Application of polyethyleneglycol (PEG)-modified liposomes for oral vaccine: effect of lipid dose on systemic and mucosal immunity

Journal of Controlled Release 89 (2003) 189–197 www.elsevier.com / locate / jconrel

Application of polyethyleneglycol (PEG)-modified liposomes for oral vaccine: effect of lipid dose on systemic and mucosal immunity Seiichiro Minato a , Kazunori Iwanaga b , Masawo Kakemi b , Shinji Yamashita c , Naoto Oku a , * a

Department of Medical Biochemistry and COE Program in the 21 st Century, University of Shizuoka School of Pharmaceutical Sciences, 52 -1 Yada, Shizuoka 422 8526, Japan b Department of Pharmaceutics, Osaka University of Pharmaceutical Sciences, 4 -20 -1 Nasahara, Takatsuki, Osaka, Japan c Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Setsunan University, 45 -1 Nagaotoge-cho, Hirakata, Osaka, Japan Received 14 August 2002; accepted 10 January 2003

Abstract To examine the systemic and mucosal immunity towards a liposomal antigen in an oral vaccine, we prepared ovalbumin (OVA)-encapsulating polyethyleneglycol (PEG)-modified liposomes and unmodified ones, and orally administered two different concentrations of them to mice. Unmodified liposomes tended to induce a stronger systemic immune response than the PEG-modified ones especially at the higher concentration of liposomes. Whereas at the lower liposome concentration the mucosal immune response was stronger for the PEG-modified liposomes than for the unmodified ones but nearly the same at the higher concentration. The relative amount of immunoglobulin G (IgG) against OVA in the plasma was 1.7-fold higher for a 12.5 mmol phospholipid dose of PEG-liposomes encapsulating OVA than for a 5.0 mmol one encapsulating the same amount of OVA. On the contrary, the relative amount of IgA in the intestinal wash was 2.6-fold higher for the 5.0 mmol phospholipid dose than for the 12.5 mmol one. These results indicate that OVA encapsulated in a small number of liposomes, especially the PEG-modified ones, is favorable for inducing a mucosal immune response and that the same amount of OVA in a large number of liposomes tends to improve the systemic immune response. A possible explanation for this tendency is the differential release rate of OVA from the liposomes at the intestinal mucosa. Our present study suggests that the dose of liposomes containing antigen is an important factor for controlling the response of systemic and mucosal immune systems.  2003 Elsevier Science B.V. All rights reserved. Keywords: Oral vaccine; Mucosal immune response; Liposome; Polyethyleneglycol (PEG)

1. Introduction

*Corresponding author. Tel.: 181-54-264-5701; fax: 181-54264-5705. E-mail address: [email protected] (N. Oku).

Vaccine is one of the best preventive strategies against infectious diseases. Infusion vaccines chiefly used are not fully beneficial, since they require trained persons to administer them. In comparison,

0168-3659 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0168-3659(03)00093-2

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oral vaccines have the advantages of self-administration at ease, low risk of contamination, and a reduced cost. Therefore, the development of oral vaccines has become noticed. Oral immunization acts mainly in the gut-associated lymphoid tissue (GALT), forms a network with other mucosal sites such as respiratory and urogenital mucosa, and represents the first line of defense against colonization by viral and bacterial pathogens. A certain foreign substance is taken up into Peyer’s patches, which are epithelia enriched with phagocytic microfold (M) cells. Then the substance is transmited to macrophages and lymphocytes following secretion of immunoglobulin A (IgA) from antigen-specific B cells at general mucosal sites. IgA can neutralize biologically active antigens to prevent pathogens from invading the body [1,2]. Oral immunization, however, has a problem of low bioavailability: antigens are degraded by gastric acidity and proteolytic enzymes in the intestinal lumen and therefore extremely large doses are required to achieve an adequate immune response. In recent years, the use of microparticles such as liposomes as a carrier of antigens for inducing immunity has been explored; and microparticles were found to prevent the degradation of antigens in the gut [3]. Oral immunization with antigen incorporated in microparticles was also reported to induce not only mucosal but also systemic immunity [4,5]. Therefore, the development of optimum microparticle formulations enabling the prevention of infections by activating the systemic and mucosal immune systems has been attempted. Among the microparticles evaluated, liposomes are notable, since they can be easily controlled in terms of size, charge, membrane fluidity, etc. The change in liposomal properties such as particle size, surface charge, saturation degree of structural lipids, and lipid doses is known to affect the pharmacokinetics of the encapsulated drugs after systemic administration [6,7]. Furthermore, particle size, surface charge and lipid composition were reported to affect liposomal uptake by Peyer’s patches [8,9]. However, the effect of liposomal lipid doses on the induction of immunity has not yet been fully examined. In the present study, the effect of two liposomal lipid doses on systemic and mucosal immunity following oral administration was examined by use

