prevention and treatment of dna vaccine encoding cockroach

ORIGINAL ARTICLE EXPERIMENTAL ALLERGY AND IMMUNOLOGY

Prevention and treatment of DNA vaccine encoding

cockroach allergen Bla g 1 in a mouse model of allergic

airway inflammation1

B. Zhou1, M. Ensell1, Y. Zhou1, U. Nair1, J. Glickstein1, M. H. Kermany1, Q. Cai1, C. Cai1, W. Liu1,Y.-P. Deng2, A. Kakigi3, M. Barbieri4, M. Mora4, S. Kanangat1 & T. J. Yoo1

1Department of Medicine, University of Tennessee Health Science Center, Memphis, TN; 2Department of Anatomy & Neurobiology,

University of Tennessee Health Science Center, Memphis, TN, USA; 3Department of Otolaryngology, University of Tokyo, Tokyo, Japan;4Department of Otolaryngology, San Martino Hospital University of Genoa, Genoa, Italy2

To cite this article: Zhou B, Ensell M, Zhou Y, Nair U, Glickstein J, Kermany MH, Cai Q, Cai C, Liu W, Deng Y-P, Kakigi A, Barbieri M, Mora M, Kanangat S,

Yoo TJ. Prevention and treatment of DNA vaccine encoding cockroach allergen Bla g 1 in a mouse model of allergic airway inflammation. Allergy 2011;

DOI: 10.1111/j.1398-9995.2011.02727.x

Exposure to cockroaches is one of the major contributing fac-

tors for asthma exacerbation among inner-city children (1–3)

where infestations of the German cockroach are common (4–

6). It was found using skin test that 26.1% of the US popula-

tion is sensitized to the German cockroach (7). The primary

German cockroach allergen, Bla g 1, was detectable in 63% of

homes and 52% of childcare facilities in the United States (8,

9). Bla g 1 from German cockroach is cross-reactive with an

American cockroach allergen, Per a 1 (10–12).

Prominent characteristics of allergic airway inflammation

(AAI) include reduced airflow, airway hyper-responsiveness,

mast cell activation, influx of eosinophils into the lung, and

hyperplasia of goblet cells with excessive mucus secretion (13,

14). These are associated with secretion of cytokines and

chemokines that stimulate a Th2- and IgE-dominated response.

Glucocorticoids are currently the most effective drugs for

treating asthma. However, side-effects remain a significant

problem (15). Allergen-specific immunotherapy (SIT) is the

only curative approach toward allergic diseases (16, 17) and

appears to reduce the incidence of asthma in later life. How-

ever, SIT also has undesirable side-effects and requires inten-

sive medical supervision (17, 18).

DNA-based vaccine strategies have been proven to

induce protective cellular and humoral immune responses

Keywords

asthma; asthma mechanisms; cytokine

biology.

Correspondence

Tai J. Yoo, Department of Medicine of

Medicine, University of Tennessee Health

Science Center, 956 Court Avenue, Room

E332, Memphis, TN 38163, USA.

Tel.: +901 448 7166

Fax: +901 448 5735

E-mail: [email protected]

Accepted for publication 3 September 2011

DOI:10.1111/j.1398-9995.2011.02727.x

Edited by: Angela Haczku

Abstract

Background: One-fourth of the US population is sensitized to the German cock-

roach. Primary German cockroach allergen Bla g 1 is detected in 63% of homes

and 52% of childcare facilities in the United States. No effective treatment or vacci-

nation strategies are yet available.

Objectives: We evaluated the prophylactic and therapeutic efficacy of a plasmid

DNA-mediated vaccination using the Bla g 1 gene in a mouse model of allergic

inflammatory airway disease.

Methods: A plasmid DNA vector coding for the Bla g 1 allergen controlled by cyto-

megalovirus promoter was constructed. To estimate the protective efficacy, BALB/c

mice were given three injections of plasmid DNA–Bla g 1 prior to sensitization with

two priming doses of recombinant Bla g 1 (rBla g 1) antigens, followed by nebulized

rBla g 1 challenge. In the therapeutic approach, sensitization was followed by

administering Bla g 1 DNA vaccine.

