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Introduction
Dental caries remains one of the most common
infectious diseases affecting humans in the world, and it is often
at epidemic proportions, especially among the poor in
developing countries[1]. For example,
three-quarters of the 5-year-old children studied had evidence of
significant dental decay,
which was revealed by an oral health survey in
China[2]. Caries immunization may be beneficial to those people in the
future.
Streptococcus mutans (S. mutans) has been
strongly implicated as a causative organism of dental
caries[3,4]. Colonization of these microorganisms in tooth surfaces initiates
the procedure of tooth decay. Two mechanisms,
sucrose-independent and sucrose-dependent, are considered to
mediate the colonization of S. mutans to tooth surfaces. A
cell surface protein (PAc) is involved in the former mechanism,
which mediates the initial adherence of S.
mutans to the
acquired pellicles on tooth
surfaces[5,6]. The latter mechanism is due to the synthesis of water insoluble glucan from
sucrose catalyzed by glucosyltrans-ferases
(GTFs)[7,8]. Due to the importance of PAc and GTFs for cariogenicity of
S. mutans, these proteins are rational candidate antigens in
developing anti-caries vaccines.
Antibody responses are desirable to prevent dental
caries. Antibodies against the recombinant protein
containing the alanine-rich (A) region of PAc and the glucan
binding (GLU) domain of GTF-I inhibit glucan synthesis, as well
as the in vitro sucrose-independent and the
sucrose-dependent adhesion of S. mutans to saliva-coated
hydroxyapatite[9]. Passive immunization with milk containing antibodies against
the A region of PAc and the GLU domain of GTF-I led to
significantly less caries than the controls in
rats[10]. We previously used porline-rich (P) region of PAc as a candidate
for a potential DNA vaccine pGLUA_P, which encodes the
GLU region of GTF-I and the A_P region of PAc. Rats
immunized with pGLUA_P following subcutaneous injection near
the submandibular gland developed significantly fewer
caries lesions compared with the
controls[11].
DNA vaccine is a promising new vaccine with many
advantages over traditional vaccines, including its ease in
preparation and administration, the ability to induce
effective immune responses, as well as being a great potential for
modification and improvement. The immune responses
induced by DNA vaccines are initiated with the activation of
antigen-presenting cells (APCs). Dendritic cells play a
critical role in inducing immune responses of DNA
vaccines[12,13]. Following genetic immunization, DNA vaccines can directly
transfect local somatic cells in vivo. APCs can capture the
antigens expressed by transfected cells, process, and then
present them as major histocompatible complexes_peptide
complexes to T cells in regional lymphoid organs where
antigen-specific T cells are
activated[14]. Cytotoxic
T-lymphocyte-associated antigen 4 (CTLA-4) is a glycoprotein mainly
expressed on activated T cells, and its extracellular V-domain
has a strong binding affinity to B7 molecules (B7-1 and B7-2)
primarily expressed on APCs[15_17]. A strategy
of directing antigens directly to APCs by CTLA-4 was applied to
enhance the efficacy of DNA vaccine. Vaccination with
DNA encoding a model antigen (human Ig) and CTLA-4 have been
shown to dramatically increase antibody responses compared
with the control plasmid expressing the antigen
alone[18].
To enhance the efficacy of anti-caries DNA vaccines,
first we constructed CTLA-4 fusion DNA vaccine pGJA_P
by cloning the signal peptide and extracellular regions of the
human CTLA-4 gene and the hinge and Fc regions of the
human Igγ1 gene into
pGLUA_P[19]. Considering the possible application to clinical trials, the skeleton of pGJA_P
was changed into the vector of pVAX1, which is especially
designed for DNA vaccines, and then pGJA_P/VAX1 was
constructed[20].
In this study, we cultured human dendritic cells (DCs),
and investigated whether the CTLA-4_Ig_GLU_A_P fusion
protein encoded by pGJA_P/VAX1 could specifically bind
to human DCs. In addition, the immunogenicity and
protective efficacy of pGJA_P/VAX1, pGJA_P, and pGLUA_P were
compared via intramuscular (im) injection or intranasal (in)
administration. Caries protection and anti-PAc-specific
antibody responses were investigated in hamster experiments.
Materials and methods
Cells, animals, and plasmids COS-7 cells were generous
gifts from Dr Hua TANG from the Institute of Immunology of
the Secondary Military Medical University (Shanghai, China).
