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The intestinal and serum humoral immune response of mice to systemically and orally administered antigens in liposomes: I. The response to liposome-entrapped soluble proteins
Veterinary Immunology and Immunopathology, 32 (1992) 125-138
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Elsevier Science Publishers B.V., Amsterdam
The intestinal and serum humoral immune response of mice to systemically and orally administered antigens in liposomes: I. The response to liposome-entrapped soluble proteins C.J.
Clarke and C.R. Stokes
Department of Veterinary Medicine, Universityof Bristol, Langford House, Langford, Bristol, BS I 8 7DU, UK (Accepted 14 May 1991 ) ABSTRACT Clarke, C.J. and Stokes, C.R., 1992. The intestinal and serum humoral immune response of mice to systemically and orally administered antigens in liposomes: I. The response to liposome-entrapped soluble proteins. Vet. Immunol. lmmunopathol., 32: 125-138. The development of oral vaccines is of great importance in veterinary medicine and new adjuvants and carriers are essential to this aim. Liposomes are effective systemic adjuvants but the relatively little data on their potential as oral adjuvants is inconclusive. Liposomes containingovalbumin (OA) were effective adjuvants when administered intraperitoneallyto mice. Feeding mice with OA or keyhole limpet haemocyanin in liposomes in a series of priming and boosting regimes failed to elicit any significant increase in serum or intestinal antibody response compared with feeding the free antigen. Oral tolerance induction to systemic challenge was also unaffected by OA entrapment in liposomes. In vitro liposome stability assays at 37°C demonstrated a substantial resistance to disruption in the presence of acidic stomach contents. However, the addition of bile caused a rapid and profound release of protein marker from the liposomes. The rate and degree of disruption was influenced by the type of phospholipid used. These results suggest that liposomes may be useful as carders for orally administered compounds but they are ineffective as adjuvants for the non-particulate, naturally weak immunogens used in this study. ABBREVIATIONS CFA = complete Freund's adjuvant; CH = cholesterol; DCP = dicetyl phosphate; DPPC = DLdipalmitoylphosphatidyl choline; ELISA = enzyme linked immunosorbent assay; E.U.s = ELISA units; i.p. = intraperitoneally; KLH = keyhole limpet haemocyanin; OA = ovalbumin; PA = Lphosphatidic acid; PBS=phosphate buffered saline; PBSB=PBS/3% NaHCO3; PBST=PBS plus 0.05% Tween 20; PC = L-phosphatidylcholine; Tc (s) = transition temperature (s). INTRODUCTION
Lip.osomes can act as adjuvants for a variety of soluble protein antigens when administered parenterally (Allison and Gregoriadis, 1974; Shek and Correspondence to: C.L Clarke, Department of Veterinary Pathology, Royal (Dick) School of Veterinary Studies, Veterinary Field Station, Easter Bush, Roslin, EH25 9RG, UK. © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00
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Sabiston, 1981 ). Their action has been attributed to depot formation, hydrophobicity, rendering soluble antigen particulate, and efficient targetting of the vesicles to antigen-presenting cells of the immune system (Desiderio and Campbell, 1985; Shahum and Therien, 1988 ). Liposome components are antigenically similar to mammalian cell membranes and are therefore degradable and only weakly immunogenic. Liposomes can also be 'tailored' to meet certain requirements by altering the ratios or types ofphospholipids used for their preparation (Gregoriadis, 1980). One influential factor concerns the transition temperature (Tc) above which the major phospholipid molecules that form the lipid bilayers become less rigid and allow leakage of contents. Orally administered liposomes used as carriers have reportedly increased the intestinal absorption or efficacy of entrapped agents such as insulin (Patel and Ryman, 1976), glucose oxidase (Dapergolas et al., 1976), and coagulation factor VIII (Hemker et al., 1980). Other studies have failed to find improved effects after liposomal entrapment of various compounds (Patel and Ryman, 1977 ). Few studies have investigated the immune responses to antigens fed in liposomes. Liposomal entrapment of streptococcal antigens increased salivary antibody responses in rats (Wachsmann et al., 1985, 1986; Gregory et al., 1986) but other studies using bacterial or parasite antigens failed to demonstrate significant adjuvant activity for oral liposomes (Pierce and Sacci, 1984; Rhalem et al., 1988; Rhodes et al., 1988). Because many future vaccine preparations are likely to be based on relatively pure, low molecular weight proteins we have used the well defined, nonparticulate protein ovalbumin (about 43 000 mol.wt. ) as well as the larger KLH protein (2 000 000-3 000 000 mol.wt. ) as our model antigens. The comparative adjuvant effect of liposomes admininstered by the parenteral and oral routes was assessed in this study. The liposomes used were based on either the L-phosphatidylcholine (PC) or DL-dipalmitoylphosphatidylcholine (DPPC) phospholipids as these have different Tcs of - 15/17 °C and 41 °C respectively and have been used in many other studies. The serum antibody response in mice to ovalbumin in liposomes administered intraperitoneally, and the serum and intestinal antibody responses in mice to ovalbumin and keyhole limpet haemocyanin (KLH) fed in liposomes was measured using a variety of feeding protocols and liposome types. The stability of the liposome preparations in vitro was also investigated under conditions similar to those in the gastrointestinal tract. MATERIALS AND METHODS
Protein antigens and radiolabelling Ovalbumin (OA) (Grade V) was from Sigma Chemical Co. (Poole, UK) and KLH was from Calbiochem (San Diego, CA, USA). Proteins were ra-
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diolabeUed with 1251 (Na salt) (Amersham International, Amersham, UK) by the chloramine-T method (Hunter and Greenwood, 1962).
Liposome preparation PC (type V-E) from egg yolk, DPPC, L-phosphatidic acid (PA) (sodium salt) from egg yolk, dicetyl phosphate (DCP) and cholesterol (CH) were all from Sigma. Large multilamellar liposomes were prepared by the dehydration-rehydration procedure based on Kirby and Gregoriadis (1984). The lipids PC or DPPC, CH, and DCP or PA were used in the molar ratio 7: 2: 1. Briefly, 100 /zmol of lipid were dissolved in chloroform, dried by rotary evaporation under reduced pressure and resuspended to a suspension with 3 ml distilled water. The suspension was left to stand for 1 h in a water bath (37 °C for PC-based and 45°C for DPPC-based liposomes). Three millilitres 15 mg OA or KLH ml-1 3.3 mM phosphate buffered saline (PBS) (pH 7.2) containing a trace label of 125I-radiolabelled protein was added and mixed and 1.5 ml aliquots of the suspension were flash frozen in liquid nitrogen, freeze-dried and then rehydrated with 150 #1 distilled water and left at 20 ° C (PC-based liposomes ) or 45°C (DPPC-based liposomes) for 1 h. Isotonic PBS (33 mM, pH 7.2) was then added to restore each aliquot volume to 1.5 ml. The pooled aliquots were centrifuged (three times at 10 000 × g for 30 min) to separate liposomeentrapped from free protein. The final liposome pellet was resuspended in 0.1 M PBS (pH 7.2) and protein entrapment was estimated from the gamma emission c.p.m. Liposomes made of PC/CH/DCP and DPPC/CH/DCP lipids entrapped mean amounts of 13.27 mg OA (8.88 mg KLH) and 4.23 mg OA (4.94 mg KLH) 100/zmol- 1lipid, respectively.
Mice and procedures CBA and Balb/c male mice were bred in the Small Animal Unit, Dept. Animal Husbandry, University of Bristol and were used at 6-8 weeks old. Mice were fed 200/zl volumes of the preparations by intraoesophageal intubation with polyethylene capillary tubing. Serum was prepared from blood collected by tail bleeding under ether anaesthesia or cardiac puncture under chloroform anaesthesia. Intestinal organ cultures were prepared based on the method of Wilson et al. (1989) adapted from Svennerholm and Holmgren (1977). Briefly, the excised small intestines were washed with Hank's balanced salt solution (Flow Labs., Irvine, UK) and 300 nag segments, without Peyer's patches, were transferred to a 6-well tissue culture plate (Linbro, Flow) containing Dutch modified RPMI (Flow) supplemented with 10% foetal calf serum (Imperial Labs., Andover, UK), 2 mM L-glutamine (Flow), 5 #g ml-1 amphotericin B
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(Gibco Ltd., Paisley, UK) and 50/zg ml-1 gentamicin (Flow). The plate was incubated for 18 h at 37°C in a humidified, 5% CO2 atmosphere and then frozen and stored at - 2 0 ° C to stop further cell activity. The antibody content of the supernatant from the incubation of the intestinal tissue segments was shown by others to be the product of de novo synthesis (Svennerholm and Holmgren, 1977). However, in our laboratory this was not demonstrable and antibody detected in the supernatant was shown to be mostly pre-formed antibody that was released from intestinal plasma cells (Wilson et al., 1989).