of ovalbumin-containing unmodified ethyleneglycol-modified liposomes.

or

poly-

2. Materials and methods

2.1. Materials Distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylethanolamine – polyethyleneglycol 2000 (DSPE–PEG) were the gifts from Nippon Fine Chemical Co., Ltd. (Hyogo, Japan). Grade V ovalbumin (OVA), bovine serum albumin (BSA), and cholesterol (Chol) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Horseradish peroxidase (HRP)-immobilized goat antibodies against mouse IgG (g-chain specific) and mouse IgA (a-chain specific) antibody were obtained from Sigma Chemical Co. and Kirkegoard Perry Laboratories (Gaithersburg, MD, USA), respectively. Sodium taurocholate, o-phenylenediamine dihydrochloride tablets, and free cholesterol E TestWako were purchased from Wako Pure Chemical Industries (Osaka, Japan). The bicinchoninic acid (BCA) protein assay reagent kit was from Pierce (Rockford, IL, USA). All other chemicals used were of analytical grade.

2.2. Preparation of liposomes Multilamellar liposomes (MLVs) composed of DSPC and Chol in a molar ratio of 10:5 (unmodified liposomes, N-Lip) or DSPC, Chol and DSPE–PEG in a molar ratio of 10:5:1 (PEG-modified liposomes, PEG-Lip) were prepared as follows: the lipids dissolved in chloroform was evaporated to dryness in a rotary evaporator, and then further dried in vacuo for 1 h to remove the solvent completely. Next the thin lipid film was hydrated with 0.15 M sodium gluconate containing 40 or 16 mg / ml OVA to form liposomes (100 mM as DSPC). After three cycles of freeze–thawing, the liposomal suspension was centrifuged three times at 50,000 g for 10 min to remove untrapped OVA, and resuspended in phosphate-buffered saline (PBS, pH 6.7) to make liposomal concentrations of 20 mM or 50 mM in terms of DSPC. Since the encapsulation efficiency was not the same for all preparations, the actual concen-

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trations of OVA in N-Lip and PEG-Lip were 3.560.1 and 4.460.3 mg / ml, respectively, for 50 mM DSPC liposomal preparations, and 2.360.1 and 3.360.2 mg / ml, respectively, for 20 mM DSPC ones. The diameter of liposomes used was 1.7–3.3 mm, as determined by dynamic laser light scattering (ELS800, Otsuka Electronics Co., Ltd., Osaka, Japan).

area under the time versus time3amount curve and the time versus time3amount 2 curve, by use of the following equations:

2.3. Release of OVA from liposomes in vitro

DTT 5 (VTT)1 / 2

Liposomes were incubated for 0.5, 1, 2 and 4 h in PBS containing 10 mM sodium taurocholate at 37 8C. Then the released OVA was separated from the liposomes by centrifugation (50,000 g, 10 min), and was measured by the BCA protein assay method.

