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Graphical Abstract
International Immunopharmacology xxx (2011) xxx xxxAntigen in chitosan coated liposomes enhances immune responses throughparenteral immunization
T. Behera a, P. Swain a,, S.K. Sahoo b
a Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar-751 002, Orissa, Indiab Nanomedicine Laboratory, Institute of Life Sciences, Bhubaneswar-751023, India
12
13Antigen incorporated in chitosan coated egg yolk lecithin based liposomes through parenteral administration
14enhances both adaptive and innate immune response in two animal models such as fish and rabbit.
International Immunopharmacology xxx (2011) xxx
INTIMP-02249; No of Pages 1
1567-5769/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2011.02.002
Contents lists available at ScienceDirect
International Immunopharmacology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
Please cite this article as: Behera T, et al, Antigen in chitosan coated liposomes enhances immune responses through parenteralimmunization, Int Immunopharmacol (2011), doi:10.1016/j.intimp.2011.02.002
http://dx.doi.org/10.1016/j.intimp.2011.02.002http://www.sciencedirect.com/science/journal/15675769http://dx.doi.org/10.1016/j.intimp.2011.02.002http://dx.doi.org/10.1016/j.intimp.2011.02.002http://www.sciencedirect.com/science/journal/15675769http://dx.doi.org/10.1016/j.intimp.2011.02.002 -
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1 Antigen in chitosan coated liposomes enhances immune responses through
2 parenteral immunization
3 T. Behera a, P. Swain a,, S.K. Sahoo b
4a Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar-751 002, Orissa, India
5b Nanomedicine Laboratory, Institute of Life Sciences, Bhubaneswar-751023, India
6
7
a b s t r a c ta r t i c l e i n f o
8 Article history:
9 Received 9 December 201010 Received in revised form 22 January 2011
11 Accepted 1 February 2011
12 Available online xxxx
131415
16 Keywords:
17 ECPs
18 Chitosan
19 Liposomes
20 Lecithin
21 Parenteral immunization
22To overcome the limitations of the use of conventional liposomes chitosan coated egg yolk lecithin based
23liposomes were used as antigen carrier through parenteral administration in two animal models such as fish
24and rabbit. Extracellular proteins (ECPs) antigen ofAeromonas hydrophila encapsulated in liposomes became
25more stable and induced better immune response. It enhanced both adaptive and innate immune responses
26than other preparations both at 21, 42 and 63 days post-immunization and suggested to be a better antigen
27delivery system.
28 2011 Elsevier B.V. All rights reserved.
2930
31
32
33 1. Introduction
34 Liposomes have represented a milestone in the field of innovative
35 vaccine/drug delivery systems for a number of years. Allison and
36 Gregoriadis have first described the immunoadjuvant potential of
37 liposomes for protein and peptide antigens [1], thereafter many
38 research groups have investigated its immunostimulatory adjuvant
39 properties, for delivery of chemotherapeutic agents, vaccines/antigens/
40 proteins, and radiopharmaceuticals for diagnostic imaging, nucleic acid-
41 based medicines for gene therapy and many other applications [26]. In
42 recent years, the use of liposomes as potential carrier for vaccines has
43 been extensively explored [7]. The immunological adjuvant property of
44 liposome extends to a large area of antigens from diverse sources such as
45 virus, bacteria, protozoa, fungi etc [8]. Liposomes carrying antigens
46 particularly for viral (koi herpes virus, measles virus through orally and
47 intranasally respectively), bacterial antigens (lipopolysaccharides
48 through orally in rainbow trout) and other antigenic preparations
49 such as rat transplantation antigen have been successfully used to
50 produce humoral or cellular immunity in both higher and lower
51 vertebrates [912].
52 However,liposomes havesome limitations.First, theygenerally show
53 a short circulation half-life, are prone to adhere to each other and fuse to
54 form larger vesicles in suspension, which may result in inclusion leakage
55[13
15]. Several factors such as stability, size, phospholipids composition,56methods of preparations, surface characteristics of liposomes along with
57antigen characteristics, route of administration and animal model are
58believed to have a major impact on immunoadjuvant potential of
59liposomes [1618]. In this context, one of the major phospholipids
60compositions is egg yolk lecithin, which is cost effective, no side effect,
61easily form liposomes by various methods, and also assist protein
62antigens to increasean immuneresponse sixtimes more potentthan any
63the other adjuvants [19,20].
