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Parenteral immunization offish, Labeo rohita with Poly D, L-lactide-co-glycolicacid (PLGA) encapsulated antigen microparticles promotes innateand adaptive immune responses

T. Behera a, P.K. Nanda a, C. Mohanty b, D. Mohapatra a, P. Swain a,*, B.K. Das a,P. Routray a, B.K. Mishra a, S.K. Sahoo b

a

Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar-751002, Indiab Nanomedicine Laboratory, Institute of Life Sciences, Bhubaneswar-751023, India

a r t i c l e i n f o

Article history:

Received 27 August 2009

Received in revised form

5 November 2009

Accepted 9 November 2009

Available online 14 November 2009

Keywords:

Aeromonas hydrophila

Immune response

MicroparticlesOuter membrane proteins

PLGA

a b s t r a c t

Immunogenicity of different antigen preparations of outer membrane proteins (OMP) of Aeromonas

hydrophila such as Poly D, L-lactide-co-glycolic acid (PLGA) microparticles, oil emulsion, neat OMP and

bacterial whole cells were compared through intra-peritoneal injection in fish, Labeo rohita. Among these

preparations, PLGA encapsulated antigen stimulated both innate and adaptive immune parameters and

the immunogenicity exhibited by PLGA microparticles was significantly higher (p < 0.05) at both 21 and

42 days post-immunization suggesting that the above delivery system would be a novel antigen carrier

for parenteral immunization in fish, Labeo rohita

2009 Elsevier Ltd. All rights reserved.

Vaccination is one of the methods for prevention of infectious

diseases of human and other animals, including fish [1]. Inefficiency

of currently used conventional vaccine is due to lack of appropriate

adjuvants and/or suitable vaccine carrier. The new generation of

vaccines mainly contain purified proteins (isolated from microor-

ganisms or recombinant proteins) and peptides (by direct chemical

peptide synthesis), which are poorly immunogenic. Therefore, the

search for harmless and effective adjuvants is urgently needed in

modern vaccinology. Use of several adjuvants like particulates

(aluminium salts, ISCOMS, virosomes, chitosan, QS-21 and lipo-

somes); microbialproducts (microbial toxins,live viraland bacterial

delivery vectors, monophosphoryl lipid A); and oil emulsions

{Freund's incomplete adjuvant (FIA), Freund's complete adjuvant

and MF-59} have been extensively reviewed [2]. Although each one

creates its own type of immune modulation, they also exert some

side effects [3]. Further, their ineffectiveness to certain antigens and

their inability to elicit cell-mediatedimmuneresponses,particularly

cytotoxic T-cell responses, limits their application against intracel-

lular parasites and viral infections. When new vaccine formulations

are being sought, there are many aspects to consider such as effec-

tiveness in inducing the correct immune response, stability in the

host, toxicity and economic aspects. One such highly promising

technology is based on polymeric microparticles, which permit

a sustained or pulsed release of encapsulated antigen thus mini-

mizing the requirements for repeated administration and dose of

antigen ensuring long-term protection.

In contrast to other carriers, microparticles are more stable and

elicit both humoral as well as cellular immunity [4]. Evidence from

mammalian studies indicate that biodegradable microparticles

may act as efficient antigen delivery vehicles due to their potential

advantages of reducing the number of injections, enhancing the

immune response and reducing the total antigen dose needed to

achieve protection [5e7]. Among the polymeric systems developed,

PLGA microspheres have been widely used for controlled delivery

of peptides [8], native and synthetic proteins [9] and nucleic acids

[10] because of their excellent tissue compatibility, biodegrad-

ability, non-toxic nature and their approval by the Food and Drug

Administration for safe use in human [6]. The PLGA can also be

made into any micro- and nano sizes, with a capability of encap-

sulating almost any molecule [11]. In vivo, the polymer undergoes

random non-enzymatic hydrolysis and forms the endogenous

metabolites, lactic and glycolic acids [7], which areinnocuous to the* Corresponding author.

E-mail address: [email protected] (P. Swain).

