1

Artifi cial Cells, Nanomedicine, and Biotechnology, 2013; Early Online: 1–11

Copyright © 2013 Informa Healthcare USA, Inc.

ISSN: 2169-1401 print / 2169-141X online

DOI: 10.3109/21691401.2013.769447

Evaluation of mucoadhesive carrier adjuvant: Toward an oral anthrax vaccine

Sharad Mangal 1,2 , Dilip Pawar 1,3 , Udita Agrawal 1 , Arvind K. Jain 1 & Suresh P. Vyas 1

1 Department of Pharmaceutical Sciences, Drug Delivery Research Laboratory, Dr. H. S. Gour University, Sagar, (M.P.), India,

2 Faculty of Pharmacy and Pharmaceutical Sciences, Drug Delivery, Disposition and Dynamics (D4), Monash University,

Parkville, VIC, Australia, and 3 Biologics Formulation Development, Syngene International Limited (A Biocon Company),

Bangalore, Karnataka, India

Introduction

Th e causative agent of anthrax, B. anthracis , invades the

host through mucosal (gastrointestinal, respiratory, and

cutaneous routes) routes. Th e optimal immune response at

both systemic and mucosal fronts is desirable to counter-

act the evasion of anthrax through cutaneous and mucosal

(inhalational or gastrointestinal) sites. It has been reported

that aluminum-based vaccine could off er signifi cant protec-

tion against anthrax infection in systemic circulation but

fails to induce specifi c mucosal immune response (Ivins

et al. 1998, Fellows et al. 2001). However, current evidence

clearly shows that only vaccines given by mucosal routes can

eff ectively stimulate two distinct layers of protection consist-

ing of both mucosal and systemic immunity (Mangal et al.

2011, Pawar et al. 2009).

Th e oral administration of the vaccine may constitute

an important advance on the prophylaxis, specially, of the

gastrointestinal anthrax. Oral route is most preferred for

immunization as it off ers the advantages of high compli-

ance, minimal side eff ects, no fear of needle-borne infection,

and induction of systemic as well as of mucosal immune

response Despite the obvious need and apparent merits, the

success in the fi eld of oral vaccination is limited due to sev-

eral constraints including, extremes of pH, enzymatic barrier

and intestinal epithelium barrier which hamper the access

of susceptible bio-macromolecule to gut immune inductive

sites (Malik et al. 2010). Th erefore, an ideally designed oral

vaccine should overcome these limitations and induce both

humoral and cellular counterparts of immunity along with

the mucosal immune response against the specifi c antigen

(Malik et al. 2010). We hypothesized that oral antigen deliv-

ery mediated through suitable carrier system may prove effi -

cient in protecting the antigen in harsh gastric environment

and inducing protective immunity in both systemic and

mucosal compartments.

Polymeric particles are of special interest for the oral

delivery of susceptible bioactive agents. Th ey are reported to

off er stability to the encapsulated content and also enhance

the uptake of drug, protein, and peptide through gut epithe-

lium (Mishra et al. 2010). It has been suggested that chitosan

might be valuable for the delivery of drugs through gastro-

intestinal tract (Lopez et al. 2000, Mangal et al. 2011, Pawar

et al. 2009, He et al. 1998, Shimoda et al. 2001), but its solubility

in acidic environment may hamper its use particularly for

oral vaccine delivery. Th e coating of chitosan particles with

an acid-resistant polymer such as alginate may prove useful

for delivering the antigens through oral route.

Th e primary aim of this study was to prepare and char-

acterize rPA-associated A-CHMp. Th e study also includes

understanding the impact of antigen dose on the magnitude

of immune response following oral vaccination. Th e effi -

cacy of the A-CHMp was tested against alum-adjuvanted

vaccine and free rPA. Th e specifi c immunity in serum and

Correspondence: Prof. Suresh P. Vyas, Department of Pharmaceutical Sciences, Drug Delivery Research Laboratory, Dr. H. S. Gour University, Sagar, (M.P.),

India, 470 003. Tel: � 91-7582-265525. Fax: � 91-7582-265525. E-mail: spvyas54@gmail.com

(Received 2 December 2012 ; revised 12 January 2013 ; accepted 21 January 2013 )

Abstract

The aim of present study was to evaluate the potential

of mucoadhesive alginate-coated chitosan microparticles

(A-CHMp) for oral vaccine against anthrax . The zeta potential

of A-CHMp was � 29.7 mV, and alginate coating could prevent

the burst release of antigen in simulated gastric fl uid. The

results indicated that A-CHMp was mucoadhesive in nature

and transported it to the peyer ’ s patch upon oral delivery. The

immunization studies indicated that A-CHMp resulted in the

induction of potent systemic and mucosal immune responses,

whereas alum-adjuvanted rPA could induce only systemic

immune response. Thus, A-CHMp represents a promising acid

carrier adjuvant for oral immunization against anthrax.

