Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat

doi:10.4236/ijcm.2012.35073

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International Journal of Clinical Medicine, 2012, 3, 387-393

http://dx.doi.org/10.4236/ijcm.2012.35073 Published Online September 2012 (http://www.SciRP.org/journal/ijcm)

Oral Administration Recombinant Bifidobacterium-LTB

(B Subunit of Heat-Labile Enterotoxin) Enhances the OVA

(Ovalbumin)-Specific sIgA in Jejunal Mucosa of

Sprague-Dawley (SD) Rat

Yong-Ping Ma*, Ya-Ning Hao, Wei Tang, Rong-Rong Wang, Fa-Ping Yi, You-Quan Bu, Lu-Yu Zhang,

Fang-Zhou Song

Key Laboratory of Biochemistry and Molecular Biology, The Molecular Medicine and Cancer Research Center, Chongqing Medical

University, Chongqing, China.

Email: *[email protected]

Received June 19th, 2012; revised July 20th, 2012; accepted July 30th, 2012

ABSTRACT

The LTB of enterotoxigenic Escherichia coli (ETEC) expressed in Bifidobacterium infantis (BI) has been testified as

mucosal adjuvant with co-vaccination BI-CfaB (the major fimbrial subunit) together in vivo in our previous study. In

order to investigate the mucosal adjuvant effect of BI-LTB to purified antigens, we oral vaccinated SD rats with recom-

binant BI-LTB plus OVA (rBI-LTB + OVA), and wild type BI plus OVA (wBI + OVA), OVA and PBS (Phosphate

buffered saline) were vaccinated as controls, respectively. The OVA-specific sIgA in jejunal mucosa and specific IgG

in serium were measured with ELISA (Enzyme-linked immunosorbent assay) and the sIgA producing cells were de-

tected with immunohistochemistry technology (IHC) and Qwin image manipulation tools subsequently. The results

shown rBI-LTB could stimulate SD rats produce high titer OVA-specific sIgA in rBI-LTB + OVA group and the

OVA-specific sIgA titer in rBI-LTB + OVA group was found significant greater than that of the wBI + OVA group or

OVA single group (p < 0.05). However no such significant difference was detected between the group wBI + OVA and

OVA. IHC results suggested that intestinal mucosa and submucosa was the main field of sIgA secretion. These results

suggested that recombinant LTB expression in BI could be used as a wide range mucosal adjuvant with different form

antigens.

Keywords: Bifidobacterium infantis; LTB; OVA; Mucosal Adjuvant

1. Introduction

The LTB of Escherichia coli is one of bacterial products

with the greatest potential to function as mucosal adju-

vant. LTB belongs to ADP-ribosylating enterotoxins

(cholera toxin and the heat-labile enterotoxin of E. coli).

Both LTB and CTB (cholera toxin B subunit) recognize

and bind to mucosal cell surface via their receptor

GM1-ganglioside existed ubiquitously on the surface of

mammalian cells [1-3]. The adjuvanticity of LTB has

been directly related to GM1-binding activity and the

interaction between LTB and the receptor activates B and

CD4+ T– cells; and enhances antigen presentation by ac-

tivating DCs (Dendritic cells) and other APCs (antigen

presenting cells) through receptor-mediated endocytosis

mechanisms [4]. Scientists explain that these molecules

exert their adjuvant function by interacting with a variety

of cell types, including epithelial cells and DCs, etc. [5].

LTB activates selective differentiation of lymphocyte

populations and increases presentation on MHC (Major

histocompatibility complex) class II, which may be the

basis for its adjuvant effect [6-8].

LTB and cholera toxin B (CTB) proteins have been

successfully expressed in different expression systems,

such as intracellular production in Bifidobacterium infan-

tis (BI), Mycobacterium bovis and Lactobacillus or Ba-

cillus brevis surface-displaced on Staphylococcus xylosus

and S. carnosus, or as a secreted protein in a yeast ex-

pression system [9-15].

Lactic acid bacteria (LAB) have proved to be effective

mucosal delivery vehicles that overcome the problem of

delivering functional proteins to the mucosal tissues. BI,

a special LAB, is a Gram-positive, probiotic and ana-

erobic bacterium belonging to Bifidobacterium in human

intestinal [16]. We testified BI is suitable to human oral

*Corresponding author.

Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances

the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat

388

vaccine development and the recombinant BI-LTB

(rBI-LTB) co-vaccination with recombinant BI-CfaB of

ETEC in vivo has mucosal adjuvant function in previous

study [9]. However, we do not know whether rBI-LTB

confers adjuvantity to purified antigens. The aim of the

present study is to investigate the mucosal adjuvant ef-

fect of oral administration rBI-LTB on purified antigen

OVA.

