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Humoral immune response of carp (Cyprinus carpio) induced by oral immunization with liposome-entrapped antigen
Developmental and Comparative Immunology 27 (2003) 413–421 www.elsevier.com/locate/devcompimm
Humoral immune response of carp (Cyprinus carpio ) induced by oral immunization with liposome-entrapped antigen Takuya Irie, Shinobu Watarai*, Hiroshi Kodama Laboratory of Veterinary Immunology, Division of Veterinary Science, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan Received 29 August 2001; revised 8 August 2002; accepted 1 November 2002
Abstract To study the value of liposomes as carriers of antigens for oral vaccination in fish, humoral immune responses were analyzed after immunizing carp (Cyprinus carpio ) with liposome-entrapped bovine serum albumin (BSA) as a model antigen. Oral immunization of BSA (100 mg)-containing liposomes that were stable in carp bile induced significant antibody responses against BSA in serum as well as in intestinal mucus and bile. By contrast, no serum antibody responses were observed when fish were orally immunized with the same dose of BSA-containing unstable liposomes or BSA alone. BSA-specific antibodysecreting lymphocytes were detected in the spleen and head kidney of immunized fish. Furthermore, when we applied Vibrio cholerae toxin B subunit (CT-B)-conjugated liposomes containing BSA for oral immunization we found significant increases of anti-BSA antibodies in serum. Our results suggest that delivery systems using liposomes or liposomes with CT-B to the intestinal tract are essential for inducing effective humoral immune responses following oral vaccination. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Antigen delivery system; Carp; Cholera toxin B subunit; Liposome; Intestinal immunity; Oral immunization
1. Introduction In many countries, intensive fish farming has become a key industry in recent decades. With the increasing scale of aquaculture, fishes are reared at high population density. Even when environmental conditions are favorable and the fish are healthy, mass mortality will occur if infectious agents are introduced into the farm, causing great financial losses. Previous treatment of diseases has been * Corresponding author. Tel./fax: þ 81-72-254-9492. E-mail address: [email protected] (S. Watarai).
focused on chemicals and antibiotics. Treatment of affected fish with antibiotics is effective, but gives rise to problems such as accumulated resistance in the bacteria, which renders the antibiotic useless. Many vaccines and different methods of mass vaccination have been developed against bacterial and viral diseases in cultured fish. In general, three different methods are employed for administration of antigens; immersion, injection, and oral vaccination. The effectiveness of a vaccine depends largely on its mode of administration [1]. Since the vaccines should be administered to a large number
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of fish irrespective of size at any time in the culture cycle and in every type of culture system, oral vaccination via feed is best suited [2]. It is the least stressful method to deliver antigens, is time and labor saving, and avoids stress caused by manipulation of the fish. To induce sufficient protective immune responses, antigens should reach the lymphoid tissue of the hindgut without destruction during passage through the gastrointestinal tract [3 – 5]. Antigens are therefore, incorporated in or adhered to the feed, or are covered with artificial constructs [6 – 10]. Liposomes are multilayer vesicles composed of amphiphilic phospholipids. They are non-toxic, biodegradable and only weakly immunogenic. They have been used to deliver a wide variety of biologically active substances to specific tissues and cells, and have also been used as immunological adjuvants [11]. In particular, liposomes entrapping antigens are protected from low pH and enzymatic attack until they reach the target sites, so that the possibility of liposomes as carriers and adjuvants for developing oral vaccines has attracted considerable interest [12]. We have recently demonstrated that liposomes consisting of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS) and cholesterol (Chol) (1:1:2, molar ratio) (PS-liposomes) are stable in acidic solution, bile, and pancreatin solution [13], and would serve as an effective oral delivery vehicle for inducing mucosal immune responses [14]. Nakhla et al. [15,16] reported serum immune responses in rainbow trout (Oncorhynchus mykiss ) after injection of Aeromonas salmonicida or lipopolysaccharide incorporated in liposomes. Rodgers [17] observed a protective effect of immersion vaccination of A. salmonicida antigen with liposomes in rainbow trout. However, there is no report concerning oral immunization by antigens trapped in liposomes in fish. Oral vaccination can induce both systemic and local intestinal immune responses [3,6,8]. In the present study, therefore, in order to examine the potency of liposomes as carriers for oral vaccines, we analyzed the serum and intestinal antibody responses of carp (Cyprinus carpio ) to bovine serum albumin (BSA) administered orally with PS-liposomes.
2. Materials and methods 2.1. Materials DPPC, DPPS, Chol, dipalmitoylphosphatidylethanolamine (DPPE) and Vibrio cholerae toxin B subunit (CT-B) were purchased from Sigma Chemical Co., St Louis, MO. N-hydroxysuccinimidyl 3-(2-pyridyldithio)-propionate (SPDP) came from Amersham Pharmacia Biotechnology, Uppsala, Sweden. BSA and horseradish peroxidase-conjugated anti-rabbit IgG came, respectively, from Wako Pure Chemical Industries, Osaka, Japan and Jackson Immuno Research, West Grove, PA. 3-(2-pyridyldithio)-propionylphosphatidylethanolamine (DTP-DPPE) was prepared by the reaction of SPDP with DPPE as described previously [18]. Carboxy fluorescein (CF) came from Eastman Kodak (Rochester, NY). Fluorescein isothiocyanate (FITC)-conjugated BSA was prepared according to the procedure described previously [19]. 2.2. Animals Common carp were provided by courtesy of the Osaka Prefectural Freshwater Fish Experiment Station, Neyagawa, Japan. They were reared in 160l plastic aquaria that were filled with dechlorinated tap water (water temperature about 23 8C) and aerated. Fishes weighting about 350 g were used for the experiments. 2.3. Preparation of liposomes Liposomes entrapping BSA were prepared by the following procedure, as described previously [13,14]. DPPC (0.5 mmol), DPPS (0.5 mmol) and Chol (1 mmol), or DPPC (3.5 mmol) and Chol (1 mmol), each dissolved in organic solvent, were mixed in a conical flask. The lipids were dried on a rotary evaporator (Shibata Science Technology, Tokyo, Japan), and were left to stand for 30 min in a high vacuum in a desiccator. These liposome preparations were designated as PS- and PC-liposomes, respectively. After addition of 2 ml of BSA solution (10 mg/ ml) and incubation at room temperature for 90 min, the lipid film was dispersed by vigorous vortexing. Unencapsulated BSA was removed by centrifuging at
