Mucosal and Systemic Immune Responses in BALB/c Mice to ...

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Aug 8, 1989 - fimbriae of Haemophilus influenzae type b. Can. J. Microbiol. 34:723-729. 6. Furuta, R., S. Kawata, S. Naruto, A. Minami, and S. Kotani. 1986.
Vol. 57, No. 11

INFECTION AND IMMUNITY, Nov. 1989, p. 3466-3471 0019-9567/89/113466-06$02.00/0 Copyright © 1989, American Society for Microbiology

Mucosal and Systemic Immune Responses in BALB/c Mice to Bacteroides gingivalis Fimbriae Administered Orally TOMOHIKO OGAWA, HIDETOSHI SHIMAUCHI, AND SHIGEYUKI HAMADA* Department of Oral Microbiology, Osaka University Faculty of Dentistry, Yamadaoka, Suita-Osaka 565, Japan Received 14 April 1989/Accepted 8 August 1989 A 41,000-molecular-weight fimbrial protein was isolated from freshly cultivated whole cells of Bacteroides gingivalis 381 and purified chromatographically. Salivary and serum antibody responses to the fimbriae, which had been orally administered in the presence of an acyl derivative of muramylpeptides, i.e., either N2-[(N-acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine [MDP-Lys(L18)] or sodium P-N-acetyl-

acid-(D)amide-D-alanine (GM-53), or in the absence of adjuvant, were examined in BALB/c mice when administered by gastric intubation on days 0 and 1 as primary immunizations and on days 27 and 28 as booster immunizations. Gastric intubation of the fimbriae with an adjuvant significantly enhanced the production of anti-fimbria immunoglobulin A (IgA) in saliva. Subcutaneous injection of fimbriae along with an adjuvant also raised anti-fimbria IgA levels, as well as IgG levels, in saliva. Both immunization procedures enhanced the levels of anti-fimbria IgG, IgA, and IgM in serum, and the major class of fimbria-specific antibody was IgG, followed by IgA and IgM. However, subcutaneous injection was more effective than gastric intubation to enhance the production of serum antibody in mice. The subclasses of IgG antibody specific for fimbriae in serum were mainly IgGl, followed by IgG2a, IgG2b, and IgG3. These results demonstrated that the combined use of B. gingivalis fimbrial antigen and either GM-53 or MDP-Lys(L18) resulted in a sharply increased IgA antibody response in saliva and a predominantly stimulated IgG antibody response in serum, respectively. Both antibodies were found to be specific for the fimbriae used for immunization.

glucosaminyl-(1->4)-N-acetylmuramyl-L-alanyl-D-isogutaminyl-(L)-stearoyl-(D)-meso-2,6-dianinopimelic

Fimbriae are hairlike microfibrils, observable by electron microscopy, on the cell surface of various bacteria. Among various cell surface components, fimbriae have been suspected to be a specific adherence factor, adhesin, in microbial ecology. In other words, specific adherence of fimbriae of certain bacteria may, at least in part, determine the ability of the organism to adhere to and colonize specific host tissue cells or to interact with other bacterial cells (27). Several studies in animals and in humans have indicated that fimbriae of various bacterial species, such as Neisseria gonorrhoeae, Haemophilus influenzae, and Escherichia coli, induce antibodies which in turn protect against infections by these potentially pathogenic organisms (15, 28, 35, 37). Because of the surface location, fimbriae of certain bacterial species have been recognized as logical candidates for antibacterial vaccines (5, 36). Bacteroides gingivalis has been recognized as a major pathogen in the development of periodontal disease (34). Strains of B. gingivalis possess virulence factors that may contribute to the disease process (40, 43). Several studies also have described the role of this species in various types of experimental infections (7, 9, 13, 17, 26). Periodontal infections by B. gingivalis are associated with elevated levels of antibodies specific for this organism in serum and gingival crevicular fluid (20, 34). This finding coincides with the observation that periodontitis lesions of patients exhibit high numbers of plasma cells producing anti-B. gingivalis antibodies in the gingivae (32). Very recently, we have shown the presence of numerous antibody-secreting cells specific for B. gingivalis fimbriae (24, 25), suggestive of local antibody production. This indicates that the fimbrial protein of B. gingivalis is immunogenic in humans and supports the theory that a vaccine utilizing such a protein may be effec*