2.4. Single-pass perfusion experiment and moment analysis In situ local intestinal perfusion was carried out according to the method of Kakutani et al. [10] with a modification. Female ddY mice, weighing 22–30 g, were obtained from Japan SLC Inc. (Shizuoka, Japan). The mice, having been fasted for 18 h, were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg / kg). An abdominal incision was made, and both the duodenum and proximal jejunum (20 cm below the duodenum) were cannulated and ligated tightly. After pre-perfusion with PBS (pH 6.7) for 10 min (0.6 ml / min), 0.15 ml of the test liposomal solution was injected into the line of the perfusion flow as a pulse by using a three-position valve. The outflow perfusate was collected into pre-weighed tubes. The samples were collected every 30 s. After each sample had been weighed, the concentration of cholesterol, which indicated the liposomal concentration, was determined by the cholesterol oxidase method (free cholesterol E Test-Wako). Statistical moment analysis [10] was applied to the data obtained from the single-pass perfusion experiment. The mean transit time (MTT) and deviation of transit time (DTT) of liposomes were calculated from their outflow patterns. Briefly, the zero-order moment (S0 ) was calculated from the area under the time versus the amount (% of dose) in the perfusate curve. Similarly, the first-order moment (S1 ) and the second-order moment (S2 ) were calculated from the

MTT 5 S1 /S0 VTT 5 (S2 /S0 ) 1 (S1 )2

where VTT is the variance of transit time. DTT, the square root of VTT, was used here, since VTT was too large.

2.5. Immunization protocol for liposomes containing OVA Female ddY mice were fasted for 18 h and then administered by gastric intubation of various liposomes or PBS containing 4 mg / ml OVA (0.25 ml / mouse). Each experimental group consisting of 5–8 mice, received two immunizations, on days 0 and 2, and a booster immunization on day 28. The animals were cared for according to the animal facility guideline of the University of Shizuoka.

2.6. Collection of biological fluids from mice immunized with OVA Blood was collected weekly from the retroorbital plexus of immunized mice for 6 weeks after the first immunization. Plasma was collected after centrifugation (600 g, 4 min) and stored at 220 8C until used for assessing the IgG antibody activity by enzymelinked immunosorbent assay (ELISA). Intestinal washes were collected by the following method: Mice were orally administered 0.25 ml of distilled water before collection of the intestinal wash. Fifteen minutes later, the mice were anesthetized with ether, and then the proximal intestine (from the top of the duodenum to 15 cm below the top of it) was excised, and the contents of this region were washed into 10 ml of PBS containing 5 mM EDTA, 0.02% sodium azide, 1% BSA, and 1 mM phenyl methyl sulphonyl fluoride. Wash samples were centrifuged at 1,000 g for 10 min, and supernatant was collected and stored at 280 8C until used for assessing IgA antibody activity in the intestinal wash.

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2.7. ELISA

2.8. Statistical analysis

The relative amount of anti-OVA antibody in mouse plasma and intestinal washes was monitored by ELISA. Each well of a 96-well culture plate (Coastar, NY, USA) was coated overnight at 4 8C with 50 ml of OVA (200 mg / ml) in 0.05 mM sodium carbonate–bicarbonate buffer (pH 9.6). After the well had been washed with PBS containing 0.1% Tween 20 (PBST), 100 ml of 1% BSA solution in PBS (PBS–BSA) was added to it; and the plate was incubated for 1 h at 4 8C. Then 100 ml of test sample diluted with PBS–BSA was added to each well, and the plate was incubated for 2 h at room temperature. Following two washings with PBST, 100 ml of HRP-conjugated goat anti-mouse IgG or IgA antibody was added to each well, and the plate was incubated for a further 2 h at 4 8C. Then, after four washings with PBST, 100 ml of citric acid–phosphate buffer (pH 5.6) containing 0.5 mg / ml ophenylenediamine dihydrochloride and 0.015% H 2 O 2 was added to each well for a 10-min reaction at room temperature. The enzyme reaction was stopped by the addition of 100 ml of 1 M HCl, and the absorbance at 492 nm was measured with a microplate reader (Corona, MTP-120). The absorbance for the pre-immunized control was similarly determined, and the test results were expressed as the relative increase in absorbance against the control.

Variance in a group was evaluated by the F-test, and differences were evaluated by Student’s t-test.