Keywords: alginate , chitosan , microparticles , mucosal

vaccination

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2 S. Mangal et al.

secretions was quantifi ed using ELISA. Th e protective

effi cacy of serum and secretory antibodies was evaluated

using toxin neutralization assay.

Materials and methods

Materials Chitosan was purchased from Fluka with the deacetylation

value of 80% (according to supplier ’ s specifi cations), and

sodium alginate was procured from Sigma Chemical Co.

(St. Louis, MO). Protein was estimated using BCA-protein

estimation kit (Bangalore Genei Pvt Ltd., India). Recombi-

nant protective antigen (rPA: Mol. Wt., 83 kDa) and lethal

factor (LF) was kindly provided by Dr. Yogendra Singh (IGIB,

New Delhi). Fluorescent isothiocynate – bovine serum albu-

min (FITC – BSA) and ELISA kits were procured from Sigma

Chemicals Co. (St. Louis, MO, USA). All the others reagents

and chemicals were of analytical grade and purchased from

the local suppliers (HiMedia Laboratories Pvt. Ltd., Central

Drug House, and Loba Chemie Pvt. Ltd.) unless otherwise

mentioned.

Preparation of A-CHMp Th e A-CHMp was prepared using the method previously

described by Borges et al. with minor modifi cations (2005).

Briefl y, 0.25% w/v chitosan was dissolved in acetic acid

solution (2% v/v). Th en, 1.8 mL of sodium sulfate solution

(10% w/v) was added with probe sonication to 100 mL of this

solution. Th e resulting suspension was then centrifuged at

3500 rpm for 30 min to collect the Mps. Th ese particles were

washed with deionized water and then freeze-dried. For

antigen loading, suspension of CHMp (0.4% w/v) was incu-

bated with rPA (0.25% w/v) in phosphate buff er saline (PBS,

pH 7.4) under mild agitation for 2 h. Th e mixture was then

centrifuged at 1600 rpm for 10 min to remove the unassoci-

ated antigen. Resultant suspension of antigen-loaded Mps

was mixed with sodium alginate (1% w/v) in equal volume

and kept under magnetic stirring for 20 min to perform alg-

inate coating. Particles were separated using centrifugation

for 10 min at 2000 rpm. Th ese particles were then suspended

in 10 mL of 0.524 mM CaCl 2 in 50 mM HEPES buff er solution

and agitated for 10 min to aff ect the cross-linking of alginate

adsorbed on to the surface of CHMp.

Alum-adsorbed rPA was formulated following the

procedure reported by Berthold et al. (2005) with slight

modifi cations. Briefl y, the rPA was added to the aluminum

hydroxide adjuvant (Al(OH) 3 ), mixed using refrigerated

shaker (MaxQ 4000) at 50 rpm (4 ° C) to aff ect adsorption,

and then incubated overnight. After adsorption, the fi nal

volume was adjusted with a required volume of 0.8% NaCl,

(pH, 6.5).

Characterization of Mps

Morphology, size, and zeta potential Th e surface morphology of the particles was observed using

scanning electron microscopy (SEM) (JEOL 6100, Japan). Th e

freeze-dried powder of Mps was placed on the sample hold-

ers, sputter coated with gold. Th e particles were observed

under scanning electron microscope. Th e zeta potential

and particle size (in quintet) were evaluated using Zetasizer

Nano ZS 90 (Malvern, UK).

FTIR spectroscopic analysis Th e freeze-dried particles were analyzed using FTIR

spectroscopy. Th e IR spectra of the samples were recorded

using a Fourier-transformed infrared spectrophotometer

instrument FT/IR Th ermo Nicolet-380 (USA).

Diff erential scanning calorimetry (DSC) DSC were recorded for further confi rmation of the presence

of alginate coating over CHMp. DSC spectra were recorded

using a diff erential scanning calorimeter (DSC-2000, Dupont,

USA). Particles were placed onto pans and heated from 25

to 350 ° C under a nitrogen infl ux of 20 cc/min. Th e tempera-

ture gradient at the rate of 10 ° C/min was maintained till it

reached 350 ° C.