Enzyme linked immunosorbant assays (ELISAs) Serum and intestinal culture supernatant samples were assayed for anti-OA and anti-KLH, IgG and IgA, antibody by ELISA based on the protocol described before (Wilson et al., 1989). Briefly, 96-well PVC Microtiter plates were coated with either 10/zg OA or 10/tg KLH ml -~ PBS (0.15 M, pH 7.2) ( 100/A per well) and washing steps were with PBS containing 0.05% Tween 20 ( Sigma ) (PBST). Hyperimmune anti-OA or anti-KLH antisera were raised in mice inoculated with OA or KLH in complete Freund's adjuvant (CFA) and used as standards after chequerboard titrations to determine optimum concentrations. Samples and standards were diluted in PBST and titrated down. Subsequent incubations were with sheep anti-mouse IgG (prepared by D. Patel of Department of Animal Husbandry, University of Bristol) or goat anti-mouse IgA (Sigma), pig anti-bovine IgG-alkaline phosphatase conjugate and finally phosphatase substrate (p-nitrophenyl phosphate disodium) (Sigma) at 1 mg ml -~ carbonate buffer (0.05 M, pH 9.6). The plates were read on a Titertek Multiscan MC (Flow) automatic plate reader and absorbance readings were converted into ELISA units (E.U.s) using the ELISANALYSIS program (Dr. J.H. Peterman, University of Alabama, AL). This plots standard and sample dilutions (log~o reciprocals) against the log~o absorbance values. Samples are assigned E.U. values by comparison with the linear part of the standard slope.
Intraperitoneal inoculation of ovalbumin in liposomes Twelve groups of five CBA mice were used. On Day 0, duplicate groups were inoculated i.p. with 100/tg OA in PBS, or entrapped in liposomes made of PC/CH/DCP or DPPC/CH/DCP. Other pairs of groups received 100/tg OA mixed with either type of 'empty' liposome, or PBS alone. Mice in one group of each pair received 100/tg OA in CFA intraperitoneally (i.p.) on Day l 0. All the mice were killed on Day 16 and serum samples were prepared and assayed for IgG anti-OA antibody using the ELISA.
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Oral administration of ovalbumin in liposomes Four groups of six CBA mice were fed for 4 consecutive days with 2 mg OA in PBS or entrapped in liposomes made of P C / C H / D C P or D P P C / C H / D C P lipids. One group was fed PBS alone as a control. On Day 17, the mice received a single feed equivalent to a daily priming dose and all the mice were killed on Day 22. Serum samples and intestinal cultures were prepared and the supernatants from the latter were concentrated 10 times in Minicon cells (Grace & Co., Danvers, MA). Samples were assayed for IgG and IgA antiOA antibody by ELISA. In a second experiment groups of six mice were primed in an identical way, but two additional groups were fed OA mixed with either type of 'empty' liposomes. The mice were tail bled on Day 21 and given a boost feed on Day 23. These mice were killed on Day 28 and serum and concentrated intestinal culture supernatant was prepared and assayed as above.
Oral tolerance induction with ovalbumin in liposomes Three groups of seven mice were fed on 5 consecutive days with either PBS or 4 mg OA in PBS or entrapped in liposomes made of P C / C H / D C P lipids. All the mice were challenged with 100/zg OA in CFA i.p. on Day 19. The mice were killed on Day 28 and serum was prepared and assayed for IgG anti-OA antibody by ELISA.
Oral administration of KLH in liposomes Seven groups of six CBA mice were fed on 4 consecutive days in a protocol involving priming alone and priming followed by boosting. Duplicate groups were fed with either 1 mg KLH, 1 nag KLH entrapped in liposomes made of the lipids P C / C H / D C P , or 'empty' liposomes followed by 1 nag KLH. All the preparations were fed in PBS/3% NaHCO3 (PBSB) and the final group was fed this buffer alone. Mice from one group of each duplicate were killed on Day 8 and serum and intestinal culture supernatants were prepared and assayed for IgG and IgA anti-KLH antibody by ELISA. The remaining four groups were tail bled on Day 13 and on Days 15-18 inclusive the mice received repeat feeds similar to those of the 4 day priming period. These mice were killed on Day 22 and serum and intestinal culture supernatants were prepared and assayed as above.
In vitro stability assay of liposomes Liposomes made of the lipids P C / C H / D C P and D P P C / C H / D C P and containing 3 mg OA m1-1 PBS with trace 125I-OA were prepared. Aliquots
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(2.5 ml) of each liposome suspension were mixed with either PBS, mouse stomach contents or fresh pig bile (neat or diluted 1:5 with PBS). Stomach contents (pH 3.0) were collected from freshly killed mice, diluted 1:2 in distilled water, vortex mixed, and the pH adjusted back to 3.0 with concentrated HC1. The relevant liposome suspensions were incubated with the equivalent stomach contents from four mice. The mixtures were incubated at 37 °C in a shaking water bath (50 c.p.m. ) to simulate physiological conditions. Control suspensions of liposomes mixed with PBS were kept at 4 ° C. At the start (to) and at intervals throughout the 4 h incubation, 400/~1 aliquots were taken from each mixture. One hundred microlitres were retained and the remainder was centrifuged to separate free and liposome-entrapped OA. The volumes of the supernatants and pellets were measured and the OA content of each of the fractions was assessed by gamma emission.
Stat&tics Data from the ELISAs were not normally distributed and were therefore analysed by non-parametric methods. The Kruskal-Wallis one-way analysis of variance by ranks was used to test the null hypothesis that the group samples had all been drawn from the same population or identical populations with the same medians. The M a n n - W h i t n e y U-test was used to determine whether two independent groups were from the same population or populations with identical medians. The Wilcoxon matched pairs signed ranks test was used to determine whether paired samples were significantly different. A level of significance of 0.05 was selected as the probability (P) at which the null hypothesis was rejected. The tables of data give the means and standard deviations of the E.U. values for each group despite them being analysed by non-parametric methods. For small groups this is more representative of the data than the median value and the standard deviations indicate the spread of individual values within the group. RESULTS
Serum IgG anti-OA antibody responses in mice inoculated i.p. with OA in liposomes (Table 1) Mice primed with OA entrapped in DPPC/CH/DCP liposomes had significantly higher serum antibody levels compared with the groups given OA alone and OA mixed with 'empty' DPPC/CH/DCP liposomes. No other group was significantly different from the OA or PBS treated groups. In the priming and boosting regime increases in serum antibody for all the groups given antigen
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TABLE 1 Serum IgG antibody responses in mice to OA, alone or in various types of liposomes, administered i.p. The results are after regimes of priming, and priming followed by systemic challenge with OA
(n=5) Treatment
PBS OA PC/CH/DCP (OA) DPPC/CH/DCP (OA) PC/CH/DCP plus OA DPPC/CH/DCP plus OA
Serum IgG anti-OA antibody Mean (SD) (logloE.U. m1-1 ) Primed*
Primed/challenged**
0.97 1.12 0.97 1.95 0.79 0.71
0.94 2.27 2.76 3.14 2.22 2.45
(0.63) (0.50) (0.40) (0.55) +'1 (0.13 ) (0.25)
(0.22) 2 (0.36) (0.33) (0.52) + (0.19 ) (0.31)
Significant differences between groups by Kruskal-Wallis test (*P< 0.05, **P< 0.001 ). Mann-Whitney U-tests: + significantly greater than OA group ( P< 0.05 ); tsignificantly greater than DPPC/CH/DCP plus OA group (P< 0.005 ); 2significantly less than all other groups (P< 0.05 ).
were detected. Only the DPPC/CH/DCP (OA) group had serum antibody levels that were significantly higher than those of the OA treated group, although the PC/CH/DCP (OA) group just failed to be significantly higher (P=0.056). For both liposome types, OA entrapment in liposomes significantly increased the serum antibody response compared with the inoculation of OA mixed with liposomes (P<0.05). All the challenged groups (except PBS treated) had significantly higher serum antibody levels compared with the primed only groups (P< 0.01 ).