Results: Bla g 1 vaccination significantly reduced allergen-induced airway inflamma-

tion, even after mice were presensitized and a Th2-dominant response was estab-

lished. The Bla g 1 vaccination significantly reduced total inflammatory cell

infiltrate, eosinophilia, secretion of Th2 cytokines IL-4 and IL-5 in bronchoalveolar

lavage fluid, allergen-induced inflammatory infiltrates in the lungs, and Bla g 1-spe-

cific IgE in serum upon challenge with rBla g 1. Importantly, Bla g 1 DNA vaccina-

tion was able to induce IL-10-secreting regulatory T cells that could suppress the

allergen-specific Th2 cells.

Conclusion: DNA vaccination showed protective and therapeutic efficacy against a

clinically relevant allergen Bla g 1.

A L L 2 7 2 7 B Dispatch: 16.9.11 Journal: ALL CE: Chandran K.

Journal Name Manuscript No. Author Received: No. of pages: 9 PE: Jeyanthi

Allergy

ª 2011 John Wiley & Sons A/S 1

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against many parasitic, bacterial, and viral diseases (19–21).

Intramuscular injection of plasmid DNA-encoding allergens

is thought to lead to the recruitment of allergen-specific

Th1 cells, which secrete interferon (IFN)-c and possibly

protect against allergic responses by inhibiting the prolifera-

tion and development of Th2 cells and IgE production

(19–21).

Regulatory T (Treg) cells secreting IL-10 (Tr1) have been

shown to have a major role in allergen tolerance (22–25). The

number of Treg cells is decreased in allergic individuals and

presumably results in AAI. Both SIT and glucocorticoids

have been shown to induce CD4+ CD25+ Foxp3+ Treg cells

and IL-10-producing Tr1 cells (26, 27).

Our results show that Bla g 1 DNA vaccination has both

prophylactic and therapeutic effects. Notably, the suppression

of AAI by Bla g 1 vaccine was associated with an induction

of CD25+ CD4+ Foxp3+ Treg cells secreting IL-10, which

could suppress the in vivo-induced Th2 responses in an in vitro

coculture assay.

Material and methods

Molecular cloning, expression, and purification of Bla g 1–

recombinant plasmid

Bla g 1-encoding fragment (GenBank accession codeAF0

72220) was amplified from German cockroach cDNA by PCR

with Bla g 1-specific primers (5‘-CACCATGCCATAGAATT

CCTCAATAA-3‘, forward, and 5‘-TTATGTTGTATAAT

TAATATCTCTTT-3‘, reverse; including Sac I and Xba I

sites). The sequence of the amplified Bla g 1 was verified by

DNA sequencing, and the Bla g 1-encoding fragment was sub-

cloned into pCI-neo expression vector. Expression of the Bla g

1 in mammalian cells was verified by transfecting 1 · 106 of

mouse macrophage cell line AMJ2-C8 (ATCC, Manassas, VA,

USA) with 30 lg of Bla g 1 vaccine by LipofectAMINETM

2000 (Promega, Madison, WI, USA). Two days after culture

in RPMI-1640 medium, culture supernatants and cell lysates

from transfected cells were analyzed by Western blotting (Invi-

trogen, Carlsbad, CA, USA) with anti-cockroach-Bla g 1

monoclonal antibody (Indoor Biotechnologies, Charlottesville,

VA, USA). Large-scale purification of the expression vectors

was conducted with Endo Free Plasmid Maxi kits (Qiagen,

Valencia, CA, USA).

Sensitization, airway challenge, and vaccination

Female BALB/c mice, 6 weeks old (Jackson Laboratory, Bar

Harbor, ME, USA), were intraperitoneally injected with

10 lg of cockroach allergen Bla g 1 (rBla g 1; Indoor Bio-

technologies) together with 4 mg Al(OH) (Serva, Heidelberg,

Germany) in a total volume of 150 ll sterile phosphate buffer

saline (PBS). The experimental mice subsequently received a

daily airway exposure to an aerosol of 1% rBla g 1 in PBS

for 30 min over five consecutive days. The DNA vaccination

was given intramuscularly with 100 lg of plasmid DNA in a

total volume of 150 ll sterile PBS.

Experimental design

The design to evaluate the prophylactic effect of DNA vac-

cination is shown in Fig. 2A. Mice were given three intra-

A B

C

Figure 16 Construction and expression of Bla g 1 DNA vaccine.

Restriction analysis of the recombinant plasmid Bla g 1 (A and B).