Loading effi ciency and loading capacity Th e CHMp and A-CHMp were evaluated for the loading

effi ciency and loading capacity. Th e aliquots of the particles

suspension were collected, centrifuged at 14,000 rpm for

30 min, and the protein in the supernatant was determined

using BCA-protein assay. Empty CHMp and A-CHMp were

also treated under similar conditions and were used as nega-

tive control to normalize the background interference for the

correction of the OD value and estimated using BCA protein

assay.

% Loading Efficiency (LE)

Total Amount of An �

ttigen-Free Antigen

Total amount of antigen100

% Loading Ca

�

ppacity (LC)

Total Amount of Antigen-Free

�AAntigen

Weight of Particles100�

SDS – PAGE analysis Th e structural integrity of rPA loaded in A-CHMp was evalu-

ated using sodium dodecyl sulfate polyacrylamide gel elec-

trophoresis (SDS – PAGE). Briefl y, rPA-loaded A-CHMp was

incubated overnight in PBS (pH 7.4) at 37 ° C under mild

agitation (50 rpm). Th e particles were centrifuged at 4000

rpm for 30 min and released antigen was separated from

supernatant and mixed with SDS loading buff er. Sample was

heated at 100 ° C for 5 min and then allowed to cool down to

an ambient temperature. SDS – PAGE was run after loading

native rPA, rPA extracted from formulation, and marker in

separate lane onto SDS electrophoresis assembly (Bio-Rad,

USA) using 5% stacking gel and 10% separation gel, run at

60 – 110 V until the dye band reached the gel bottom. After

migration, the gel was removed and stained with Coomassie

blue to locate the respective position of proteins, which was

then destained.

In vitro release study Th e release rate of antigen from CHMp and A-CHMp was

determined in simulated intestinal fl uid (SIF: pH, 6.8) and

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Oral Anthrax Vaccine 3

simulated gastric fl uid (SGF: pH, 1.2) prepared according

to USP25 NF20. Th e Mps were incubated with the release

medium (SIF and SGF) and kept at 37 ° C in a shaker bath at

50 rpm. Th e samples were withdrawn at regular time inter-

vals, and the amount of sample withdrawn was replaced with

the similar volume of particles suspension kept under simi-

lar conditions to maintain the sink condition. Th e samples

were centrifuged at 14,000 rpm for 30 min and the protein

content was determined using BCA-protein assay. Simulta-

neously, control CHMp and A-CHMp suspensions were also

subjected to the same conditions and kept as blank so as to

remove background interference and estimated using the

BCA-protein assay.

In vitro mucoadhesion measurements A Franz diff usion cell (Permeager, USA) with a donor cham-

ber was modifi ed as described by Rossi et al. (1999) and

Bonferoni et al. (1999). Briefl y, in the donor chamber, a stream

of buff er was maintained through two holes. Th e incoming

buff er fl ux was regulated by means of an HPLC pump (model

300, Gynkotek, Munich, Germany). Th e outcoming buf-

fer was collected in a beaker and continuously stirred. Rat

jejunum tissue was placed between the donor and acceptor

chambers of the cell laying on a fi lter paper disc imbibed in

HBSS, in turn, placed on a Parafi lm membrane (impermeable

to fl uids). Th e receptor chamber of the cell was fi lled with

distilled water whose only function was to keep the jejunum

tissue thermoregulated. Th e A-CHMp suspension (500 μ l)

was placed on the excised rat jejunum (area � 2 cm 2 ) tissue,

and physiologic solution (NaCl 0.9% w/v) at 37 ° C was fl uxed

at 0.7 mL/min over the formulation to mimic the washing

action of intestine fl uids. Five hundred-microliter samples

of the fl uid outcoming from the donor chamber were with-

drawn at fi xed times. Th e amount of FITC-BSA ‘ ‘ washed

away ’ ’ was determined in a receptor beaker at defi ned times

by means of a spectrofl uorimetric method. Th e amount of

FITC-BSA was not removed by the buff er stream adhered

and interacted with the biological substrate providing an

indirect measure of the mucoadhesion.

Peyer ’ s patch uptake study Fluorescence microscopy was performed to confi rm the

deposition of particulate carriers in peyer ’ s patch. FITC-BSA

was used as a fl uorescent marker and was entrapped into

the Mps. Mice were administered with 100 μ l of 7.5% w/v

sodium bicarbonate solution to neutralize the gastric acid.

Th e fl uorescent-loaded A-CHMp was then administered

orally to BALB/c mice. After 2 h the mice was sacrifi ced, its

intestine was removed, and peyer ’ s patches were identifi ed

as gray nodule and isolated to determine the uptake of the

carrier. Th e peyer ’ s patch-dominated region of the intestine

was microtomed, and the section(s) were observed under a

fl uorescent microscope (Nikon Eclips E-200, Japan). Mice

receiving FITC-BSA in PBS (pH 7.4) was kept as control.