Serum and intestinal anti-OA antibody responses in mice fed OA in liposomes
(Table 2) Serum IgG antibody responses were elicited by OA fed and PC/CH/DCP (OA) fed groups in the Day 17 boosting regime but these two responding groups were not significantly different from each other. Intestinal IgA antibody was detected in the culture supernatant from one individual in the group fed DPPC/CH/DCP (OA) ( l0 E.U. ml- 1) and two animals in the group fed PC/CH/DCP (OA) (17.0 and 9.4 E.U. ml -~ ). There were also detectable IgG antibody responses in individuals from the groups fed OA, PC/CH/DCP (OA) and DPPC/CH/DCP (OA) (8.2, 13.6 and 12.4 E.U. ml -~ respectively). All these culture supernatant samples were from individuals with high serum IgA or IgG antibody levels. Serum antibody levels were not significantly different between groups sampled in the Day 23 boosting regime and antibody was not detected in intestinal samples.
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TABLE2 Serum antibody responses in mice fed 8 mg OA, alone or in various types of liposome, over 4 days. Mice received a boost feed (2 mg OA) on Day 17 or Day 23 and were killed on Day 22 or Day 28, respectively ( n = 6 ) Treatment
Serum anti-OA antibody. Mean (SD) (log~oE.U.ml-t ) Boost dose on Day 17
Boost dose on Day 23
Sampled on Day 22*
Sampled on Day 21
Sampled on Day 28
0.83 (0.26) 1.63 (0.90) + 2.39 (0.57) ++ 1.74 (1.16) n.d. n.d.
0.81 0.86 0.74 0.86 0.63 0.95
(0.29) (0.22) (0.44) (0.34) (0.19) (0.41)
0.93 (0.33) 0.70 (0.22) 0.92 (0.27) 0.81 (0.17) 0.71 (0.20) 1.18 (0.36)
1.00 (0.37) 0.66 (0.21) 1.38 (0.66) 1.16 (0.44) n.d. n.d.
1.03 (0.25) 1.08 (0.26) 1.03 (0.51) 1.12 (0.12) 1.24 (0.34) 1.01 (0.23)
1.30 (0.23) 1.31 (0.45) 1.84 (0.86) 1.18 (0.86) 1.19 (0.50) 1.10 (0.27)
lgG PBS OA
PC/CH/DCP (OA) DPPC/CH/DCP (OA) PC/CH/DCPplusOA DPPC/CH/DCP plus OA
lgA PBS OA PC/CH/DCP (OA) DPPC/CH/DCP (OA) PC/CH/DCP plus OA DPPC/CH/DCPplus OA
n.d., Not detected. Significant differences between groups by Kruskai-Wailis test (*P< 0.01 ). Significantly greater than PBS group by Mann-Whitney U-test ( +P< 0.05, + +P< 0.002 ).
Serum and intestinal anti-KLH antibody responses in mice fed KLH in liposomes (Table 3) Groups fed KLH alone and in liposomes produced elevated serum IgG and IgA antibody responses in the priming and boosting regimes. Despite some group differences in these levels of significance there were no significant differences between these two responding groups under any regime. Serum antibody responses of the group fed liposomes and KLH separately were notably low in all the regimes. Intestinal culture supernatant IgG and IgA antibody responses were not significantly above background levels in the primed and boosted mice. However in the samples from the boosted mice one half of the L (KLH) fed group and three-fifths of the KLH fed group had IgA antibody levels obviously above background. No significant difference was detected between the serum antibody levels of the Day 13 and Day 22 samples for any feeding regime (P> 0.1 for both isotypes), nor was there any significant change in antibody response
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TABLE 3 Serum antibody responses in mice fed 4 mg KLH, alone or with P C / C H / D C P liposomes (L) over 4 days. A priming regime alone and a similar regime followed by a boost feed (4 mg KLH) was investigated ( n = 6 ) Treatment
Serum anti-KLH antibody. Mean (SD) (logloE.U. m l - 1) Primed
Primed and boosted Days 15-18
Sampled on Day 8**
Sampled on Day 13**
Sampled on Day 22**
-
-
0.30 (0.17)
2.18 (0.68) ++ 1.68 (1.20) ++ 0.17 (0.17)
2.26 (1.29) 2.15 (1.02) + 0.42 (0.34)
2.90 (1.26) + 2.43 (1.20) + 0.93 (1.04)
1.67 (1.11) 2.25 (0.47) ++ 0.98 (0.67)
1.41 2.86 2.32 1.42
IgG PBSB KLH L (KLH) L plus KLH
IgA PBSB KLH L (KLH) L plus KLH
. 2.23 1.72 1.39
. . (0.34) ++ (0.70) (0.14)
.
(0.37) (0.97) 1 (0.82) 1 (0.42)
Significant differences between groups by Kruskal-Wallis tests (*P< 0.05, **P< 0.01 ). Mann-Whitney U-tests: +significantly greater than PBSB ( P < 0.005); + +significantly greater than PBSB and greater than L plus KLH (both P < 0.005); t significantly greater than PBSB and L plus KLH (both P<0.05).
for any group fed similar material between the serum collected on Days 8 and 13 or Days 8 and 22.
Tolerance induction experiment (Table 4) Following parenteral challenge with OA, the group fed PBS had significantly higher levels of antibody than the groups fed OA alone and OA in liposomes and there was no significant difference between the latter two groups.
Liposome in vitro stability assay Measurement of the gamma emission from the fractions of the aliquots taken at timed intervals during the incubations allowed the percentage of OA released from the P C / C H / D C P and D P P C / C H / D C P liposomes to be calculated (Figs. 1 and 2). Negligible release of entrapped OA from the liposomes kept at 4 °C was observed ( < 1% for both types). However, similar mixtures incubated at 37 °C steadily released OA, until at the end of the 4 h period the D P P C / C H / D C P liposomes had released about 20% and the PC/ C H / D C P liposomes about 15% of the entrapped OA. Incubation with the
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TABLE 4 Serum antibody responses in mice fed 20 mg OA, alone or in liposomes, over 5 days. Mice were challenged with OA i.p. on Day 19 and killed on Day 28 (n = 7 ) Treatment
Serum IgG anti-OA antibody Mean (SD) (logloE.U. ml- t ),
PBS OA PC/CH/DCP (OA)
1.91 (0.27) + 1.62 (0.14) 1.72 (0.16)
*Significant differences between groups by Kruskal-Wallis test (P< 0.05 ). + Significantly greater than other groups by Mann-Whitney U-test (P< 0.05 ).
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Fig. 1. OA release from PC/CH/DCP liposomes. OA with traces of radiolabelled protein was entrapped in P C / C H / D C P liposomes and incubated at 37 °C in PBS, acidic stomach contents or bile. OA release was measured by the gamma emission values of the free and liposome-associated fractions collected at timed intervals.
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Fig. 2. OA release from D P P C / C H / D C P liposomes. OA with traces of radiolabelled protein was entrapped in D P P C / C H / D C P liposomes and incubated at 37°C in PBS, acidic stomach contents or bile. OA release was measured by the gamma emission values of the free and liposome-associated fractions collected at timed intervals.