When the recombinant plasmid Bla g 1 was cut with Sac I and Xba I,

218-bp Bla g 1 and 5500-bp pCI-neo bands were observed (A), (1)

DNA marker; (2) pCI-neo–Bla g 1 cut with Sac I and Xba I; however,

when it was cut with Xba I, a band of approximately 8 kb was

observed (B). (3) DNA marker; (4) pCI-neo–Bla g 1 cut with Xba I only.

Western blot analysis using Bla g 1-specific antibody (C). The culture

supernatants of AMJ2-C8 cells transfected with either empty vector

(1) or pCI-neo–Bla g 1 (2) were run on a 15% polyacrylamide gel. The

proteins were transferred onto a nitrocellulose membrane. The mem-

branes were probed with mouse anti-cockroach-Bla g 1 monoclonal

antibody, followed by sheep anti-mouse peroxidase-conjugated anti-

body and enhanced chemiluminescence detection; b-actin served as

loading controls.

LOW

RESOLUTIO

NFIG

Genetic vaccination against Bla g 1 Zhou et al.

2 ª 2011 John Wiley & Sons A/S

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muscular injections (days 0, 7, and 14) of Bla g 1 vaccine.

On days 28 and 35 after the first injection, mice were sensi-

tized with rBla g 1. The design to study the therapeutic effi-

cacy of DNA vaccination is shown in Fig. 3A. Mice were

given two intraperitoneal injections (days 0 and 7) of rBla g

1; the nonsensitized mice received PBS instead of rBla g 1/

alum solution. Subsequently, the nonsensitized and sensi-

tized mice were treated with Bla g 1 vaccine on days 21, 28,

and 35. On days 51–55 after the first injection, all mice were

challenged with rBla g 1 by exposing them to an aerosol of

0.1% rBla g 1 for 30 min over a period of five consecutive

days.

Collection of bronchoalveolar lavage (BAL) fluid

Mice were euthanized 24 h after the last challenge. The tra-

chea was cannulated, and the lungs were lavaged twice with

0.7 ml of ice-cold PBS. After centrifugation, the supernatant

was collected, and the levels of cytokines in the BAL fluid

were measured by enzyme-linked immunosorbent assay

(ELISA) (R&D Systems, Minneapolis, MN, USA). The cells

were counted using a hemocytometer. Bronchoalveolar lavage

cell differentials were determined using Wright–Giemsa stain;

‡200 cells were differentiated using conventional morphologic

criteria.

In vitro cytokine production and lymphocytes proliferation

Splenocytes (3 · 105 cells/well) were cultured in 96-well flat-

bottomed plates (Costar, Corning, NY, USA) in RPMI

1640 complete medium supplemented with 5% fetal calf

serum (Gibco, Paisley, UK) and stimulated with rBla g 1

(10 lg/ml). Positive control wells contained 2 lg/ml anti-

mouse CD3 (eBioscience, San Diego, CA, USA), and nega-

tive control wells contained only PBS. Supernatants were

harvested after 48 h for their cytokine measurement (R&D

Systems). Cells were pulsed with [3H]-thymidine during the

last 18 h of the 72-h assay, harvested, and counted for [3H]-

thymidine incorporation (Packard Instrument, Boston, MA,

USA).

Flow cytometry

The CD4+ CD25+ Foxp3+-expressing T cells were identi-

fied by staining splenocytes with PE 3-labeled anti-CD4 and

APC-labeled anti-CD25 (eBioscience). For intracellular

A

B C

D E F

Figure 27 Prophylactic effects of DNA vaccine on rBla g 1-induced

AAI. (A) Prophylactic DNA vaccination scheme. BALB/c mice were

vaccinated on days 0, 7, and 14 with plasmid DNA coding for the

allergen Bla g 15 (pCI-Bla g 1) at doses of 25, 50, 100, 200, and

400 lg or pCI-neo blank vector. On days 28 and 35, mice were

sensitized with rBla g 1. From day 51 to 55, mice were exposed to

an aerosol of 1% rBla g 1 in phosphate buffer saline for 30 min.

Mice were killed 24 h after the last challenge, and serum and

bronchoalveolar lavage (BAL) fluids were collected for IgE and cyto-

kine measurements and cellular analysis. (B–E) BAL fluid was col-

lected and centrifuged. Cells were resuspended to count total cells

(B), and eosinophils (C) were determined using Wright–Giemsa

stain, *P < 0.005 vs control DNA mice; supernatant was collected

for enzyme-linked immunosorbent assay (ELISA) of IL-4 (D) and IL-

5 (E). Serum Bla g 1-specific IgE (F) was determined by ELISA

(modified BD OptEIA ELISA kit; BD Pharmingen, San Jose, CA,

USA). The data represent the means ± SE from five mice/group. P

value vs control DNA mice.

