intimp_2249

8/6/2019 INTIMP_2249

1/10

AUTHOR QUERY FORM

Journal: INTIMP Please e-mail or fax your responses and any corrections to:E-mail: [email protected]: +1 619 699 6721

Article Number: 2249

Dear Author,

Any queries or remarks that have arisen during the processing of your manuscript are listed below and highlighted by flags in

the proof. Please check your proof carefully and mark all corrections at the appropriate place in the proof (e.g., by using on-

screen annotation in the PDF file) or compile them in a separate list.

For correction or revision of any artwork, please consult http://www.elsevier.com/artworkinstructions.

Any queries or remarks that have arisen during the processing of your manuscript are listed below and highlighted by flags in

the proof. Click on the Q link to go to the location in the proof.

Locationin article

Query / Remark: click on the Q link to goPlease insert your reply or correction at the corresponding line in the proof

Q1 Please check if the page range provided here is correct.



Q4 Figure 2-5,7,10 contain cut-off data while figures 4 and graphical abstract are of poor quality. Please check

and provide replacement as deemed necessary.

Thank you for your assistance.

Our reference: INTIMP 2249 P-authorquery-v8

Page 1 of 1
http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-

8/6/2019 INTIMP_2249

2/10

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

8/6/2019 INTIMP_2249

3/10

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

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

8/6/2019 INTIMP_2249

4/10

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

http://dx.doi.org/10.1016/j.intimp.2011.02.002http://dx.doi.org/10.1016/j.intimp.2011.02.002

8/6/2019 INTIMP_2249

5/10

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


8/6/2019 INTIMP_2249

6/10

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.



8/6/2019 INTIMP_2249

7/10

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.



8/6/2019 INTIMP_2249

8/10

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.



8/6/2019 INTIMP_2249

9/10

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.

433 References

434 [1] Allison AC, Gregoriadis G. Liposomes as immunological adjuvants. Nature 1974:435 252-252.436 [2] Mishra N, Gupta PN, Mahor S, Khatri K, Goyal AK, Vyas SP. Liposomes as adjuvant437 for combination vaccines. Indian J Exp Biol 2007;45:23741.438 [3] Harrington KJ, Syrigos KN, Vile RG. Liposomally-targeted cytotoxic drugs in the439 treatment of cancer. J Pharm Pharmacol 2002;54:1573600.440 [4] Zho F, Neutra MR. Antigen delivery to mucosa-associated lymphoid tissues using441 liposomes as a carrier. Biosci Rep 2002;22:35569.442 [5] Matteucci ML, Thrall DE. The role of liposomes in drug delivery and diagnostic443 imaging: a review. Vet Radiol Ultrasound 2000;4:1007.

444 [6] Mady MM, Ghannam MM, KhalilWA, Repp R, MarkusM, Rascher W, etal. Efficient445 gene delivery with serum into human cancer cells using targeted anionic446 liposomes. J Drug Target 2004;12:118.447 [7] Wassef NM, Alving CR, Richards RL. Liposomes as carriers for vaccines. Immunol448 Methods 1994;4:21722.449 [8] Gregoriadis G. Immunological adjuvants: a role for liposomes. Immunol Today450 1990;11:8997.451 [9] Yasumoto S, Yoshitaka K, Yasuda M, Yoshimura T, Miyazaki T. Oral Immunization452 of common carp with a liposome vaccine fusing koi herpesvirus antigen. Fish453 Pathol 2006;41:1415.454 [10] Haan de A, Geerligs HJ, Huchshorn JP, van Scharrenburg GJM, Palache AM,455 Wilschut J. Mucosal immunoadjuvant activity of liposomes: induction of systemic456 IgG and secretory IgA response in mice by intranasal immunization with an457 influenza subunit vaccine and coadministred liposomes. Vaccine 1995;13:155.458 [11] Nakhla N, Szalai AJ, Banoub JH, Keough KMW. Serum anti-LPSantibody production459 by rainbow trout (Oncorhynchus mykiss) in response to the administration of free460 and liposomally-incorporated LPS from Aeromonas salmonicida. Fish Shellfish461 Immunol 1997;7:387401.462 [12] Hedlund G, Jansson B, Sjogren HO. Comparision of immune response induced by463 rat RT-1 antigens presented as inserts into liposomes, as protein micelles and464 intact cells. Immunology 1984;53:6978.465 [13] Bakker-Woudenberg A, Storm G, Woodle MC. Liposomes in the treatment of466 infections. J Drug Target 1994;2:36371.467 [14] Taylor TM, Davidson PM, Bruce DB, Weiss J. Liposomal nanocapsules in food468 science and agriculture. Crit Rev Food Sci Nutr 2005;45:119.469 [15] Zhang LF, Granick S. How to stabilize phospholipid liposomes (using nanoparticles).470 Nano Lett 2006;6:6948.471 [16] Graff A, WinterhalterM, MeierW. Nanoreactorsfrom polymer-stabilized liposomes.472 Langmuir 2001;17:91923.473 [17] Yan W, Chen W, Huang L. Mechanism of adjuvant activity of cationic liposome:474 phosphorylation of a MAPkinase, ERK and induction of chemokines. MolImmunol475 2007;44:367281.476 [18] Baca-Estrada ME, Foldvari M, Snider M, Harding K, Kournikakis B, Babiuk LA, et al.477 Intranasal immunization with liposome-formulated Yersinia pestis vaccine478 enhances mucosal immune responses. Vaccine 2000;18:2203-2011.479 [19] Ishikawa H, Shimoda Y, Matsumoto K. Preparation of liposomal microcapsules by480 proliposome method with soybean lecithin. J Fac Agric Kyushu Univ 2004;49(1):