The animals were purchased from Wuhan Institute of
Biological Products (Wuhan, Hubei, China), and bred and
maintained in the Hubei Medical Laboratory Animal Center
(Wuhan, China). Five plasmids were used in the study: 2
CTLA-4 fusion anti-caries DNA vaccines (pGJA_P/VAX1
and pGJA_P), 1 non-fusion DNA construct (pGLUA_P),
negative controls including pCI (Promega, Madison, WI,
USA), and pVAX1 (Invitrogen, San Diego, CA, USA). As
shown in Figure 1, pGLUA_P[11] encoded the GLU domain of
the gtfB gene from S. mutans GS-5 and the A_P fragment of
the Pac gene from S. mutans MT8148.
pGJA_P[19] was constructed by inserting the signal peptide and extracellular
regions of the human CTLA-4 gene and the hinge and Fc
region of the human Igγ1 gene into pGLUA_P. The vector of
pCI was the skeleton of pGLUA_P and pGJA_P.
pGJA_P/VAX1[20] was obtained by cloning genes encoding the
CTLA-4_Ig_GLU_A_P fusion protein from pGJA_P
into the vector of pVAX1.
Plasmid preparation The plasmids were prepared with
Endo-free plasmid mini kit II and maxi kit (Omega Biotek,
Norcross, GA, USA). For immunization via in administration,
DNA-bupivacaine complexes were prepared by adding bupivacaine hydrochloride (Sigma, St. Louis, MO, USA) to
the aqueous DNA solution according to the fast mixing
method[21]. The final complexes contained 0.25% bupivacaine.
Transfection and preparation of the fusion protein
In the 6-well plates, the COS-7 cells were cultured at a
concentration of 2×105 cells/well in RPMI-1640 (Gibco, Carlsbad,
CA, USA) with 10% fetal calf serum (FCS, Gibco, USA). The
cells were transfected with pGJA_P/VAX1 or pVAX1 using
Sofast transfection reagent (Sunma Biotech, Xiamen, China)
and cultured for 48 h before collecting the culture
super-natant. The supernatant was concentrated by
Amicon ultra-15 centrifuge filter devices (Millipore, Bedford, MA, USA),
and stored at -70 oC.
The concentration of the fusion protein in the
supernatant was determined by ELISA. Polyclonal goat anti-human
IgG Ab (5 µg/mL, Southern Biotech, Birmingham, AL, USA)
and peroxidase-conjugated goat anti-human IgG Ab
(1:10,000, VectorLabs, Burlingame, CA, USA) were used to
detect the fusion protein in the supernatant. Both were
directed against the human Ig determinant in the fusion
protein. Standard curves were generated by serially diluted
human IgG (Sigma, USA) in the Ab-coated plates at a
concentration range of 1.25_80 ng/mL.
Generation of human monocyte-derived DCs
The monocytes were purified from human peripheral blood mononuclear
cells by negative sorting using magnetic microbeads (Dynal,
Oslo, Norway). The acquisition and treatment protocols of
human blood were approved by Wuhan Blood Center. DCs
were generated as described[22] by culturing the monocytes
in complete medium supplemented with
granulocyte_macrophage colony stimulating factor (GM_CSF, 50 ng/mL,
R&D, Minneapolis, MN, USA) and interleukin (IL)-4 (50 ng/mL,
R&D, USA). The complete medium included RPMI-1640,
1 mmol/L sodium pyruvate (Sigma, USA), 50 µmol/L
2-merca-ptothanol (Merck, West Point, PA, USA), 100 U/mL
penicillin/ streptomycin, and 10% FCS. On d 5,
the cells were stimulated by the addition of
TNF-α (100 ng/mL, PeproTech, Rocky Hill, NJ, USA).
The phenotype of the cells was assayed by flow cytometry. The Abs used for analysis were
PE-conjugated anti-human CD14, anti-human CD80, anti-human CD86,
and anti-human HLA-DR (eBioscience, San Diego, CA, USA).
The isotype control used was PE-conjugated mouse
IgG1 (eBioscience, USA).