LOW

RESOLUTIO

NFIG

Zhou et al. Genetic vaccination against Bla g 1


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staining of Foxp3, cells were fixed and permeabilized

before incubation with FITC4 -labeled anti-mouse Foxp3

(eBioscience). Appropriate isotype-matched control

antibodies were used to determine nonspecific staining.

Samples were analyzed on a FACScan flow cytometer

(Becton, Dickinson and Company, Franklin Lakes, NJ,

USA).

In vitro suppression assay

CD4+, CD4+CD25+, and CD4+ CD25) T cells were iso-

lated using a mouse Treg isolation kit (Miltenyi Biotec, Berg-

isch Gladbach, Germany). Proliferation assays were

performed by culturing CD4+ CD25) cells (responder,

5 · 104 from splenocytes of rBla g 1-sensitized mice) and

CD4+ CD25+ T cells (suppressor, 5 · 104 from splenocytes

of Bla g 1-vaccinated mice, DNA control mice, PBS-treated

mice, the nonsensitized mice received Bla g 1 DNA, and

naıve mice) in 96-well plates with irradiated antigen-present-

ing cells (APCs, 5 · 104 from splenocytes of normal BALB/c

mice) for 72 h at 37�C in complete medium. Cultures were

stimulated with rBla g 1 (10 lg/ml), and some cocultures

were treated with anti-IL-10 antibodies (10 lg/ml; eBio-

science). After 3 days, 1 lCi/well [3H]-thymidine was added.

Incorporation of [3H]-thymidine was assessed after 18 h of

additional culturing.

Statistical analysis

Statistical analyses were performed using ANOVA or Student’s

t-test.

Results

Expression of Bla g 1 DNA vector

The plasmid DNA vector, the Bla g 1 DNA fragment, and the

expressed 55-kD protein of Bla g 1 are shown in Fig. 1. The

expression levels of the Bla g 1 DNA construct were tested in

AMJ2-C8 cells. Culture supernatants and cell lysates from

transfected cells were analyzed by Western blotting using Bla g

1-specific antibody. The protein was detected in the culture

supernatants (Fig. 1C-2). No Bla g 1 protein was present in

control samples transfected with the empty vector (Fig. 1C-1).

A

B D

C E

Figure 38 Therapeutic effects of DNA vaccine on rBla g 1-induced

AAI. (A) Therapeutic treatment scheme. BALB/c mice received

two intraperitoneal injections (days 0 and 7) of rBla g 1; the non-

sensitized mice received phosphate buffer saline (PBS) instead of

rBla g 1/alum solution. Subsequently, the nonsensitized and sensi-

tized mice were treated with plasmid DNA coding for Bla g 1 or

mock vector at days 21, 28, and 35. From day 51 to 55, mice

were exposed to an aerosol of 1% rBla g 1 in PBS for 30 min.

Mice were killed 24 h after the last challenge, and serum and

bronchoalveolar lavage (BAL) fluids were collected for IgE and

cytokine measurements and cellular analysis. The lungs were

fixed for histological analysis. (B) Absence of cellular infiltration in

the BAL. Bronchoalveolar lavage was extracted, and total cells in

all the groups were counted. (C) Prevention of eosinophilic infiltra-

tion in the lungs. The cells from the BAL fluid were stained with

Wright–Giemsa for differential counts. Data are depicted as the

percentage of eosinophils compared with the total number of

cells counted. (D and E) IL-4 and IL-5 in the BAL. The amounts

of IL-4 and IL-5 in the BAL were determined by enzyme-linked

immunosorbent assay. The data are represented as the mean-

s ± SE from each group containing five mice/group. P value vs

control DNA mice.

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Bla g 1 vaccine prevents cockroach allergen–induced pulmo-

nary manifestations

In the prophylactic approach (Fig. 2A), BAL fluid showed

that the infiltration of total cells (Fig. 2B) and eosinophils

(Fig. 2C) was significantly inhibited at a Bla g 1 vaccine

dose of 100 lg and reached maximal inhibition at 200 lg.

Doses of 25 and 50 lg, however, exhibited a minimal or

low effect on inhibition of infiltration, respectively. In con-

trast, the control mice exhibited a high level of infiltration.