64Chitosan, which is a biocompatible, biodegradable, and nontoxic
65cationic polysaccharide [21] already used as a vaccine/protein carrier
66[22] in several animal models has been used to improve the efficiency
67of conventional liposomes [2325]. Appropriate combining of the
68liposomal and chitosan characteristics has produced the liposomes
69more stable, prolonged specific, controlled and targeted drug
70deliveries [23,2630]. Chitosan as a coating agent for liposomal is
71restricted only to nasal administration in mice and rabbit but the
72formulation, immunization condition and results are not so clear
73which interfereits expansionto human use [31,32]. Moreover,there is
74no such report on parenteral administration this chitosan coated egg
75yolk lecithin based liposomes as antigen carrier in any species.
76Therefore, the present study was undertaken to evaluate the
77potential use of chitosan coated lecithin based liposome through
78parenteral route as antigen carrier by taking a bacterial antigen
79(extracellular proteins, which acts as a major virulent factor of
80Aeromonas hydrophila [33]) in both the model animals suchas fishand
81rabbit.
International Immunopharmacology xxx (2011) xxxxxx
Corresponding author. Tel.: +91 943 723 1099; fax: +91 674 2465407.
E-mail address: [email protected] (P. Swain).
INTIMP-02249; No of Pages 8
1567-5769/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2011.02.002
Contents lists available at ScienceDirect
International Immunopharmacology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
Please cite this article as: Behera T, et al, Antigen in chitosan coated liposomes enhances immune responses through parenteralimmunization, Int Immunopharmacol (2011), doi:10.1016/j.intimp.2011.02.002
http://dx.doi.org/10.1016/j.intimp.2011.02.002http://dx.doi.org/10.1016/j.intimp.2011.02.002http://dx.doi.org/10.1016/j.intimp.2011.02.002mailto:[email protected]://dx.doi.org/10.1016/j.intimp.2011.02.002http://www.sciencedirect.com/science/journal/15675769http://dx.doi.org/10.1016/j.intimp.2011.02.002http://dx.doi.org/10.1016/j.intimp.2011.02.002http://www.sciencedirect.com/science/journal/15675769http://dx.doi.org/10.1016/j.intimp.2011.02.002mailto:[email protected]://dx.doi.org/10.1016/j.intimp.2011.02.002 -
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2. Materials and methods
2.1. Bacteria
The bacteria Aeromonas hydrophilic is an opportunistic pathogen,
ubiquitous in nature and is known to cause infection in many animal
populations mostly in fish, reptiles, amphibians and human [34,35].
A. hydrophila strain (Ahv) originally isolated from Carassius auratus
species showing septicemia conditions was preserved in a lyophilizedcondition in this laboratory.
2.2. Extraction of extracellular proteins (ECPs)
Bacterial extracellular proteins (ECPs) were recovered by a modifi-
cation of the methods of Suprapto et al. and Mekuchi et al. [36,37]. An
isolate ofA. hydrophilawascultured on nutrient agar mediumat 25 C for
48 h, and these bacterial cells were harvested by rinsing with sterile
phosphate buffered saline (PBS, pH=7.2), and the bacterial colonies
agitated to remove them from the agar. The bacterial suspension was
centrifuged at 12,000g for 20 min, 10 C to remove the cells. The
supernatant wasfiltered through 0.22 m pore membrane, andstored at
80 C until use. The protein concentration of each ECP sample was
determined using Genei protein estimation kit (Bangalore genei,
India) by the BCA method.
2.3. Preparation of liposome encapsulating ECPs by proliposome method
Liposomal microcapsules were prepared according to the method
described byPerrett etal. andRengel etal. [38,39] withlittle modification.
100 mg of purified egg yolk lecithin (iodine value75%, Himedia, India)
and ethanol (200 l) was stirred and heated to 60 C for 5 min. After
cooling to room temperature,500 l of aqueous solution containing ECPs
(25 mg) was added. The proliposome mixture was converted to a
liposomesuspensionby dropwise additionof 20 ml of 10 mM phosphate
buffer (pH 7.0) under the controlled drop rate (1.5 ml/min) and stirred
for30 min. Theprepared liposome suspension was left to hydrate at 2 C.
Proliposome-capsules were obtained by ultracentrifugation at 80,000g
for 45 min at 4 C.