481 119

27.482 [20] Topfer Chris. Nanoparticle-based adjuvant of lecithin to boost vaccine. Drug483 delivery, medical nanotechnology; October 6, 2009 http://medicalnanotec.com/484 drug-delivery/nanoparticle-based-adjuvant-of-lecithin-to-boost-vaccine.485 [21] Hirano S, Seino H, Akiyama Y, Nonaka I. Biocompatibility of chitosan by oral and486 intravenous administration. Polym Eng Sci 1988;59:897901.487 [22] Nagamoto T, Yoshiyuki H, Takayama K, Maitani Y. Novel chitosan particles and488 chitosan-coated emulsions inducing immune response via intranasal vaccine489 delivery. Pharmaceut Res 2004;21:6714.490 [23] Henriksen, Smistad G, Karlsen J. Interaction between liposomes and chitosan. Int J491 Pharm 1994;101:22736.492 [24] Takeuchi H, Matsui Y, Yamamoto H, Kawashima Y. Mucoadhesive properties of493 carbopol or chitosan-coated liposomes and their effectiveness in the oral494 administration of calcitonin to rats. J Control Release 2003;86:23542.495 [25] Khatri K, Goyal AK, Gupta PN, Mishra N, Mehta A, Vyas SP. Surface modified496 liposomes for nasal delivery of DNA vaccine. Vaccine 2008;26:222533.497 [26] Alamelu S, Panduranga K. Studies on the carboxymethyl chitosan containing498 liposomes for their stability and controlled release of dapsone. J Microencapsul499 1991;8:50519.

500[27] Dong, Rogers JA. Polymer-coated liposomes: stability and release of ASA from501carboxymethyl chitin-coated liposomes. J Control Release 1991;17:21724.502[28] Henriksen, Vagen SR, Sande SA, Smitad G, Karlsen J. Interactions between503liposomes and chitosan II: effect of selected parameters on aggregation and504leakage. Int J Pharm 1997;146:193204.505[29] Takeuchi H, Matsui Y, Yamamoto H, Kawashima Y. Mucoadhesive properties of506carbopol or chitosan-coated liposomes and their effectiveness in the oral507administration of calcitonin to rats. J Control Release 2003;86:23542.508[30] Guo J, Ping Q, Jiang G, Huang L, Tong Y. Chitosan-coated liposomes: characterization509and interaction with leuprolide. Int J Pharm 2003;260:16773.510[31] Alpar HO, Eyles JE, Williamson ED, Somavarapu S. Intranasal vaccination against