Moreover, Bla g 1-vaccinated mice showed significantly

reduced levels of IL-4 and IL-5 (Fig. 2D,E), which finding

correlated with significantly reduced infiltration of total cells

and eosinophils in the lungs. Furthermore, Bla g 1 vaccine

was able to prevent the induction of Bla g 1-specific IgE

(Fig. 2F). In contrast, the levels of Bla g 1-specific IgE were

high in the control group.

Bla g 1 vaccine suppresses ongoing cockroach allergen–

induced pulmonary manifestations indicating therapeutic

potentials

In the therapeutic approach (Fig. 3A), Bla g 1-vaccinated

mice had markedly lower cellular infiltrates in BAL com-

pared with those in control mice (Fig. 3B). The number of

cells recovered from the BAL of Bla g 1-vaccinated mice was

comparable to the number recovered from normal, untreated

mice and the nonsensitized mice that received Bla g 1 DNA.

Moreover, there was no detectable eosinophilic infiltration in

the mice treated therapeutically with Bla g 1 DNA (Fig. 3C).

This result was also reflected by a decrease in IL-4 and IL-5

levels in the lungs of the vaccinated mice (Fig. 3D,E). Histo-

logical analysis of the lungs showed that the cockroach-sensi-

tized and cockroach-challenged mice that received control

DNA or PBS (Fig. 4B,C) demonstrated the cardinal features

A B

C

E

D

Figure 4 Histological analysis of allergen-challenged mice demon-

strates reduced inflammation in Bla g 1-vaccinated mice. All mice

were sensitized and challenged with rBla g 1 using the therapeutic

approach described in Fig. 3A, with the exception of the mouse

shown in A, which was untreated and unchallenged. Lungs from all

mice were fixed in 10% formalin and embedded in paraffin; sec-

tions were stained with H&E and examined by light microscopy.

Data shown are representative of (A) naive BALB/c mice; (B) BALB/

c mice receiving PBS; (C) BALB/c mice receiving control DNA; (D)

BALB/c mice receiving Bla g 1 vaccine; (E) the nonsensitized

BALB/c mice that received Bla g 1 DNA. 11

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of AAI, including clusters of inflammatory cells surrounding

the airways and bronchiolar epithelium showing columnar

epithelial cell hyperplasia, while the Bla g 1-vaccinated mice

nearly restored these features (Fig. 4D); lungs of naıve

(untreated, unchallenged) mice (Fig. 4A), the nonsensitized

mice that received Bla g 1 DNA (Fig. 4E), and Bla g 1-vacci-

nated mice (Fig. 4D) were indistinguishable, demonstrating

the therapeutic effects of the Bla g 1 vaccine.

To investigate whether Bla g 1 vaccine induced T-cell

and B-cell responses, lymphoproliferation assays were per-

formed 24 h after the last challenge. The Bla g 1-specific

proliferative response was elevated five- to six-fold in Bla g

1-vaccinated mice compared with that of control mice

(Fig. 5A). The culture supernatants of Bla g 1-vaccinated

mice produced significant levels of IFN-c compared with

those in control DNA mice (Fig. 5B) along with significant

reduction in IL-4 and IL-5 levels (Fig. 5C,D), indicating

that the DNA vaccine profoundly altered cytokine secretion

patterns in cockroach-sensitized and cockroach-challenged

mice. Most interesting was the finding that splenocytes from

the Bla g 1-vaccinated group exhibited a significant increase

in the levels of IL-10 (Fig. 5E), indicating that this treat-

ment may induce the development of Tr1-like cells. Further-

more, the Bla g 1-vaccinated group had significantly

reduced levels of Bla g 1-specific IgE (Fig. 5F) compared

with those in the control DNA group, indicating that the

DNA vaccine was able to suppress the production of Bla g

1-specific IgE, even after a Th2-polarized immune response

was established.

Treg cells were recruited by Bla g 1 vaccine

Previous studies have demonstrated that Treg cells can con-

fer significant protection against AAI by suppressing the

Th2 responses (26–28). Therefore, we compared the propor-

tion and suppressive activity of Treg cells between experi-

mental and control groups. The Bla g 1-vaccinated group

had a significantly higher percentage of CD4+ CD25+

Foxp3+ cells (11.5 ± 1.72%) than did the DNA control

group (6.6 ± 0.49%, P = 0.02). Moreover, we examined

the suppressive activity of Bla g 1-specific Treg cells on

allergen-induced Th2 cells obtained from rBla g 1-sensitized

mice. Figure 6B shows that CD4+ CD25+ T cells from Bla

g 1-vaccinated mice suppressed the proliferation of rBla g

1-activated CD4+ CD25) T cells, and this effect was signif-

icantly reversed by anti-IL-10 antibodies. Thus, the Bla g 1

vaccine could be inducing Treg cells secreting IL-10, which

suppresses the allergen-induced Th2 activity.