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acidic stomach contents had a very similar effect to PBS alone on the liposome integrity. The greatest changes were seen with the bile mixtures. DPPC/CH/DCP liposomes released about 30% of the OA immediately on mixing and continued to release the marker throughout the incubation. After 4 h about 60% and 75% of the OA was not associated with the liposomes in the 10% and 50% bile mixture, respectively. PC/CH/DCP liposomes were markedly disrupted upon mixing with the bile. Immediate release accounted for about 80% and 95% of the OA from the 10% and 50% bile mixtures, respectively. At the end of the incubation almost 100% of the OA was no longer associated with the liposomes. DISCUSSION
An adjuvant effect for OA entrapped in DPPC liposomes and administered i.p. was demonstrated. The superior adjuvarit action of liposomes made with phospholipids with a high transition temperature may be related to bilayer stability (Kinsky, 1978) but the higher lipid to protein mass ratio of the DPPC-based ( 15.26:1 ), compared with the PC-based liposomes (4.97:1 ) could also have influenced the adjuvant activity (Davis and Gregoriadis, 1987 ). Memory induction, thymic dependency and the advantage of a physical association between the antigen and liposome were also demonstrated in this experiment (Shek and Sabiston, 1981 ). Feeding OA entrapped in liposomes did not produce significantly elevated serum or intestinal antibody responses to OA compared with the same dose of antigen fed alone. Preliminary experiments (data not shown ) investigated the response to feeding doses of 0.4-8 mg OA in priming regimes (over 1-6 days) as well as using different strains of mouse (Balb/c), and omitting or using a different charged lipid (PA). In all cases no significant serum or intestinal antibody responses was detected in any group fed OA alone or in liposomes. The regime of early boosting (on Day 17) did induce serum IgG antibody production in the groups fed OA and OA in PC-based liposomes but when boosting was delayed the response was no longer apparent, possibly indicating that the original immune response was exhausted or that any memory induction was weak and transient. Serum antibody responses to fed soluble proteins are well documented (Hanson et al., 1979) although regimes of single high doses or multiple feeds are usually required. Intestinal antibody responses in this study were weak and this has been reported previously for fed soluble antigens. Pilot experiments and other reports (Hamilton et al., 1981; Wilson et al., 1989 ) showed that fed KLH was able to induce serum and intestinal antibody responses and indicated that adjuvant effects may be easier to assess. Serum and some intestinal antibody levels in the groups fed KLH alone or in lipo-
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somes were elevated compared with the control groups but no adjuvant action could be attributed to the liposomal entrapment. Feeding 'empty' liposomes just prior to KLH abolished any serum or intestinal antibody response, possibly by inhibiting transit of the protein through the stomach and allowing greater proteolysis. Both types of liposome were substantially unaffected by incubation at 37 °C in acidic conditions confirming that entrapped antigen may be protected from proteolytic degradation during transit through the stomach (Rowland and Woodley, 1980; Chiang and Weiner, 1987a). In contrast, the detergent action of bile salts caused OA release that was almost immediate and total for the PC-based liposomes. Richards and Gardner (1978) also found that vesicle disruption was most apparent in 'fluid' liposomes with a low Tc. The failure to improve intestinal IgA antibody levels after feeding antigen in liposomes is of relevance for the development of oral vaccines, particularly those based on refined proteins. In this study antigen size did not appear to influence adjuvant activity as neither OA nor the larger KLH protein was able to elicit significantly improved levels of local or serum antibody. Entrapment of protein in liposomes also failed to abrogate the induction of tolerance to systemic challenge. This correlates with the lack of an intestinal i m m u n e response observed after feeding and shows that the liposome is not sufficiently effective as an oral adjuvant to reverse the toleragenic nature of a fed soluble protein. Furthermore this finding may suggest that feeding antigen in liposomes does not significantly alter the mechanism of antigen handling within the intestine. Our data indicate that significant liposomal disruption occurs in the gut. Other studies have failed to find evidence for the intestinal absorption of liposomes or non-absorbable, liposome-entrapped markers (Whitmore and Wheeler, 1979; Chiang and Weiner, 1987b ). Together these suggest that the properties that are responsible for the adjuvant action of liposomes administered parenterally may be inapplicable to the induction of an i m m u n e response in the intestine. In conclusion, liposomes would appear to be useful as carriers for drugs or antigens that are susceptible to degradation in the gastric environment, but they failed to improve the intestinal i m m u n e response for the soluble proteins used in this study. REFERENCES Allison, A.C. and Gregoriadis, G., 1974. Liposomesas immunologicaladjuvants. Nature, 252: 252. Chiang, C. and Weiner, N., 1987a. Gastrointestinal uptake of liposomes. I. In vitro and in situ studies. Int. J. Pharm., 37: 75-85. Chiang, C. and Weiner, N., 1987b.Gastrointestinal uptake ofliposomes. II. In vivo studies. Int. J. Pharm., 40: 143-150. Dapergolas, G., Neerunjun, E.D. and Gregoriadis, G., 1976. Penetration of target areas in the rat by liposome-associatedbleomycin,glucoseoxidase and insulin. FEBS Lett., 63: 235-239.
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Davis, D. and Gregoriadis, G., 1987. Liposomes as adjuvants with immunopurified tetanus toxoid: influence of liposomal characteristics. Immunology, 61: 229-234. Desiderio, J.V. and Campbell, S.G., 1985. Immunization against experimental murine salmonellosis with liposome-associated O-antigen. Infect. Immun., 48:658-663. Gregoriadis, G., 1980. Tailoring liposome structure. Nature, 283:814-815. Gregory, R.L., Michalek, S.M., Richardson, G., Harmon, C., Hilton, T. and McGhee, J.R., 1986. Characterisation of immune response to oral administration of Streptococcus sobrinus ribosomal preparations in liposomes. Infect. Immun., 54: 780-786. Hamilton, S.R., Keren, D.F., Yardley, J.H. and Brown, G.D., 1981. No impairment of local intestinal immune response to KLH in the absence of Peyer's patches. Immunology, 42:431435. Hanson, D.G., Vas, N.M., Maia, L.G.S. and Lynch, J.M., 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: 2337-2342. Hemker, H.C., Hermans, W.Th., Muller, A.D. and Zwaal, R.F.A., 1980. Oral treatment of haemophilia A by gastrointestinal absorption of factor VIII entrapped in liposomes. Lancet, 1: 70-71. Hunter, W.M. and Greenwood, F.C.I., 1962. Preparation of iodine-31 labelled human growth hormone of high specific activity. Nature, 194: 495. Kinsky, S.C., 1978. Immunogenicity ofliposomal model membranes. Ann. NY Acad. Sci., 308: lll-ll6. Kirby, C. and Gregoriadis, G., 1984. Dehydration-rehydration vesicles: A simple method for high yield drug entrapment in liposomes. Biotechnology, 2: 979-984. Patel, H.M. and Ryman, B.E., 1976. Oral administration of insulin by encapsulation within liposomes. FEBS Lett., 62: 60-63. Patel, H.M. and Ryman, B.E., 1977. Orally administered liposomally entrapped insulin. Biochem. Soc. Trans., 5: 1739-1741. Pierce, N.F. and Sacci, J.B., 1984. Enhanced mucosal priming by cholera toxin and procholeragenoid with a lipoidal amine adjuvant (avridine) delivered in liposomes. Infect. Immun., 44: 469-473. Rhalem, A., Bourdieu, C., Luffau, G. and Pery, P., 1988. Vaccination of mice with liposomeentrapped antigens ofNippostrongylus brasiliensis. Ann. Inst. Pasteur, Immunol., 139: 157163. Rhodes, M.B., Baker, P.K., Christenson, D.L. and Anderson, G.A., 1988. Ascaris suum antigens incorporated into liposomes used to stimulate protection to migrating larvae. Vet. Parasitol., 26: 343-349. Richards, M.H. and Gardner, C.R., 1978. Effects of bile salts on the structural integrity of liposomes. Biochim. Biophys. Acta, 543: 508-522. Rowland, R.N. and Woodley, J.F., 1980. The stability ofliposomes in vitro to pH, bile salts and pancreatic lipase. Biochim. Biophys. Acta, 620: 400-409. Shahum, E. and Therien, H-M., 1988. Immunopotentiation of the humoral response by liposomes: encapsulation versus covalent linkage. Immunology, 65:315-317. Shek, N. and Sabiston, B.H., 1981. Immune response mediated by liposome-associated protein antigens. I. Potentiation of the plaque-forming cell response. Immunology, 45: 349-356. Svennerholm, A-M. and Holmgren, J., 1977. Immunoglobulin and specific antibody synthesis in vitro by enteral and non-enteral lymphoid tissues after subcutaneous cholera immunization. Infect. Immun., 15: 360-369. Wachsmann, D., Klein, J.P., Scholler, M. and Frank, R.M., 1985. Local and systemic immune response to orally administered liposome-associated soluble S. mutans cell wall antigen. Immunology, 54: 189-193. Wachsmann, D., Klein, J.P., Scholler, M., Ogler, M., Ackeramans, F. and Frank, R.M., 1986.
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Serum and salivary antibody responses in rats orally immunised with Streptococcus mutans carbohydrate protein conjugate associated with liposomes. Infect. Immun., 52:408-413. Whitmore, D.A. and Wheeler, K.P., 1979. The fate of liposomes in the rat small intestine. Biochem. Soc. Trans., 7: 929-931. Wilson, A.D., Stokes, C.R. and Bourne, F.J., 1989. Adjuvant effect of cholera toxin on the mucosal immune response to soluble proteins. Differences between mouse strains and protein antigens. Scand. J. Immunol., 29" 739.