Immunization studies Female BALB/c mice (CDRI, Lucknow) 8 – 10 weeks old,

weighing 20 – 25 g, were used to assess the immunogenicity of

developed formulations, as they are well established for the

immunization studies. Mice were housed in group (n � 6)

one week before the experiments for acclimatization, with

free access to food and water. Th e Institutional Animals Ethi-

cal Committee of Dr. Hari Singh Gour University approved

the protocols which were conducted following the guidelines

of the Council for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Ministry of Social Justice

and Empowerment, Government of India. Animals were

withdrawn of any food intake 2 h before immunization. Mice

were pre-administered with 100 μ l of 7.5% sodium bicar-

bonate solution prior to the administration of rPA-loaded

A-CHMp in order to neutralize the gastric acid. Group I was

kept control, and rPA-loaded A-CHMp (PBS: pH, 7.4) was

administered orally in four diff erent doses (equivalent to 10,

25, 40 and 55 μ g of rPA) to four separate groups (II – V); group

VI received 100 μ g of soluble rPA (PBS: pH, 7.4) orally, while

group VII was administered with 10 μ g of alum-adsorbed

rPA subcutaneously (s.c.). All the groups received a booster

dose on day 21.

Sample collection Blood samples were collected periodically from the retro-

orbital plexus under mild ether anesthesia on days 0, 14,

28, and 42. Serum was separated and stored at � 40 ° C until

tested using ELISA for antibody titer. Th e gastric, vaginal,

and salivary secretions were collected on days 0, 21, and 42.

Th e vaginal washes were obtained according to the method

described by Debin et al. (2002). Briefl y, the vaginal tract of

non-anesthetized mice was fl ushed with 50 μ l of PBS con-

taining 1% (w/v) BSA using a Gilson pipette. Th ese 50- μ l

aliquots were withdrawn and used for repeated vaginal

fl ushing of nine times. Intestinal lavage was performed

using the technique given by Elson et al. (1984). Briefl y,

four doses of lavage solution [NaCl (25 mM), Na 2 SO 4

(20 mM), and polyethylene glycol of molecular weight,

3350 (48.5 mM)] were given orally at 15-min interval using

a blunt-tipped needle. After 30 min of the last dose of

lavage solution, the mice were injected intraperitoneally

(i.p.) with 0.2 mL pilocarpine (10 mg/mL) to induce the

gastric motility, causing discharge of intestinal contents.

Th e intestinal discharges occurred for next 20 min were

collected regularly and carefully. Salivary secretion was

collected using the method developed in our laboratory

with a minor modifi cation (Jain et al. 2005). A volume of

0.2 mL of pilocarpine (10 mg/mL) was given intraperitone-

ally to induce salivation. After 20 min, the salivary samples

were then collected from mice using capillary tube. All

secretions were stored with 100 mM phenylmethyl sulfonyl

fl uoride (PMSF) at � 40 ° C until tested for secretory anti-

body (sIgA) levels using ELISA.

Measurement of anti-PA IgG and IgA antibodies Specifi c anti-PA antibodies in serum/secretions were esti-

mated using ELISA kit (Sigma, USA). Each well of 96-well

fl at-bottom immunoplate (Nunc-Immuno Plate ® Fb 96

Mexisorb, NUNC) was coated with 100 ng of rPA in 100 μ l

of carbonate buff er (pH, 9.6) after overnight incubation at

4 ° C. Th e plates were then washed thrice with PBS-Tween

20 (PBS-T). Th e free sites were blocked by incubating with

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4 S. Mangal et al.

resulted in the complete inversion of zeta potential of the

CHMp (�29.7 � 5.5 mV). Th is gives a qualitative indication

of coating of a positively charged CHMp with a negatively

charged alginate. Th e antigen was loaded onto preformed

CHMp, and high percent antigen loading was recorded

(Table I). Th is adsorption of antigen was mainly caused by

the ionic interaction between amine group of positively

charged chitosan and carboxyl group of negatively charged

antigen substrate. Th e results also indicated that the alginate

coating over antigen-bearing CHMp did not aff ect the

antigen loading to a large extent (Table I).

FTIR analysis Th e FTIR spectrum of native chitosan showed peaks near

1650 cm � 1 and 1430 cm � 1 indicating the presence of C � O

stretching and N-H stretching of amide, respectively. Th e

CHMp also demonstrated an absorption peak near 1650

cm � 1 , whereas the peak due to N-H stretching was almost

disappeared (Figure 2A), which may be attributed to the

interaction of counter ion with the primary amino groups

of the chitosan (Xu and Du 2003). Th e native sodium alg-

inate demonstrated a strong peak near 1640 cm � 1 and 1450

cm � 1 which correspond to the carboxylate salt present in

the gluronic and mannuronic acid residue and carboxylate

group, respectively. Th e similar peaks were observed in the

case of A-CHMp, which indicates the association of alginate

with CHMp.