Discussion

There are no specific therapeutic strategies to treat Bla g 1-

mediated AAI. In this study, we tested the efficacy of a DNA

vaccine against the common household cockroach allergen,

A D

B E

C F

Figure 59 Induction of Bla g 1-specific immune responses after Bla

g 1 vaccination. To investigate whether Bla g 1 vaccine induces T-

cell and B-cell responses, splenocytes were harvested 24 h after

the last challenge and stimulated in vitro with rBla g 1 to study

their recall response to allergen challenge. The culture supernatants

were tested by enzyme-linked immunosorbent assay (ELISA) for

the presence of cytokines. Sera were tested for Bla g 1-specific

IgE. (A) Spleen cells from mice that received Bla g 1 DNA vaccina-

tion displayed stronger proliferative responses to secondary rBla g

1 challenge. (B–E) Bla g 1 vaccination increased the production of

Th1-type cytokine interferon-c, decreased secretion of Th2-type

cytokines IL-4 and IL-5, and increased the level of IL-10. F. Bla g

1-specific IgE concentrations in serum were measured by Bl g

1-specific ELISA. The data depict the means ± SE from five mice/

group. P value vs Bla g 1-vaccinated mice.

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Bla g 1. Our data showed that the DNA vaccine has both

prophylactic and therapeutic effects.

Allergen-rBla g 1-sensitized and allergen-rBla g 1-chal-

lenged mice therapeutically vaccinated with Bla g 1 vaccine

significantly reduced the total inflammatory cell infiltrate and

eosinophilia, correlating with decreased levels of IL-4 and

IL-5 in the BAL of vaccinated mice. IL-4 is critical to the ini-

tial induction of Th2-dependent allergic responses and

recruitment of effector cells, causing the influx of lympho-

cytes and eosinophils into the airways (28, 29). Eosinophils

play a central role in the pathogenesis of AAI. Their recruit-

ment, activation, differentiation, and survival depend on IL-5

production by Th2 cells that migrate to the airways following

allergen exposure (29, 30). Eosinophils may further exacer-

bate the inflammation by secreting IL-5 and possibly as

APCs on activation (29, 30). Thus, the decrease in eosinoph-

ils recruited into the airways following DNA vaccination

may ameliorate the allergic responses. The histological evi-

dence of the Bla g 1 vaccine’s ability to abrogate lung inflam-

mation was noteworthy. The lungs of naıve (untreated,

unchallenged) mice, the nonsensitized mice that received Bla

g 1 DNA, and Bla g 1-vaccinated mice appeared indistin-

guishable, while the mice that received control DNA or PBS

demonstrated a significant influx of leukocytes around the

airways and epithelial cell hyperplasia. These histological

findings correlate very well with the cellular infiltration pro-

file of the Bla g 1-vaccinated and control mice. These data

demonstrate the ability of Bla g 1 vaccine to suppress an

ongoing Th2-mediated, allergen-induced pathologic inflam-

mation, leading to the healing of the injuries. Furthermore,

there was significantly enhanced Bla g 1-specific lymphopro-

liferation in Bla g 1-vaccinated mice compared with the con-

trols, indicating the potential of this vaccine to stimulate

cellular responses against allergens. Similarly, the levels of

Bla g 1-specific IL-4 and IL-5 were significantly lower in Bla

g 1-vaccinated mice. So the Bla g 1 vaccine is inducing an

antigen-specific immune response that is not Th2 dominated.

Also, the statistically significant increases in IFN-c produc-

tion that we observed in the Bla g 1-vaccinated mice could

have been augmented also by the CpG motifs of the vector.

IL-10 is an important factor for Treg cells, which play a

key role in the control of AAI and the induction of periph-

A

B

Figure 610 Induction of Treg cells with suppressive functions after Bla

g 1 vaccination. Splenocytes were harvested 24 h after the last chal-

lenge. (A) Frequency of CD4+ CD25+ Foxp3+ T cells was assessed

by flow cytometry. Live lymphocytes from the spleen of mice were

gated. The cell surfaces were stained with CD4 (PE) and CD25 (APC)

Ab in accordance with the manufacturer’s recommended protocols.