Flow cytometric analysis of the fusion protein encoded
by pGJA_P/VAX1 binding to human DCs Before the
binding assay, the concentration of the fusion protein of the
concentrated supernatant from pGJA_P/VAX1-transfected
cells was determined by ELISA. It was equivalent to 0.22
µg/mL human IgG calibrated by the standard curve
(R=
0.998). There was no fusion protein that was detected in the
supernatant from pVAX1-transfected cells.
On d 7, the DCs were harvested. The supernatant from
pVAX1-transfected cells was used as the negative control to
determine whether the fusion protein could bind to DCs.
One group of DCs were blocked with B7-1 and B7-2
monoclonal antibodies (SBA, Birmingham, AL USA) to determine
whether the binding activity was due to the interaction of
CTLA-4 and B7 molecules on DCs. Three groups were
divided as follows: group A (DCs vs pVAX1), group B (DCs
vs pGJA_P/VAX1), and group C (B7-blocked DCs
vs pGJA_P/VAX1). Briefly, the three groups of DCs were mixed with
an equal volume (300 µL) of the concentrated supernatant,
respectively, which were from either
pGJA_P/VAX1-transfected cells or pVAX1-transfected cells. After being
incubated for 1 h at 4 oC, the DCs were washed twice with binding
buffer [phosphate buffered saline (PBS) plus 2% FCS].
Then FITC-conjugated goat anti-human IgG (Sigma, USA)
was added (1:100 dilution in binding buffer) and incubated
for 45 min at 4 oC. The DCs were washed and resuspended
in 200 µL of 1% formaldehyde in PBS. The mean
fluorescence intensities (MFIs) of the cells in the assay were
analyzed under the same setting in a FACScan flow cytometer
( Becton, Dickinson and Company, San Jose, CA, USA,)
which had been calibrated with standard beads (Becton,
Dickinson and Company, USA).
Animal immunizations The hamsters were divided into
12 groups, with 8 hamsters per group.
Experimental hamster caries models were created as
described[11]. Briefly, newborn, female golden hamsters were weaned on d 23 and raised on
a cariogenic diet (Keyes 2000)[23]. Antibiotics (ampicillin,
chloramphenicol, and carbenicillin, 1.0 g/kg diet) were added
to the diet on d 24_26, and the hamsters were then orally
challenged with 2×109 colony forming units (CFU) of
S. mutans Ingbritt on d 28_30. Bacterial samples from the
occlusal surfaces of each hamster were examined to make
sure that all were infected with S. mutans.
After 3 d of S. mutans infection, the 12 groups of
hamsters were immunized by quadriceps muscle injection or in
administration. The immunization protocols were reviewed
and approved by the review board of Hubei Medical
Laboratory Animal Center. Six groups were immunized by im
injection. Each hamster was injected into the quadriceps
muscle of 1 leg with 100 µg plasmid in 100 µL saline solution
under ether vapor anesthesia. The sham group was only
injected with 100 µL/hamster saline solution. Six groups
were immunized by in administration. Plasmid 100 µg in
the solution of 50 µL DNA_bupivacaine complexes was
deposited into both nostrils with the aid of a micropipette
(25 µL/per nostril). The sham group was immunized with
50 µL/hamster saline solution containing 0.25% bupivacaine.
The same procedure of immunization employing the same
dose of immunogen was used for boost immunizations 2 weeks
later. On d 70, serum and saliva samples were collected as
described[24], and then hamsters were killed. The mandibles
were removed, cleaned, and stained with murexide. The teeth
were sectioned and caries levels were scored by the Keyes
method[25].
Antibody analysis An ELISA was established to
determine anti-PAc IgG in the serum and anti-PAc IgA in saliva
samples. Each well of a polystyrene microtiter plate
was coated with 100 µL rPAc (10 µg/mL, provided by Prof
Takahiko OHO) in carbonate buffer (pH 9.6) and incubated
overnight at 4 oC. Non-specific binding sites were blocked
with 3% bovine serum albumin in phosphate-buffered saline
containing 0.05% Tween 20 (PBST) for 2 h at room
tempera-ture. Diluted sera or saliva (1:100 or 1:10 dilution, respectively,
in blocking buffer) were added in triplicate to individual wells
and incubated for 2 h at 37 oC. After washing with PBST, the
amount of bound Abs was detected with
peroxidase-conjugated goat anti-mouse IgG (1:2500, VectorLabs, USA) or
peroxidase-conjugated goat anti-mouse IgA (1:1000, Sigma,
USA) diluted in blocking buffer, followed by the addition of
O-phenylenediamine substrate (40 mg/mL) in a solution
containing 0.1 mol/L citric acid, 0.2 mol/L
Na2HPO4, and 3%
H2O2 to the plates. The reactions were quenched in 50 µL 2 mol/L
sulfuric acid, and absorbance was measured at 490 nm. The
background values were subtracted from the values of the
experimental samples. The data were simplified by
calculating the mean±SD of the absorbance of each sample,
determined in triplicate.