3. Results

3.1. Release of OVA from liposomes in vitro To examine the integrity of liposomal OVA during passage through the oral–gastrointestinal route, we studied the release of OVA from liposomes in artificial gastric fluid. The release of OVA from liposomes in HCl solution containing 0.9% NaCl at pH 2.0, which solution mimics gastric fluid, was not observed with any liposomal compositions or concentrations tested (data not shown). On the contrary, OVA was released from liposomes in 10 mM sodium taurocholate, which concentration is usually used as an artificial intestinal medium [11,12], although the release was only partial (Fig. 1). OVA release from liposomes with a the concentration of 50 mM as DSPC (50 mM liposomes) was less than that from those having 20 mM (20 mM liposomes). Interestingly, OVA release was enhanced by the modification of liposomes with PEG at both concentrations of the liposomes. These results suggest that the liposomes are essentially stable in the gastric

Fig. 1. Release of OVA from liposomes in 10 mM sodium taurocholate. Unmodified liposomes (N-Lip; m) and PEG-modified liposomes (PEG-Lip; ♦) were incubated for 0.5, 1, 2, and 4 h in 10 mM sodium taurocholate. The released OVA was determined as described in Materials and methods. (A) 50 mM liposomes; (B) 20 mM liposomes. Data are expressed as the mean6S.E. of three separate experiments. Significant differences between the release from PEG-Lip and that from N-Lip are indicated (*, P,0.05; **, P,0.01).

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lumen and that the surface modification of the liposome partially affects the permeability properties of the liposomal membrane.

3.2. Intestinal transit of liposomes Next, the intestinal transit of liposomes was examined. The outflow pattern of liposomes is shown in Fig. 2. Unmodified liposomes showed sharp peaks of liposomal outflow, whereas a broad peak was observed after administration of PEGmodified liposomes. Outflow of N-Lip was detectable up to 315 s after the administration. On the contrary, PEG-Lip was continuously detected until the last sampling point. Moment analysis was applied to these data to compare the difference in the intestinal transit patterns of the liposomes, and the results are summarized in Table 1. PEG-Lip showed an approx. 2.3-fold longer MTT than N-Lip, reflecting the slow transit of PEG-Lip. The PEG-Lip also had a larger DTT, indicating the wide spreading of these liposomes in the intestinal lumen. These results suggest that modification of liposomes with PEG

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Table 1 Moment parameters for intestinal transit of liposomes Formulation

Recovery (%)

MTT (s)

DTT (s)

N-Lip PEG-Lip

96.961.3 72.565.1

134.25613.65 302.99624.62*

248.55617.30 476.83634.13*

Data are expressed as the mean6S.E. of three separate experiments. An asterisk indicates a significant difference between the two liposomal preparations (P,0.05).

prolonged the residence time of the liposomes in the intestinal lumen.

3.3. Production of plasma anti-OVA IgG antibody Fig. 3 shows the level of OVA-specific IgG antibody in the plasma of mice that had received oral immunization with various formulations of OVA. When 50 mM liposomes were used (12.5 mmol as DSPC), the ability for production of plasma antiOVA IgG antibody was the highest for unmodified liposomes, next for PEG-Lip, and the lowest for the OVA solution (Fig. 3A). On the other hand, improvement of the systemic immune response by administration of 20 mM liposomes (5.0 mmol as DSPC) was not observed especially when PEG-Lip was used (Fig. 3B). The P-values of the difference between 50 mM and 20 mM liposomes were 0.330 and 0.352 for N-Lip and PEG-Lip, respectively. These results suggest that the systemic immune response was induced more effectively by the administration of a high dose of liposomes containing antigen than by that of a low dose of such liposomes.

3.4. Production of intestinal wash anti-OVA IgA antibody

Fig. 2. Typical outflow pattern of liposomes obtained from the single-pass perfusion experiment. An abdominal incision was made, and both the duodenum and proximal jejunum (20 cm below the duodenum) were cannulated and ligated tightly. N-Lip (m) and PEG-Lip (♦) were injected into the line of the perfusion flow as a pulse by using a three-position value. The outflow perfusate was collected into tubes. A sample was collected every 30 s.