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The intestinal and serum humoral immune response of mice to systemically and orally administered antigens in liposomes: I. The response to liposome-entrapped soluble proteins C.J.
Clarke and C.R. Stokes
Department of Veterinary Medicine, Universityof Bristol, Langford House, Langford, Bristol, BS I 8 7DU, UK (Accepted 14 May 1991 ) ABSTRACT Clarke, C.J. and Stokes, C.R., 1992. The intestinal and serum humoral immune response of mice to systemically and orally administered antigens in liposomes: I. The response to liposome-entrapped soluble proteins. Vet. Immunol. lmmunopathol., 32: 125-138. The development of oral vaccines is of great importance in veterinary medicine and new adjuvants and carriers are essential to this aim. Liposomes are effective systemic adjuvants but the relatively little data on their potential as oral adjuvants is inconclusive. Liposomes containingovalbumin (OA) were effective adjuvants when administered intraperitoneallyto mice. Feeding mice with OA or keyhole limpet haemocyanin in liposomes in a series of priming and boosting regimes failed to elicit any significant increase in serum or intestinal antibody response compared with feeding the free antigen. Oral tolerance induction to systemic challenge was also unaffected by OA entrapment in liposomes. In vitro liposome stability assays at 37°C demonstrated a substantial resistance to disruption in the presence of acidic stomach contents. However, the addition of bile caused a rapid and profound release of protein marker from the liposomes. The rate and degree of disruption was influenced by the type of phospholipid used. These results suggest that liposomes may be useful as carders for orally administered compounds but they are ineffective as adjuvants for the non-particulate, naturally weak immunogens used in this study. ABBREVIATIONS CFA = complete Freund's adjuvant; CH = cholesterol; DCP = dicetyl phosphate; DPPC = DLdipalmitoylphosphatidyl choline; ELISA = enzyme linked immunosorbent assay; E.U.s = ELISA units; i.p. = intraperitoneally; KLH = keyhole limpet haemocyanin; OA = ovalbumin; PA = Lphosphatidic acid; PBS=phosphate buffered saline; PBSB=PBS/3% NaHCO3; PBST=PBS plus 0.05% Tween 20; PC = L-phosphatidylcholine; Tc (s) = transition temperature (s). INTRODUCTION
Lip.osomes can act as adjuvants for a variety of soluble protein antigens when administered parenterally (Allison and Gregoriadis, 1974; Shek and Correspondence to: C.L Clarke, Department of Veterinary Pathology, Royal (Dick) School of Veterinary Studies, Veterinary Field Station, Easter Bush, Roslin, EH25 9RG, UK. © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00
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Sabiston, 1981 ). Their action has been attributed to depot formation, hydrophobicity, rendering soluble antigen particulate, and efficient targetting of the vesicles to antigen-presenting cells of the immune system (Desiderio and Campbell, 1985; Shahum and Therien, 1988 ). Liposome components are antigenically similar to mammalian cell membranes and are therefore degradable and only weakly immunogenic. Liposomes can also be 'tailored' to meet certain requirements by altering the ratios or types ofphospholipids used for their preparation (Gregoriadis, 1980). One influential factor concerns the transition temperature (Tc) above which the major phospholipid molecules that form the lipid bilayers become less rigid and allow leakage of contents. Orally administered liposomes used as carriers have reportedly increased the intestinal absorption or efficacy of entrapped agents such as insulin (Patel and Ryman, 1976), glucose oxidase (Dapergolas et al., 1976), and coagulation factor VIII (Hemker et al., 1980). Other studies have failed to find improved effects after liposomal entrapment of various compounds (Patel and Ryman, 1977 ). Few studies have investigated the immune responses to antigens fed in liposomes. Liposomal entrapment of streptococcal antigens increased salivary antibody responses in rats (Wachsmann et al., 1985, 1986; Gregory et al., 1986) but other studies using bacterial or parasite antigens failed to demonstrate significant adjuvant activity for oral liposomes (Pierce and Sacci, 1984; Rhalem et al., 1988; Rhodes et al., 1988). Because many future vaccine preparations are likely to be based on relatively pure, low molecular weight proteins we have used the well defined, nonparticulate protein ovalbumin (about 43 000 mol.wt. ) as well as the larger KLH protein (2 000 000-3 000 000 mol.wt. ) as our model antigens. The comparative adjuvant effect of liposomes admininstered by the parenteral and oral routes was assessed in this study. The liposomes used were based on either the L-phosphatidylcholine (PC) or DL-dipalmitoylphosphatidylcholine (DPPC) phospholipids as these have different Tcs of - 15/17 °C and 41 °C respectively and have been used in many other studies. The serum antibody response in mice to ovalbumin in liposomes administered intraperitoneally, and the serum and intestinal antibody responses in mice to ovalbumin and keyhole limpet haemocyanin (KLH) fed in liposomes was measured using a variety of feeding protocols and liposome types. The stability of the liposome preparations in vitro was also investigated under conditions similar to those in the gastrointestinal tract. MATERIALS AND METHODS
Protein antigens and radiolabelling Ovalbumin (OA) (Grade V) was from Sigma Chemical Co. (Poole, UK) and KLH was from Calbiochem (San Diego, CA, USA). Proteins were ra-
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diolabeUed with 1251 (Na salt) (Amersham International, Amersham, UK) by the chloramine-T method (Hunter and Greenwood, 1962).
Liposome preparation PC (type V-E) from egg yolk, DPPC, L-phosphatidic acid (PA) (sodium salt) from egg yolk, dicetyl phosphate (DCP) and cholesterol (CH) were all from Sigma. Large multilamellar liposomes were prepared by the dehydration-rehydration procedure based on Kirby and Gregoriadis (1984). The lipids PC or DPPC, CH, and DCP or PA were used in the molar ratio 7: 2: 1. Briefly, 100 /zmol of lipid were dissolved in chloroform, dried by rotary evaporation under reduced pressure and resuspended to a suspension with 3 ml distilled water. The suspension was left to stand for 1 h in a water bath (37 °C for PC-based and 45°C for DPPC-based liposomes). Three millilitres 15 mg OA or KLH ml-1 3.3 mM phosphate buffered saline (PBS) (pH 7.2) containing a trace label of 125I-radiolabelled protein was added and mixed and 1.5 ml aliquots of the suspension were flash frozen in liquid nitrogen, freeze-dried and then rehydrated with 150 #1 distilled water and left at 20 ° C (PC-based liposomes ) or 45°C (DPPC-based liposomes) for 1 h. Isotonic PBS (33 mM, pH 7.2) was then added to restore each aliquot volume to 1.5 ml. The pooled aliquots were centrifuged (three times at 10 000 × g for 30 min) to separate liposomeentrapped from free protein. The final liposome pellet was resuspended in 0.1 M PBS (pH 7.2) and protein entrapment was estimated from the gamma emission c.p.m. Liposomes made of PC/CH/DCP and DPPC/CH/DCP lipids entrapped mean amounts of 13.27 mg OA (8.88 mg KLH) and 4.23 mg OA (4.94 mg KLH) 100/zmol- 1lipid, respectively.
Mice and procedures CBA and Balb/c male mice were bred in the Small Animal Unit, Dept. Animal Husbandry, University of Bristol and were used at 6-8 weeks old. Mice were fed 200/zl volumes of the preparations by intraoesophageal intubation with polyethylene capillary tubing. Serum was prepared from blood collected by tail bleeding under ether anaesthesia or cardiac puncture under chloroform anaesthesia. Intestinal organ cultures were prepared based on the method of Wilson et al. (1989) adapted from Svennerholm and Holmgren (1977). Briefly, the excised small intestines were washed with Hank's balanced salt solution (Flow Labs., Irvine, UK) and 300 nag segments, without Peyer's patches, were transferred to a 6-well tissue culture plate (Linbro, Flow) containing Dutch modified RPMI (Flow) supplemented with 10% foetal calf serum (Imperial Labs., Andover, UK), 2 mM L-glutamine (Flow), 5 #g ml-1 amphotericin B
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(Gibco Ltd., Paisley, UK) and 50/zg ml-1 gentamicin (Flow). The plate was incubated for 18 h at 37°C in a humidified, 5% CO2 atmosphere and then frozen and stored at - 2 0 ° C to stop further cell activity. The antibody content of the supernatant from the incubation of the intestinal tissue segments was shown by others to be the product of de novo synthesis (Svennerholm and Holmgren, 1977). However, in our laboratory this was not demonstrable and antibody detected in the supernatant was shown to be mostly pre-formed antibody that was released from intestinal plasma cells (Wilson et al., 1989).