2.3.1. Chitosan coating of ECP encapsulated proliposome microcapsules
Each chitosan was dissolved in 50 mM acetic acid solution, and then
the pH of the solution was adjusted to 5.0 by the addition of 0.5 M Tris
solution. Liposome solution (1 ml) was drop wise added to the chitosan
solution (4 ml). Final concentration of chitosan was set at 0.3%. After
stirring of the mixed solution for 2 h, the solution was stored in a
refrigerator overnight. Proliposome-capsules with chitosan coating
were obtained by ultracentrifugation at 80,000gfor 45 min at 4 C.
2.3.2. Encapsulation efficiency measurement
The antigen loaded in liposomes was determined from the total
amount of antigen added in the formulation and the antigen amount
thatwas notencapsulatedleftin supernatant.For this, theconcentrationof antigen in the supernatant was analyzed using Genei protein
estimation kit (Bangalore genei,India) by the BCA method to determine
free antigen concentration and encapsulation efficiency of antigen was
calculated using the following formula:
Encapsulation efficiency % = fTotal antigen mg added
antigen in supernatant of liposome suspensionmg
= Totalantigenmg addedg 100:
0
2.3.3. Transmission electron microscopy (TEM)
The samples were prepared by coating a copper grid (200 mesh
covered with Formvar/carbon) with a thin layer of dilute liposome
135suspension. After negative staining with 2% (w/v) phosphotungstic
136acid for 2 min, the copper grid was then dried at room temperature
137before measurement. Liposomes were investigated using transmis-
138sion electron microscopy (TEM, Zeiss EM 10) at an accelerating
139voltage of 300 kV.
1402.3.4. Zeta potential analysis
141Dynamic light scattering (DLS) was used to measure the hydrody-
142namic diameter along with size distribution (polydispersity index, PDI).143The DLS analysis was performed using a Zetasizer Nano ZS (Malvern
144Instruments, Malvern, UK). The DLS measurements were done with a
145wavelength of 532 nm at 25 C with an angle detection of 90.
146Approximately, 1 mg of the lyophilized samples was dissolved in 1 ml
147MilliQwaterand 100 l of these solutionswerefurtherdiluted to 1 ml for
148the measurements of particle size. All measurementswere performed in
149triplicate.
1502.4. Emulsification with FIA
151ECPs (40 g and 100 g for fish and rabbit respectively) in 1 ml PBS
152was emulsified with an equal volumeof Freund's incomplete adjuvant
153(IFA) and stored at 4 C until further use.
1542.5. Immunization protocol forfish
155Indian major carp, Labeo rohita juveniles of average weight ranging
156from 30 to 40 g were acclimatized in the wet laboratory of Fish Health
157Management Division of Central Institute of Freshwater Aquaculture
158(CIFA),Kausalyaganga, India,15 days prior to thestart of theexperiment.
159Ten juveniles per tank were maintained in 1000 l cemented tanks of the
160wet laboratory. The fish were fed with artificial carp diet with constant
161aeration and daily one-third water exchange. Water temperature of the
162experimental tanks was 27 C to 30 C. Fish were divided into 4 groups,
16310 fish in each group were intraperitonially injected separately with
1640.1 ml of different liposome preparations such as ECP encapsulated
165liposomes, ECP encapsulated chitosan coated liposomes and IFA-ECPs at
16640 g of ECPs, whereas control group was injected withPBS ingroup 1 to1674 respectively. The fish of all the treated groups including the control
168group were bled at an interval of 3-weeks up to 63 days of post-
169immunization to study various immune parameters.
1702.5.1. Preparation of anti-rohu-globulin rabbit serum
171The rabbit anti-rohu globulin was prepared as per the standard
172method Swain et al. [40] using sera obtained from healthy adult rohu
173of average weight 250300 g. Briefly, serum was collected from
174healthy rohu and pooled to 1015 ml. An equal volume of saturated
175ammonium sulfate solution was mixed with the pooled sera drop by
176drop and then placed on a magnetic stirrer overnight at 4 C. The
177sample mixture was centrifuged at 10,000g for 10 min at 4 C and
178the precipitate was dissolved with 5 ml carbonate bicarbonate buffer
179(pH 9.6). The sample was then centrifuged at 10,000gfor 10 min at1804 C. The pellet was collected and the volume was made to 2 ml with
181carbonate bicarbonate buffer (pH 9.6). The globulin solution was
182dialyzed using dialysis membrane (Snakeskin, Pierce Chemical
183Company, USA) with 7000 molecular weight cut off against PBS (pH
1847.2) for 72 h at 4 C, after which the globulin was collected. The anti-
185rohu globulin sera were raised in a New Zealand white rabbit as per
186the method of Lund et al. [41].
