholerae-derived cholera toxin (CT) or the related protein, heat labile enterotoxin (LT) from Escherichia coli to mouse skin for 2 h results in a potent (titers r103) anti-CT or LT IgG response in the sera within 2 weeks of the immunization and that the response is boosted with subsequent immunizations [5]. Application of CT with other proteins including tetanus toxoid, diphtheria toxoid, and bovine serum albumin also results in a systemic IgG response against the coadministered antigen [4,5] indicating that this method has broad utility for the induction of host immunity through the skin. The immune response induced using CT as an immunogen or adjuvant for TCI appears to be physiologically relevant since vaccinated mice have been shown to be
0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 2 3 3 - 9
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protected from a lethal intranasal challenge with CT and, in the case of animals immunized against tetanus, from a lethal challenge with tetanus toxin [5,6]. The adjuvant properties of CT and LT are most widely applied to oral and intranasal uses [7], however, the toxicity of the proteins has restricted their widespread use and led to development of strategies using non-toxic (and often less immunogenic) mutants and subunits [8±11]. In contrast, application of intact CT and LT to unbroken skin does not induce the systemic toxicities that are associated with their use via oral, nasal, or parenteral routes [4]. To better understand the potential applications and mechanism of TCI we have evaluated the immunological rules which in¯uence the success of this method. In this study we demonstrate that CT can function as a transcutaneous adjuvant in MHC-disparate mouse strains including lipopolysaccharide (LPS) responsive and non-responsive animals. The ability to induce immune responses in animals which are traditionally considered low-responders to CT (C3H) suggests that the TCI method has potential for broad use in outbred populations. Furthermore, we describe optimal methods for use of the ear, rather than the back, as a site for TCI. The latter studies, using the ear method which does not involve shaving of the skin surface, argue against a role for inadvertent abrasion or penetration in the TCI process. Finally, the ability to introduce antigen through the ear may assist in the discovery of the mechanisms involved in TCI as study of skin immune function is routinely conducted using mouse ear tissue. 2. Materials and methods 2.1. Immunization and antigen Cholera toxin and diphtheria toxoid (DT) were obtained from LIST Biologicals (Cambell, CA) and bovine serum albumin (BSA) and hen egg lysozyme (HEL) from Sigma (St. Louis, MO). BALB/c and C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and C3H/HeN and C3H/HeJ animals from Harlan Sprague-Dawley, Inc. (Frederick, MD). Mice (6±10 weeks of age) were maintained in pathogen-free conditions and fed rodent chow and water ad libitum. 2.1.1. Back immunization Mice were shaved on the dorsum with a No. 40 clipper and rested for 48 h. Groups of three to ®fteen mice were anaesthetized in the hind thigh intramuscularly with 110 mg/kg ketamine mixed with 11 mg/kg xylazine during the immunization procedure, to prevent grooming. The skin was wetted with 100 ml of
immunizing solution [CT (100 mg), CT/BSA (100/200 mg), DT (100 mg), CT/DT (100/100 mg), HEL (100 mg), or CT/HEL (100/100 mg)] placed on the shaved skin over a 2 cm2 area for 2 h. The mice were then extensively washed with approximately 1 l of lukewarm tap water, patted dry and washed again. No adverse eects from either the shaving, anesthesia, immunization, or washing procedures were observed. Neither erythema nor induration was seen at the immunization site for up to 72 h after the antigen exposure.
2.1.2. Ear immunization Mice were anaesthetized as above, with ketamine/ xylazine cocktail. As indicated antigen solution was either placed on the dorsal (outer) or ventral (inner) surface of the ear. For dorsal immunizations, the ear was treated by gently rubbing the outer skin surface with a cotton tipped applicator containing either 70% isopropanol or sterile water. After 5 min the excess water was blotted from water-treated ears and antigen [CT (50 mg), CT+BSA (50/100 mg) or BSA (50 mg)] in 50 ml phosphate buered saline (PBS; Biowhittaker, Walkersville, MD) was painted onto the prepared skin of all ears using a pipetter. At 2.5 h, the ears were rinsed and blotted dry twice. Mice were boosted in a similar fashion 4 and 8 weeks later. For ventral vaccinations, the ear was treated by gently rubbing the ear with a cotton tipped applicator wetted with sterile water. After 5 min, excess water was blotted from the skin and antigen (CT+HEL (100/100 mg)) in 25 ml PBS was painted onto the prepared skin using a pipetter. After 2 h the ears were rinsed and blotted dry twice. Mice were boosted 5 and 8 weeks later.