Corresponding author.

tive. We report here salivary and serum antibody responses to B. gingivalis fimbriae administered either orally or subcutaneously (s.c.), with an acyl derivative of muramylpeptides, to BALB/c mice. MATERIALS AND METHODS Animals. Six-week-old male BALB/c mice were used in all experiments. These mice were purchased from Charles River Japan (Atsugi City, Japan). Cultivation of B. gingivalis. B. gingivalis 381 was grown in GAM broth (Nissui, Tokyo, Japan) supplemented with hemin (5 mg/liter; Wako Pure Chemical Industries, Osaka, Japan) and menadione (10 ,ug/liter; Wako). After 26 h of incubation at 37°C in an anaerobic chamber (model 1024; Forma Scientific, Marietta, Ohio) containing 5% C02, 5% H2, and 90% N2, organisms were harvested by centrifugation at 10,000 x g for 30 min at 25°C. Preparation of fimbriae. Fimbriae were prepared on a large scale by modification of a method previously described by Yoshimura et al. (41). Briefly, bacterial cells collected from a 16-liter culture (ca. 120 g [wet weight]) were suspended in 2 liters of 20 mM Tris hydrochloride (Tris-HCl) buffer (pH 7.4)-0.15 M NaCl-10 mM MgCl2, and 200-ml aliquots of the suspension were gently pipetted. The aliquots were combined, gently agitated further with a stirring bar for 15 min at 25°C, and centrifuged at 10,000 x g for 30 min at 25°C. Ammonium sulfate was added to the centrifuged supernatant to a 40% saturation. The precipitate was collected by centrifugation and resuspended in 50 ml of 20 mM Tris-HCl buffer (pH 8.0). This material was dialyzed against 25 liters of the same buffer. The dialysate was clarified by centrifugation at 10,000 x g for 15 min, and the supernatant was applied to a column (5 by 15 cm) of DEAE-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden) which had been equilibrated with the same buffer. The column was washed with 3466

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1.5 liters of 20 mM Tris-HCl buffer (pH 8.0) and eluted with a stepwise gradient of 0 to 0.15 M NaCl in 20 mM Tris-HCl buffer (pH 8.0). The fimbrial protein was eluted from the column with 0.15 M NaCl. The fractions containing the fimbrial protein were combined, concentrated by ammonium sulfate precipitation, and dialyzed against 10 liters of 20 mM Tris-HCl buffer (pH 8.0). The procedures described above were repeated 10 times to obtain sufficient amounts of fimbriae; 1.3 g of the purified protein was obtained from a total of 160 liters of B. gingivalis 381 culture. It was found that the purified fimbrial protein and crude whole-cell extract produced a fused single precipitin band against a rabbit anti-fimbria-specific antiserum by double diffusion in an agar plate. No contaminating proteins were present. SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a 1.0-mmthick slab gel (12% polyacrylamide) as described by Neville (22). The samples were heated with SDS at 100°C for 5 min. Adjuvants. N2-[(N-Acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine [MDP-Lys(L18); muroctasin] (14) and sodium ,-N-acetylglucosaminyl-(1-- -4)-N-acetyl-