Statistical analysis The statistical analysis of the
ELISA antibody data and caries scores was performed with
SPSS 10.0 software (SPSS Inc, Chincago, IL, USA) .
Differences among the test and control groups were determined
by ANOVA, followed by a multiple-mean comparison
using Student_Newman_Keuls test. The value of
P<0.05 was considered significant.
Results
Generation of human monocyte-derived DCs After 7 d
culture with GM_CSF and IL-4, 70%_80% of the cells
appeared as isolated or clustered floating cells with the
typical dendritic morphology (Figure 2). An analysis of surface
markers showed that the cells were homogeneous and
expressed high levels of HLA-DR, CD80, and CD86, and had
low level expressions of CD14 (Figure 3). The molecular
profile of the cells is consistent with the results of
Pickl et al[26] and Meierhoff
et al[27].
Binding of CTLA-4 fusion protein encoded by
pGJA_P/VAX1 to human DCs In the assay, group A (DCs
vs pVAX1) served as the negative control. The MFI of group A was 8.87
and 6.11% of the cells showed fluorescence. The MFI of
group B (DCs vs pGJA_P/VAX1) was 12.09 and 12.69% of
the cells showed fluorescence. The MFI of group C (blocked
DCs vs pGJA_P/VAX1) was 7.68 and 2.29% of the cells
showed fluorescence. As evidenced by an increased shift in
fluorescence compared with negative control, the
CTLA-4_Ig_GLU_A_P fusion protein encoded by pGJA_P/VAX1 was
able to bind to human DCs (Figure 4). The specificity was
confirmed by blockade of B7 molecules expressed on human
DCs (Figure 4). The experiment was repeated 3 times.
Specific anti-PAc antibody responses in hamsters
The hamsters immunized with CTLA-4 fusion anti-caries DNA
vaccines pGJA_P/VAX1, pGJA_P, and non-fusion DNA
construct pGLUA_P showed significantly higher serum and
salivary specific anti-PAc antibody responses than the
negative controls and sham group (Figure 5). Furthermore, the
hamsters immunized with pGJA_P/VAX1 and pGJA_P via
the in route showed significantly higher serum and salivary
anti-PAc-specific antibody responses than that of
pGLUA_P (Figure 5). For immunization via the im route, serum IgG
antibody responses induced by CTLA-4 fusion anti-caries
DNA vaccines were significantly higher than that of
non-fusion DNA construct pGLUA_P. There was no significant
difference in anti-PAc antibody responses between the 2
CTLA-4 fusion anti-caries DNA vaccines.
Caries protection pGJA_P/VAX1-, pGJA_P-, and
pGLUA_P-immunized hamsters displayed significantly fewer
enamel (E), dentinal slight (Ds), and dentinal moderate (Dm)
lesions than that of the negative controls and sham group
(Figure 6). pGJA_P/VAX1 and pGJA_P-immunized hamsters
displayed significantly fewer E and Ds lesions than
pGLUA_P_immunized hamsters (Figure 6). No significant difference
in the caries score level was found between pGJA_P/VAX1
and pGJA_P-immunized hamsters.
Discussion
Many studies have demonstrated that targeting an
antigen as a CTLA-4 fusion protein to APCs can significantly
enhance the magnitude of antibody
responses[17,18,28]. We first employed the strategy to enhance the efficacy of
anti-caries vaccines. It has been shown that the strategy is
applicable to increase the magnitude of antibody responses
against dental caries and significantly reduce the caries
lesions. The biological activity of the CTLA-4 fusion
protein is directly related with the efficacy of DNA vaccines or
recombinant protein vaccines. Huang et
al[17] have shown that mice immunized twice with
Id_CTLA-4Y104A, a fusion protein with mutant CTLA-4 at residue 104
(Tyr®Ala) which loses binding activity to B7 molecules, induced only low
titers of anti-Id Ab similar to the titers achieved by Id
immuni-zation, whereas a single immunization with Id_CTLA-4 was
able to produce significantly high titers of the anti-Id
antibody. Deliyannis et
al[28] demonstrated that the fusion
protein of CTLA-4_hIg-hemagglutinin (HA) encoded by a
HA-based influenza DNA vaccine was capable of binding to
B7-NIT cells expressing membrane-bound B7-1 molecules.