2.2. Antibody assay Antibody levels against CT, DT, BSA, and HEL were determined using ELISA. Immulon-2 polystyrene plates (Dynatech Laboratories, Chantilly, VA) were coated with 0.1 mg/well of antigen in saline, incubated at room temperature overnight, blocked with 0.5% casein-Tween-20, washed, serial dilutions of serum applied, and the plates incubated for 2 h at room temperature. Speci®c IgG (H+L) antibody was detected using HRP-linked goat anti-mouse IgG (H+L) (Biorad, Richmond VA) and revealed after 30 min using 2,2 '-azino-di (3-ethyl benzthiazolone) sulphonic acid substrate (ABTS; Kirkegaard and Perry, Gaithersburg, MD) and the reaction stopped using 1% SDS. The plates were read at 405 nm. Results are reported in ELISA Units which are de®ned as the inverse dilution of the sera that yields an OD of 1.0 at 405 nm.
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2.3. T cell proliferation C3H/HeJ mice were immunized with CT or CT/DT on the back at 0, 4, and 8 weeks as described above. Tissues were removed 19 weeks after the primary immunization spleens, and draining lymph node (LN; inguinal) and single cell suspensions prepared using a manual tissue grinder. Red blood cells were lysed with ACK lysis buer (Biowhittaker, Walkerville, MD). Spleen and LN cells from four mice were pooled and cultured at 4 105 cells per well in 96-well plates for 5 days at 378C, 5% CO2 in the presence or absence of 10 mg/ml of DT. Concanavalin A (ConA) at 5 mg/ml was used as a positive control. Culture media contained RPMI 1640 (BioWhittaker), 10% Fetal calf serum (GibcoBRL, Grand Island, NY), penicillin (10 U/ml; BioWhittaker), streptomycin (100 mg/ml; BioWhittaker), L-glutamine (2 mM; Sigma), Hepes (10 mM; BioWhittaker) and 2-mercaptoethanol (50 mM; BioRad, Hercules, CA). [3 H]-thymidine (1 mCi/ well) was added to the cultures during the last 20 h of the 5 day culture period. Thymidine incorporation was assessed by harvesting cellular DNA onto glass-®ber ®lters followed by liquid scintillation counting.
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C57BL/6 or BALB/c mice suggested that the former mice are superior responders to transcutaneously administered CT as determined by both the plasma anti-CT IgG titer and the number of responding animals. To formally test this concept we evaluated the immune response to CT administered by the transcutaneous route in BALB/c, C57BL/6, C3H/HeN and C3H/HeJ mice. Animals were exposed to 100 mg of CT on the back at 0 weeks and boosted 4 and 8 weeks later. Serum was collected 2, 8 and 12 weeks after the primary immunization. As shown in Fig. 1, the plasma IgG response to CT was distinct among the strains at the 2 and 8 week time points with the geometric mean plasma IgG response in C57BL/6> BALB/c r C3H. Although the hierarchy of responders persisted for 8 weeks, the degree of dierence was most apparent 2 weeks after the primary immunization, when the geometric mean titer in the C57BL/6 mice was more than 1 log of that observed in the other strains, and the number of C57BL/6 responders (12 of 12) was greater than that in BALB/c (13 of 15), C3H/HeJ (6 of 12) or C3H/HeN (1 of 9) animals. One hypothesis which might explain the dierences in the immune response following primary and booster
2.4. CD4+ T cell puri®cation CD4+ T cells were isolated from pooled spleen cells from the CT/DT immunization group (see above) using a CD4+ T cell selection column as per the manufacturer's instructions (R and D systems, Minneapolis, MN). Brie¯y, cells eluted from the column (CD4+) were cultured in 96-well plates at 1 105 cells per well in the presence or absence of 3 105 irradiated (3000 RAD) feeder cells from naive mice. Proliferation assays were conducted as described in detail above. 2.5. Statistical analysis Unless otherwise indicated, the data shown are the geometric means of values from individual animals. Comparisons between antibody titers in groups were performed using an unpaired, two-tailed Student's ttest and p-values <0.05 regarded as signi®cant. 3. Results and discussion 3.1. Transcutaneous delivery of cholera toxin as antigen in C57BL/6, BALB/c and C3H mouse strains Over the course of developing the transcutaneous immunization (TCI) method we have evaluated the immunogen and adjuvant activities of CT in several inbred mouse strains. Numerous studies employing