Diff erential scanning calorimetry Th e DSC spectrum of chitosan (Figure 3) revealed an endo-

thermic peak near 100 ° C and an exothermic peak near

200 μ l of 1% w/v solution of BSA in PBS-T (Blocking buff er)

for 2 h at 37 � 1 ° C. Th e Maxisorb plates were then washed 6

times with PBS-T. One hundred microliters of serially diluted

mice sera/secretions samples (PBS: pH, 7.4; 0.05% Tween-20;

and 1% BSA) was added to each well and incubated for 2 h

at 37 ° C. Th e wells were then washed with washing buff er,

and specifi c antibodies in the serum/secretion samples

were determined by the addition of 100 μ l of horse-radish

peroxidase-conjugated goat anti-mouse IgG and IgA (diluted

to 1:1000 in PBS – BSA), respectively. Th e wells were washed

fi ve times with washing buff er. Th en, 100 μ l of tetramethyl-

benzidine (TMB-H 2 O 2 ) was added as a substrate to produce

color. After 15 – 20 min of incubation, 50 μ l of H 2 SO 4 (1N)

was added in each well to stop the reaction. Color produced

within 15 min was measured using ELISA plate reader (Bio-

Rad) at 450 nm. End-point titer was expressed as the log

reciprocal of the last dilution, which gave an optical density

(OD) at 450 nm above the optical density (OD) of negative

controls. Similarly, antibody isotyping response (IgG1 and

IgG2a) was determined by ELISA using sigma isotyping kit

(type II) following specifi cations as per the manufacturer ’ s

recommendations.

Toxin neutralization assay (TNA) Th e lethal toxin (Letx) neutralization ability of various sera

and secretion samples was measured by determining the

capacity of anti-PA antibody to prevent cytotoxicity of Letx

to J774A.1 cells (Singh et al. 1989). A volume of 0.2 mL of

cell suspension (6 � 10 5 �8 � 10 5 cells/mL) was plated onto

96-well cell culture plates. Samples were serially diluted

with PBS (pH 7.4) containing 0.05% Tween-20 and 1% BSA.

Th e protective antigen (rPA) and lethal factor (LF) were

added to the antiserum dilutions at fi nal concentrations

of 5 and 2 μ g/mL, respectively. After incubation, 10 μ l of

antiserum – toxin mixture was added to J774A.1 cell suspen-

sion. Th e plates were incubated for 5 h at 37 ° C under 5%

CO 2 . MTT assay was conducted in order to monitor the cell

viability by taking the absorbance at 540 nm (Mosmann

1983). Th e end point was defi ned as the highest antibody

sample dilution that exhibits OD above that of the control.

Neutralizing-antibody titer was expressed as the reciprocal

end-point dilution.

Statistical analysis Th e results were expressed as mean � standard deviation.

Student ’ s t -test was carried out for statistical analysis of data

obtained, and the statistical signifi cance was designated as

p � 0.05. Multiple comparisons were made using one-way

analysis of variance (ANOVA) followed by post hoc analysis

by applying Tukey – Karmer post-test.

Results

Size, morphology, and zeta potential analysis Th e SEM photomicrograph revealed that both uncoated

and coated CHMp had smooth surface and almost spherical

shape (Figures 1A and 1B). Th e particle size and zeta potential

of CHMp were found to be 667 � 119 nm and 34.22 � 3.98 mV,

respectively (Table I). Th e coating CHMp with alginate Figure 1. SEM photomicrograph. (A) CHMps, (B) A-CHMp.

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Oral Anthrax Vaccine 5

250 ° C, which may be due to the evaporation of water and the

onset of the chitosan degradation, respectively. Th e CHMp

demonstrated two endothermic peaks at 235 ° C and 275 ° C,

which may be related to the breakdown of weak unspecifi c

interaction and the cleavage of electrostatic bond between

sulfate ion and polymer, respectively. Th e native sodium

alginate demonstrated an exothermic peak near 250 ° C

which may be attributed to the breakdown of alginate. On

the other hand, A-CHMp revealed no peak in this region;

instead, a slow exothermic raise was manifested near 200 ° C,

which may be attributed to the contribution of two phenom-

ena: the exothermic behavior of alginate and the endother-

mic behavior of the CHMp. Th e results obtained were in line

with the report previously published (Borges et al. 2005,

Gonzalez-Rodriguez et al. 2002).