T. Irie et al. / Developmental and Comparative Immunology 27 (2003) 413–421
14,000g for 20 min at 4 8C in 150 mM phosphatebuffered saline (PBS, pH 7.2). The resulting pellet of liposomes was suspended in saline and used in the immunization. CT-B-conjugated PS-liposomes containing BSA for immunization were prepared from the lipid mixture solution containing DPPC (0.3 mmol), DPPS (0.3 mmol), Chol (0.6 mmol) and DTP-DPPE (0.015 mmol) as described above. The amount of BSA trapped in liposomes was determined by the following method. Ninety microlitre of isopropyl alcohol was added to a 10 ml suspension of liposome-entrapped BSA (1:3 dilution in PBS), and vortex mixing was done. The protein concentration of the resulting solutions was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA), with bovine plasma gamma globulin used as a standard. PS- and PC-liposomes entrapping CF were prepared as described previously, and were used for studying the stability of liposomes [13]. 2.4. Immunization of carp Carps (n ¼ 3 or 4) were immunized orally with liposome-entrapped BSA or with BSA alone (100 mg protein/500 ml saline). Immunization was repeated two or three times at 2 week intervals. Sera, bile and intestinal tract were collected 2 weeks after the last immunization. Sera and bile obtained were used for antibody assay. Intestinal tract was used to prepare intestinal mucus. 2.5. Preparation of intestinal mucus samples Intestinal mucus was prepared as follows. Intestinal tract was cut with scissors to open the intestinal lumen. Then, intestinal mucus was collected from the intestine by gentle scraping with spatula and transferred to a tube. After addition of 500 ml of PBS, the tube was vortexed for 30 s, and centrifuged at 20,000g for 20 min at 4 8C. The supernatant was collected and assayed for antibody activity. 2.6. Preparation of anti-carp IgM antibody The procedure for purification of carp IgM from carp serum followed by the method described by Yamaguchi et al. [20].
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Antibody against the purified carp IgM was raised in a Japanese white rabbit. Briefly, 1 mg of purified carp IgM was mixed with Freund’s complete adjuvant (1:1) and injected subcutaneously into a rabbit on days 0 and 14. The animal was given booster with the purified carp IgM in PBS (no adjuvant) 2 weeks after the second immunization. At 1 week after the booster immunization, the animal was bled out and serum was collected. The IgG fraction was purified from the immune serum by precipitation with 50% ammonium sulfate, followed by affinity chromatography on AffiGel Protein A MAPS II (Bio-Rad Laboratories). The specificity of anti-carp IgM rabbit IgG was analyzed by enzyme-linked immunosorbent assay (ELISA). Anti-carp IgM rabbit IgG gave positive reaction only with carp IgM. 2.7. Antibody assay Specific antibodies against BSA were determined by ELISA. BSA (20 mg/ml) diluted with PBS was dispensed at 50 ml/well into 96-well microtiter plates (Iwaki Glass Co. Ltd., Funabashi, Japan). The plates were left overnight at 4 8C, and were washed five times with PBS containing 0.1% Tween 20 (washing solution). Next, 100 ml of PBS containing 0.5% horse serum (blocking solution) was added to each well. Following incubation for 60 min at 25 8C the wells were washed with washing solution, and 50 ml of test serum, intestinal mucus or bile, each diluted with blocking solution, was then added to each well. The plates were incubated for 90 min at 25 8C, washed with washing solution, and 50 ml of anti-carp IgM rabbit IgG (1:100 dilution in PBS) was added. After incubation for 90 min at 25 8C the wells were washed with washing solution and 50 ml of horseradish peroxidase-conjugated anti-rabbit IgG (1:8000 dilution in PBS) was added. The plates were then incubated for 90 min at 25 8C and washed with washing solution. Next, 100 ml of substrate solution (0.4 mg/ml o-phenylenediamine and 0.2 ml/ml of 30 % H2O2 in citrate buffer) was added and left to react for 10 min at 25 8C. The enzyme reaction was stopped by adding 100 ml of 2N H2SO4, and the absorbance at 492 nm was measured with a microplate reader (Model 450; Bio-Rad Laboratories). Antibody titers are represented as the reciprocal of
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endpoint dilution exhibiting A492 more than three times that of background. 2.8. Separation of cells Preparation of head kidney and spleen lymphocytes from carp was performed according to the method of Yamaguchi et al. [20] with slight modifications. Briefly, the head kidney and the spleen of the carp were all cut into small pieces with scissors in cold Eagle’s minimum essential medium (MEM, pH 7.4, Nissui Pharmaceutical Co., Tokyo, Japan). Cells were passed through a stainless steel mesh, and cell suspensions were carefully overlayered on a 1.060 and 1.070 discontinuous density Percoll solution (Amersham Pharmacia Biotechnology). After centrifuging at 1200g for 20 min at 20 8C, the cells above Percoll solution of 1.070 were collected and washed three times with MEM. More than 90% of cells were lymphocytes [20]. Cell viability, according to the trypan blue dye exclusion test, was more than 90%. 2.9. Flow cytometric analysis Head kidney and spleen lymphocytes (5 £ 106 cells) from carp were incubated with FITC-conjugated BSA (1:100 dilution in PBS) on ice for 90 min. Cells from non-immunized carp were used as control. The cells were then washed three times with PBS, and fixed with 1% paraformaldehyde solution. Fluorescence-positive cells were measured in a flow cytometer (CytoACE-150, Japan Spectroscopic Co., Tokyo, Japan) equipped with a 10 mW argon ion laser tuned at 488 nm. Fluorescence histograms were analyzed gating on both forward and rectangular light scatter characteristics to eliminate the influence of cell debris. Fluorescence intensity was represented in log units over a 4-log scale. All the data are based on 10,000 gated cell count with 256 channels.