Contents lists available at ScienceDirect

Fish & Shellfish Immunology

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 / f s i

1050-4648/$ e see front matter 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.fsi.2009.11.009

Fish & Shellfish Immunology 28 (2010) 320e325

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body and are eliminated by the Kreb's cycle as carbon dioxide and

in urine [12]. Several encouraging trials have been conducted

providing data to confirm the superior efficacy of this system over

other adjuvants in several vaccines [13e15]. Today PLGA particles

are used in different marketed products, medical and pharmaceu-

tical fields, and are capable of releasing peptides and proteinsslowly and continuously from 1 to 4 months [11]. Further, PLGA

particles have been shown to be taken up in vivo by the main

antigen presenting cells (APC) in mammals, dendritic cells, and

enhance antigen-presentation efficacy by 10e100 fold [16e18]. The

easy manufacture of this microparticle and the possibility of

administration by different routes offered the additional advan-

tages for its use as a vaccine carrier [19].

Fish vaccinology faces similar problems that are encountered in

vaccine design for human and mammals,as they are considered to be

very important in reducing economic losses in aquaculture caused by

some diseases [20e22]. This must be considered for developing

effective vaccines to face old challenges with new possibilities in the

21st century. In this regard, the use of biodegradable microparticles

as an antigen delivery system in fish is a relatively new area of

research. Among the biodegradable microparticles, PLGA micropar-

ticles as a vaccine carrier infish have only been investigated through

oral vaccination in rainbow trout [23]. Moreover, PLGA microparti-

cles alone also stimulate certain non-specific immune parameters

and pro-inflammatory cytokine production through intraperitonial

injection in fish [24], but there is no report for PLGA as an antigen

carrier in fish through parenteral administration.

Hence the present study was undertaken to evaluate PLGA

microparticles as an antigen carrier using low molecular weight

protein antigen such as outer membrane proteins (OMP) of Aero-

monas hydrophila, a known bacterial pathogen of fish and the

results obtained herewith found that it stimulated both specific and

innate immune parameters.

1. Materials and methods

1.1. Bacteria

Aeromonas hydrophila strain (Ahv), isolated from Channa striatus

showing dropsy conditions was preserved in a lyophilized condi-

tion in our laboratory and was used throughout the study.

1.2. Isolation of outer membrane proteins

The OMP ofA. hydrophila (Ahv) were prepared according to the

method of Nikaido [25] with minor modifications. The pellet

obtained from 1 L culture ofA. hydrophila was washed twice in40 ml

of0.15 M phosphate buffered saline (PBS, pH 7.2), once in 40 ml of

20 mM Trise

HCl (pH 7.5) and centrifuged at 13,000 g for 10 min.Thecells were then resuspended in 20 ml TriseHCl and disrupted by

sonication for 30 min at 10 W at an interval of 30 s. Unbroken cells

and cellular debris were removed by centrifugation at 4000 g for

15 min. The supernatant was then further centrifuged at 10,000 g

for 1 h at 4 C. The pellet was suspended in 20 ml of 2% (w/v) sar-

cosine and incubated at room temperature for 30 min to solubilize

the inner membrane. The solution was then centrifuged at

10,000 g f o r 1 h a t 4 C. The resultant pellet of sarcosine-insoluble

components wasfreeze-driedand stored at20 C until use.Protein

concentration of OMP was determined using Genei TM protein esti-

mation kit (Bangalore genei, India) by the BCA method.

1.3. Antigen formulations

Three forms of antigen preparations were done as described

below.

1.3.1. Preparation of PLGA microparticle encapsulated OMP

PLGA (copolymer ratio 50:50, I.V 0.76) was purchased from Bir-

mingham polymers, Inc. (Birmingham, AL). Polyvinyl alcohol (PVA,

average MW 30,000e70,000) was purchased from SigmaeAldrich Co.

(St Louis, MO 63195, USA). All reagents used were of analytical grade

from E-merck, India and organic solvents used were of HPLC grade.Microparticles containing different antigens were formulated using

a double emulsion-solvent evaporation technique [26] with little

modification. In this method, aqueous solution of antigen dissolved in

300 ml of distilled water was emulsified with 100 mg of PLGA in

chloroform solution (3% w/v) followed by vortexing for 2 min to get

a primary emulsion. The primary emulsion was further emulsified in

an aqueous PVA solution (12 ml, 5% w/v) to form an oil-in-water

emulsion. For preparation of microparticles, the emulsion was

homogenized for 2 min andstirred forovernight at room temperature

to allow the evaporation of organic solvent. Microparticles were

recovered by normal centrifugation at 5000 g for 10 min using

SIGMA 3K30 (Germany) centrifuge. The process of centrifugation was

repeated three times to remove excess PVA and un-encapsulated

antigens. The recovered microparticle suspensions were lyophilized

for two days (80 C and

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buffered saline (PBS, pH 7.2). Finally, the bacteria were sus-

pended at 109 cells ml1 in PBS.