SDS – PAGE analysis Previous studies have reported the use of biodegradable

particles of PLGA or PLA as a vaccine carrier. Encapsula-

tion of antigen in these carriers requires the use of organic

Table I. Table showing particle size, zeta potential, % loading effi ciency and % loading capacity.

System Zeta potential (mV) Particle size (nm) P.D.I * % LE % LC

Chitosan particles 34.22 � 3.98 667 � 119 0.241 � 0.092 � � Antigen-adsorbed particles 28.82 � 5.27 691 � 125 0.272 � 0.072 79.44 � 7.11 36.78 � 7.34Alginate-coated chitosan particles � 29.7 � 5.5 714 � 167 0.293 � 0.132 70.23 � 4.13 31.61 � 2.11

Results are expressed as mean � SD (n � 5). P.D.I * : polydispersity index; % LE: % loading effi ciency; % LC: % loading capacity.

Figure 2. FT-IR spectra. (A) Figure Ccomparing FTIR spectra of chitosan and CHMps; (B) Figure Ccomparing FTIR spectra of CHMps, sodium alginate, and A-CHMps.

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6 S. Mangal et al.

into the release medium. However, A-CHMp could prevent

the burst release of antigen in both SIF and SGF (Figure 5).

In vitro mucoadhesion measurements Figure 6 shows the amount of FITC-BSA washed away vs

time of FITC-BSA and FITC-BSA associated with A-CHMp

using the rat jejunum tissue as a biological substrate. Th e

results showed that FITC – BSA washed away rapidly indicat-

ing no mucoadhesion. On the other hand, the washing rate

of FITC – BSA associated with A-CHMp was signifi cantly low

as compared to that of FITC – BSA. Th is study indicated that

the A-CHMp was mucoadhesive in nature and could prolong

the residence time of antigen in gastrointestinal tract.

Peyer ’ s patch uptake study It has been reported that particles smaller than 10 μ m are

selectively taken up by M cells (Eldridge et al. 1990, Smith

et al. 1995). Th ese M cells transport the antigen-loaded car-

rier to the underlying gut-associated lymphoid tissue (GALT),

that is, peyer ’ s patch. Th e results indicated that orally admin-

istered soluble FITC – BSA (PBS, pH 7.4) could not produce

any fl uorescence in the peyer ’ s patch (Figure 7A). However,

orally administered A-CHMp-associated FITC-BSA could

demonstrate fl uorescence in peyer ’ s patch indicating the

uptake/deposition of dye-loaded A-CHNp into the peyer ’ s

patch (Figure 7B).

Anti-PA antibody titer Th e anti-PA IgG titer of animals immunized with diff erent

formulations is shown in Figure 8A.

We fi rst determined the optimal dose of rPA for oral

immunization of mice. It was observed that oral delivery of

free rPA-induced low plasma anti-PA antibody responses

indicating that rPA itself was poorly immunogenic when

administered orally. However, rPA in A-CHMp demonstrated

substantially higher antibody titer in serum when compared

to free rPA at all tested dose. It was also observed that the

antibody response with A-CHMp was typically dose depen-

dent and higher rPA dose resulted in the higher antibody

titer. Th e ELISA results indicated that the serum antibody

titer increased signifi cantly when the dose of A-CHMp-

associated rPA was increased from 10 to 25 μ g. On the other

hand, 25, 40, and 55 μ g of A-CHMp-associated rPA demon-

strated comparable serum IgG titer. Th e alum-adjuvanted

rPA resulted in the induction of strongest antibody titer in

serum when compared to all other formulations.

Specifi c mucosal immunity structured as sIgA can pre-

vent the attachment of infectious pathogen to gastrointesti-

nal (GI) mucosa (Jain et al. 2006, Medina and Guzm á n 2000).

Both nasal and oral immunizations are well established to be

the most reliable strategy for inducing mucosal immunity for

optimal protection of mucosal surfaces. Th e results indicated

that alum-adsorbed rPA and free rPA could induce minimal

antibody titer in all examined (local and distal) secretions.

Th e A-CHMp-associated rPA resulted in the induction of

substantially higher secretory antibody titer when compared

to free rPA and alum-adsorbed rPA. Th e secretory antibody

titer was also found to be dose dependent. It was observed

that the secretory antibody titer was substantially higher in

solvent and high shearing conditions that may require the

use of additional care to protect antigen (Jaganathan et al.