3. Results 3.1. Serum antibody responses in carp orally immunized with BSA or liposome-entrapped BSA In the first experiment, we examined the stability of PS- and PC-liposomes by measuring the leakage of CF from liposomes. PS-liposomes were stable in 20% carp bile (leakage 13 ^ 10 %), whereas PC-liposomes were unstable (92 ^ 14 %) when incubated for 60 min at 37 8C. In the next experiment, serum antibody titers were measured in carp immunized orally with 100 mg of BSA entrapped in PS- or PC-liposomes. Control fish were administered the same dose of BSA alone. Antibody titers increased significantly 14 days after secondary immunization (28 days after primary immunization) when fish were immunized with BSA-PS-liposome as shown in Fig. 1. Fourteen days after the third immunization (42 days after primary immunization), the highest antibody responses were observed. By contrast, no obvious increase of antibody titer was noted in fish orally immunized with BSA entrapped in unstable PC-liposomes or immunized with BSA alone (Fig. 1). Antibody titers in fish immunized with BSA-PS-liposomes were significantly higher ( p , 0.05) after the third immunization when compared to other groups. 3.2. Antibody production in intestinal mucosa and in bile To study the presence of antigen-specific antibodies in intestinal mucosa and bile, carp were orally administered with BSA-PS-liposomes three times. Though the titers were relatively low, a clear increase of anti-BSA antibody titers was observed after the immunization (Fig. 2). In particular, the titers in bile of immunized fish were significantly higher ( p , 0.001) than those of non-immunized control.
2.10. Statistical analysis
3.3. Serum antibody responses in carp immunized orally with BSA entrapped in CT-B-conjugated liposomes
The student’s t-test was used to evaluate of the results.
For effective immune responses, efficient delivery of liposomes to the inductive sites of the intestinal
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Fig. 1. Serum antibody responses in carp immunized with BSA entrapped in liposomes. Three groups of four fish were immunized orally with BSA entrapped in stable PS-liposomes (B), unstable PC-liposomes (O), or with BSA alone (V), respectively. Antibody titers were measured 2 weeks after primary, secondary or third immunization, respectively. ( p ) p , 0.05 vs BSA alone and PC-liposomes. Data are expressed as mean values and standard errors of the mean from four individual experiments.
immune system is vital. Accordingly, liposomes coupled with a ligand for which a receptor is expressed on the mucosal epithelium are suitable for the enhancement of immune responses. More
recently, we demonstrated the presence of ganglioside GM1 on the epithelial cell surface of carp intestinal mucosa (Irie et al., submitted for publication). We therefore, used CT-B, which has a high affinity for
Fig. 2. Antibody production in intestinal mucosa (A) and bile (B). Four individual fish were immunized orally with BSA-PS-liposomes three times, and antibody titers were measured 2 weeks after the third immunization. Titers were significantly higher than in no treatment ((#) P , 0.1; ( p ) P , 0.001). Data are expressed as mean values and standard errors of the mean from four fish.
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Fig. 3. Serum antibody responses in carp immunized orally with BSA in CT-B-conjugated PS-liposomes. Three groups of three were immunized with BSA in CT-B-conjugated PS-liposomes (X), BSA-PS-liposomes (B), or BSA only (V). Antibody titers were measured 2 weeks after primary and secondary immunization, respectively. ( p ) p , 0.05 vs BSA alone. (#) p , 0.05 vs PS-liposomes. ($) p , 0.05 vs BSA alone. Results obtained from three individual tests are expressed as mean values and standard errors of the mean from three fish.
ganglioside GM1, as a ligand to carp intestinal mucosa. CT-B-conjugated PS-liposomes containing BSA were administered orally to carp. The antibody responses to BSA were evaluated 2 weeks after secondary immunization. As shown in Fig. 3, oral administration of CT-B-conjugated PS-liposomes containing BSA induced significant antibody responses. Antibody titers were significantly higher compared to those of fish immunized with BSA-PSliposomes or non-immunized control ( p , 0.05). This result indicates that the coupling of CT-B to PS-liposomes is more effective at inducing of the immune responses in carp. 3.4. Detection of BSA-specific antibody-secreting lymphocytes We attempted to detect lymphocytes producing BSA-specific antibody in the spleen and head kidney. Fish were administered three times with BSA (100 mg) incorporated in PS-liposomes. Table 1 shows the extent of lymphocytes secreting BSA-specific Ig. In immunized fish, BSA-specific
Ig-secreting lymphocytes were detected in 18.6 ^ 1.9 and 9.1 ^ 4.6% of spleen and head kidney lymphocytes, respectively ( p , 0.05 in comparison with non-immunized fish).
4. Discussion The mucosal surfaces of fish intestines are a major place of attack by many infectious agents. Therefore, to protect fish from enteropathogenic agents, the Table 1 Detection of BSA-specific antibody-secreting lymphocytes in spleen and head kidney Lymphocytesa
Immunizedb
Non-immunized
Spleen Head kidney
18.6 ^ 1.9c ( p , 0.05) 9.1 ^ 4.6 ( p , 0.05)
3.1 ^ 0.7 2.1 ^ 0.4
a
BSA-specific antibody-producing lymphocytes were detected by flow cytometry by staining the cells with FITC-conjugated BSA. b Fish were immunized orally with 100 mg of BSA-PS-liposomes three times at 2 week intervals. c % Fluorescence positive cells ^ SD from four fish.