1872.6. Immunization protocol for rabbit
188White albino rabbits weighing 22.5 kg (3 animals per group)
189were intramuscularly immunized with 0.51 ml of different prepara-
190tions at 100 g of ECPs. Three different preparations are ECP
191encapsulated liposomes, ECP encapsulated chitosan coated liposomes
2 T. Behera et al. / International Immunopharmacology xxx (2011) xxxxxx
Please cite this article as: Behera T, et al, Antigen in chitosan coated liposomes enhances immune responses through parenteralimmunization, Int Immunopharmacol (2011), doi:10.1016/j.intimp.2011.02.002
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192 and FIA-ECPs. Each animal was bled at an interval of 3 weeks (up to
193 63 days post immunization).
194 2.7. Non-specific immune parameters
195 2.7.1. Myeloperoxidase activity
196 For determination of myeloperoxidase activity, 15 l of serum was
197 diluted in 135 l of Hank's balanced salt solution (Ca2+, Mg2+ free)
198
and then 50 l [of 20 mM, TMB (3, 30,5,50-tetra methyl benzidine)199 and 5 mM H2O2] were added. The reaction was stopped after 2 min by
200 adding 50 l of 4 M sulfuric acid and the optical density (OD) wasread
201 at 450 nm using UVVIS spectrophotometer, Thermo Spectronic, U.K.
202 [42].
203 2.7.2. Respiratory burst activity
204 The respiratory burst activity was measured by the reduction of
205 nitro blue tetrazolium (NBT) by intracellular superoxide radicals [43].
206 Briefly, 50 l of heparinised blood from each group was mixed with
207 50 l of 0.2% NBT (Sigma, USA) solution for 30 min at 25 C. After
208 incubation, 50 l from the above mixture was added with 1 ml of N, N
209 diethylmethyl formamide (Qualigens, India) and then centrifuged at
210 6000 g for 5 min. The OD of the supernatant was measured at
211 540 nm.
212 2.7.3. Serum antiprotease activity
213 Total antiprotease activity was determined as indicated by the
214 capacity of serum to inhibit trypsin activity [44,45]. Briefly, 20 l of
215 serum was incubated with 20 l of standard trypsin solution (1000
216 2000 BAEE, 5 mg ml/1; Sigma-Aldrich T-7409) for 10 min at 22 C in
217 Eppendorf tubes. Then, 200 l of 0.1 M PBS (pH 7.0) and 250 l of 2%
218 (w/v) azocasein (Sigma-Aldrich) in PBS were added, and incubated
219 for 1 h at 22 C. The reaction was stopped by the addition of 500 l of
220 10% (v/v) trichloroacetic acid (TCA, Sigma-Aldrich), incubated for
221 30 min at 22 C, and then centrifuged at 6000g for 5 min. The
222 supernatants (100 l) were transferred to a 96-well microtitre plate
223 (Nalge Nunc) containing 100 l well1 of 1 N sodium hydroxide
224 (NaOH, BDH). The OD was read at 450 nm using a plate reader. For a
225 positive (100%) control, buffer replaced the serum, and for a negative226 control, buffer replaced both serum and trypsin. The inhibitory ability
227 of antiprotease was expressed in terms of percentage trypsin
228 inhibition using following formula:
% Trypsin inhibition = Trypsin ODSample ODf g = Trypsin OD 100:
229230
231 2.7.4. Bacterial agglutination activity
232 The agglutination test was conducted in U shaped microtiter
233 plates. Two-fold serial dilution of 25 l fish serum was made with an
234 equal volume of PBS in each well, to which 25 l of formalin-killed A.
235 hydrophila (107 cells ml1) suspension was added. The plates were
236 incubated overnight at room temperature. The titer was calculated as237 the reciprocal of the highest dilution of serum showing complete
238 agglutination of the bacterial cells.
239 2.7.5. Hemaggultination activity
240 The hemagglutination activity of serum samples was carried out
241 using a standardized method Blazer et al. [46]. This assay was done in
242 U-shaped microtitre plates by serial two-fold dilution of 50 l serum
243 (inactivated at 45 C for 30 min) with PBS (pH 7.2). Then 50 l of
244 freshly prepared 1% New Zealand white rabbit red blood cell (RBC)
245 suspension was added to each well. The plates were kept at room
246 temperature (2830 C) for 2 h or over night at 4 C, in case if
247 agglutination was not visible within 2 h. The titre was calculated as
248 the reciprocal of the highest dilution of serum showing complete
249 agglutination of RBC.
