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ffects of targeted fusion anti-caries DNA vaccine pGJA-P/VAX in rats with caries
hang Liu, Mingwen Fan ∗, Zhuan Bian, Zhi Chen, Yuhong Liey Laboratory for Oral Biomedical Engineering Ministry of Education, School and Hospital of Stomatology,uhan University, Luoyu Road 237, 430079 Wuhan, Hubei, China
r t i c l e i n f o
rticle history:eceived 9 July 2008eceived in revised form 24 August 2008
a b s t r a c t
Our previously prophylactic studies have proved that the anti-caries DNA vaccine pGJA-P/VAX could gener-ate effective immune response by intramuscular (i.m.) and intranasal (i.n.) administration in rats without
ccepted 25 August 2008
eywords:NA vaccineental caries
caries. In the present, we determine whether it also could produce efficacy in rats with caries. By immu-nized with pGJA-P/VAX, rats were elicited both significantly higher anti-Streptococcus mutans serum IgGand salivary SIgA responses, compared to those with pVAX1. Correspondingly, rats immunized with pGJA-P/VAX via i.n. displayed significantly fewer enamel, dentinal lesions compared to those with pVAX1 viai.n. However, there was no significant difference in dental caries lesions between pGJA-P/VAX (i.m.) and
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. Introduction
Dental caries is the most common global oral health problemn humans, especially in school-aged children [1]. In most develop-ng countries, both dental caries prevalence rates and dental cariesxperience are increasing. Studies show that caries can proceed toseries of complicated oral diseases, including endodontitis and
eriapical periodontitis, which can negatively impact a person’suality of life [2].
It is generally accepted that methods used to treat caries causedy infections are classified as either surgical or medical. The tra-itional conception is that surgical removal of the carious tissue isufficient; however, it is now known that surgical treatment with-ut considering caries risk factors are responsible for both newesions and restorative failures [3]. For this low-to-moderate cariesisk population, aggressive surgical management could not stoparies development completely [4]. Moreover, clinical evidenceuggests that non-cavitated lesions, even those extending into theentin, can be managed by non-surgical means [5].
Poor compliance is considered to be one of the main causesor caries treatment failure [6], along with a variety of difficulties.
ost children and adults feel stress and anxiety toward conven-
ional treatment procedures. In addition, the procedure will likelyause considerable discomfort. Since the operative procedures areompleted in the oral cavity, and require a series of complexechanical equipment, the risk of both oral mucosal injury and
nstrument swallow will also increase. Furthermore, it has beenlinically demonstrated that medical treatment, with a few side-ffects, seems to be less effective for caries-active patients [7].herefore, it is necessary to develop an improved strategy whichould be conducted quickly outside the oral cavity and with littleiscomfort.
When specific immune responses are ineffective during theatural course of the disease, and when a conventional antimi-robial therapy is not sufficient, vaccines could cure a chronicnfection by either reinforcing or broadening defenses [8]. Vac-ines might be a promising alternative, especially for a patientith poor compliance. For lesions which require surgical removal
ut surgical procedures are not universally effective for the recur-ence, DNA vaccine in conjunction with surgery could help withesions [3]. In previous research, an anti-caries DNA vaccine pGJA-/VAX was designed according to caries-associated bacteria [9]. Itas been proved that intramuscular or intranasal immunization ofhe targeted fusion anti-caries DNA vaccine pGJA-P/VAX could bothffectively induce specific immune responses and protect againstental caries [10].
Thus, in the present study, the DNA vaccine pGJA-P/VAX wasurther studied in a rat caries model to confirm an effect, such asnducing antibody protective responses, and delaying or preventingaries process.
. Materials and methods
.1. Experimental rat caries model
Thirty-two specific pathogen-free (SPF) female Wistar ratsMedical Laboratory Animal Center, Wuhan, Hubei, China) were
ig. 1. Diagram showing the infection, immunization, and sampling protocols used tor three consecutive days; (�) oral infection for five consecutive days with 1 × 109
f blood and saliva samples; (�) immunization.
eaned at 18 days of age. To temporarily suppress oral flora andacilitate the cariogenic bacterial colonization, the rats were treatedith cariogenic diet [11] containing antibiotics (ampicillin, chlo-
amphenicol, and carbencillin 1.0 g/kg diet) for three consecutiveays. Before infection, oral swabs were taken from individual ratso check for bacteria suppression. From days 24 to 28, the rats wererally challenged with 300 �l of an Streptococcus mutans Ingbrittell suspension containing 1 × 109 colony forming units (CFU) usingwabs presoaked with the bacterial suspension. To ensure all werenfected, on day 29, S. mutans infection levels were assessed bywabbing each rat’s occlusal surfaces.