muramyl-L-alanyl-D-isoglutaminyl-(L)-stearoyl-(D)-meso-2,6diaminopimelic acid-(D)-amide-D-alanine (GM-53) (6) were generously supplied by Daiichi Seiyaku Co., Tokyo, Japan, and Shigeo Kawata, Research Laboratories, Dainippon Pharmaceutical Co., Osaka, Japan, respectively. Preparation of antifimbria-specific antiserum. Antiserum was raised in rabbits against purified fimbrial protein. Fimbriae (1.0 mg of protein) were mixed with GM-53 and Freund incomplete adjuvant (Difco Laboratories, Detroit, Mich.), and the mixture was s.c. injected into rabbits three times at 2-week intervals. Five days after the final injection, serum was collected. Incorporation of fimbriae and adjuvant into liposomes. Lecithin (DL-a-phosphatidylcholine, dipalmitoyl, Grade I; Sigma Chemical Co., St. Louis, Mo.) and cholesterol (Sigma) (30 ,umol of each) were dissolved in 3 ml of chloroform in a 25-ml round-bottomed flask. The chloroform was then evaporated in vacuo at room temperature, leaving a thin film consisting of lecithin and cholesterol on the inside wall of the flask. B. gingivalis fimbriae with or without an adjuvant in 20 mM Tris-HCl buffer (pH 8.0) were added to the flask, and the mixture was shaken at 55°C. The contents of the flask were then ultrasonicated at 40°C by use of an ultrasonic cleaner (model UT-51; Sharp Electronic Inc., Osaka, Japan) to obtain a homogeneous suspension of small unilamellar liposome vesicles (10). In some experiments, B. gingivalis fimbriae dissolved in 20 mM Tris-HCl buffer (pH 8.0) were used as an immunogen without being incorporated into liposomes. Experimental design of oral immunization. Groups of 8 to 12 BALB/c mice were orally given the fimbriae with or without an adjuvant or were given liposomes alone or Tris-HCl buffer (0.25 ml per mouse) with the aid of an intubation needle (Natsume Co., Ltd., Tokyo, Japan) on days 0 and 1. The mice received the secondary (booster) oral administration of immunogens on days 27 and 28 in the same manner that the primary immunization had been given. The mice were bled from the inferior ophthalmic vein by use of a capillary glass tube on day 33 after the primary immunization. Saliva samples were then collected from the mice after they were treated with pilocarpine hydrochloride (Wako) under anesthesia with pentobarbital sodium (Nembutal; Dainippon Pharmaceutical Co.). In some experiments, BALB/c mice were injected s.c. with the same immunogens described above on days 0 and 28. On day 33, serum and