2. Materials and Methods

Bacterial strains and growth conditions. E. coli DH5α

strain was propagated in LB medium (Luria-Bertani) and

used as host cell for molecular cloning; pBEX-LTB was

constructed as described previously [9].

BI strain was cultured in MRS broth (Difco) con-

taining 0.25% l-cysteine-HCl (w/v, pH 7), at 37˚C under

anaerobic conditions. 50 μg·mL–1 of ampicillin was

added to both recombinant BI and E. coli strains when

required.

Animals and Immunization. Sprague-Dawley (SD)

rats of 3 - 4 weeks of age (male) were obtained from the

experimental animal center (Chongqing Medical Univer-

sity, China) and divided into four groups as A, B, C and

D. After treating with saturated sodium bicarbonate (0.3

ml each rat), the rats were intragastrically vaccinated

three times on day 0, 7 and 14 with rBI-LTB + OVA,

wBI + OVA, OVA and PBS, The BI dose was 1.0  1010

CFU mL–1 and that of OVA was 10 μg per rat on 3 occa-

sions (Table 1).

Immunological assay (IMA). Blood samples were

individually collected from immunized rats by a tail

bleed on days 0, 7, 14 and 21 for the analysis of systemic

OVA-specific antibodies (10e10 bateria, on 3 occasions).

Fresh fecal pellets were individually collected lyophi-

lized from the same rat groups on days 0, 7, 14 and 21.

The samples were treated as previous described [9] (Ma

et al., 2011). The supernatants were analyzed for

OVA-specific sIgA levels to study the secretory mucosal

immune response. Both the serum was analyzed with

goat anti-rat IgG and the fecal specific antibody titers

were analyzed with goat anti-rat IgA by ELISA, respec-

tively. OVA (1.0 μg·mL–1) was diluted in a coating

buffer (50 mM sodium carbonate, 30 mM sodium bicar-

bonate, pH 9.6) and allowed to adsorb to 96-well plates

Table 1. The SD rats and the vaccination groups.

Groups (rats) Vaccinations agents Dose ((C.F.U. mL–1) + μg OVA)

A (n = 12) rBI-LTB + OVA 1.0  1010 + 10 μg

B (n= 12) wBI + OVA 1.0  1010 + 10 μg

C (n = 12) OVA 10 μg

D (n = 10) PBS /

(1.0 μg each well) overnight at 4˚C. The plates were

blocked with 5% skim milk for 1h at room temperature

and a twofold serial dilution of serum was added. After

incubation, the plates were washed three times with PBS

with 0.05% Tween 20 (PBS-Tween), and then 1.0

mg· mL –1 of goat anti-rat IgG mAb or anti-rat sIgA mAb

conjugated to HRP (1.0 μg·mL–1, BOSTER, China) was

added and incubated again.

The OD450 value was measured on a Molecular De-

vices SpectroMax Plus spectrophotometer. Endpoint titers

were determined as the dilution of each sample showing

a 2.1-fold higher level of absorbance at 450 nm than that

of the negative control samples. Average OD450 values

for the animals were calculated.

Tissue samples preparation. Following collection of

blood and fecal samples, two of vaccinated rats were

randomly anaesthetised and sacrificed from each group

and the jejunum samples were isolated from each rat on

days 7, 14 and 21, respectively. Following wash with

PBS, the jejunum samples were fixed with 4% polyoxy-

methylene for 24 h. Then the fixed jejunums were rinsed

with 95% ethyl and successively dehydrated 10 m with

ethyl from concentration 90%, 95% and 100 %, respec-

tively. After treating with dimethylbenzene, the samples

were embedded 4 h with paraffins at 56˚C and cut into

slices (<5 μm). Then the slices were treated with acetone

containing 1% APES (3-aminopropyl triethoxysilane)

and dried at 37˚C.

Immunohistochemical staining sections (IHCS). 1)

The prepared slice samples were twice treated 40 m with

dimethylbenzene to dewax and then successively dehy-

drated with ethyl from concentration 100%, 95%, 90%,

80% to 70 % for 30 s, respectively; 2) Following rinse 5

m with PBS three times, each slice was incubated 30 m

with 50 μL peroxidase blocking solution (198 mL metha-

nol + 2 mL 30 % hydrogen peroxide) at 25˚C; 3) After

rinsing 5 m with distilled water, the slices were put into

0.01 M citric acid solution (pH 6.0) and heat 15 m at

95˚C. Then, the slices were naturally cooled to 25˚C and

rinsed with PBS three times; 4) Incubated with OVA (1.0

μg·mL–1) at 37˚C for 3 h and then rinsed 5 m with PBS

three times; 5) Following blocked with 5% skim milk,

each slice was incubated at 37˚C for 30 m and then

sucked up the serum with filter paper; 6) Incubated with

OVA antibody (goat anti-OVA/HRP) at 37˚C for 3 h and

then rinsed 5 m with PBS three times; 7) Incubated 3 m

with 100 μL DBA solution and rinsed with tap water; 8)

Following counterstain 3 m with hematine, then rinsed

with tap water and overnight at 50˚C; 9) Coverslip with

neutral balsam.