1.4. Emulsification with FIA

OMP(20 mg) in1 mlPBSwas emulsified with an equal volume ofFIA and stored at 4 C until further use.

1.5. Immunization protocol

Indian major carp, Labeo rohita (rohu), juveniles of average

weight ranging from 30 to 40 g were acclimatized in the wet

laboratory of Fish Health Management Division of Central Institute

of Freshwater Aquaculture (CIFA), Kausalyaganga, India, 15 days

prior to the start of the experiment. The fish were fed with artificial

carp diet with constant aeration and daily one-third water

exchange. Water temperature of the experimental tanks was 27 C

to 30 C. Fish were separated into 5 groups, 10 fish in each group

were immunized separately with 0.1 ml of different preparations @

20 mg of OMP and 0.1 ml (109 cells ml1) bacterial whole cell

antigen (WC), whereas controls were injected with PBS. The fish of

all the treated groups including the control group were bled at an

interval of 3-weeks (at 21 and 42 days) post-injection to study

various immune parameters.

1.6. Preparation of anti-rohu-globulin rabbit serum

The rabbit anti-rohu globulin was prepared as per a standard

method [28] using sera obtained from healthy adult rohu of

average weight 250e300 g. Briefly, serum was collected from

healthy rohu and pooled to 10e15 ml. An equal volume of satu-

rated ammonium sulphate solution was mixed with the pooled

sera drop by drop and then placed on a magnetic stirrer overnight

at 4 C. The sample mixture was centrifuged at 10,000 g for

10 min at 4 C and the precipitate was dissolved with 5 ml car-

bonateebicarbonate buffer (pH 9.6). The sample was then centri-

fuged at 10,000 g for 10 min at 4 C. The pellet was collected and

the volume was made to 2 ml with carbonateebicarbonate buffer

(pH 9.6). The globulin solution was dialyzed using dialysis

membrane (Snakeskin, Pierce Chemical Company, USA) with 7000

molecular weight cut off against PBS (pH 7.2) for 72 h at 4 C, after

which the globulin was collected. The anti-rohu globulin sera were

raised in a New Zealand white rabbit as per the method of Lund

et al. [29].

1.7. Immunoresponse studies

1.7.1. Myeloperoxidase activity

For determination of myeloperoxidase activity, 15 ml of serumwas diluted in 135 ml of Hank's balanced salt solution (Ca2, Mg2

free) and then 50 ml of 20 mM, TMB (3, 30,5,50-tetra methyl

benzidine) and 5 mM H2O2 were added. The reaction was stopped

after 2 min by adding 50 ml of 4 M sulphuric acid and the optical

density (O.D) was read at 450 nm [30] using UVeVIS spectropho-

tometer, Thermo Spectronic, UK.

1.7.2. Respiratory burst assay

The respiratory burst activity was measured by the reduction of

nitro blue tetrazolium (NBT) by intracellular superoxide radicals

[31]. Briefly,100 ml of heparinised blood fromfish of eachgroup was

mixed with 100 ml of 0.2% NBT (Sigma, USA) solution and incubated

for 30 min at 25 C. After incubation, 50 ml from the above was

mixed with 1 ml of N, N diethylmethyl formamide (Qualigens,India) and then centrifuged at 6000 g for 5 min. The O.D of the

supernatant was measured at 540 nm.

1.7.3. Bacterial agglutination activity

The agglutination test was conducted in U-shaped microtitre

plates. Two-fold serial dilution of 25 ml fish serum was made with

an equal volume of PBS in each well, to which 25 ml of formalin-

killed A. hydrophila (107 cells ml1) suspension was added. The

plates were incubated overnight at room temperature. The titrewas calculated as the reciprocal of the highest dilution of serum

showing complete agglutination of the bacterial cells.