Fig. 1. Serum anti-cholera toxin titers in C57BL/6, BALB/c and C3H mice following TCI with 100 mg cholera toxin on the back at 0 and 4 weeks. Serum was collected at 0, 2 and 8 weeks after the primary immunization and analyzed for CT speci®c IgG by ELISA as described in the Materials and Methods. Results shown are the IgG titers in individual animals (open circles) and the geometric mean for the group (solid bars) in ELISA Units. ELISA units are de®ned as the inverse dilution at which the OD is 1.0 at 405 nm. Statistical signi®cance between C57BL/6 values and other mouse strains are indicated.
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exposures to CT by the transcutaneous route is the eects of the H-2 MHC genes of the strains tested; C3H (H-2k), C57BL/6 (H-2b) and BALB/c (H-2d). Our ®nding of a hierarchy among these strains is consistent with a series of experiments by Elson et al. which clearly show that the plasma IgG response to CT following oral or parenteral administration is under the genetic control of the I-A region of the H-2 major histocompatibility complex [12±14]. Thus, inbred mice with H-2b or H-2q haplotypes were high responders, strains with the H-2s and H-2k low responders and those with the H-2d haplotype intermediate responders. Further study in H-2 congenics on similar backgrounds (i.e., B10 (H-2b) vs B10.BR (H-2k) vs B10.D2 (H-2d) is necessary to formally test the in¯uence of MHC genes on the transcutaneous method. The rise in anti-CT titers in C3H strains following the second immunization suggested that the inferior response in these animals is transient. Accordingly, the animals were boosted a second time and, after the third immunization, the resulting plasma anti-CT levels were comparable to the other strains (Fig. 2A). Thus the quantitative dierence in antigenicity of CT administered by TCI is not absolute and may be overcome by additional exposure to CT. Oral administration of CT to lipopolysaccharide (LPS) congenic mice (C57BL/10 SnJ (lps+) and ScN (lpsÿ)) has been shown to result in a higher plasma
anti-CT IgG titer in the endotoxin responsive strain suggesting a role for the LPS genes in controlling this response [14]. In contrast, our studies with LPS congenics on the C3H background (HeJ (lpsÿ), and HeN(lps+)) failed to reveal an in¯uence of the LPS genes on CT IgG levels following TCI (Fig. 1). The cause of the discrepancy between these studies is unclear and may re¯ect true dierences in the genetic control of oral and TCI procedures or relate to background genes in the C3H and C57BL/10 mouse strains employed. 3.2. Transcutaneous adjuvant activity of CT for diphtheria toxoid and hen egg lysozyme in C57BL/6, C3H and BALB/c mice To assess the adjuvant activity of CT delivered by the transcutaneous route, mice were exposed to adjuvant (CT), antigen (DT or HEL), or adjuvant + antigen at 0, 4 and 8 weeks. After 4 weeks of the ®nal immunization the animals were bled and the plasma anti-CT, DT and HEL titers measured by ELISA. Neither DT nor HEL were able to induce immune responses to themselves after application to the skin (Fig. 2B and C). As stated above, all three mouse strains raised similar anti-adjuvant (CT) responses after three exposures to this protein. However, the antigen (DT and HEL) speci®c IgG titers showed con-
Fig. 2. Serum anti-CT, DT, and HEL titers in C57BL/6, BALB/c and C3H mice following TCI with CT and/or antigen on the back at 0, 4 and 8 weeks. Serum was collected 0 and 12 weeks after the primary immunization and analyzed for CT speci®c IgG by ELISA. Results shown are the IgG titers in individual animals (open circles) and the geometric mean for the group (bars) in ELISA units.