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induction of mucosal immunity, and especially an intestinal immune response, is very important. Development of oral vaccines is crucial since the oral route of immunization can induce not only mucosal but also systemic immune responses [2]. However, the problem arises of antigen degradation by gastric acidity and proteolytic enzymes in the intestinal lumen, and extremely large doses are required to achieve satisfactory immune responses. New adjuvant and carriers are therefore, essential. We have previously reported that PS-liposomes act as an effective oral delivery vehicle for inducing mucosal immune responses, and also act as an adjuvant for antibody production on the intestinal mucosa [13,14]. However, we know of no report on antigen trapped in liposomes as an oral vaccine in fish. In the present study, we therefore, studied the value of PS-liposomes as carriers for oral vaccines in carp. We found that PS-liposomes showed stability to carp bile. Furthermore, when BSA entrapped in PSliposomes was administered orally to carp, significant antibody responses were induced in serum. On the other hand, no obvious antibody responses to BSA were induced when BSA alone or entrapped in unstable PC-liposomes was administered (Fig. 1). These results suggest that PS-liposomes are able to deliver antigens to immune responsive areas of the intestinal tract without leakage or degradation of incorporated antigens following oral administration, and then induce immune responses against antigens incorporated into PS-liposomes. We conclude that PSliposomes have great potential as carriers for oral vaccines in fish. It is known that the hindgut of fish adsorbs proteins and particulate antigen from the lumen [2,6]. Although fish lack Peyer’s patches and antigentransporting M cells, enterocytes in the hindgut show an antigen-transporting ability, and many macrophages and lymphoid cells are present in gut mucosa [21]. Antigens reaching the posterior segment of the gut are transferred either to intracellular spaces or to macrophages or enterocytes [5,6]. Azad et al. [6] observed that biofilm antigens of Aeromonas hydrophila administered orally to carp appeared in melanomacrophage centers in the kidney and spleen. In the present study, specific antibody-secreting cells were detected in lymphocyte preparations separated from the head kidney and spleen of carp that had been
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immunized with BSA entrapped in PS-liposomes (Table 1). Since liposomes are avidly taken up by phagocytic cells such as macrophages, it is reasonable to suppose that uptake of BSA-PS-liposomes by macrophages acting as antigen-presenting cells in the posterior part of the gut induces to the stimulation of the immunocytes associated with mucosal membranes, leading to systemic and intestinal antibody responses. In this study, the percentage of BSA-specific antibody-secreting cells in spleen and head kidney lymphocytes was significantly higher in the immunized carp than in the non-immunized carp (Table 1). This result suggests the induction of lymphocytes producing BSA-specific antibody in the spleen and head kidney following oral immunization. However, it is possible that FITC-conjugated BSA non-specifically binds to other cells in separated lymphocytes used in flow cytometric analysis. If this were so, it would be expected that a number of fluorescencepositive cells were detected in the lymphocytes from non-immunized carp. However, the experimental result indicated that the number of fluorescencepositive cells in both spleen and head kidney lymphocytes was significantly more in the immunized carp than in the non-immunized carp (Table 1). It has also been reported that fish lymphocytes have an Igbinding capacity [22]. Although, thus, it cannot be excluded that, in immunized fish, immune complexes are formed and bound to lymphocytes, it is not too far from the truth to say that many of the fluorescencepositive cells would be BSA-specific antibodysecreting lymphocytes. However, further studies are required to fully clarify the lymphocytes having antiBSA antibody at their surface. To promote uptake of antigens-containing liposomes by immunocompetent phagocytic cells in the intestinal tract, coupling to a ligand which is recognized by a receptor on the surface of the epithelial cells in the intestine should be effective. Recently we demonstrated the presence of ganglioside GM1, which serves as a receptor of CT-B in carp intestinal mucosa [Irie et al., submitted for publication]. Accordingly, in the present study, CTB having the ganglioside GM1 binding property was coupled to the outer surface of PS-liposomes in order to target delivery of incorporated antigens to the carp intestinal epithelium. As expected, oral
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administration of BSA-containing CT-B-conjugated PS-liposomes induced a significant higher antibody response compared to responses cooked by BSA entrapped in PS-liposomes (Fig. 3). This suggests enhanced binding by an interaction between liposomal CT-B and ganglioside GM1 on the epithelial cell surface of the intestinal mucosa. The delivery of antigen in combination with CT-B has been shown to increase binding of CT-Bantigen complex to the GM1 gangliosides, increasing uptake of the antigen by M cells in humans [23]. CT-B has also been used as an adjuvant for the oral delivery of antigens in tilapia (Oreochromis mossambicus ) [4]. As stated, an increase in the permeability of the intestinal epithelium facilitates binding and uptake of antigens by the epithelial cells. Davidson et al. [3] detected antigen-specific antibody-secreting cells in the head kidney and intestinal mucosa of rainbow trout after peroral intubation of A. salmonicida. Joosten et al. [8] also identified specific plasma cells in the gut of carp immunized orally with Vibrio anguillarum. In our study, BSA-specific antibodies were detected in the intestinal mucosa and bile of fish immunized orally with BSA entrapped in liposomes, but not in fish immunized with BSA alone (Fig. 2), suggesting that specific antibodies against BSA might be also produced by lymphocytes located in the intestinal epithelium, and that the intestinal epithelium is an active site of antibody secretion. However, further research is necessary to clarify these points. In conclusion, oral administration of liposomeentrapped BSA antigens effectively induced both serum and intestinal antibody responses directed against BSA. It follows that PS-liposomes have great potential in developing oral vaccine for fish. The induction of protective immune responses by administration of bacterial antigens entrapped in PSliposomes is now under study in freshwater and seawater fish, and could help to prevent infectious diseases in fish culture.