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saliva samples were collected in the same way. These serum and saliva specimens were divided into small amounts and stored at -80°C until use. ELISA. The isotypes and subclasses of IgG and the levels of anti-fimbria antibodies in the serum and saliva of mice were determined by enzyme-linked immunosorbent assay (ELISA) as described by Voller et al. (39) with a minor modification. In brief, a solution of fimbriae (0.01 mg/ml) dissolved in 0.1 M carbonate buffer (pH 9.6) (100 ,l per well) was added to wells of a 96-well flat-bottomed micro-ELISA plate (M129; Dynatech Laboratories Ltd., Billingshurst, United Kingdom). The plates were incubated overnight at 4°C and washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 0.2% sodium azide (PBS-T) to remove the unbound fimbriae from the wells. The plates were then incubated with 1% bovine serum albumin (Sigma) for 18 h at 4°C. Unless otherwise stated, PBS-T was used to dilute serum and saliva and to wash plates. After being washed, serum and saliva specimens (100 RI), appropriately diluted, were added to each well. After overnight incubation at 4°C, the plates were washed three times and alkaline phosphatase-labeled goat anti-mouse IgA-, IgG-, or IgMheavy-chain-specific antibody or rabbit anti-mouse IgGl-, IgG2a-, IgG2b-, or IgG3-heavy-chain-specific antibody (Zymed Laboratories Inc., San Francisco, Calif.) was added to the test wells. The plates were incubated at 37°C for 2 h and washed, and p-nitrophenylphosphate (Phosphatase Substrate 104; Sigma) dissolved in diethanolamine solution (pH 9.8; 1 mg/ml) was added at a concentration of 100 ,ug per well. After incubation at 25°C for 30 min, the enzyme reaction was stopped by adding 50 RI of 3 N sodium hydroxide, and the A405 was read with a Titertek Multiskan MC photometer (Flow Laboratories, Inc., McLean, Va.). The values obtained from quadruplicate wells were averaged to obtain the mean values and their standard errors. To determine the antibody concentration in the serum and saliva from mice that had been immunized with B. gingivalis fimbriae, the 96-well flat-bottomed plates (Dynatech) were coated with 100 ,u of goat anti-mouse IgA-, IgG-, or IgMheavy-chain-specific antibody (0.01 mg/ml) (Zymed) for 18 h at 4°C. After the plates were washed with PBS-T, they were incubated in 100 ,ul of 1% bovine serum albumin (Sigma) for 18 h at 4°C and washed again with PBS-T. Calibration curves were obtained by using twofold serial dilutions of purified mouse IgG (Zymed) for the IgG standard, mouse IgA (mouse myeloma protein MOPC315; Organon Teknika Corporation, West Chester, Pa.) for the IgA standard, and mouse IgM (TEPC185, Cappel) for the IgM standard. Then, IgGl (MOPC21; Organon Teknika), IgG2a (RPC5; Organon Teknika), IgG2b (MOPC195; Organon Teknika), and IgG3 (FLOPC21; Organon Teknika) were added as standards, respectively, for assay of IgG subclasses. Namely, purified mouse IgG or monoclonal antibodies in hybridoma culture supernatants were incubated with plates for 18 h at 4°C. After being washed again with PBS-T, alkaline phosphataselabeled goat anti-mouse IgA-, IgG-, or IgM-heavy-chainspecific antibody or rabbit anti-mouse IgGl-, IgG2a-, IgG2b-, or IgG3-heavy-chain-specific antibody (Zymed) was added for 2 h at 37°C, followed by washing with PBS-T. pNitrophenylphosphate was added to the plates. The degree of the reaction was determined by measuring the A405 in a micro-ELISA reader. The calibration curves and interpolation of unknown samples were obtained by use of a PC9801vm personal computer (NEC Corporation, Tokyo, Japan) by using a program based on an equation of log-logit transformation (30) and fitted to a linear equation of the

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Elustion volume (ml) FIG. 1. Isolation and purification of B. gingivalis fimbriae by using a DEAE-Sepharose Fast Flow column and SDS-PAGE analysis of the purified fimbriae. Crude fimbrial extract was precipitated with 40% saturated ammonium sulfate, and the precipitate was dialyzed against 20 mM Tris-HCl (pH 8.0). The dialysate was applied to a DEAE-Sepharose Fast Flow ion-exchange column and eluted with a stepwise gradient of 0 to 0.15 M NaCl in the same buffer. The fimbrial protein was eluted with 0.15 M NaCl. The SDS-PAGE profile is shown within the elution pattern.

regression with correlation coefficients of higher than 96%B. The titers of anti-fimbria antibodies in serum and saliva were expressed as micrograms per milliliter and nanograms per milliliter, respectively. Statistics. Comparisons between groups were done by Student's t test for independent samples. RESULTS Serum immune responses to B. gingivalis fimbriae. Crude fimbrial extract isolated from freshly grown cells of B. gingivalis 381 was chromatographically purified by passage through a column of DEAE-Sepharose Fast Flow. The protein peak which specifically reacted with anti-fimbria serum was identified as a 41,000-molecular-weight protein by SDS-PAGE (Fig. 1). Oral administration of the fimbrial antigen incorporated into liposomes resulted in production of significantly increased levels of anti-fimbra antibodies in the serum of BALB/c mice. Similar administration of liposomes alone did not induce significant antibody responses. It was also found that incorporation of adjuvant GM-53 into the fimbriae-liposomes complex enhanced immune responses, and the antibody levels increased significantly as compared with those in the fimbra-alone group. Such an effect was observed following oral administration of adjuvant MDPLys(L18) with fimbrial antigen in liposomes, although there was a statistically significant increase in anti-fimbria IgG relative to the sham controls (Fig. 2). The major isotype of antibody specific for fimbrial antigen in serum was IgG. In the second series of experiments, fimbrial antigen was found to markedly enhance anti-fimbria antibody levels in serum, particularly of the IgG class, when it was given s.c. in liposomes (Fig. 2). Definitive immunoadjuvant activity was noted following the use of GM-53 or MDP-Lys(L18) together with the fimbriae-liposomes complex. It was of interest to compare the distribution of fimbrial antigen-specific IgG subclass antibodies in serum from mice immunized orally or s.c. with the fimbriae-adjuvant-lip-

