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)
1
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.
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
0
Sorum l
g
G tite
r
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)
60
50
40
30
20
10
0
Fecal sIgA tite
r
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)
(c) (d)
(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
0
sIgA secretion cells (gray value)
Group
A
Group B
Group C
Group
D
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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