1.7.4. Haemagglutination activity

The haemagglutination activity of serum samples was carried

out using a standard method [32]. This assay was done in

U-shaped microtitre plates by serial two-fold dilution of 50 ml

serum with PBS (pH 7.2). Then 50 ml of freshly prepared 1% New

Zealand white rabbit red blood cell (RBC) suspension was added

to each well. The plates were kept at room temperature

(28e30 C) for 2 h or overnight at 4 C if agglutination was not

visible within 2 h. The titre was calculated as the reciprocal of

the highest dilution of serum showing complete agglutination

of RBC.

1.7.5. Haemolytic activity

The haemolytic titre of serum was determined in a similar

manner as described for HA titre [32] by using fresh sera from

all the groups. Titre was expressed as the reciprocal of the

highest dilution of serum showing complete haemolysis of the

rabbit RBC.

1.8. Triple antibody indirect enzyme linked immunosorbent assays

The triple antibody indirect ELISA was conducted as per the

method of Swain et al. [28] with slight modifications using 96

well microtitre polystyrene plates (Nunc, Denmark). The wells

were separately coated with 50 ml of purified outer membrane

proteins from A. hydrophila (Ahv) (1e

2 mg/well) diluted in car-

bonateebicarbonate buffer (pH 9.6) overnight at 4 C. The plates

were then washed in PBS containing Tween-20 (PBS-T, pH 7.2)

and blocked with 100 ml of 3% skim milk powder for 2 h at 37 C.

The wells were further washed in PBS-T. The fish sera raised

against several antigens was two fold diluted after initial dilu-

tion of 1:10 with PBS (pH 7.2) as first antibody and added to

homologous antigen-coated wells in duplicate per serum dilu-

tion. The plates were incubated at 37 C for 45 min and washed

thrice in PBS-T. Rabbit anti-rohu sera (the second antibody) at

a dilution of 1:20 was added to each well and incubated at 37 C

for 45 min. Then the anti-rabbit-HRPO conjugated goat serum

(the third antibody) was added and incubated for 45 min. The

wells were then thoroughly washed and added with 50 ml of

substrate solution (5 mg of O-phenylene diamine tetra hydro-chloride and 10 ml of H2O2 (38%, v/v) in 5 ml of acetate buffer,

pH 5.0). The plates were incubated at 37 C for 5 min in a dark

chamber and finally O.D was recorded at 450/655 nm in

a microplate reader (BIO-RAD, USA). The antibody activity was

expressed in terms of O.D value after subtracting the values

obtained by unimmunized healthy sera.

1.9. Statistical analysis

The statistical analysis system (SAS) software (version 6.12) was

used to analyse all the data [33]. One-way analysis of variance

followed by Duncan's multiple range tests were done to compare

the variations in various immuneparameters at significance level of

difference (p < 0.05) in different injected groups. The mean stan-dard error (S.E) of assayed parameters was calculated for each

group offish.

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2. Results

2.1. Physico-chemical characterization of antigen

loaded microparticles

Antigen loaded microparticles were prepared from the PLGA

polymer using double emulsion method. Antigen was efficiently

loaded in the PLGA microparticles, reaching encapsulation effi-ciency of 25 1.3%. DLS analysis revealed that the formulated

microparticles had an average diameter of 1121 10.6 nm (Fig. 1).

Topology and size of the microparticle as observed by SEM analysis

confirmed the smooth and spherical nature of OMP-loaded PLGA

microparticles (Fig. 2). The average size of these microparticles was

in the range of 1 mm.

2.2. Immune response studies

2.2.1. Non-specific immune responses

The non-specific immune parameters of fish following the

injection with different antigen preparations(PLGA-OMP, FIA-OMP,

OMP and WC), at 21 and 42 days post-immunization are presented

in Figs. 3e7. All these parameters i.e. myeloperoxidase, respiratory

burst activity, haemagglutination, hemolytic and bacterial aggluti-nation titrewere significantly higher (p< 0.05) in alltreated groups

than the control. The PLGA-OMP treated group showed signifi-

cantly higher (p< 0.05) titre than other groups (FIA-OMP, OMP and

WC). These parameters did not vary much in all treated groups at

21 and 42 days post-immunization.

2.2.2. Specific immune responses

The serum antibody titre, as measured by indirect ELISA, was

expressed in terms of mean OD values (after subtracting the values

obtained by unimmunized healthy sera) (S.E.) and presented in

Fig. 8. The antibody titres at 21 and 42 days post-immunization

were significantly higher (p < 0.05) in the PLGA- encapsulated

antigen and FIA treated groups than the OMP and WC treated

groups. However, no significant difference (p > 0.05) in the anti-

body level at 21 and 42 days post-immunization was recorded

between these two groups.