2502.7.6. Hemolytic activity
251The hemolytic titre of serum was determined in a similar manner
252as described for HA titre [46] by using fresh sera from all the groups.
253Titre was expressed as the reciprocal of the highest dilution of serum
254showing complete hemolysis of the rabbit RBC.
2552.8. Triple antibody indirect enzyme linked immunosorbent assays (ELISA)
256offish serum
257The triple antibody indirect ELISA was conducted as per the method
258of Swain et al. [40] with slight modifications using 96 well microtitre
259polystyrene plates (Nunc, Denmark). The wells were separately coated
260with 50 l of purified ECPs from A. hydrophila (Ahv) (12 g/well)
261diluted in carbonatebicarbonate buffer (pH 9.6) overnight at 4 C. The
262plates were then washed in PBS containing Tween-20 (PBS-T, pH 7.2)
263and blocked with 100 l of 3% skim milk powder for 2 h at 37 C. The
264wells were further washed in PBS-T. The fish sera raised against several
265antigens was twofold diluted after initial dilution of 1:10 with PBS (pH
2667.2) asfirst antibody and added to homologous antigen-coated wells in
267duplicate per serum dilution. The plates were incubated at 37 C for
26845 min and washed thrice in PBS-T. Rabbit anti-rohu sera (the second
269antibody) at a dilution of 1:20 wasadded to each well and incubated at
27037 C for45 min. Then theanti-rabbit-HRPO conjugatedgoat serum (the
271third antibody) was added and incubated for 45 min. The wells were
272then thoroughly washed and added with 50 ml of substrate solution
273(5 mg of O-phenylene diamine tetra hydrochloride and 10 ml of H2O2274(38%, v/v) in 5 ml of acetate buffer, pH 5.0). Theplateswereincubatedat
27537 C for 5 min in a dark chamber and finally O.D was recorded at 450/
276655 nm in a microplate reader (BIO-RAD, USA). The antibody activity
277was expressed in terms of O.D value after subtracting the values
278obtained by unimmunized healthy sera.
2792.9. Challenge study in fish
280For the challenge, 24 h culture of virulent A. hydrophila (challenge
281strain: 28v/08) was used. Two days after the last bleeding, fish from
282each group (containing 20 fish in each group) were injected intra-
283peritoneally with 0.1 ml (109 CFU ml1) of virulent A. hydrophila.284Clinical signs or mortalities were monitored for 7 days. The cause of
285death and pathological signs were verified by re-isolation of bacteria
286from samples of freshly dead/infected fish. Relative percent survival
287(RPS) was calculated according to the formula: RPS= [1% of
288mortality in immunized group/% of mortality in control group]100.
2892.10. Indirect enzyme linked immunosorbent assays of rabbit serum
290ELISA was conducted as per the method of Swain et al. [40] with
291slight modifications using 96 well microtitre polystyrene plates
292(Nunc, Denmark). The wells were separately coated with 50 l of
293purified ECPs from A. hydrophila (Ahv) (12 g/well) diluted in
294carbonatebicarbonate buffer (pH 9.6) overnight at 4 C. The plates
295were then washed in PBS containing Tween-20 (PBS-T, pH 7.2) and296blocked with 100 l of 3% skim milk powder for 2 h at 37 C. The wells
297were further washed in PBS-T. The rabbit sera raised against different
298antigenic preparations was twofold diluted after initial dilution of
2991:10 with PBS (pH 7.2) added to homologous antigen-coated wells in
300duplicate per serum dilution. The plates were incubated at 37 C for
30145 min and washed thrice in PBS-T. Then the anti-rabbit-HRPO
302conjugated goat serum wasaddedand incubatedfor 45 min. The wells
303were then thoroughly washed and added with 50 l of substrate
304solution (5 mg of O-phenylene diamine tetra hydrochloride and 10 ml
305of H2O2 (38%, v/v) in 5 ml of acetate buffer, pH 5.0). The plates were
306incubated at 37 C for 5 min in a dark chamber and finally O.D was
307recorded at 450/655 nm in a microplate reader (BIO-RAD, USA). The
308antibody activity was expressed in terms of O.D value after
309subtracting the values obtained by unimmunized healthy sera.