Mice receiving the targeted vaccine developed accelerated
and increased antibody responses as compared with those
receiving the nontargeted control. In this study, we chose
DCs as the target cells and investigated the biological
activity of the antigen encoded by CTLA-4 fusion DNA vaccines.
The enhancement of immune response may be due to the
targeting property of the CTLA-4 fusion
protein. It has been hypothesized that directly targeting antigens to APCs
through the interaction of CTLA-4 and B7 causes the
antigen to be processed and presented to T cells with much
higher efficiency, therefore leading to a stronger immune
response[17]. T-independent B cells may also contribute to
the enhanced immune responses. However, the mechanisms
still deserve further investigation.
In the present study, we also provide evidence that
pGJA_P/VAX1 was able to induce high levels of specific anti-PAc
antibody responses comparable with the levels induced by
DNA vaccine pGJA_P. Furthermore, significant inhibition
of dental caries was also found in hamsters immunized with
pGJA_P/VAX1. Investigators have doubted the
effectiveness of pVAX1 as a DNA vaccine delivery vector for it failed
to express substantial levels of protein compared with other
mammalian expression vectors in
vitro[29]. However, our study showed that the skeleton of the pVAX1 vector did not
affect the potency of DNA vaccines in vivo. This will be
helpful for the application of CTLA-4 fusion anti-caries DNA
vaccines to be used by mankind in the future.
Salivary IgA is thought to be a key inhibitor of
S. mutans infection[30]. Zhang
et al[31] have reported that a chimeric
protein SBR_GLU induced significant salivary anti-SBR and
anti-GLU IgA responses after in immunization in mice, and
that the inhibition of S. mutans colonization was in
agreement with the salivary antibody responses. In that study,
CTLA-4 fusion DNA vaccines pGJA_P/VAX1 or
pGJA_P-immunized hamsters via the in route showed significantly
higher levels of salivary anti-PAc IgA responses associated
with fewer E, Ds, and Dm lesions than pGLUA_P-immunized
groups. These results indicated that the degree of inhibition
of dental caries may be intimately related to the levels of
secretory IgA (S-IgA) responses. In the present study , the
highest titer of S-IgA and the lowest caries scores were
observed on hamsters immunized with CTLA-4 fusion
anti-caries DNA vaccines via the in route. As a non-invasive
delivery method, in delivery will be desirable for the application
of anti-caries DNA vaccines.
The results from the hamster experiments may help us to
learn more about the immune responses induced by DNA
vaccine. The systemic immune and mucosal immune
systems are 2 distinct compartments of the immune system.
Antibodies associated with the systemic compartment are
mainly of the IgG isotype. In contrast, antibodies in the
mucosa are primarily S-IgA[32]. It has been generally regarded
that im injection cannot induce effective mucosal immune
responses. However, in the study, anti-caries DNA
vaccines delivered via the im route induced significantly higher
salivary specific anti-PAc IgA responses than the negative
controls. We think this can be explained as
follows: first, the secreted fusion antigen from transfected cells in local
tissues was transferred by the circulating system to the
mucosal immune system and captured by DCs to initiate the
immune responses; and second, the immune responses may
be initiated by antigen-loaded DCs migrated from the
muscular tissue to the mucosal immune system.
Enioutina et al[33] found that antigen-pulsed DCs injected into
UVB-exposed peripheral skin sites were able to migrate to the
Peyer's patches and stimulate both mucosal and systemic
immune responses. However, it needs further study to
explore the precise mechanism.
In conclusion, we demonstrated that CTLA-4 fusion
anti-caries DNA vaccines had good immunogenicity and potent
capacities against caries in gnotobiotic hamsters. The
efficacy of anti-caries DNA vaccine has been markedly improved
by fusing antigen to CTLA-4, which directs the antigen to
APCs.
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