T. Scharton-Kersten et al. / Vaccine 17 (1999) S37±S43
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Table 1 Proliferative response to DT in C3H micea Immunization proteins
CT CT/DT CT CT DT
Source of cells
spleen spleen CD4+APC APC LN LN
In vitro stimulation media (cpm)
(cpm)
1486 236 43 3700 6061
2766 9512 46 4407 55056
DT
SI
(cpm)
1.9 40.3 1.1 1.2 9.1
27725 20247 176 23425 27696
ConA
SI 18.7 85.8 4.1 6.3 4.6
a
Mice were immunized with CT (adjuvant) or CT (adjuvant) and DT (antigen) at 0.4, and 8 weeks as described in the Materials and Methods. Spleen and lymph node cells were collected nineteen weeks after the primary immunization. Data shown are the proliferation values (cpm) obtained from cultures of pooled cells stimulated with media alone DT (10 mg/ml) or the lectin concanavalin A (5 mg/ml). The stimulation index (SI) was calculated with the formula:cpm (experimental)/cpm (media).
siderable variation among the three mouse strains (Fig. 2B and C). BALB/c mice displayed potent titers against both HEL and DT and, in the case of HEL, the antibody response in mice immunized transcutaneously was comparable to that observed in animals given HEL in alum by the intramuscular route. C57BL/6 mice responded well to DT but failed to display detectable anti-HEL responses even after three immunizations. In contrast, C3H mice (HeN and HeJ) displayed high anti-HEL IgG responses and minimal anti-DT titers. The variable response to antigen among the strains appears to be in¯uenced by several factors. First, it is well established that mice bearing the H-2b haplotype (i.e., C57BL/6) fail to produce detectable antibody titers after injection of HEL as they do not bear appropriate MHC molecules to successfully present this antigen [15]. On this basis it was not unexpected that C57BL/6 mice immunized with CT and HEL by the transcutaneous route did not raise an anti-HEL antibody response. One implication of this result is that TCI using CT does not overcome established MHC restriction in the host and thus is unlikely to cause undesirable immune responses against endogenous skin antigens. Consistent with this result we failed to see a rise in titers against cholesterol, a common skin lipid in mice which were treated with CT at doses between 0 and 500 mg (Nabila Wassef, personal communication). C3H mice, on the other hand, did not exhibit substantial IgG titers against DT and the response to this antigen is not known to be restricted by particular MHC H-2 haplotypes [16]. One possible explanation for the de®cient DT response in these animals is that, the less potent immune response to the adjuvant (CT; Fig. 1) contributes to the diminished antigen speci®c anti-DT IgG response in these animals. Detectable anti-DT IgG titers were observed in sera from three of ®ve C3H mice and puri®ed splenic CD4+ T cells and
LN cells from the C3H/HeJ mice clearly showed antigen speci®c proliferative responses to DT (Table 1). Thus, while the antibody response was poor, the C3H mice clearly responded to the antigen. 3.3. Ability to immunize in the ear Immunization on the back has been routinely conducted after shaving of the skin surface to remove hair. The shaving procedure assists in the controlled delivery of the antigen and allows visual assessment of the applied solution. However, one concern with this method has been that small abrasions resulting from the shaving process itself may contribute to the potent systemic immune response observed, following TCI.