511plague, tetanus and diphtheria. Adv Drug Deliv Rev 2001;51:173.512[32] Amina M, Jaafari MR, Tafaghodia M. Impact of chitosan coating of anionic liposomes513on clearance rate, mucosal and systemic immune responses following nasal514administration in rabbits. Colloid Surf B 2009;74:2259.515[33] Kanai K, Wakabayashi H. Purification and some properties of protease from516Aeromonas hydrophila. Bull Jpn Soc Sci Fish 1984;50:136774.517[34] Gold WL, Slit IE. Aeromonas hydrophila infections of skin and soft tissue: report of51811 cases and review. Clin Infect Dis 1993;16:6974.519[35] George WL, Nakata MM, Thompson J, White ML. Aeromonas related diarrhea in520adults. Arch Intern Med 1985;145:2207-2011.521[36] Suprapto H, Nakai T, Muroga K. Toxicity of extracellular products and intracellular522components ofEdwardsiella tarda in the Japanese eel and flounder. J Aquat Anim523Health 1995;7:2927.524[37] Mekuchi T, Kiyokawa T, Honda K, Nakai T, Muroga K. Vaccination trials in the525Japanese flounder against Edwardsiellosis. Fish Pathol 1995;30:2516.526[38] Perrett S, Golding M, Williams WP. A simple method for the preparation of527liposomes for pharmaceutical applications: characterization of liposomes. J Pharm528Pharmacol 1991;43:15461.529[39] Rengel RG, BarisicK, Pavelic Z, Grubisic TZ, Cepelak I, Filipovic-GrcicJ. Hlgh efficiency

530entrapment of superoxide dismutase into mucoadhesive chitosan-coated liposome.531Eur J Pharm Sci 2002;15:4418.532[40] Swain P, Nayak SK,Sahu A, MohapatraBC, Meher PK. Bath immunization of spawns,533fries and fingerlings of Indian major carps using a particulate antigen and534determinationof age, doseand duration ofantigen exposure.Fish Shellfish Immunol5352002;13:13340.536[41] Lund V, Jorgensen T, Holm KO, Eggset G. Humoral immune response in Atlantic537salmon, Salmo salar L., to cellular and extracellular antigens of Aeromonas538salmonicida. J Fish Dis 1991;14:44352.539[42] Quade MJ, Roth JA. A rapid, direct assay to measure degranulation of bovine540neutrophil primary granules. Vet Immunol Immunopathol 1997;58:23948.541[43] Anderson P, Siwicki AK. Duration of protection against Aeromonas salmonicida in542brook trout immunostimulated with glucan or chitosan by injection and543immersion. Prog Fish Cult 1994;56:25861.544[44] Ellis AE. Serum antiproteases infish and lysozymeassays. In:Stolen JS, Fletcher TC,545Anderson DP, Roberson BS, van Muiswinkel WB, editors. Techniques in fish546immunology. Fair Haven NJ: SOS Publications; 1990. p. 95103.547[45] Magnado ttir B, Jonsdottir H, Helgason S, Bjornsson B, Jrgensen T, Pilstrom L.548Humoral immune parameters in Atlantic cod (Gadus morhua L.). I. The effects of549environmental temperature. Comp Biochem Physiol 1999;122:17380.550[46] Blazer VS, Wolke RE. The effects of a-tocopherol on the immune response and551non-specific resistance factors of rainbow trout (Salmo gairdneri Richardson).552Aquaculture 1984;37:19.553[47] SAS Institute Inc. SASR system for regression. 2nd ed. Cary, NC: SAS Institute Inc;5541991. p. 10.555[48] Dwivedi N, Arunagirinathan MA, Sharma S, Bellare J. Silica-coated liposomes for556insulin delivery. J Nanomater 2010, doi:10.1155/2010/652048.557[49] Mady MM, DarwishMM, KhalilS, KhalilWM. Biophysical studieson chitosan-coated558liposomes. Eur Biophys J 2009;38:112733.559[50] Rockstad AM, Bogwald J, Jorgensen T. Microsilica as antigen carrier and adjuvant560in the Atlantic salmon (Salmo salarL.). Fish Shellfish Immunol 1996;6:56780.561[51] Zheng CH, Gao JQ, Zhang YP, Liang WQ. A protein delivery system: biodegradable562alginate-chitosan-poly (lactic-co-glycolic acid) composite microspheres. Biochem563Biophys Res Commun 2004;323:13217.564[52] Zaharoff DA,Rogers CJ, Hance KW,Schlom J, Greiner JW.Chitosan solution enhances565both humoral and cell-mediated immune responses to subcutaneous vaccination.566Vaccine 2007;25:208594.