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[2] Quentel C, Vegneulle M. Antigen uptake and immune responses after oral vaccination. Dev Biol Stand 1997;90: 69–78. [3] Davidson GA, Ellis AE, Secombes CJ. Route of immunization influences the generation of antibody secreting cells in the gut of rainbow trout (Oncorhynchus mykiss ). Dev Comp Immunol 1993;17:373–6. [4] Jenkins PG, Wrathmell AB, Harris JE, Pulsford AL. The effects of different adjuvants on intestinal antigen absorption and subsequent immune responses of the tilapian Oreochromis mossambicus. Fish Shellfish Immunol 1994;4:167–77. [5] Rombout JHWM, Lamers CHJ, Helfrich MH, Dekker A, Taverne-Thiele JJ. Uptake and transport of intact macromolecules in the intestinal epithelium of carp (Cyprinus carpio L.) and the possible immunological implications. Cell Tissue Res 1985;239:519–30. [6] Azad IS, Shankar KM, Mohan CV, Kalita B. Uptake and processing of biofilm and free-cell vaccines of Aeromonas hydrophila in Indian major carps and common carp following oral vaccination—antigen localization by a monoclonal antibody. Dis Aquat Org 2000;14:103 –8. [7] Wong G, Kaattari LS, Christensen MJ. Effectiveness of an oral enteric coated Vibrio vaccine for use in salmonid fish. Immunol Invest 1992;21:353–64. [8] Joosten PHM, Tiemersma E, Threels A, Caumartin-Dhieux C, Rombout JHWM. Oral vaccination of fish against Vibrio anguillarum using alginate microparticles. Fish Shellfish Immunol 1997;7:471– 85. [9] Joosten PHM, Aviles-Trigueros M, Sorgeloos P, Rombout JHWM. Oral vaccination of juvenile carp (Cyprinus carpio ) and gilthead seabream (Sparus aurata ) with bioencapsulated Vibrio anguillarum bacterin. Fish Shellfish immunol 1995;5: 289 –99. [10] Campbell R, Adams A, Tatner MF, Chair M, Sorgeloos P. Uptake of Vibrio anguillarum vaccine by Artemia salina as a potential oral delivery system to fish fry. Fish Shellfish immunol 1993;3:451–9. [11] Gregoriadis G. Immunological adjuvants: a role for liposomes. Immunol Today 1990;11:89–97. [12] Alving CR. Liposomes as carriers of antigens and adjuvants. J Immunol Methods 1991;140:1 –13. [13] Han M, Watarai S, Kobayashi K, Yasuda T. Application of liposomes for development of oral vaccines: study of in vitro stability of liposomes and antibody response to antigen associated with liposomes after oral immunization. J Vet Med Sci 1997;59:1109–14. [14] Watarai S, Han M, Tana, Kodama H. Antibody response in the intestinal tract of mice orally immunized with antigen associated with liposomes. J Vet Med Sci 1998;60: 1047–50. [15] Nakhla AN, Szalai AJ, Banoub JH, Keough KMW. Uptake and biodistribution of free and liposomally incorporated lipopolysaccharide of Aeromonas salmonicida administered via different routes to rainbow trout (Oncorhynchus mykiss ). J Liposome Res 1994;4:1029–48. [16] Nakhla AN, Szalai AJ, Banoub JH, Keough KMW. Serum antiLPS antibody production by rainbow trout (Oncorhynchus
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[21] Rombout JHWM, Van Den Berg AA. Immunological importance of the second gut segment of carp I. Uptake and processing of antigens by epithelial cells and macrophages. J Fish Biol 1989;35:13 –22. [22] Koumans-van Diepen JC, van de Lisdonk MH, Taverne-Thiele AJ, Verburg-van Kemenade BM, Rombout JHWM. Characterisation of immunoglobulin-binding leucocytes in carp (Cyprinus carpio L.). Dev Comp Immunol 1994;18:45–56. [23] Quiding M, Nordstrom I, Kilander A, Anderson G, Hanson LA, Holmgren J, Czerkinsky C. Intestinal immune responses in humans: oral cholera vaccination induces strong intestinal antibody responses and interferon-gamma production and evokes local immunological memory. J Clin Invest 1991;88:143– 8.
Humoral immune response of carp (Cyprinus carpio ) induced by oral immunization with liposome-entrapped antigen Takuya Irie, Shinobu Watarai*, Hiroshi Kodama Laboratory of Veterinary Immunology, Division of Veterinary Science, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan Received 29 August 2001; revised 8 August 2002; accepted 1 November 2002
Abstract To study the value of liposomes as carriers of antigens for oral vaccination in fish, humoral immune responses were analyzed after immunizing carp (Cyprinus carpio ) with liposome-entrapped bovine serum albumin (BSA) as a model antigen. Oral immunization of BSA (100 mg)-containing liposomes that were stable in carp bile induced significant antibody responses against BSA in serum as well as in intestinal mucus and bile. By contrast, no serum antibody responses were observed when fish were orally immunized with the same dose of BSA-containing unstable liposomes or BSA alone. BSA-specific antibodysecreting lymphocytes were detected in the spleen and head kidney of immunized fish. Furthermore, when we applied Vibrio cholerae toxin B subunit (CT-B)-conjugated liposomes containing BSA for oral immunization we found significant increases of anti-BSA antibodies in serum. Our results suggest that delivery systems using liposomes or liposomes with CT-B to the intestinal tract are essential for inducing effective humoral immune responses following oral vaccination. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Antigen delivery system; Carp; Cholera toxin B subunit; Liposome; Intestinal immunity; Oral immunization
1. Introduction In many countries, intensive fish farming has become a key industry in recent decades. With the increasing scale of aquaculture, fishes are reared at high population density. Even when environmental conditions are favorable and the fish are healthy, mass mortality will occur if infectious agents are introduced into the farm, causing great financial losses. Previous treatment of diseases has been * Corresponding author. Tel./fax: þ 81-72-254-9492. E-mail address: [email protected] (S. Watarai).
focused on chemicals and antibiotics. Treatment of affected fish with antibiotics is effective, but gives rise to problems such as accumulated resistance in the bacteria, which renders the antibiotic useless. Many vaccines and different methods of mass vaccination have been developed against bacterial and viral diseases in cultured fish. In general, three different methods are employed for administration of antigens; immersion, injection, and oral vaccination. The effectiveness of a vaccine depends largely on its mode of administration [1]. Since the vaccines should be administered to a large number
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of fish irrespective of size at any time in the culture cycle and in every type of culture system, oral vaccination via feed is best suited [2]. It is the least stressful method to deliver antigens, is time and labor saving, and avoids stress caused by manipulation of the fish. To induce sufficient protective immune responses, antigens should reach the lymphoid tissue of the hindgut without destruction during passage through the gastrointestinal tract [3 – 5]. Antigens are therefore, incorporated in or adhered to the feed, or are covered with artificial constructs [6 – 10]. Liposomes are multilayer vesicles composed of amphiphilic phospholipids. They are non-toxic, biodegradable and only weakly immunogenic. They have been used to deliver a wide variety of biologically active substances to specific tissues and cells, and have also been used as immunological adjuvants [11]. In particular, liposomes entrapping antigens are protected from low pH and enzymatic attack until they reach the target sites, so that the possibility of liposomes as carriers and adjuvants for developing oral vaccines has attracted considerable interest [12]. We have recently demonstrated that liposomes consisting of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS) and cholesterol (Chol) (1:1:2, molar ratio) (PS-liposomes) are stable in acidic solution, bile, and pancreatin solution [13], and would serve as an effective oral delivery vehicle for inducing mucosal immune responses [14]. Nakhla et al. [15,16] reported serum immune responses in rainbow trout (Oncorhynchus mykiss ) after injection of Aeromonas salmonicida or lipopolysaccharide incorporated in liposomes. Rodgers [17] observed a protective effect of immersion vaccination of A. salmonicida antigen with liposomes in rainbow trout. However, there is no report concerning oral immunization by antigens trapped in liposomes in fish. Oral vaccination can induce both systemic and local intestinal immune responses [3,6,8]. In the present study, therefore, in order to examine the potency of liposomes as carriers for oral vaccines, we analyzed the serum and intestinal antibody responses of carp (Cyprinus carpio ) to bovine serum albumin (BSA) administered orally with PS-liposomes.