Fig. 3. Serum anti-CT titers of BALB/c mice immunized transcutaneously on the ear with 50 ug CT following pretreatment with water (A) or isopropanol (B). Mice were immunized at 0, 4 and 8 weeks and serum collected 0, 4 and 12 weeks after the primary immunization. Anti-CT IgG titers were analyzed by ELISA. Data shown are titers in individual mice (gray bars) and the geometric mean of the group (black bars) in ELISA units. Numeric values for the geometric mean of the group are shown above the black bars.
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HEL as antigen. Thus, the penetration and immunization of the host with topically applied antigens is not dependent upon shaving related insults to the stratum corneum.
Fig. 4. Serum antibody titers of individual BALB/c mice immunized transcutaneously with CT and BSA: (A) on the back following pretreatment by hydration or (B) on the ear following pretreatment with isopropanol. Mice were immunized at 0, 4 and 8 weeks. Serum was collected 10 weeks after the primary immunization and assayed for CT and BSA speci®c IgG by ELISA. Values shown are the prebleed (gray bars) and ten week (black bars) titers for individual mice in ELISA units. NSA = no serum available.
To exclude this possibility we have transcutaneously immunized mice on the ear which does not require shaving because of its naturally hairless surface. As shown in Figs. 3±5 we have successfully achieved TCI on the ear utilizing CT as adjuvant and both BSA and
Fig. 5. Serum antibody titers of individual C3H mice immunized transcutaneously with 100 ug CT and 100 ug HEL: (A) on the back or (B) on the ventral surface of the ear following simple water hydration. Mice were immunized at 0, 4 and 8 weeks. Serum was collected 12 weeks after the primary immunization and assayed for CT and HEL speci®c IgG by ELISA. Values shown are the prebleed (grey bars) and twelve week (black bars) titers for individual mice in ELISA units.
3.3.1. Immunization on the dorsal ear surface The mechanism by which TCI permits induction of a systemic immune response against topically administered antigen remains to be de®ned. One hypothesis is that hydration of the skin allows penetration of antigen into the epidermis where Langerhans cells (LCs) subsequently pick up and convey antigen to the draining lymph node for presentation to T cells [4]. To test this hypothesis we have optimized TCI methods for the ear since it is a convenient source of LCs for study of their function. Our initial attempts to immunize on the ear utilized the dorsal (outer) surface. Although we achieved a detectable antibody response using this approach, the magnitude of the response was considerably less than that achieved with TCI on the back (Fig. 1 vs Fig. 3A). In this regard, the antibody titers against CT were dramatically improved when the area for immunization was gently wiped with isopropanol and allowed to dry, prior to application of the antigen (Fig. 3). This procedure is thought to dilapidate the aected skin and appeared to enhance penetration, as the immunizing solution was absorbed more rapidly as compared to our standard procedure which involves hydration with water prior to immunization. To evaluate the eectiveness of TCI on the ear, BALB/c mice were immunized with CT and BSA on the dorsal surface of the ear or on the back at 0, 4, and 8 weeks and the serum anti-adjuvant and antigen IgG titers determined 10 weeks after the primary immunization. The immune response to adjuvant (CT) and antigen (BSA) was comparable in both groups (Fig. 4). 3.3.2. Immunization on the ventral ear surface Although the dorsal ear method described achieved high titer antibodies, we were concerned that treatment of the skin with chemical agents can alter Langerhans cell function [17]. Thus, we have continued to search for alternate methods which require only minimal modi®cation of the skin surface. To this end we tested whether TCI could be used to introduce antigen through the inner (ventral) ear surface. C3H/HeN animals were immunized with CT and HEL on the back or on the ventral surface of the ear without alcohol swabbing at 0, 4 and 8 weeks and the serum anti-adjuvant and antigen IgG titers determined 12 weeks after the primary immunization. The immune response to the adjuvant (CT) and antigen (HEL) was comparable in both groups (Fig. 5). Thus, with minimal manipulation of the inner ear (i.e., water hydration) animals can be eectively vaccinated using TCI. Together the series of experiments on the ear pro-