567[53] Ishikawa H, Shimoda Y, MatsumotoK. Liposomal microencapsulationof enzymes by568proliposomemethodwith chitosan-coating. J Fac AgricKyushu Univ2005;50:1419.569[54] Raz A, Bucana C, Fogler WE, Poste G, Fidler IJ. Biochemical, morphological, and570ultrastructural studies on theuptake of liposomesby murine macrophages.Cancer571Res 1981;41:48794.572[55] Hsu MJ, Juliano RL. Interactions of liposomes with the reticuloendothelial system.573II. Nonspecific and receptor-mediated uptake of liposomes by mouse peritoneal574macrophages. Biochim Biophys Acta 1982;720:4119.575[56] Foged C, Arigita C, Sundblad A, Jiskoot W, Storm G, Frokjaer S. Interaction of576dendritic cells with antigen-containing liposomes: effect of bilayer composition.577Vaccine 2004;22:190313.578[57] Vangasseri DP, Cui Z, Chen W, Hokey DA, Falo LD, Huang L. Immunostimulation of579dendritic cells by cationic liposomes. Mol Membr Biol 2006;23:38595.580[58] Ahsan, Rivas IP, Khan MA, Torres Suarez AI. Targeting to macrophages: role of581physicochemicalpropertiesof particulatecarriersliposomes and microsphereson582the phagocytosis by macrophages. J Control Release 2002;79:2940.583[59] Gopalakannan A, Arul V. Immunomodulatory effects of dietary intake of chitin,584chitosan and levamisole on the immune system of Cyprinus carpio and control of585Aeromonas hydrophila infection in ponds. Aquaculture 2006;255:17987.


http://medicalnanotec.com/drug-delivery/nanoparticle-based-adjuvant-of-lecithin-to-boost-vaccinehttp://medicalnanotec.com/drug-delivery/nanoparticle-based-adjuvant-of-lecithin-to-boost-vaccinehttp://medicalnanotec.com/drug-delivery/nanoparticle-based-adjuvant-of-lecithin-to-boost-vaccinehttp://dx.doi.org/10.1155/2010/652048http://dx.doi.org/10.1016/j.intimp.2011.02.002http://dx.doi.org/10.1016/j.intimp.2011.02.002http://dx.doi.org/10.1155/2010/652048http://medicalnanotec.com/drug-delivery/nanoparticle-based-adjuvant-of-lecithin-to-boost-vaccinehttp://medicalnanotec.com/drug-delivery/nanoparticle-based-adjuvant-of-lecithin-to-boost-vaccine

8/6/2019 INTIMP_2249

10/10

[60] Nishimura K, Nishimura S, Nishi N, Numata F, Tone Y, Tokura S, et al. Adjuvantactivity of chitin derivatives in mice and guinea-pigs. Vaccine 1985;3:37984.

[61] Mutoloki S, Reite OB, Brudeseth B, Tverdal A, Evensen Q. A comparative immuno-pathological study of injection site reactions in salmonids followingintraperitonealinjection with oil-adjuvanted vaccines. Vaccine 2006;24:57888.

[62] Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends.Immunol Cell Biol 2004;82:48896.

[63] Bollinger JN. Metabolic fate of mineral oil adjuvants using 14C-labeled tracers. I.Mineral oil. J Pharm Sci 1970;59:10848.

[64] Garcon NM, Six HR. Universal vaccine carrier. Liposomes that provide T-dependenthelp to weak antigens. J Immunol 1991;146:3697702.

[65] Nkhala AN,SzalaiAJ, BanoubJH, KeoughKMW.Serumanti-LPS antibodyproduction byrainbow trout (Oncorhynchus mykiss) in responses to the administration of free andliposomally-incorporated LPS from Aeromonas salmonicida. Fish Shellfish Immunol1997;7:387401.

601[66] Hardling CV, Collins DS, Slot JW, Geuze HJ, Unanue ER. Liposome-encapsulated602antigens are processed in lysosomes, recycled and presented to T cells. Cell6031991;64:393401.604[67] Alving CR. Immunologic aspects of liposomes: presentation and processing of605liposomal protein and phospholipid antigens. Biochim Biophys Acta 1992;1113:60630722.607[68] Vallejo AN, Miller NW, Clem LW. Cellular pathway(s) of antigen processing in fish608APC: effect of varying in vitro temperatures on antigen catabolism. Dev Comp609Immunol 1991;16:67381.610[69] Amina M, Jaafari MR, Tafaghodia M. Impact of chitosan coating of anionic611liposomes on clearance rate, mucosal and systemic immune responses following

612nasal administration in rabbits. Colloid Surf B 2009;74:225

9.613[70] Alpar HO, Eyles JE, Williamson ED, Somavarapu S. Intranasal vaccination against614plague, tetanus and diphtheria. Adv Drug Deliv Rev 2001;51:173.


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

intimp_2249

Documents