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FIG. 2. Serum antibody responses in the mice immunized either orally or subcutaneously with B. gingivalis fimbriae and adjuvant in liposomes. Four groups of 8 to 12 BALB/c mice (male, 6 weeks old) were immunized on days 0, 1, 27, and 28 by oral administration of fimbriae + GM-53 ( ), fimbriae + MDP-Lys(L18) ( ), fimbriae alone ( 1111 ), or liposomes alone (control) (LI). Another four groups of BALB/c mice were immunized by s.c. administration of liposomes containing fimbriae in the presence or absence of adjuvant on days 0 and 28. Serum anti-fimbria antibody levels were determined by ELISA 5 days after the booster immunization. Values (mean ± standard error [SE]) are expressed as micrograms per milliliter of serum, as described in detail in Materials and Methods. Symbols: **, statistical difference from the value for the control (liposome alone) group at P < 0.01; t, statistical difference from the value of the group given fimbriae alone at P < 0.05.

osomes complex. The most prevalent subclass following oral administration of the complex was IgGl (68.4% total IgG), followed by IgG2b (14.0%), IgG2a (13.6%), and IgG3 (4.0%) (Fig. 3). Enhanced levels of fimbria-specific antibodies in serum were observed in IgGl and IgG2b subclasses when mice were orally given fimbrial antigen with GM-53 in liposomes. On the other hand, s.c. immunization of mice with fimbriae with or without GM-53 in liposomes induced fimbria-specific antibodies that were predominantly of the IgGl subclass (86.5%), followed by IgG2a (8.1%), IgG2b (5.0%), and IgG3 (0.4%) subclasses (Fig. 3). The total IgG levels induced were clearly higher following s.c. immunization than following oral immunization (Fig. 3). Salivary antibody responses of fimbrial antigen. Oral administration of fimbrial antigen in liposomes caused negligible production of anti-fimbria antibodies in saliva, even when a dose as high as 2 mg per mouse (four times) was given (data not shown). However, when GM-53 (500 ,ug) was incorporated into liposomes together with fimbriae (500 ,ug), salivary anti-fimbria IgA levels were significantly raised and were higher than those following s.c. administration of fimbrial antigen with GM-53 in liposomes (Fig. 4). MDPLys(L18), unlike GM-53, was found to exhibit a weak but definite adjuvant effect under the same conditions. Saliva from mice orally immunized with fimbriae and either MDPLys(L18) or GM-53 in liposomes had approximately 7- to 17-fold-higher levels of anti-fimbria IgA, but not IgG, antibody than did saliva from animals immunized with fimbriae alone. The adjuvant effect of GM-53 appeared to be more prominent than that of MDP-Lys(L18). On the other hand, mice that had been immunized s.c. with fimbriae together with either GM-53 or MDP-Lys(L18) in liposomes raised