Next, we determined the ability of orally delivered liposomal OVA to elicit local immunity in the intestinal mucosa. Fig. 4 shows the level of OVAspecific IgA antibody in the intestinal wash of the individual mice that had been orally administered various liposomes containing OVA. Administration of free OVA solution resulted in an approx. fourfold increase in the absorbance in the ELISA compared

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Fig. 3. Production of OVA-specific IgG antibody after oral immunization with various liposomes containing OVA. Mice were immunized with 50 mM liposomes (A) or 20 mM liposomes (B) containing OVA by gastric intubation on days 0, 2, and 28. Plasma was collected every 7 days after immunization with OVA and assayed for OVA-specific IgG antibody by the ELISA method. Data were converted to ratios of OVA-specific IgG antibody to that on day 0. Horizontal bars express the mean; and symbols, the data of individual mouse.

with that for the samples from the non-immunized mice. The amount of anti-OVA IgA antibody in the intestinal wash of mice immunized with 50 mM liposomes was similar to that found with free OVA (Fig. 4A). On the contrary, the relative amount of anti-OVA IgA antibody was increased in mice immunized with liposomal OVA of 20 mM liposomes, especially PEG-Lip was used (Fig. 4B): the P-values of the difference between 20 mM and 50 mM liposomes were 0.014 and 0.148 for N-Lip and PEG-Lip, respectively. These data suggest that the mucosal immune response is induced more effectively by the administration of a low dose of liposomes containing antigen than by that of a high dose, the reverse of the case for systemic immunity.

4. Discussion Since oral vaccines have the attractive attributes of easy administration, safety, and low cost, many trials to develop such vaccines have been performed. However, for the development of oral vaccines, one of the utmost hurdles is their instability in the gastrointestinal lumen. Therefore, to overcome this hurdle, microparticles such as liposomes [3] and biodegradable microspheres [13] have been tried as a carrier for antigens in order to protect them from degradation in the gastrointestinal lumen. Among these microparticles, liposomes are notable, because they can be prepared easily and their surface can be modified in various ways. In fact, immunization with

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Fig. 4. Production of OVA-specific IgA antibody after oral immunization with various liposomes containing OVA. Mice were immunized with 50 mM liposomes (A) or 20 mM liposomes (B) containing OVA by gastric intubation on days 0, 2 and 28. Intestinal wash was collected at 42 days after immunization with OVA and assayed for OVA-specific IgA antibody by the ELISA method. Data were converted ratios of OVA-specific IgA antibody to that determined non-immunized mice. Horizontal bars express the mean, and symbols indicate the data of individual mice.

liposomes that had been modified with specific ligands for homing to Peyer’s patches, e.g., cholera toxin B subunit or Ulex Europaeus Agglutinin I [14,15], elevated systemic immunity [16]. In the present study, we used unmodified and PEG-modified liposomes as a carrier of OVA, a model antigen which has been widely used in studies on oral immunization or tolerance. As carrier liposomes, we selected multilamellar liposomes without reducing their size by sonication or extrusion, since multilamellar liposomes are more resistant than unilamellar ones to detergents [17] and are able to incorporate a larger number of antigens. Furthermore, multilamellar liposomes of 1.7 to 3.3 mm in diameter are considered to be suitable for oral vaccines, since particles between 1 and 10 mm are the most favorable for being taken up by Peyer’s patches, the lymphoid tissue facing the intestinal lumen and responsible for the acquisition of local immunity [8,18]. The liposomes used in this study

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were rather stable under low pH conditions and even in the presence of a bile salt. Even though the amount of OVA released from the liposomes was quite low, the amount of OVA released from the PEG-Lip was greater than that from the N-Lip, which is inconsistent with a previous report that PEG modification enhanced liposomal resistance against bile salts [11]. This discrepancy might be caused by the difference in the molar ratio of the lipids composing the liposomes between the two studies. Next, we examined the immune responses against OVA contained in various liposomes. Our findings, as summarized in Table 2, showed that when mice were immunized with 12.5 mmol liposomes (50 mM liposomes) containing about 1 mg OVA, the systemic immune response of the mice was improved, especially when N-Lip was used. Since the systemic immune response may depend on the uptake OVA by Peyer’s patches, liposomal formulation enhanced the systemic immune response. In fact, the induction of systemic immunity by oral administration of microparticles containing antigen requires ingestion of the microparticles by MAC-1-positive cells located in the neighborhood of Peyer’s patches, followed by their transfer through the mesenteric lymph nodes to the systemic lymphoid tissues such as the spleen [18,19]. The systemic immunity-inducing ability of PEG-Lip was less than N-Lip. A possible explanation of this phenomenon is that PEG-modification suppressed the uptake by Peyer’s patches, since it is generally known that PEG modification suppresses Table 2 Systemic and mucosal immunity after oral administration of liposomal OVA Ig subclass