ICH analysis (ICHA). All slices were surveyed and

took pictures with Leica DM2000 microscope. Each slice

randomly selected ten visual fields to statistical analysis

Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances

the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat

389

by Qwin image manipulation tools. The yellowish-brown

value stands for the positive cell area.

Statistical analysis. The data was statistically evalu-

ated by the SPSS 19.0 statistical software package (SPSS

Inc., Chicago, IL) and a value of p < 0.05 was considered

significant.

3. Results

IMA. Serum and mucosal OVA-specific antibody levels

were measured by ELISA. High levels of serum OVA-

speciﬁc IgG were observed on day 14 after the secondary

booster vaccination, with an endpoint titer of 206 and

reached a novel increased level in following booster dose

on day 21 with the titer of 566 in groups A (n = 12).

However, in group B (n = 12), the OVA-speciﬁc IgG

titer just reached to 95 on 14 d and to 183 on 21 d. In the

other way, we did not find the OVA-speciﬁc IgG in

group C (n = 12) and D (n = 10). Statistically, there was

a significant difference in the IgG titer between the

groups A and B for the adjuvant function of LTB (p <

0.05) (Figure 1). The level of variation in the responses

was 20.48 in group A and 20.18 in group B between in-

dividual animals on day 21.

Similar to the serum IgG titer variation, the fecal sIgA

titer in group A was greater than that in group B. High

levels of OVA-speciﬁc sIgA was observed on day 14

after the secondary booster vaccination, with an endpoint

titer of 7.57 and reached a novel increased level in fol-

lowing booster dose on day 21 with the titer of 54.67 in

groups A (n = 12). However, in group B (n = 12), the

OVA-speciﬁc sIgA titer just reached to 3.02 on 14 d and

to 9.70 on 21 d. However, we did not find the OVA-

speciﬁc sIgA in group C (n = 12) and D (n = 10). The

Group A

Group B

Group C

Group D

7 14 21

day (d)

700

600

500

400

300

200

100

Sorum l

G tite

Figure 1. The serum IgG titer in different groups post-im-

munization. The group A rats were immunized orally with

rBI-LTB + OVA, group B with wBI + OVA, group C with

OVA, and group D were treated with PBS (10e10 bateria,

on 3 occasions). The group A shown significant difference,

compared with group B (p < 0.05). However, there was no

statistically significant difference IgG titer between the

group C and group D for no detectable spec i fic IgG.

results suggest that rBI-LTB expressed in BI had muco-

sal adjuvant activity in group A. Statistically, there was a

significant difference in the sIgA titer between the

groups A and B for the adjuvant function of LTB (p <

0.05) (Figure 2). The level of variation in the responses

was 10.88 in group A and 0.61 in group B between indi-

vidual animals on day 21.

ICHA. Examined under a microscope, the OVA-spe-

cific sIgA producing cells were stained in yellow-

ish-brown and distributed in jejunal mucosa, submucosa,

external and internal of intestinal crypt, intestinal lamina

propria and serous membrane (Figures 3 (a)-(h)). How-

ever, the colored positive cells in serous membrane were

obviously fewer than that in other type cells. So the re-

sults suggest that intestinal mucosa and submucosa was

the main field of sIgA secretion.

Analysis the specific sIgA producing cells on 7 d

post-vaccination statistically, we found that the positive

cells were increased appreciably group A than that in

group B, but there was no significant difference each

other. However, the positive cells were increased sig-

nificantly from secondary and third booster in group A

than that in group B (p < 0.05). As was expected, the

positive cells did not find in group C and D (Figure 4).

4. Discussion

Appropriate mucosal adjuvant is essential for oral immu-

nization to elicit immune response. Besides of the

ADP-ribosylating enterotoxins (CT and LTB), the other

two bacterial products, synthetic oligodeoxy-nucleotides

containing unmethylated CpG dinucleotides (CpG ODN),

and mono-phosphoryl lipid A (MPL), were used As

mucosal adjuvants. Both MPL and CpG act through

Group A

Group B

Group C

Group D

7 14 21

day (d)

Fecal sIgA tite

Figure 2. The fecal sIgA titer in different immunization

groups in SD rat. The group A rats were immunized orally

with rBI-LTB + OVA, group B with wBI + OVA, group C

with OVA, and group D were treated with PBS (10e10

bateria, on 3 occasions). The group A shown significant

difference, compared with group B (p < 0.05). However,

there was no statistically significant difference sIgA titer

between the group C and group D for no detectable specific

sIgA.

Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances

the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat

390

(a) (b)

(e) (f)

(g) (h)

Figure 3. Immunohistochemic al evaluation of OVA-specific producing cells in jenunal mucosa. (a), (b) Jenunal mucosal sec-

tions on 14 d and 21 d in group A (magnification 200 and 400, respectively). (c), (d) Sections on 14 d and 21 d in group B

(magnification 200 and 400, respectively). (e), (f) Sections on 14 d and 21 d in group C (magnification 200 and 400, respec-

tively). (g), (h) sections on 14 d and 21 d in group D (magnification 400 and 200, respectively). The OVA-specific sIgA pro-

ducing cells were stained with goat anti-OVA/HRP in yellowish-brown and distributed in jejunal mucosa, submucosa (arrows

indicating mucosal locations of sIgA producing cells).

Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances

the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat

391

7 14 21

day (d)

0.06

0.05

0.04

0.03

0.02

0.01

sIgA secretion cells (gray value)

Group

Group B

Group C

Group

Figure 4. The specific sIgA secretion cells measured with

yellow-brownish value. The positive cells, stained with goat

anti-OVA/HRP, were increased significantly from sec ondar y

and third booster in group A than that in group B ( p < 0.05).

However, the positive cells did not find in group C and D.

MyD88-dependent and -independent pathways and the

adjuvant activities of CpG and MPL are due to several

different effects they have on innate and adaptive im-

mune responses [5].

LTB is a nontoxic molecule with potent biological

properties and is a powerful mucosal and parenteral ad-

juvant that induces a strong humoral and mucosal im-

mune response against co-administered antigen or cou-

pled antigens [8,9,15,17-19]. The adjuvant mechanism of

LTB appears to be related with its capacity to: 1) en-

hance antigen presentation via MHC class I and MHC II;

2) activate selective differentiation of lymphocytes; 3)

influence DCs maturation and activation; 4) induce B7-2

expression on APCs for subsequent co-stimulatory sig-

naling to CD4+ T cells and; 5) increase the expression of

activation markers on B lymphocytes (MHC class II, B7,

CD40, CD25 and ICAM-1) [4,20].

Oral vaccination is based on antigen delivery to the

gastrointestinal tract, the largest mucosal surface and the

central site of IgA secretion [21]. Since the intestinal

mucosa is the natural site of BI colonization, rLTB pro-

ducing in BI easily crosses the epithelial layer to the area

rich in cells of the mucosal immune system. BI-LTB

appears to be one of the best forms of LTB adjuvant de-

livery system [9]. Because of the same reason, the sev-

eral strains LAB has been genetically modified as a

promising oral recombinant vaccine delivery system for

inducing efficient mucosal immunity as well as system-

atic immunity [9,22-26].

Bifidobacterial cell wall preparation (whole pepti-

doglycan, WPG) has been documented with adjuvant

activity and the activity of WPG is related to their ability

to induce a reduction in regulatory T cells (Tregs) activ-

ity [27,28]. A previous document was demonstrated that

LAB might be promising adjuvants in vaccines due to

their capability to reduce functional activity of Tregs,

thereby speeding up vaccine-induced immune responses

[29]. Another document suggests that WPG of bifido-

bacterium induces IL-12 secretion in DCs from bone

marrow and rhIL-12, as adjuvant, has been shown to

augment both cellular and humoral immunity [30-33]. In

this study, we find that wild type BI performed weak

adjuvant activity to OVA oral administration, which

suggests that WPG of BI might be promising adjuvant

activity in OVA.

There are many more experiments have been done in

mice where there is more immunological reagents for

these types of investigation. In this study, we use of the

rat model is novel for oral vaccination. One reason for

choosing rat is that a report used of rat for investigation

of the role of intestinal bifidobacteria on immune system

development [34]. They found that intestinal bifidobacte-

ria plays an important role of development of both the

gut and systemic immunity in early life. Neonatal SD rats

supplemented daily with bifidobacteria could promote

the maturation of DCs and its expression of IL-12 locally

in the gut, favour the development of Th1 response by

increasing the local and systemic expression of IFNγ and

ensure the intestinal Treg response by promoting the lo-

cal expression of IL-10 [34].

There is no purified LT-B plus OVA as a comparable

control to compare the organism expressing LT-B in this

study. One reason is that the nontoxic CT plus OVA or

LT-B plus OVA confirmed that the enterotoxin B subunit

acts as a mucosal adjuvant intranasally immunized

[35,36]. Oral vaccination is not an economically afford-

able way for purified LT-B or other enterotoxin B sub-

unit because it needs high dose enterotoxin B subunit.

However, BI expressing LT-B can easily get over it.

5. Acknowledgements

This study was funded by the National Natural Science

Foundation of China (No. 30972585).

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