Enzyme linked immunosorbant assays (ELISAs) Serum and intestinal culture supernatant samples were assayed for anti-OA and anti-KLH, IgG and IgA, antibody by ELISA based on the protocol described before (Wilson et al., 1989). Briefly, 96-well PVC Microtiter plates were coated with either 10/zg OA or 10/tg KLH ml -~ PBS (0.15 M, pH 7.2) ( 100/A per well) and washing steps were with PBS containing 0.05% Tween 20 ( Sigma ) (PBST). Hyperimmune anti-OA or anti-KLH antisera were raised in mice inoculated with OA or KLH in complete Freund's adjuvant (CFA) and used as standards after chequerboard titrations to determine optimum concentrations. Samples and standards were diluted in PBST and titrated down. Subsequent incubations were with sheep anti-mouse IgG (prepared by D. Patel of Department of Animal Husbandry, University of Bristol) or goat anti-mouse IgA (Sigma), pig anti-bovine IgG-alkaline phosphatase conjugate and finally phosphatase substrate (p-nitrophenyl phosphate disodium) (Sigma) at 1 mg ml -~ carbonate buffer (0.05 M, pH 9.6). The plates were read on a Titertek Multiscan MC (Flow) automatic plate reader and absorbance readings were converted into ELISA units (E.U.s) using the ELISANALYSIS program (Dr. J.H. Peterman, University of Alabama, AL). This plots standard and sample dilutions (log~o reciprocals) against the log~o absorbance values. Samples are assigned E.U. values by comparison with the linear part of the standard slope.
Intraperitoneal inoculation of ovalbumin in liposomes Twelve groups of five CBA mice were used. On Day 0, duplicate groups were inoculated i.p. with 100/tg OA in PBS, or entrapped in liposomes made of PC/CH/DCP or DPPC/CH/DCP. Other pairs of groups received 100/tg OA mixed with either type of 'empty' liposome, or PBS alone. Mice in one group of each pair received 100/tg OA in CFA intraperitoneally (i.p.) on Day l 0. All the mice were killed on Day 16 and serum samples were prepared and assayed for IgG anti-OA antibody using the ELISA.
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Oral administration of ovalbumin in liposomes Four groups of six CBA mice were fed for 4 consecutive days with 2 mg OA in PBS or entrapped in liposomes made of P C / C H / D C P or D P P C / C H / D C P lipids. One group was fed PBS alone as a control. On Day 17, the mice received a single feed equivalent to a daily priming dose and all the mice were killed on Day 22. Serum samples and intestinal cultures were prepared and the supernatants from the latter were concentrated 10 times in Minicon cells (Grace & Co., Danvers, MA). Samples were assayed for IgG and IgA antiOA antibody by ELISA. In a second experiment groups of six mice were primed in an identical way, but two additional groups were fed OA mixed with either type of 'empty' liposomes. The mice were tail bled on Day 21 and given a boost feed on Day 23. These mice were killed on Day 28 and serum and concentrated intestinal culture supernatant was prepared and assayed as above.
Oral tolerance induction with ovalbumin in liposomes Three groups of seven mice were fed on 5 consecutive days with either PBS or 4 mg OA in PBS or entrapped in liposomes made of P C / C H / D C P lipids. All the mice were challenged with 100/zg OA in CFA i.p. on Day 19. The mice were killed on Day 28 and serum was prepared and assayed for IgG anti-OA antibody by ELISA.
Oral administration of KLH in liposomes Seven groups of six CBA mice were fed on 4 consecutive days in a protocol involving priming alone and priming followed by boosting. Duplicate groups were fed with either 1 mg KLH, 1 nag KLH entrapped in liposomes made of the lipids P C / C H / D C P , or 'empty' liposomes followed by 1 nag KLH. All the preparations were fed in PBS/3% NaHCO3 (PBSB) and the final group was fed this buffer alone. Mice from one group of each duplicate were killed on Day 8 and serum and intestinal culture supernatants were prepared and assayed for IgG and IgA anti-KLH antibody by ELISA. The remaining four groups were tail bled on Day 13 and on Days 15-18 inclusive the mice received repeat feeds similar to those of the 4 day priming period. These mice were killed on Day 22 and serum and intestinal culture supernatants were prepared and assayed as above.
In vitro stability assay of liposomes Liposomes made of the lipids P C / C H / D C P and D P P C / C H / D C P and containing 3 mg OA m1-1 PBS with trace 125I-OA were prepared. Aliquots
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(2.5 ml) of each liposome suspension were mixed with either PBS, mouse stomach contents or fresh pig bile (neat or diluted 1:5 with PBS). Stomach contents (pH 3.0) were collected from freshly killed mice, diluted 1:2 in distilled water, vortex mixed, and the pH adjusted back to 3.0 with concentrated HC1. The relevant liposome suspensions were incubated with the equivalent stomach contents from four mice. The mixtures were incubated at 37 °C in a shaking water bath (50 c.p.m. ) to simulate physiological conditions. Control suspensions of liposomes mixed with PBS were kept at 4 ° C. At the start (to) and at intervals throughout the 4 h incubation, 400/~1 aliquots were taken from each mixture. One hundred microlitres were retained and the remainder was centrifuged to separate free and liposome-entrapped OA. The volumes of the supernatants and pellets were measured and the OA content of each of the fractions was assessed by gamma emission.
Stat&tics Data from the ELISAs were not normally distributed and were therefore analysed by non-parametric methods. The Kruskal-Wallis one-way analysis of variance by ranks was used to test the null hypothesis that the group samples had all been drawn from the same population or identical populations with the same medians. The M a n n - W h i t n e y U-test was used to determine whether two independent groups were from the same population or populations with identical medians. The Wilcoxon matched pairs signed ranks test was used to determine whether paired samples were significantly different. A level of significance of 0.05 was selected as the probability (P) at which the null hypothesis was rejected. The tables of data give the means and standard deviations of the E.U. values for each group despite them being analysed by non-parametric methods. For small groups this is more representative of the data than the median value and the standard deviations indicate the spread of individual values within the group. RESULTS
Serum IgG anti-OA antibody responses in mice inoculated i.p. with OA in liposomes (Table 1) Mice primed with OA entrapped in DPPC/CH/DCP liposomes had significantly higher serum antibody levels compared with the groups given OA alone and OA mixed with 'empty' DPPC/CH/DCP liposomes. No other group was significantly different from the OA or PBS treated groups. In the priming and boosting regime increases in serum antibody for all the groups given antigen
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TABLE 1 Serum IgG antibody responses in mice to OA, alone or in various types of liposomes, administered i.p. The results are after regimes of priming, and priming followed by systemic challenge with OA
(n=5) Treatment
PBS OA PC/CH/DCP (OA) DPPC/CH/DCP (OA) PC/CH/DCP plus OA DPPC/CH/DCP plus OA
Serum IgG anti-OA antibody Mean (SD) (logloE.U. m1-1 ) Primed*
Primed/challenged**
0.97 1.12 0.97 1.95 0.79 0.71
0.94 2.27 2.76 3.14 2.22 2.45
(0.63) (0.50) (0.40) (0.55) +'1 (0.13 ) (0.25)
(0.22) 2 (0.36) (0.33) (0.52) + (0.19 ) (0.31)
Significant differences between groups by Kruskal-Wallis test (*P< 0.05, **P< 0.001 ). Mann-Whitney U-tests: + significantly greater than OA group ( P< 0.05 ); tsignificantly greater than DPPC/CH/DCP plus OA group (P< 0.005 ); 2significantly less than all other groups (P< 0.05 ).
were detected. Only the DPPC/CH/DCP (OA) group had serum antibody levels that were significantly higher than those of the OA treated group, although the PC/CH/DCP (OA) group just failed to be significantly higher (P=0.056). For both liposome types, OA entrapment in liposomes significantly increased the serum antibody response compared with the inoculation of OA mixed with liposomes (P<0.05). All the challenged groups (except PBS treated) had significantly higher serum antibody levels compared with the primed only groups (P< 0.01 ).