Formulation

Liposomal preparation 50 mM liposomes

20 mM liposomes

IgG

N-Lip PEG-Lip Solution

11.1765.27 3.6061.27 3.6060.82

4.6360.70 2.0760.33

IgA

N-Lip PEG-Lip Solution

0.8560.17 0.9260.12 0.5960.09

1.8860.28 2.3860.84

Plasma IgG and intestinal wash IgA were measured by the ELISA method at day 42. Data are expressed as mean arbitrary unit mean6S.E. (n55 for the 50 mM liposomal group, n58 for the 20 mM liposomal group).

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the uptake of liposomes by macrophages [20– 22].The administration of 5.0 mmol liposomes (20 mM liposomes) did not much improve the systemic immune response. The reason is not clear at present; however, it may be that the availability of OVA decreased when a lower dose of liposomes is taken up by Peyer’s patches, perhaps due to less efficient uptake. Moreover, the release of OVA from a lower dose of liposomes would in itself further decrease the availability. On the other hand, the level of OVA-specific IgA antibody in the intestinal wash, which was use as an index of the mucosal immune response, was only slightly elevated by the administration of 12.5 mmol liposomes in comparison with that of free OVA. However, the administration of 5.0 mmol liposomes improved the mucosal immune response. The mucosal immune response is reported to require the ingestion of free antigen by Peyer’s patches after the release of the antigen from microparticles [18,19]. Therefore, we speculate the following: high-dose liposomes tend to remain intact in the intestinal lumen; and most of the antigen is ingested in its liposomal form by Peyer’s patches, resulting in improved systemic immunity. On the other hand, low-dose liposomes would be more easily degraded by bile salts; and the amount of antigen taken up as free form would thus increase, thus inducing mucosal immunity. Furthermore, PEG modification might increase the local concentration of liposomes near the Peyer’s patches where the liposomes release OVA. In fact, PEG-Lip increased the liposomal transit time in the intestinal lumen (Fig. 2). The reason for this increase in transit time might be due to the stronger interaction of PEG-Lip with the mucous layer than that of N-Lip with it. Such an effect of PEG-modification was observed previously [23]; and the reduced recovery of PEG-Lip after the single-pass perfusion might have occurred for the same reason. This tendency of PEG-Lip is also favorable for the entry of orally administered substances into the bloodstream [11,23]. Currently, vaccines are being investigated for their ability to prevent many diseases. As targets of vaccines, there are various diseases that exert harmful effects in the gastrointestinal tract. For example, glucosyltransferase causes dental caries [24,25], and Helicobactor pylori causes stomach diseases [26]. In

these diseases, the induction of a systemic immune response is not required. On the other hand, against pathogens that invade through mucosal sites such as influenza virus [27] and human immunodeficiency virus [28], induction of both systemic and mucosal immunity is desirable. Thus, the appropriate control of the immune system induced by oral vaccines enhances the usefulness of them, and our present study provides the possibility of such control.

5. Conclusions In the application of liposomes for use as oral vaccines, the relations between liposomal formulations and immune responses should be clarified. In the present study, we examined the effect of liposomal doses on the immune response. As a result, a small number of liposomes with concentrated OVA tend to improve mucosal immunity, perhaps due to the moderate release of antigen from the liposomes at the immune site. For this purpose, PEG-modification is useful because it endows liposomes with a long transit time in the intestine. On the other hand, a large number of liposomes with diluted OVA tends to stimulate the systemic immune system, possibly because of increased uptake of liposomes in Peyer’s patches in their intact form, although further experiments are needed to clarify the details. The present results suggest that the doses of liposomes are an important factor for designing oral vaccines.

Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science.

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