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FIG. 3. Comparison of IgG subclass antibodies specific for B. gingivalis fimbriae in serum from the mice either orally or s.c. immunized with fimbriae in the presence or absence of GM-53 in liposomes. Three groups of 8 to 12 BALB/c mice were immunized by either oral or subcutaneous administration of fimbriae + GM-53 ( u ), fimbriae alone ( M ), or liposomes alone (control) (LII). The experimental protocols and the antibody assay methods are the same as described in the legend to Fig. 2. Symbols: * and **, statistical differences from the value for the control group at P < 0.05 and P < 0.01, respectively; t, statistical difference from the value of the group given fimbriae alone at P < 0.05.

approximately four- to fivefold-higher levels of IgG antibodies to fimbriae in saliva than did animals given fimbriae alone. However, no adjuvant effect was noted in the production of anti-fimbria IgA when the complex of fimbriae and either GM-53 or MDP-Lys(L18) in liposomes was administered to mice s.c. (Fig. 4). DISCUSSION The present study demonstrates that B. gingivalis fimbriae administered orally together with an adjuvant led to specific antibody production in serum as well as in saliva of BALB/c mice. This is in contrast to the previous findings by Ogawa et al. (23) that a similar manner of oral immunization with the same dose of bovine serum albumin as an antigen caused no significant increase in anti-bovine serum albumin antibodies in saliva. It has been recognized empirically that many antigens, particularly those from nonintestinal pathogens, are not good immunogens for the gut mucosal immunocytes (4, 8, 21). These results suggest, however, that the nature of the protein antigen, the combination of antigen and adjuvant, the immunization schedule, and the route of administration may be of importance with regard to induction of salivary antibodies in orally immunized mice. We have found that GM-53, a semisynthetic adjuvant (6), induced a salivary antibody response to B. gingivalis fimbriae. Furthermore, we compared the induction of salivary antibody to B. gingivalis fimbriae following either oral or s.c. immunization of mice with the fimbriae-GM-53-liposomes complex. It is of interest that the combination of fimbrial antigen and GM-53 resulted in a superior effect compared with MDP-Lys(L18); however, the mechanisms of the difference in the adjuvant effects are not known at present. In saliva, higher IgA

0

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FIG. 4. Salivary antibody responses in the mice either orally or s.c. immunized with B. gingivalis fimbriae in the presence or absence of adjuvant in liposomes. Four groups of 8 to 12 BALB/c mice were immunized by either oral or s.c. administration of fimbriae + GM-53 (E), fimbriae + MDP-Lys(L18) (M), fimbriae alone (1m ), or liposomes alone (control) (LII). Values (mean ± SE) are expressed as nanograms per milliliter of saliva, as described in Materials and Methods. The experimental protocols are the same as described in the legend to Fig. 2. Symbols: **, statistical difference from the value for the control group at P < 0.01; t, statistical difference from the value of the group given fimbriae alone at P < 0.05.

responses, but not IgG anti-fimbria responses, were seen in the orally immunized mice. Serum IgG antibodies followed by lower levels of IgA and IgM antibodies were noted. No correlation between salivary and serum antibody levels was observed in terms of either IgA or IgG antibodies. When these results are considered together, it appears that salivary IgA antibodies specific for B. gingivalis fimbriae are locally produced. Reynolds and Thompson (29) further reported on some functional differences between IgG and IgA antibodies in the secretions of rabbits. They demonstrated that agglutinating activity was found in both IgG and IgA antibodies, but IgA was found to have an inhibitory effect on the growth of Pseudomonas aeruginosa, whereas IgG antibodies exhibited stronger opsonizing activities when compared with IgA. These data indicated that, in addition to secretory IgA, secretory IgG may play an important, but different, role in mucosal immunity. Similar findings were reported by other groups, who showed that oral immunization with Streptococcus mutans (1) and E. coli (12) led to antibody responses not only in the saliva but also in the serum of humans. Bergmann and Waldman (3) stated that they could not determine whether the origin of specific antibody in secretions was locally produced or exuded from diffusion through gingival blood circulation. Our present study demonstrates that levels of IgA antibodies to B. gingivalis fimbriae in saliva and also levels of IgG antibodies in serum increase following oral immunization with this antigen. It can be assumed that oral immunization of B. gingivalis fimbriae with an adjuvant stimulated production of IgA antibody to