Serum and intestinal anti-OA antibody responses in mice fed OA in liposomes
(Table 2) Serum IgG antibody responses were elicited by OA fed and PC/CH/DCP (OA) fed groups in the Day 17 boosting regime but these two responding groups were not significantly different from each other. Intestinal IgA antibody was detected in the culture supernatant from one individual in the group fed DPPC/CH/DCP (OA) ( l0 E.U. ml- 1) and two animals in the group fed PC/CH/DCP (OA) (17.0 and 9.4 E.U. ml -~ ). There were also detectable IgG antibody responses in individuals from the groups fed OA, PC/CH/DCP (OA) and DPPC/CH/DCP (OA) (8.2, 13.6 and 12.4 E.U. ml -~ respectively). All these culture supernatant samples were from individuals with high serum IgA or IgG antibody levels. Serum antibody levels were not significantly different between groups sampled in the Day 23 boosting regime and antibody was not detected in intestinal samples.
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TABLE2 Serum antibody responses in mice fed 8 mg OA, alone or in various types of liposome, over 4 days. Mice received a boost feed (2 mg OA) on Day 17 or Day 23 and were killed on Day 22 or Day 28, respectively ( n = 6 ) Treatment
Serum anti-OA antibody. Mean (SD) (log~oE.U.ml-t ) Boost dose on Day 17
Boost dose on Day 23
Sampled on Day 22*
Sampled on Day 21
Sampled on Day 28
0.83 (0.26) 1.63 (0.90) + 2.39 (0.57) ++ 1.74 (1.16) n.d. n.d.
0.81 0.86 0.74 0.86 0.63 0.95
(0.29) (0.22) (0.44) (0.34) (0.19) (0.41)
0.93 (0.33) 0.70 (0.22) 0.92 (0.27) 0.81 (0.17) 0.71 (0.20) 1.18 (0.36)
1.00 (0.37) 0.66 (0.21) 1.38 (0.66) 1.16 (0.44) n.d. n.d.
1.03 (0.25) 1.08 (0.26) 1.03 (0.51) 1.12 (0.12) 1.24 (0.34) 1.01 (0.23)
1.30 (0.23) 1.31 (0.45) 1.84 (0.86) 1.18 (0.86) 1.19 (0.50) 1.10 (0.27)
lgG PBS OA
PC/CH/DCP (OA) DPPC/CH/DCP (OA) PC/CH/DCPplusOA DPPC/CH/DCP plus OA
lgA PBS OA PC/CH/DCP (OA) DPPC/CH/DCP (OA) PC/CH/DCP plus OA DPPC/CH/DCPplus OA
n.d., Not detected. Significant differences between groups by Kruskai-Wailis test (*P< 0.01 ). Significantly greater than PBS group by Mann-Whitney U-test ( +P< 0.05, + +P< 0.002 ).
Serum and intestinal anti-KLH antibody responses in mice fed KLH in liposomes (Table 3) Groups fed KLH alone and in liposomes produced elevated serum IgG and IgA antibody responses in the priming and boosting regimes. Despite some group differences in these levels of significance there were no significant differences between these two responding groups under any regime. Serum antibody responses of the group fed liposomes and KLH separately were notably low in all the regimes. Intestinal culture supernatant IgG and IgA antibody responses were not significantly above background levels in the primed and boosted mice. However in the samples from the boosted mice one half of the L (KLH) fed group and three-fifths of the KLH fed group had IgA antibody levels obviously above background. No significant difference was detected between the serum antibody levels of the Day 13 and Day 22 samples for any feeding regime (P> 0.1 for both isotypes), nor was there any significant change in antibody response
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TABLE 3 Serum antibody responses in mice fed 4 mg KLH, alone or with P C / C H / D C P liposomes (L) over 4 days. A priming regime alone and a similar regime followed by a boost feed (4 mg KLH) was investigated ( n = 6 ) Treatment
Serum anti-KLH antibody. Mean (SD) (logloE.U. m l - 1) Primed
Primed and boosted Days 15-18
Sampled on Day 8**
Sampled on Day 13**
Sampled on Day 22**
-
-
0.30 (0.17)
2.18 (0.68) ++ 1.68 (1.20) ++ 0.17 (0.17)
2.26 (1.29) 2.15 (1.02) + 0.42 (0.34)
2.90 (1.26) + 2.43 (1.20) + 0.93 (1.04)
1.67 (1.11) 2.25 (0.47) ++ 0.98 (0.67)
1.41 2.86 2.32 1.42
IgG PBSB KLH L (KLH) L plus KLH
IgA PBSB KLH L (KLH) L plus KLH
. 2.23 1.72 1.39
. . (0.34) ++ (0.70) (0.14)
.
(0.37) (0.97) 1 (0.82) 1 (0.42)
Significant differences between groups by Kruskal-Wallis tests (*P< 0.05, **P< 0.01 ). Mann-Whitney U-tests: +significantly greater than PBSB ( P < 0.005); + +significantly greater than PBSB and greater than L plus KLH (both P < 0.005); t significantly greater than PBSB and L plus KLH (both P<0.05).
for any group fed similar material between the serum collected on Days 8 and 13 or Days 8 and 22.
Tolerance induction experiment (Table 4) Following parenteral challenge with OA, the group fed PBS had significantly higher levels of antibody than the groups fed OA alone and OA in liposomes and there was no significant difference between the latter two groups.
Liposome in vitro stability assay Measurement of the gamma emission from the fractions of the aliquots taken at timed intervals during the incubations allowed the percentage of OA released from the P C / C H / D C P and D P P C / C H / D C P liposomes to be calculated (Figs. 1 and 2). Negligible release of entrapped OA from the liposomes kept at 4 °C was observed ( < 1% for both types). However, similar mixtures incubated at 37 °C steadily released OA, until at the end of the 4 h period the D P P C / C H / D C P liposomes had released about 20% and the PC/ C H / D C P liposomes about 15% of the entrapped OA. Incubation with the
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TABLE 4 Serum antibody responses in mice fed 20 mg OA, alone or in liposomes, over 5 days. Mice were challenged with OA i.p. on Day 19 and killed on Day 28 (n = 7 ) Treatment
Serum IgG anti-OA antibody Mean (SD) (logloE.U. ml- t ),
PBS OA PC/CH/DCP (OA)
1.91 (0.27) + 1.62 (0.14) 1.72 (0.16)
*Significant differences between groups by Kruskal-Wallis test (P< 0.05 ). + Significantly greater than other groups by Mann-Whitney U-test (P< 0.05 ).
loo 80'
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40
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-
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60 80 100 120 140 160 180 200 220 240 Incubation time {mins.)
Fig. 1. OA release from PC/CH/DCP liposomes. OA with traces of radiolabelled protein was entrapped in P C / C H / D C P liposomes and incubated at 37 °C in PBS, acidic stomach contents or bile. OA release was measured by the gamma emission values of the free and liposome-associated fractions collected at timed intervals.
100 80
•- - - O - ~. --
PBS Stomach contents 10% Bile 50% Bile
60
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20 4'o 60 "80 ,~o 1~o 1;o t;o,~o 2~o2~o2;o I n c u b a t i o n
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Fig. 2. OA release from D P P C / C H / D C P liposomes. OA with traces of radiolabelled protein was entrapped in D P P C / C H / D C P liposomes and incubated at 37°C in PBS, acidic stomach contents or bile. OA release was measured by the gamma emission values of the free and liposome-associated fractions collected at timed intervals.