3. Discussion

PLGA has been proven to be a very useful antigen delivery

system in mammals since it provides long lasting immunity

[34e36]. So, we attempted to evaluate the potential use of PLGA

microparticles as antigen carrier in fish using the OMP of Gram-

negative bacteria, A. hydrophila.

The isolated OMP of A. hydrophila was encapsulated in PLGA

microparticles and the resulted microspheres were tested for

immunogenicity in fish along with other antigenic preparations.

0

10

20

30

40

1 10 100 1000 10000

)%(ytisnetnI

Size (d.nm)

Fig.1. Typical size distribution of PLGA microparticles as determined by dynamic light

scattering.

Fig. 2. Scanning electron micrograph of outer membrane proteins loaded PLGA

microparticles (bar 1 mm).

c

c

c

b

a

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

CONTROL PLGA-

OMP

FIA-OMP OMP WC

Treatment

mn045ta

eulavDO

21 days

42 days

Fig. 3. The respiratory burst activity (measured by NBT assays) of blood ofLabeo rohita

in different treated groups at 21 and 42 days post-immunization (Values are mean OD

values S.E.). Mean values bearing same superscript are not statistically significant

(p > 0.05) at 21 and 42 days post-immunization.

cc

c

b

a

0

0.05

0.1

0.15

0.2

CONTROL PLGA-

OMP

FIA-OMP OMP WC

Treatment

mn054taeulavDO

21 days

42 days

Fig. 4. The myeloperoxidase activity of sera of Labeo rohita in different treated groups

at 21 and 42 days post-immunization (Values are mean OD values S.E.). Mean values

bearing same superscript are not statistically significant (p > 0.05) at 21 and 42 days

post-immunization.

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A successful micro particulate system has a high loading capacity to

reduce the quantity of the carrier required for administration. In

this study, antigen was efficiently loaded in PLGA microparticles,

reaching encapsulation efficiency of 25 1.3% with average size

diameter of 1121 10.6 nm. In an earlier study, similar results e.g.

size rangew1080 nm with encapsulation efficiency ofw25% were

obtained by using bovine serum albumin as a model protein

antigen in PLGA microparticles [37].

Both specific andnon-specific immune parameters were studied

and all four preparations of vaccines used (PLGA-OMP, FIA-OMP,

OMP and WC) elicited immune responses in the fish at varying

levels as compared to the control. The PLGA-OMP treated groupshowed significantly higher immune responses than the other

groups at both 21 and 42 days post-immunization. PLGA micro-

particles have been found to enhance phagocytosis by macro-

phages [38], and neutrophils [18] in mouse and human,

respectively. Comparable results were also found by the use of

PLGA alone in mammal [16] as well as in fish, rainbow trout [24].

The specific antibody titres of the sera were higher in both FIA-

OMP and PLGA-OMP treated groups with no significant difference

(p < 0.05) between them. Similar results were also found when

PLGA was used as carrier for peptide vaccine in mammals [39] and

in mice through subcutaneous route using BSA as a model antigen

[40]. Moreover, the superiority of PLGA microspheres over alum

adjuvant in eliciting high antibody responses was seen in mice

through subcutaneous administration [41]. In this study the supe-riority of PLGA microparticles was recorded without any side

effects, which is usually noticed in FIA formulation [3]. The serum

antibody titres detected at 21 days persisted for at least 42 days.

According to O'Hagan et al. [42], the level of antibody remained

high even one year after injection through subcutaneous route in

mice which indicates the injectable PLGA microparticles control the

release of antigen over a period of several weeks. So, the use of

PLGA microparticles as a vaccine carrier can reduce the number of

administrations and induce both innate and adaptive immunity.

Among these four preparations of OMP of A. hydrophila, PLGA

microparticles showed encouraging results without any adverse

effects on fish health. Therefore, it can be considered to be a useful

antigen carrier for inducing both cellular and humoral immune

response in fish through parenteral immunization.

Acknowledgements

The authors are thankful to the Director, Central Institute of

Freshwater Aquaculture, Bhubaneswar and the Director, Institute of

Life Sciences, Bhubaneswar for providing all necessary facilities to

carry out the above study.

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