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fimbriae in secretion by way of a possible common mucosal immune system; antibody-forming cells in gut-associated lymphoid tissues would be stimulated by the antigen-adjuvant-liposomes complex and then disseminated close to various exocrine organs, including salivary glands (18), as would be synthesis of IgG antibody to fimbriae in serum through a systemic immune system. Combined use of B. gingivalis fimbriae and an adjuvant may induce significant serum antibody responses comparable to the level following administration of live vaccines. Mechanisms of the adjuvant effect of an acyl derivative of muramylpeptides with protein antigens are not known well at present. We are currently investigating the mode of stimulation of antibody-forming cells in various lymph nodes in mice after oral immunization with B. gingivalis fimbrial antigen and an adjuvant under the same conditions. We also investigated the efficacy of liposomes as a carrier and an adjuvant for enhancing the production of antibodies against the B. gingivalis fimbriae. We found that oral administration of liposomes containing fimbriae and GM-53, but not Tris-HCl buffer solution, led to a significant enhancement of salivary antibodies that reacted with fimbriae (data not shown). Liposomes have been reported previously to be an "adjuvant" to enhance the immunogenicity of watersoluble antigens, such as diphtheria toxin (2), human serum albumin (38), and bovine serum albumin (23). Rowland and Woodley (31) showed that orally administered liposomes could resist extremes of pH in the stomach and the presence of bile salts and pancreatic lipase in the duodenum. It appears that liposomes may be effective adjuvants for stimulating the mucosal immune system when administered by the oral route, possibly because liposomes will enhance and prolong the contact of the fimbrial antigen and adjuvant with the afferent immune system. Both oral and non-oral administrations of fimbrial antigens to mice induced mainly IgGl antibodies, along with far less amounts of IgG2 and IgG3 subclasses. In this regard, it has been reported that, in adults, protein antigens, i.e., tetanus toxin (33), and diphtheria toxin (19), induced mainly IgGl antibodies, while carbohydrate antigens (11, 16, 42) preferentially induced IgG2 antibodies. LITERATURE CITED 1. Allansmith, M. R., J. L. Ebersole, and C. A. Burns. 1983. IgA antibody levels in human tears, saliva, and serum. Ann. N.Y. Acad. Sci. 409:766-768. 2. Allison, A. C., and G. Gregoriadis. 1974. Liposomes as immunological adjuvants. Nature (London) 252:252. 3. Bergmann, K.-C., and R. H. Waldman. 1988. Stimulation of secretory antibody following oral administration of antigen. Rev. Infect. Dis. 10:939-950. 4. Clancy, R., and A. Pucci. 1978. Sensitisation of gut-associated lymphoid tissue during oral immunization. Aust. J. Exp. Biol. Med. Sci. 56:337-340. 5. Erwin, A. L., G. E. Kenny, A. L. Smith, and T. L. Stull. 1988. Human antibody response to outer membrane proteins and fimbriae of Haemophilus influenzae type b. Can. J. Microbiol. 34:723-729. 6. Furuta, R., S. Kawata, S. Naruto, A. Minami, and S. Kotani. 1986. Synthesis and biological activities of N-acetylglucosaminyl-P(1--*4)-N-acetylmuramyl tri- and tetrapeptide derivatives. Agric. Biol. Chem. 50:2561-2572. 7. Grenier, D., and D. Mayrand. 1983. Etudes d'infections mixtes anadrobies comportant Bacteroides gingivalis. Can. J. Microbiol. 29:612-618. 8. Hanson, D. G., N. M. Vaz, L. C. S. Maia, and J. M. Lynch. 1979. Inhibition of specific immune responses by feeding protein antigens. III. Evidence against maintenance of tolerance to ovalbumin by orally induced antibodies. J. Immunol. 123:

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