2. Materials and methods 2.1. Materials DPPC, DPPS, Chol, dipalmitoylphosphatidylethanolamine (DPPE) and Vibrio cholerae toxin B subunit (CT-B) were purchased from Sigma Chemical Co., St Louis, MO. N-hydroxysuccinimidyl 3-(2-pyridyldithio)-propionate (SPDP) came from Amersham Pharmacia Biotechnology, Uppsala, Sweden. BSA and horseradish peroxidase-conjugated anti-rabbit IgG came, respectively, from Wako Pure Chemical Industries, Osaka, Japan and Jackson Immuno Research, West Grove, PA. 3-(2-pyridyldithio)-propionylphosphatidylethanolamine (DTP-DPPE) was prepared by the reaction of SPDP with DPPE as described previously [18]. Carboxy fluorescein (CF) came from Eastman Kodak (Rochester, NY). Fluorescein isothiocyanate (FITC)-conjugated BSA was prepared according to the procedure described previously [19]. 2.2. Animals Common carp were provided by courtesy of the Osaka Prefectural Freshwater Fish Experiment Station, Neyagawa, Japan. They were reared in 160l plastic aquaria that were filled with dechlorinated tap water (water temperature about 23 8C) and aerated. Fishes weighting about 350 g were used for the experiments. 2.3. Preparation of liposomes Liposomes entrapping BSA were prepared by the following procedure, as described previously [13,14]. DPPC (0.5 mmol), DPPS (0.5 mmol) and Chol (1 mmol), or DPPC (3.5 mmol) and Chol (1 mmol), each dissolved in organic solvent, were mixed in a conical flask. The lipids were dried on a rotary evaporator (Shibata Science Technology, Tokyo, Japan), and were left to stand for 30 min in a high vacuum in a desiccator. These liposome preparations were designated as PS- and PC-liposomes, respectively. After addition of 2 ml of BSA solution (10 mg/ ml) and incubation at room temperature for 90 min, the lipid film was dispersed by vigorous vortexing. Unencapsulated BSA was removed by centrifuging at
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14,000g for 20 min at 4 8C in 150 mM phosphatebuffered saline (PBS, pH 7.2). The resulting pellet of liposomes was suspended in saline and used in the immunization. CT-B-conjugated PS-liposomes containing BSA for immunization were prepared from the lipid mixture solution containing DPPC (0.3 mmol), DPPS (0.3 mmol), Chol (0.6 mmol) and DTP-DPPE (0.015 mmol) as described above. The amount of BSA trapped in liposomes was determined by the following method. Ninety microlitre of isopropyl alcohol was added to a 10 ml suspension of liposome-entrapped BSA (1:3 dilution in PBS), and vortex mixing was done. The protein concentration of the resulting solutions was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA), with bovine plasma gamma globulin used as a standard. PS- and PC-liposomes entrapping CF were prepared as described previously, and were used for studying the stability of liposomes [13]. 2.4. Immunization of carp Carps (n ¼ 3 or 4) were immunized orally with liposome-entrapped BSA or with BSA alone (100 mg protein/500 ml saline). Immunization was repeated two or three times at 2 week intervals. Sera, bile and intestinal tract were collected 2 weeks after the last immunization. Sera and bile obtained were used for antibody assay. Intestinal tract was used to prepare intestinal mucus. 2.5. Preparation of intestinal mucus samples Intestinal mucus was prepared as follows. Intestinal tract was cut with scissors to open the intestinal lumen. Then, intestinal mucus was collected from the intestine by gentle scraping with spatula and transferred to a tube. After addition of 500 ml of PBS, the tube was vortexed for 30 s, and centrifuged at 20,000g for 20 min at 4 8C. The supernatant was collected and assayed for antibody activity. 2.6. Preparation of anti-carp IgM antibody The procedure for purification of carp IgM from carp serum followed by the method described by Yamaguchi et al. [20].
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Antibody against the purified carp IgM was raised in a Japanese white rabbit. Briefly, 1 mg of purified carp IgM was mixed with Freund’s complete adjuvant (1:1) and injected subcutaneously into a rabbit on days 0 and 14. The animal was given booster with the purified carp IgM in PBS (no adjuvant) 2 weeks after the second immunization. At 1 week after the booster immunization, the animal was bled out and serum was collected. The IgG fraction was purified from the immune serum by precipitation with 50% ammonium sulfate, followed by affinity chromatography on AffiGel Protein A MAPS II (Bio-Rad Laboratories). The specificity of anti-carp IgM rabbit IgG was analyzed by enzyme-linked immunosorbent assay (ELISA). Anti-carp IgM rabbit IgG gave positive reaction only with carp IgM. 2.7. Antibody assay Specific antibodies against BSA were determined by ELISA. BSA (20 mg/ml) diluted with PBS was dispensed at 50 ml/well into 96-well microtiter plates (Iwaki Glass Co. Ltd., Funabashi, Japan). The plates were left overnight at 4 8C, and were washed five times with PBS containing 0.1% Tween 20 (washing solution). Next, 100 ml of PBS containing 0.5% horse serum (blocking solution) was added to each well. Following incubation for 60 min at 25 8C the wells were washed with washing solution, and 50 ml of test serum, intestinal mucus or bile, each diluted with blocking solution, was then added to each well. The plates were incubated for 90 min at 25 8C, washed with washing solution, and 50 ml of anti-carp IgM rabbit IgG (1:100 dilution in PBS) was added. After incubation for 90 min at 25 8C the wells were washed with washing solution and 50 ml of horseradish peroxidase-conjugated anti-rabbit IgG (1:8000 dilution in PBS) was added. The plates were then incubated for 90 min at 25 8C and washed with washing solution. Next, 100 ml of substrate solution (0.4 mg/ml o-phenylenediamine and 0.2 ml/ml of 30 % H2O2 in citrate buffer) was added and left to react for 10 min at 25 8C. The enzyme reaction was stopped by adding 100 ml of 2N H2SO4, and the absorbance at 492 nm was measured with a microplate reader (Model 450; Bio-Rad Laboratories). Antibody titers are represented as the reciprocal of
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endpoint dilution exhibiting A492 more than three times that of background. 2.8. Separation of cells Preparation of head kidney and spleen lymphocytes from carp was performed according to the method of Yamaguchi et al. [20] with slight modifications. Briefly, the head kidney and the spleen of the carp were all cut into small pieces with scissors in cold Eagle’s minimum essential medium (MEM, pH 7.4, Nissui Pharmaceutical Co., Tokyo, Japan). Cells were passed through a stainless steel mesh, and cell suspensions were carefully overlayered on a 1.060 and 1.070 discontinuous density Percoll solution (Amersham Pharmacia Biotechnology). After centrifuging at 1200g for 20 min at 20 8C, the cells above Percoll solution of 1.070 were collected and washed three times with MEM. More than 90% of cells were lymphocytes [20]. Cell viability, according to the trypan blue dye exclusion test, was more than 90%. 2.9. Flow cytometric analysis Head kidney and spleen lymphocytes (5 £ 106 cells) from carp were incubated with FITC-conjugated BSA (1:100 dilution in PBS) on ice for 90 min. Cells from non-immunized carp were used as control. The cells were then washed three times with PBS, and fixed with 1% paraformaldehyde solution. Fluorescence-positive cells were measured in a flow cytometer (CytoACE-150, Japan Spectroscopic Co., Tokyo, Japan) equipped with a 10 mW argon ion laser tuned at 488 nm. Fluorescence histograms were analyzed gating on both forward and rectangular light scatter characteristics to eliminate the influence of cell debris. Fluorescence intensity was represented in log units over a 4-log scale. All the data are based on 10,000 gated cell count with 256 channels.