T. Scharton-Kersten et al. / Vaccine 17 (1999) S37±S43
vide several key insights related to the TCI method. The observation that animals can be immunized on the ear indicates that the shaving procedure used for TCI on the back is not required for the introduction of antigens to the host through the skin. Moreover, experiments to evaluate the cellular mechanisms of TCI can now be designed, given the body of research available, regarding Langerhans cell morphology and function in the mouse ear and our ability to eectively transcutaneously immunize these animals via the ear. Finally, these data provide strong rationale for integration of TCI into domestic and production animal medicine where the ear is a desirable site for immunization. Acknowledgements We thank Elaine Morrison and Julie Weitz for technical assistance and Dr. Nabila Wassef for measuring the anti-cholesterol antibodies and critically evaluating the manuscript. The work presented was supported and performed under a Cooperative Research and Development Agreement between Walter Reed Army Institute of Research and Medical Technologies and Practice Patterns Institute, Washington DC. References [1] Katz SL. Future vaccines and a global perspective. Lancet 1997;13:1767±70. [2] Reis EC, Jacobson RM, Tarbell S, Weninger BG. Taking the sting out of shots: control of vaccination-associated pain and adverse reactions. Pediatr Ann 1998;27:375±86. [3] Liu MA. Vaccine developments. Nature Med 1998;4:515±9. [4] Glenn GM, Rao M, Matyas G, Alving CR. Skin immunization made possible by cholera toxin. Nature 1998;391:851.
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[5] Glenn GM, Scharton-Kersten T, Vassell R, Matyas GR, Alving CR. Transcutaneous immunization using bacterial ADP-ribosylating exotoxins as antigens and adjuvants. Infection and Immunity 1999; 67:1100±1106. [6] Glenn GM, Scharton-Kersten T, Vassell R, Mallett CP, Hale TL, Alving CR. Transcutaneous immunization with cholera toxin protects mice against lethal mucosal toxin challenge. J Immunol 1998;161:3211±4. [7] Lycke N. The mechanism of cholera toxin adjuvanticity. Res Immunol 1997;148:504±20. [8] Marchetti M, Rossi M, Giannelli V, Giuliani MM, Pizza M, Censini S, Covacci A, Massari P, Pagliaccia C, Manetti R, Telford JL, Douce G, Dougan G, Rappuoli R, Ghiara P. Protection against Helicobactor pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant. Vaccine 1998;16:33±7. [9] Komase K, Tamura S, Matsuo K, Watanabe K, Hattori N, Odaka A, Suzuki Y, Kurata T, Aizawa C. Mutants of Escherichia coli heat-labile enterotoxin as an adjuvant for nasal in¯uenza vaccine. Vaccine 1998;16:248±54. [10] Rappuoli R, Pizza M. Novel molecular biology approaches to acellular vaccines. Biotech Ann 1996;2:391±408. [11] O' Hagan DT. Recent advances in vaccine adjuvants for systemic and mucosal administration. J Pharm Pharmacol 1997;49:1±10. [12] Elson CO, Ealding W. Genetic control of the murine immune response to cholera toxin. J Immunol 1985;135:930±2. [13] Elson CO, Ealding W. Ir gene control of the murine secretory IgA response to cholera toxin. Eur J Immunol 1987;17:425±8. [14] Elson CO. Cholera toxin as a mucosal adjuvant: eects of H-2 major histocompatibility complex and lps genes. Inf Immun 1992;60:2874±9. [15] Hill SW, Sercarz EE. Fine speci®city of the H-2 linked immune response gene for the gallinaceous lysozymes. Eur J Immunol 1975;5:317±24. [16] Kameyama S, Nagaoka F, Matuhasi T. Comparison of immune responses to diphtheria and tetanus toxoids of various mouse strains. Japan J Med Sci Biol 1989;42:83±99. [17] Katoh N, Hirano S, Kishimoto S, Yasuno H. Acute cutaneous barrier perturbation induces maturation of Langerhans' cells in hairless mice. Acta Dermato-Venereologica 1997;77:365±9.