3T. Behera et al. / International Immunopharmacology xxx (2011) xxxxxx
Please cite this article as: Behera T, et al, Antigen in chitosan coated liposomes enhances immune responses through parenteralimmunization, Int Immunopharmacol (2011), doi:10.1016/j.intimp.2011.02.002
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2.11. Statistical analysis
The statistical analysis system (SAS) software (version 6.12) was
used to analyze all the data [47]. One-way analysis of variance
313followed by Duncan's multiple range tests (DMRT) was done to
314compare the variations in various immune parameters at significance
315level of difference (pb0.05) in different injected groups. The mean
316standard errors(SE) of assayed parameters were calculated for each
317group offish.
3183. Results
3193.1. Characteristics of liposomes
320Liposomes prepared by the proliposome method were morpho-
321logically multilamellar vesicles as revealed by TEM study (Fig. 1(A)
322and (B)). Mean diameter of liposomes was determined to be 0.5 m
323and 1.2 m for non-coated (Fig. 1B) and chitosan coated liposomes324(Fig. 1A), respectively with significant difference in size between
325coated and non-coated liposomes. The encapsulation efficiency of
326ECPs in liposomes was80% and 70%in non-coated andchitosancoated
327liposomes, respectively and the liposomal Zeta potential became more
328positive by coating with chitosan.
Fig. 1. Transmission electron micrograph of (A) ECP encapsulated liposomes
(bar=0.5 m) and (B) ECP encapsulated chitosan coated liposomes (bar=1.2 m).
Fig. 2. The mean OD values [Standard Error (S.E.)] of specific antibody level in
different treated groups of Labeo rohita detected through indirect ELISA at 21, 42 and
63 days post-immunization. Mean values bearing same superscript are not statistically
significant (pN
0.05) at 21, 42 and 63 days post-immunization.
Fig. 3. The mean OD values (S.E.) of specific antibody level in differenttreated groups
of rabbit detected through indirect ELISA at 21, 42 and 63 days post-immunization.
Meanvalues bearingsame superscript are notstatistically significant(pN0.05) at21,42
and 63 days post-immunization.
Fig. 4. TheMyeloperoxidase activity of seraof Labeo rohita in differenttreatedgroups at
21 and 42 days post-immunization (values are mean OD values S.E). Mean values
bearing same superscript are not statistically significant (pN0.05) at 21 and 42 days
post-immunization.
4 T. Behera et al. / International Immunopharmacology xxx (2011) xxxxxx
Please cite this article as: Behera T, et al, Antigen in chitosan coated liposomes enhances immune responses through parenteralimmunization, Int Immunopharmacol (2011), doi:10.1016/j.intimp.2011.02.002
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329 On evaluation of physical stability of liposomes that were stored
330 for 2 months in 4 C, more than 70% and 50% of ECPs remained
331
encapsulated during this time in coated and non-coated liposomes,332 respectively.
333 3.2. Serum antibody response in fish
334 Antibody response increased significantly in all the treated groups
335 than the control (Fig. 2). The coated-liposome showed significantly
336 higher antibody response (Pb0.05) than the liposomes and FIA-ECP
337 treated groups and there was no significant difference among them in
338 21, 42 and 63 days post immunization. In case of coated liposome
339 significantly higher antibody response was detected in 63 and 42 d.p.i
340 than the 21 d.p.i in contrast no significant difference was shown in
341 case of other treatments (Fig. 2). Whereas, at 63 d.p.i the FIA-ECP
342 treated group showed significantly (Pb0.05) lower antibody response
343 than the other treated groups.
344 3.3. Serum antibody response in rabbit
345 Antibody response increased significantly in all the treated groups
346 than the control (Fig. 3) where as the chitosan coated liposome
347 showed comparatively higher antibody response than the liposomes
348 and FIA-ECP treated groups and there was no significant among them,
349 excluding control in 21, 42 and 63 d.p.i. The antibody response was
350 also significantly higher in 63 and 42 d. p. i than the 21 d.p.i only in
351case of coated liposomes besides other treatments. In addition the
352FIA-ECP treated group showed significantly lower antibody response
353at 63 d.p.i than other treated groups.