2005). However, in this study, the antigen was loaded onto

chitosan particles by simple incubation of antigen with pre-

formed particles to avoid encounter of antigen with such

harsh conditions. SDS – PAGE (Figure 4) of the rPA isolated

from the Mps demonstrated a band at the position (83 kDa)

identical to that of native antigen. Th is confi rmed that the

preparation conditions did not cause any irreversible aggre-

gation or cleavage of the protein.

In vitro release studies Th e in vitro release study indicated that CHMp exhibited

burst release in both SGF and SIF (Figure 5). It was observed

that approximately 80% and around 60% of the antigen was

released within fi rst 30 min in both SGF and SIF, respectively.

Th is may be attributed to the rapid desorption of antigen

Figure 3. DSC spectra. Figure comparing DSC spectra of chitosan, CHMps, sodium alginate, and A-CHMps.

Figure 4. SDS – PAGE Analysis. SDS – PAGE showing stability of antigen isolated from particulate formulation. Lane 1: Marker proteins (205-kDa myosin, rabbit muscle; 97-kDa phosphorylase B; 67-kDa BSA; 43-kDa ovalbumin; and 29-kDa carbonic anhydrase); Lane 2: native rPA (83 kDa); and Lane 3: rPA isolated from A-CHMps (83 kDa).

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Oral Anthrax Vaccine 7

Antibody subtyping To determine the type of immune response being stimu-

lated, the levels of two subtypes of antibodies, that is,

IgG1 and IgG2a, were assessed in mice sera on day 42

(Figure 10). IgG2a is indicative of Th 1-type (cellular)

response, whereas IgG1 is indicative of the presence of Th 2

(humoral) response. Th e results indicated that IgG1 subtype

constitute the major fraction and was signifi cantly higher

as compared to IgG2a subtype ( p � 0.05). Th is indicated

that the immune response was Th 2 based (humoral)

regardless of the formulation composition and the route of

immunization in this study.

Discussion

It has been found that the upper respiratory tract and other

mucosal tissues are aff ected after anthrax spore exposure

through mucosal sites (Abramova et al. 1993, Grinberg

et al. 2001). Th erefore, optimal immune protection against

anthrax (inhalational and gastrointestinal) requires two lay-

ers of defense in the form of mucosal and systemic immune

response to check the infection in early stages (Welkos et al.

2001, 2002) and the cutaneous anthrax. In this study, we

tested the effi cacy of a carrier-based subunit vaccine for

oral administration with the intent to induce mucosal and

systemic immunity against anthrax.

It has been reported that recombinant antigens are less

immunogenic in nature, and hence, they need adjuvant

to enhance their immunogenicity (Boyaka et al. 2003).

Polymeric particulate carriers are considered to be of spe-

cial interest for this purpose as these carriers are reported

to enhance the uptake of loaded antigen by antigen-

presenting cells (APCs) and consequently the presentation

other cells of immune system for the elicitation of strong

immune response. Th e results in this study indicated that

soluble rPA was poorly immunogenic when administered

orally even at higher dose (100 μ g). Our results showed that

the case of CN25 when compared to CN10. However, CN25,

CN40, and CN55 demonstrated comparable secretory anti-

body titer in local and distal secretions.

Neutralizing antibody titer Th e functional signifi cance of both plasma and secretory

anti-PA antibody was analyzed using the in vitro toxin

neutralization assay (Figures 9A and 9B) (Reuveny et al.

2001, Williamson et al. 1999). It was observed that A-CHMp

demonstrated strong neutralizing antibody titer in serum

as compared soluble rPA (Figure 9A). Th e neutralizing anti-

body titer increased amongst the group fed with various

dosage of Mps-associated rPA following the order: soluble

rPA � CN10 � CN25 � CN40 � CN55. Results indicated

that the alum-adjuvanted rPA could induce substantially

high neutralizing antibody titer as compared to A-CHMp at

equivalent dose. Th e neutralizing ability of anti-PA sIgA of

various secretions was also determined (Figure 9B). It was

observed that A-CHMp could induce signifi cantly high neu-

tralizing antibody titer in various local and distal mucosal

secretions when compared to alum-adjuvanted rPA and

soluble rPA.

Figure 5. In vitro release . Graph showing percentage of antigen release with respect to time in SGF (pH, 1.2) and SIF (pH, 6.8).

Figure 6. In vitro Mucoadhesion Measurement. Graph showing FITC – BSA amount washed away vs time.

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8 S. Mangal et al.

Figure 7. Fluorescence photograph of mice peyer ’ s patch. (A). Mice treated with FITC-BSA solution; (B). Mice treated with A-CHMps loaded with FITC – BSA. Hot-Spot indicated by arrows showing the uptake of A-CHMps by peyer ’ s patch of mice.