After permeabilization buffer treatment, the cells were stained with

anti-mouse Foxp3 (FITC) Ab. Treg cell populations in the spleen were

significantly increased by Bla g 1 vaccine. (B) The suppressive func-

tion of CD4+ CD25+ Treg cells. CD4+ CD25+ T cells purified from

splenocytes of Bla g 1-vaccinated mice (B), DNA control mice (D),

phosphate buffer saline-treated mice (P), the nonsensitized mice that

received Bla g 1 DNA (S), and naıve mice (N) by magnetic antibody

cell sorting (MACS). CD4+ CD25) T cells isolated from rBla g 1-sensi-

tized mice as responders. CD4+ CD25+ T cells alone, CD4+ CD25) T

cells alone, and CD4+ CD25+ T cells cocultured with CD4+ CD25) T

cells stimulated with allergen-rBla g 1 in the presence of antigen-pre-

senting cells, and some cocultures treated with neutralizing antibod-

ies to IL-10 (anti-IL-10). After 3 days, 1 lCi/well [3H]-thymidine was

added. Incorporation of [3H]-thymidine was assessed after 16 h of

additional culturing. Values are the means ± SE of five mice/group.

P = 0.006 vs control DNA mice; P = 0.012 vs Bla g 1 vaccination

group.

POOR

QUALITY

COLOR

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eral tolerance in vivo (24–27). The upregulation of Bla g

1-specific IL-10 production suggests the possibility that Bla g

1 vaccine may induce IL-10-producing Treg cells (31, 32) that

suppress the effector responses in the lungs. We therefore

examined this possibility in vivo and found a significantly

elevated percentage of CD4+ CD25+ Foxp3+ cells in Bla g

1-vaccinated mice. Also, CD4+ CD25+ cells isolated from

Bla g 1-vaccinated mice showed profound immunosuppres-

sive activities on rBla g 1-specific effector T cells, which were

significantly abrogated by anti-IL-10 antibodies. Therefore,

Bla g 1 vaccination could induce IL-10-producing Treg cells

in allergic mice that suppress effector T cells and mediate

T-cell tolerance. Also, IL-10 could exhibit a number of regu-

latory mechanisms, including downregulation of mast cell

function, suppression of IgE, eosinophil-sensitive chemokine

production, and reduced eosinophil survival (33, 34). There-

fore, the presence of IL-10-producing CD4+ CD25+ Foxp3+

Treg cells might explain the absence of inflammation in the

lungs of Bla g 1-vaccinated mice sensitized and challenged

with experimental allergen.

Allergic diseases are all characterized by elevated levels of

serum IgE and show a positive, close relationship with clinical

symptoms. Bla g 1 vaccination after systemic allergen sensitiza-

tion significantly reduced Bla g 1-specific IgE production,

whereas vaccination before sensitization almost completely

diminished Bla g 1-specific IgE production. Also, Treg cells

can directly inhibit B-cell response and Ig class switch recombi-

nation (35). In contrast to CD4+ CD25+ Foxp3+ Treg cells,

Tr1 cells also cross-talk to B cells by producing IL-10, which

leads to the regulation of antibody production. Therefore,

IL-10 not only generates T-cell tolerance but also regulates spe-

cific Ig isotype formation of B cells.

In summary, the current study demonstrates that Bla g 1

vaccine, beyond its protective properties, also displays a thera-

peutic potential, suggesting that DNA vaccines may provide a

novel therapeutic approach for allergic diseases. Mechanisti-

cally, our results indicate that Bla g 1 vaccine might be induc-

ing IL-10-producing Bla g 1-specific CD4+ CD25+ Foxp3+

Treg cells through which both prophylactic and therapeutic

potentials are mediated.

Acknowledgments

Supported by the Allergy support Fund, project no.

R073375003.

Author’s contributions

BZ and ME conceived of the study, participated in its design

and coordination, and helped to draft the manuscript. BZ,

YZ, UN, and YPD participated in the analysis and interpre-

tation of data and performed the statistical analyses. BZ,

ME, YZ, JG, MHK, QC, CC, and WL carried out the

assays. AK, MB, MM, and SK drafted the article or revised

it critically for important intellectual content. TJY provided

final approval of the version to be published.

Conflict of interests

The authors declare that they have no competing interests.

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