3543.4. Non-specific immune parameters in both fish and rabbit
355The non-specific immuneparametersof differentantigenpreparations
356(Chitosan coated liposome, liposomes and FIA-ECPs), at 21 and 42 days
357post-immunization are presented in Figs. 49 and Table 1 offish and
358rabbit,respectively. All these parameters i.e. myeloperoxidase, respiratory
359burst, hemagglutination, hemolytic, antiprotease activity and bacterial
360agglutinationtitre were significantlyhigher (pb0.05) in all treatedgroups
361than the control. The coated liposome showed significantly higher
362(pb0.05) response than other groups (FIA-ECPs, liposomes). However,
363theseparametersdid notvary muchin alltreated groups at21 and 42 days
364post-immunization. The non-specific immune responsesalso persistedup365to 63 d.p.i in all treated groups than the control group.
3663.5. Challenge study in fish
367In challenge study, no clinical sign and symptom of A. hydrophila
368infection was recorded in all the treated groups where as with typical
369symptoms and 90% mortality was recorded unimmunized control
370group during 15 days post-challenge (Fig. 10).
Fig. 5. The respiratory burst activity (measured by NBT assays) of blood of Labeo rohita
in different treated groups at 21 and 42 days post-immunization (values are mean OD
valuesS.E). Mean values bearing same superscript are not statistically significant
(pN0.05) at 21 and 42 days post-immunization.
a
b70
80
90
bb
c
30
40
50
60
21 days
42 days
0
10
20
ECPs-Chitosan
coated liposomes
ECPs-liposomes FIA-ECPs Control
%o
finhibitio
n
Treatments
Fig. 6. Serumtotal antiprotease activity of serain differenttreated groups ofLabeorohita at
21 and 42 days post-immunization (values are mean % of inhibition valuesS.E). Mean
valuesbearingsame superscriptare notstatisticallysignificant (pN0.05)at 21and42 days
post-immunization.
Fig.7. Thehemagglutinatingactivity of seraofLabeo rohita in differenttreated groups at
21 and 42 days post-immunization (values are mean log2 titre valuesS.E). Mean
values bearing same superscript are not statistically significant (pN0.05) at 21 and
42 days post-immunization.
a
b
b8
9
b
c4
5
6
7
21 days
42 days
0
1
2
3
Ttitervalueinl
og2
ECPs-Chitosancoated
liposomes
ECPs- liposomes FIA-ECPs Control
Treatments
Fig. 8. The heamolysin titre of sera ofLabeo rohita in different treated groups at 21 and
42 days post-immunization (values are mean log2 titre valuesS.E). Mean values
bearing same superscript are not statistically significant (pN0.05) at 21 and 42 days
post-immunization.
5T. Behera et al. / International Immunopharmacology xxx (2011) xxxxxx
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4. Discussions
Liposomes have been receiving a lot of interest in the field of
vaccine delivery but due to several limitations its use is restricted for
vaccine/protein/drug delivery in several species. One of the major
limiting factors is the stability of liposomes in various adverse
conditions. To overcome this various materials are used to coat the
liposome microcapsules such as silica and chitosan [48,49]. Moreover,
the chitosan have antibacterial, immune stimulatory effect and most
effectiveto increase theefficiency of conventional liposomes [49] than
the silica which have some toxic side effect in fish [50]. In our study,
we used chitosan to coat the liposome microcapsules used, the results
obtained from parenteral immunization studies in fish showed that,
chitosan coated liposome induced both specific and non-specific
immune responses in fish, in comparison to other antigen prepara-
tions such as liposomes alone and FIA. It was also found that antibody
response gradually increased and showed significantly more
responses in both 63 and 42 d.p.i, than that of the 21 d.p.i only incase of chitosan coated liposomes. This indicated that chitosan
retarded the instant release of encapsulated antigens and helped in
controlled release [51], ability to forman antigen depot [52] andmade
the liposomes more stable [53]. Upon chitosan coating liposomes
became cationic which deliver their content more efficiently to
macrophages, stimulating antigen presenting cells [5457]. Liposome
by themselves acted as immunostimulatory adjuvants and perhaps
activated the antigen presenting cells [5658]. In addition, Chitosan
also act as a immunostimulant, enhances innate immune parameters
through versatile route of administration in fish as well as in other
higher vertebrates [59,60]. So both the combination of liposome and
399chitosan in this preparation increase both specific and non-specific
400immune parameters in fish. Although comparable non specific and
401specific immune responses were found in case of liposomes and FIA
402treated groups, the toxic side effects and is not readily biodegradable
403limited the clinical use of FIA [6163]. In addition, the present study
404shows that theantibodyresponsedid notpersistsup to 63 d.p.i in case
405of FIA adjuvant as compared to liposomes and chitosan coated
406liposomes. Upon challenge, all treated groups were protected from
407A. hydrophila infections.