Figure 8. Anti-PA Antibody Titer. Graph showing anti-PA antibody titer in serum [A] and secretion [B]. Anti-PA antibody titer is expressed as the reciprocal dilution titers � SE (n � 6), which gave an optical density (OD) above negative control. Abbreviation used in graph indicated the various groups used for the study and are as follows: rPA – Group fed with soluble rPA (100 μ g), CN10 – Group fed with A-CHMps containing 10 μ g rPA, CN25 – Group fed with A-CHMps containing 25 μ g rPA; CN40 – Group fed with A-CHMps containing 40 μ g rPA and CN55 – Group fed with A-CHMps containing 55 μ g rPA, Alum-adsorbed rPA – Group injected s.c. with 10 μ g of alum-adjuvanted rPA. Asterisk over bars indicated degree of signifi cance. Where, [ * � p � 0.05; * * � p � 0.01; * * * � p � 0.001; ns � not signifi cant].

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Oral Anthrax Vaccine 9

indicated that A-CHMp demonstrated mucoadhesive ability

and may prolong the residence time of the antigen in GIT

and subsequently promote the uptake of A-CHMp into

GALT. Our study also confi rmed the deposition of the selected

carrier in peyer ’ s patch. Th erefore, It was concluded that

alum-adjuvanted rPA could induce substantially higher

antibody titer in serum but it fails to induce specifi c secre-

tory immune response (sIgA) at the mucosal level. However,

A-CHMp could induce strong immunological response

in both systemic and mucosal compartments. Th e results

Figure 9. Neutralizing Antibody Titer. Graph showing neutralizing antibodies titer in serum [A] and secretions [B]. End-point titer is expressed as the reciprocal dilution titers � SE (n � 6), which gave an optical density (OD) above negative control. Note: Free rPA and alum-adsorbed rPA could not generate detectable neutralizing antibody titer in examined secretions.

Figure 10. Antibody Subtyping. Graph showing anti-PA IgG1 and IgG2a titers. End-point titer is expressed as the reciprocal dilution titers � SE (n � 6), which gave an optical density (OD) above negative control.

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10 S. Mangal et al.

the A-CHMp may enhance the uptake, presentation, and

processing of antigen in the gut immune inductive sites for

effi cient immune induction. It is worth noting that strong

neutralizing antibody titer was determined in serum and

secretions of mice fed with A-CHMp-associated rPA. How-

ever, neither alum-adjuvanted rPA nor soluble rPA could

induce neutralizing antibody titer in mucosal secretions.

Th us, the potential benefi t of the developed vaccine is the

induction of specifi c s-IgA in various secretions that may also

interfere with the germination of B. anthracis spores and/or

favor spore uptake by phagocytic cells (Boyaka et al. 2003).

It was also observed that the magnitude of immunological

response in mice immunized with A-CHMp was typically

antigen dose dependent and higher dose of rPA resulted into

the potentiation of the anti-PA antibody titer in serum and

secretions. To further characterize the nature of immune

response, we analyzed the antibody sub-typing pattern. It was

observed that IgG1 was the predominant subtype obtained

in the serum of all animal groups immunized with rPA. Th is

indicates that the developed vaccine resulted in the

induction of humoral-based immune response. It was con-

cluded that the immune mechanisms stimulated remain

identical in mice immunized with diff erent rPA-based

formulations.

Conclusion

In summary, A-CHMp is an effi cient mucoadhesive car-

rier adjuvant, which could prolong the residence time of

loaded antigen in the GIT and subsequently resulted in

better uptake by M cells. Th is consequently results in the

prolonged activation of professional APC in GALT and

much effi cient immune induction. Our results clearly

indicate that A-CHMp represents a promising mucoad-

hesive carrier adjuvant for eff ective anthrax vaccine that

provides strong immune response in systemic and mucosal

compartments.

Acknowledgement

We are thankful to Dr. Yogendra Singh (IGIB, New Delhi)

for providing recombinant Protective Antigen and Lethal

Factor and Raj Kurupati (IGIB, New Delhi) for his coopera-

tion, support, and help. We are thankful to Indian Institute

of Technology (Bombay), All India Institute of Medical

Sciences (AIIMS) and Punjab University (PU) for providing

SAIF facility. Th e authors would also like to acknowledge

All India Council of Technical Education (AICTE) for pro-

viding Junior Research Fellowship (JRF) to carry out the

research work.

Declaration of interest

Th e authors report no declarations of interest. Th e authors

alone are responsible for the content and writing of the

paper.

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