3. Results 3.1. Serum antibody responses in carp orally immunized with BSA or liposome-entrapped BSA In the first experiment, we examined the stability of PS- and PC-liposomes by measuring the leakage of CF from liposomes. PS-liposomes were stable in 20% carp bile (leakage 13 ^ 10 %), whereas PC-liposomes were unstable (92 ^ 14 %) when incubated for 60 min at 37 8C. In the next experiment, serum antibody titers were measured in carp immunized orally with 100 mg of BSA entrapped in PS- or PC-liposomes. Control fish were administered the same dose of BSA alone. Antibody titers increased significantly 14 days after secondary immunization (28 days after primary immunization) when fish were immunized with BSA-PS-liposome as shown in Fig. 1. Fourteen days after the third immunization (42 days after primary immunization), the highest antibody responses were observed. By contrast, no obvious increase of antibody titer was noted in fish orally immunized with BSA entrapped in unstable PC-liposomes or immunized with BSA alone (Fig. 1). Antibody titers in fish immunized with BSA-PS-liposomes were significantly higher ( p , 0.05) after the third immunization when compared to other groups. 3.2. Antibody production in intestinal mucosa and in bile To study the presence of antigen-specific antibodies in intestinal mucosa and bile, carp were orally administered with BSA-PS-liposomes three times. Though the titers were relatively low, a clear increase of anti-BSA antibody titers was observed after the immunization (Fig. 2). In particular, the titers in bile of immunized fish were significantly higher ( p , 0.001) than those of non-immunized control.
2.10. Statistical analysis
3.3. Serum antibody responses in carp immunized orally with BSA entrapped in CT-B-conjugated liposomes
The student’s t-test was used to evaluate of the results.
For effective immune responses, efficient delivery of liposomes to the inductive sites of the intestinal
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Fig. 1. Serum antibody responses in carp immunized with BSA entrapped in liposomes. Three groups of four fish were immunized orally with BSA entrapped in stable PS-liposomes (B), unstable PC-liposomes (O), or with BSA alone (V), respectively. Antibody titers were measured 2 weeks after primary, secondary or third immunization, respectively. ( p ) p , 0.05 vs BSA alone and PC-liposomes. Data are expressed as mean values and standard errors of the mean from four individual experiments.
immune system is vital. Accordingly, liposomes coupled with a ligand for which a receptor is expressed on the mucosal epithelium are suitable for the enhancement of immune responses. More
recently, we demonstrated the presence of ganglioside GM1 on the epithelial cell surface of carp intestinal mucosa (Irie et al., submitted for publication). We therefore, used CT-B, which has a high affinity for
Fig. 2. Antibody production in intestinal mucosa (A) and bile (B). Four individual fish were immunized orally with BSA-PS-liposomes three times, and antibody titers were measured 2 weeks after the third immunization. Titers were significantly higher than in no treatment ((#) P , 0.1; ( p ) P , 0.001). Data are expressed as mean values and standard errors of the mean from four fish.
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Fig. 3. Serum antibody responses in carp immunized orally with BSA in CT-B-conjugated PS-liposomes. Three groups of three were immunized with BSA in CT-B-conjugated PS-liposomes (X), BSA-PS-liposomes (B), or BSA only (V). Antibody titers were measured 2 weeks after primary and secondary immunization, respectively. ( p ) p , 0.05 vs BSA alone. (#) p , 0.05 vs PS-liposomes. ($) p , 0.05 vs BSA alone. Results obtained from three individual tests are expressed as mean values and standard errors of the mean from three fish.
ganglioside GM1, as a ligand to carp intestinal mucosa. CT-B-conjugated PS-liposomes containing BSA were administered orally to carp. The antibody responses to BSA were evaluated 2 weeks after secondary immunization. As shown in Fig. 3, oral administration of CT-B-conjugated PS-liposomes containing BSA induced significant antibody responses. Antibody titers were significantly higher compared to those of fish immunized with BSA-PSliposomes or non-immunized control ( p , 0.05). This result indicates that the coupling of CT-B to PS-liposomes is more effective at inducing of the immune responses in carp. 3.4. Detection of BSA-specific antibody-secreting lymphocytes We attempted to detect lymphocytes producing BSA-specific antibody in the spleen and head kidney. Fish were administered three times with BSA (100 mg) incorporated in PS-liposomes. Table 1 shows the extent of lymphocytes secreting BSA-specific Ig. In immunized fish, BSA-specific
Ig-secreting lymphocytes were detected in 18.6 ^ 1.9 and 9.1 ^ 4.6% of spleen and head kidney lymphocytes, respectively ( p , 0.05 in comparison with non-immunized fish).