408In mammal, liposomal encapsulated antigens enhance antigen
409presentation and uptake by macrophages, reticuloendothelial system
410compared to free antigens resulting T-cell mediated responses [6467].
411Antigen presentation system in fish is believed to be similar to those in412mammals, although it hasnot been extensively studied [68]. Similarlyin
413the present immunization studies in rabbit we also found comparable
414results with that offish in response to both specific and non-specific
415immune responses and in this case also chitosan coated liposome was
416more stable and showed significantly higher immune response.
417However, some contrasting results are found in case of chitosan coated
418liposomes through nasal and mucosal routs [69,70]. This indicated that
419route of administration and lipid composition of liposome affected the
420immune response better.
421The data presented here suggested that chitosan coated lecithin
422based liposomes have a promising effect than other antigenic
a
b7
8
b
c
2
3
4
5
6
Titervaluelog2
21 days
42 days
0
1
2
ECPs-Chitosancoated liposomes
ECPs-li posomes FIA-ECPs Co ntrol
Treatments
Fig. 9. The bacterial agglutination activity of sera in different treated groups of Labeo
rohita at 21 and 42 days post-immunization (values are mean log2 titre valuesS.E).
Mean values bearing same superscript are not statistically significant (pN0.05) at 21
and 42 days post-immunization.
Table 1
Differentnon-specific immune parameters in differenttreated groups of rabbitat 21 and42 dayspost-immunization. Means bearingsame superscripts are notstatisticallysignificant
(pN0.05) at 21 and 42 days post-immunization.
I mmune par amete rs ECP-Chitosan c oat ed
liposomes
ECP-Liposomes FIA-ECPs Control
21 days 42 days 21 days 42 days 21 days 42 days 21 days 42 days
Myeloperoxidase activity (OD) 0.56 0.02a 0.510.003a 0.360.02b 0.290.02b 0.30.01 b 0.250.02 b 0.180.02c 0.15 0.01 c
Hemaggultination titer (log2) 6.33 0.333a 5.3330.333a 4.00.333 b 3.6660.333 b 3.6660.333 b 3.6660.333 b 2 0 c 2 0 c
Hemolytic titer (log2) 6 0.577a 5.6660.333a 5.10.33 b 4.80.33 b 3.6660 b 4.90.005 b 2 0 c 2 0 c
Bacterial agglutination titer (log2) 6 0a 6.330.333a 5.330.005 b 4.00.003 b 5.330 b 5.330.005 b 1.6660.666 c 2.3330.333 c
Antiprotease activity (% of
inhibition)
71.02.081a 700a 50.00 b 48.3331.666 b 53.333.333 b 49.3331.666 b 40.6662.333 c 42.02.886 c
0 Respiratory burst activity
(measured by NBT assays) (OD)
0.4010.047a 0.4010.045a 0.2280.005 b 0.2210.011 b 0.1980.015 b 0.1880.010 b 0.1240.011 c 0.1590.015 c
Means bearing same superscripts are not statistically significant (pN
0.05) at 21 and 42 days post-immunization.1
Fig. 10. The Relative percentage of survivability of immunized rohu (Labeo rohita) after
15th day post challenge [values are mean % values (of each group containing 20 fish)
Standard Error (S.E)]. Mean values bearing same superscript are not statistically
significant (pN0.05) at 15th day post challenge.
6 T. Behera et al. / International Immunopharmacology xxx (2011) xxxxxx
Please cite this article as: Behera T, et al, Antigen in chitosan coated liposomes enhances immune responses through parenteralimmunization, Int Immunopharmacol (2011), doi:10.1016/j.intimp.2011.02.002
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423 preparations in enhancing both specific and non-specific immunity and
424 can be safely administered to fish as well as other mammalian systems
425 through parenteral route and had brought some hope for its use in
426 human.
427 Acknowledgements
428 The authors are thankful to the Head of SAIF, All India Institute of
429 Medical Science (AIIMS), NewDelhifor providing TEMfacility,Head of the430 department, Fish Health Management Division and Director of Central
431 Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar for
432 providing necessary facilities to carry out the research work.
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