PROTEIN ANTIGENSIN ORALLIPOSOMES
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acidic stomach contents had a very similar effect to PBS alone on the liposome integrity. The greatest changes were seen with the bile mixtures. DPPC/CH/DCP liposomes released about 30% of the OA immediately on mixing and continued to release the marker throughout the incubation. After 4 h about 60% and 75% of the OA was not associated with the liposomes in the 10% and 50% bile mixture, respectively. PC/CH/DCP liposomes were markedly disrupted upon mixing with the bile. Immediate release accounted for about 80% and 95% of the OA from the 10% and 50% bile mixtures, respectively. At the end of the incubation almost 100% of the OA was no longer associated with the liposomes. DISCUSSION
An adjuvant effect for OA entrapped in DPPC liposomes and administered i.p. was demonstrated. The superior adjuvarit action of liposomes made with phospholipids with a high transition temperature may be related to bilayer stability (Kinsky, 1978) but the higher lipid to protein mass ratio of the DPPC-based ( 15.26:1 ), compared with the PC-based liposomes (4.97:1 ) could also have influenced the adjuvant activity (Davis and Gregoriadis, 1987 ). Memory induction, thymic dependency and the advantage of a physical association between the antigen and liposome were also demonstrated in this experiment (Shek and Sabiston, 1981 ). Feeding OA entrapped in liposomes did not produce significantly elevated serum or intestinal antibody responses to OA compared with the same dose of antigen fed alone. Preliminary experiments (data not shown ) investigated the response to feeding doses of 0.4-8 mg OA in priming regimes (over 1-6 days) as well as using different strains of mouse (Balb/c), and omitting or using a different charged lipid (PA). In all cases no significant serum or intestinal antibody responses was detected in any group fed OA alone or in liposomes. The regime of early boosting (on Day 17) did induce serum IgG antibody production in the groups fed OA and OA in PC-based liposomes but when boosting was delayed the response was no longer apparent, possibly indicating that the original immune response was exhausted or that any memory induction was weak and transient. Serum antibody responses to fed soluble proteins are well documented (Hanson et al., 1979) although regimes of single high doses or multiple feeds are usually required. Intestinal antibody responses in this study were weak and this has been reported previously for fed soluble antigens. Pilot experiments and other reports (Hamilton et al., 1981; Wilson et al., 1989 ) showed that fed KLH was able to induce serum and intestinal antibody responses and indicated that adjuvant effects may be easier to assess. Serum and some intestinal antibody levels in the groups fed KLH alone or in lipo-
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somes were elevated compared with the control groups but no adjuvant action could be attributed to the liposomal entrapment. Feeding 'empty' liposomes just prior to KLH abolished any serum or intestinal antibody response, possibly by inhibiting transit of the protein through the stomach and allowing greater proteolysis. Both types of liposome were substantially unaffected by incubation at 37 °C in acidic conditions confirming that entrapped antigen may be protected from proteolytic degradation during transit through the stomach (Rowland and Woodley, 1980; Chiang and Weiner, 1987a). In contrast, the detergent action of bile salts caused OA release that was almost immediate and total for the PC-based liposomes. Richards and Gardner (1978) also found that vesicle disruption was most apparent in 'fluid' liposomes with a low Tc. The failure to improve intestinal IgA antibody levels after feeding antigen in liposomes is of relevance for the development of oral vaccines, particularly those based on refined proteins. In this study antigen size did not appear to influence adjuvant activity as neither OA nor the larger KLH protein was able to elicit significantly improved levels of local or serum antibody. Entrapment of protein in liposomes also failed to abrogate the induction of tolerance to systemic challenge. This correlates with the lack of an intestinal i m m u n e response observed after feeding and shows that the liposome is not sufficiently effective as an oral adjuvant to reverse the toleragenic nature of a fed soluble protein. Furthermore this finding may suggest that feeding antigen in liposomes does not significantly alter the mechanism of antigen handling within the intestine. Our data indicate that significant liposomal disruption occurs in the gut. Other studies have failed to find evidence for the intestinal absorption of liposomes or non-absorbable, liposome-entrapped markers (Whitmore and Wheeler, 1979; Chiang and Weiner, 1987b ). Together these suggest that the properties that are responsible for the adjuvant action of liposomes administered parenterally may be inapplicable to the induction of an i m m u n e response in the intestine. In conclusion, liposomes would appear to be useful as carriers for drugs or antigens that are susceptible to degradation in the gastric environment, but they failed to improve the intestinal i m m u n e response for the soluble proteins used in this study. REFERENCES Allison, A.C. and Gregoriadis, G., 1974. Liposomesas immunologicaladjuvants. Nature, 252: 252. Chiang, C. and Weiner, N., 1987a. Gastrointestinal uptake of liposomes. I. In vitro and in situ studies. Int. J. Pharm., 37: 75-85. Chiang, C. and Weiner, N., 1987b.Gastrointestinal uptake ofliposomes. II. In vivo studies. Int. J. Pharm., 40: 143-150. Dapergolas, G., Neerunjun, E.D. and Gregoriadis, G., 1976. Penetration of target areas in the rat by liposome-associatedbleomycin,glucoseoxidase and insulin. FEBS Lett., 63: 235-239.
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Davis, D. and Gregoriadis, G., 1987. Liposomes as adjuvants with immunopurified tetanus toxoid: influence of liposomal characteristics. Immunology, 61: 229-234. Desiderio, J.V. and Campbell, S.G., 1985. Immunization against experimental murine salmonellosis with liposome-associated O-antigen. Infect. Immun., 48:658-663. Gregoriadis, G., 1980. Tailoring liposome structure. Nature, 283:814-815. Gregory, R.L., Michalek, S.M., Richardson, G., Harmon, C., Hilton, T. and McGhee, J.R., 1986. Characterisation of immune response to oral administration of Streptococcus sobrinus ribosomal preparations in liposomes. Infect. Immun., 54: 780-786. Hamilton, S.R., Keren, D.F., Yardley, J.H. and Brown, G.D., 1981. No impairment of local intestinal immune response to KLH in the absence of Peyer's patches. Immunology, 42:431435. Hanson, D.G., Vas, N.M., Maia, L.G.S. and Lynch, J.M., 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: 2337-2342. Hemker, H.C., Hermans, W.Th., Muller, A.D. and Zwaal, R.F.A., 1980. Oral treatment of haemophilia A by gastrointestinal absorption of factor VIII entrapped in liposomes. Lancet, 1: 70-71. Hunter, W.M. and Greenwood, F.C.I., 1962. Preparation of iodine-31 labelled human growth hormone of high specific activity. Nature, 194: 495. Kinsky, S.C., 1978. Immunogenicity ofliposomal model membranes. Ann. NY Acad. Sci., 308: lll-ll6. Kirby, C. and Gregoriadis, G., 1984. Dehydration-rehydration vesicles: A simple method for high yield drug entrapment in liposomes. Biotechnology, 2: 979-984. Patel, H.M. and Ryman, B.E., 1976. Oral administration of insulin by encapsulation within liposomes. FEBS Lett., 62: 60-63. Patel, H.M. and Ryman, B.E., 1977. Orally administered liposomally entrapped insulin. Biochem. Soc. Trans., 5: 1739-1741. Pierce, N.F. and Sacci, J.B., 1984. Enhanced mucosal priming by cholera toxin and procholeragenoid with a lipoidal amine adjuvant (avridine) delivered in liposomes. Infect. Immun., 44: 469-473. Rhalem, A., Bourdieu, C., Luffau, G. and Pery, P., 1988. Vaccination of mice with liposomeentrapped antigens ofNippostrongylus brasiliensis. Ann. Inst. Pasteur, Immunol., 139: 157163. Rhodes, M.B., Baker, P.K., Christenson, D.L. and Anderson, G.A., 1988. Ascaris suum antigens incorporated into liposomes used to stimulate protection to migrating larvae. Vet. Parasitol., 26: 343-349. Richards, M.H. and Gardner, C.R., 1978. Effects of bile salts on the structural integrity of liposomes. Biochim. Biophys. Acta, 543: 508-522. Rowland, R.N. and Woodley, J.F., 1980. The stability ofliposomes in vitro to pH, bile salts and pancreatic lipase. Biochim. Biophys. Acta, 620: 400-409. Shahum, E. and Therien, H-M., 1988. Immunopotentiation of the humoral response by liposomes: encapsulation versus covalent linkage. Immunology, 65:315-317. Shek, N. and Sabiston, B.H., 1981. Immune response mediated by liposome-associated protein antigens. I. Potentiation of the plaque-forming cell response. Immunology, 45: 349-356. Svennerholm, A-M. and Holmgren, J., 1977. Immunoglobulin and specific antibody synthesis in vitro by enteral and non-enteral lymphoid tissues after subcutaneous cholera immunization. Infect. Immun., 15: 360-369. Wachsmann, D., Klein, J.P., Scholler, M. and Frank, R.M., 1985. Local and systemic immune response to orally administered liposome-associated soluble S. mutans cell wall antigen. Immunology, 54: 189-193. Wachsmann, D., Klein, J.P., Scholler, M., Ogler, M., Ackeramans, F. and Frank, R.M., 1986.
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Serum and salivary antibody responses in rats orally immunised with Streptococcus mutans carbohydrate protein conjugate associated with liposomes. Infect. Immun., 52:408-413. Whitmore, D.A. and Wheeler, K.P., 1979. The fate of liposomes in the rat small intestine. Biochem. Soc. Trans., 7: 929-931. Wilson, A.D., Stokes, C.R. and Bourne, F.J., 1989. Adjuvant effect of cholera toxin on the mucosal immune response to soluble proteins. Differences between mouse strains and protein antigens. Scand. J. Immunol., 29" 739.