4. Discussion The mucosal surfaces of fish intestines are a major place of attack by many infectious agents. Therefore, to protect fish from enteropathogenic agents, the Table 1 Detection of BSA-specific antibody-secreting lymphocytes in spleen and head kidney Lymphocytesa
Immunizedb
Non-immunized
Spleen Head kidney
18.6 ^ 1.9c ( p , 0.05) 9.1 ^ 4.6 ( p , 0.05)
3.1 ^ 0.7 2.1 ^ 0.4
a
BSA-specific antibody-producing lymphocytes were detected by flow cytometry by staining the cells with FITC-conjugated BSA. b Fish were immunized orally with 100 mg of BSA-PS-liposomes three times at 2 week intervals. c % Fluorescence positive cells ^ SD from four fish.
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induction of mucosal immunity, and especially an intestinal immune response, is very important. Development of oral vaccines is crucial since the oral route of immunization can induce not only mucosal but also systemic immune responses [2]. However, the problem arises of antigen degradation by gastric acidity and proteolytic enzymes in the intestinal lumen, and extremely large doses are required to achieve satisfactory immune responses. New adjuvant and carriers are therefore, essential. We have previously reported that PS-liposomes act as an effective oral delivery vehicle for inducing mucosal immune responses, and also act as an adjuvant for antibody production on the intestinal mucosa [13,14]. However, we know of no report on antigen trapped in liposomes as an oral vaccine in fish. In the present study, we therefore, studied the value of PS-liposomes as carriers for oral vaccines in carp. We found that PS-liposomes showed stability to carp bile. Furthermore, when BSA entrapped in PSliposomes was administered orally to carp, significant antibody responses were induced in serum. On the other hand, no obvious antibody responses to BSA were induced when BSA alone or entrapped in unstable PC-liposomes was administered (Fig. 1). These results suggest that PS-liposomes are able to deliver antigens to immune responsive areas of the intestinal tract without leakage or degradation of incorporated antigens following oral administration, and then induce immune responses against antigens incorporated into PS-liposomes. We conclude that PSliposomes have great potential as carriers for oral vaccines in fish. It is known that the hindgut of fish adsorbs proteins and particulate antigen from the lumen [2,6]. Although fish lack Peyer’s patches and antigentransporting M cells, enterocytes in the hindgut show an antigen-transporting ability, and many macrophages and lymphoid cells are present in gut mucosa [21]. Antigens reaching the posterior segment of the gut are transferred either to intracellular spaces or to macrophages or enterocytes [5,6]. Azad et al. [6] observed that biofilm antigens of Aeromonas hydrophila administered orally to carp appeared in melanomacrophage centers in the kidney and spleen. In the present study, specific antibody-secreting cells were detected in lymphocyte preparations separated from the head kidney and spleen of carp that had been
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immunized with BSA entrapped in PS-liposomes (Table 1). Since liposomes are avidly taken up by phagocytic cells such as macrophages, it is reasonable to suppose that uptake of BSA-PS-liposomes by macrophages acting as antigen-presenting cells in the posterior part of the gut induces to the stimulation of the immunocytes associated with mucosal membranes, leading to systemic and intestinal antibody responses. In this study, the percentage of BSA-specific antibody-secreting cells in spleen and head kidney lymphocytes was significantly higher in the immunized carp than in the non-immunized carp (Table 1). This result suggests the induction of lymphocytes producing BSA-specific antibody in the spleen and head kidney following oral immunization. However, it is possible that FITC-conjugated BSA non-specifically binds to other cells in separated lymphocytes used in flow cytometric analysis. If this were so, it would be expected that a number of fluorescencepositive cells were detected in the lymphocytes from non-immunized carp. However, the experimental result indicated that the number of fluorescencepositive cells in both spleen and head kidney lymphocytes was significantly more in the immunized carp than in the non-immunized carp (Table 1). It has also been reported that fish lymphocytes have an Igbinding capacity [22]. Although, thus, it cannot be excluded that, in immunized fish, immune complexes are formed and bound to lymphocytes, it is not too far from the truth to say that many of the fluorescencepositive cells would be BSA-specific antibodysecreting lymphocytes. However, further studies are required to fully clarify the lymphocytes having antiBSA antibody at their surface. To promote uptake of antigens-containing liposomes by immunocompetent phagocytic cells in the intestinal tract, coupling to a ligand which is recognized by a receptor on the surface of the epithelial cells in the intestine should be effective. Recently we demonstrated the presence of ganglioside GM1, which serves as a receptor of CT-B in carp intestinal mucosa [Irie et al., submitted for publication]. Accordingly, in the present study, CTB having the ganglioside GM1 binding property was coupled to the outer surface of PS-liposomes in order to target delivery of incorporated antigens to the carp intestinal epithelium. As expected, oral
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administration of BSA-containing CT-B-conjugated PS-liposomes induced a significant higher antibody response compared to responses cooked by BSA entrapped in PS-liposomes (Fig. 3). This suggests enhanced binding by an interaction between liposomal CT-B and ganglioside GM1 on the epithelial cell surface of the intestinal mucosa. The delivery of antigen in combination with CT-B has been shown to increase binding of CT-Bantigen complex to the GM1 gangliosides, increasing uptake of the antigen by M cells in humans [23]. CT-B has also been used as an adjuvant for the oral delivery of antigens in tilapia (Oreochromis mossambicus ) [4]. As stated, an increase in the permeability of the intestinal epithelium facilitates binding and uptake of antigens by the epithelial cells. Davidson et al. [3] detected antigen-specific antibody-secreting cells in the head kidney and intestinal mucosa of rainbow trout after peroral intubation of A. salmonicida. Joosten et al. [8] also identified specific plasma cells in the gut of carp immunized orally with Vibrio anguillarum. In our study, BSA-specific antibodies were detected in the intestinal mucosa and bile of fish immunized orally with BSA entrapped in liposomes, but not in fish immunized with BSA alone (Fig. 2), suggesting that specific antibodies against BSA might be also produced by lymphocytes located in the intestinal epithelium, and that the intestinal epithelium is an active site of antibody secretion. However, further research is necessary to clarify these points. In conclusion, oral administration of liposomeentrapped BSA antigens effectively induced both serum and intestinal antibody responses directed against BSA. It follows that PS-liposomes have great potential in developing oral vaccine for fish. The induction of protective immune responses by administration of bacterial antigens entrapped in PSliposomes is now under study in freshwater and seawater fish, and could help to prevent infectious diseases in fish culture.
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