.2. Caries assessment
Before immunization (on day 87), to ensure the rats sufferedrom slight dental caries, 12 rats were sacrificed. The mandiblesere then removed, cleaned, and stained with murexide (0.4% in
0% ethanol). Using the Keyes’ method, the mandibles were hemi-ectioned, and the buccal, sulcal, and proximal molar caries werecored [12]. At the end of the experiment, the caries levels werelso determined by this method.
.3. Preparation of DNA vaccine
Plasmid pGJA-P/VAX was constructed in our laboratory as pre-iously described [9]. Briefly, pGJA-P was comprised of four proteinncoding sequences: the signal peptide and extracellular regions ofhe human CTLA4 gene; the hinge and Fc regions of the human Ig�ene; the GLU of the gtfB gene from S. mutans; and the A-P frag-ent of the PAc gene from S. mutans. Next, the plasmid was coloned
nto the vector pVAX1 containing an anti-kana gene (Invitrogen) toreate the pGJA-P/VAX construct. DNA vaccine was prepared usingn EndoFree Plasmid Maxi-prep Kit (Tiangen, Beijing, China). ForNA immunization, bupivacaine hydrochloride (Sigma, St. Louis,O, USA) was used as a delivery system. For the bupivacaine, DNA
omplexes were prepared by adding the aqueous DNA solutionsnd using the fast mixing method previously used [13]. The finalupivacaine concentration was 0.25%. The final DNA concentrationas 1 �g/�l.
.4. DNA immunization
Apart from the 12 rats sacrificed for caries assessment beforemmunization, the remaining rats were divided randomly into fourroups, with 5 rats per group. Group A and B rats were intra-uscularly administered with 200 �g pGJA-P/VAX and pVAX1,
espectively, while, group C and D were intranasally admin-stered with 200 �g pGJA-P/VAX–bupivacaine complexes andVAX1–bupivacaine complexes. All rats received the first DNA
accine immunization at 89 days of age. Vaccination was con-ucted on four occasions at 1-week intervals. For intramusculardministration, 50 �l of bupivacaine hydrochloride (Sigma, USA,.5%) was injected into the thigh muscle. Three days later, aterilized injection was administered into the middle of the
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y in the experimental rat models as described in Section 2: (�) antibiotic treatmentans Ingbritt C; (�) rat sacrificed and molars scored for caries activity; (♦) collection
ame thigh muscle. For intranasal immunization, 200 �g ofGJA-P/VAX–Bupivacaine complexes in 200 �l of saline was admin-
stered with a micropipette, drop by drop, into the nostrils. Equalostrils received equal doses.
.5. Sample collection
Both serum and saliva samples were collected before immuniza-ion (on day 88), as well as in weeks 2, 4, 6, 8, 10, 12, 14, 16, 18, 20fter the last immunization (Fig. 1), as previously described [14].riefly, peripheral blood was collected from the posterior auricularein with a syringe. Serum was then obtained by centrifugation.n intraperitoneal (i.p.) 0.5 ml solution of 0.2% pilocarpine injec-
ion was used to stimulate saliva flow, which was then collected.ext, 1 mg/ml of approtinin was added to the tubes as a protease
nhibitor. All samples were stored at −70 ◦C.
.6. Antibody analysis
An enzyme-linked immunosorbent assay (ELISA) was used toetermine the levels of specific antibodies in the samples. Individ-al wells of polyvinylbenzene 96-well flat-bottom plates (Greiner,erman) were coated at 4 ◦C overnight with 2 �g of rPAc (giftedindly by Prof. Takahiko Oho) or Glu (gifted kindly by Prof. Zhang) from S. mutans. The levels of total IgA in saliva were detectedn plates coated with an optimal concentration of unconjunctedoat anti-rat IgA antibodies. After non-specific binding sites werelocked with 3% bovine serum albumin (BSA) in PBS containing.05% Tween 20 (PBST) for 2 h at room temperature, the platesere washed five times. Diluted saliva (dilution 1:5 for detect-
ng specific SIgA, dilution 1:800 for detecting total IgA) or serumdilution 1:2000) were added in duplicate to each well and incu-ated at 37 ◦C for 2 h. To establish a standard curve, the affinityurified and unconjuncted goat anti-rat IgG and SIgA were coated.he antibody amount for each well was 1 �g. Then, a serial dilutedeference serum, used to create a standard curve, was added inuplicate into each well and incubated at 37 ◦C for 2 h. The platesere then washed five times each for 3 min with PBST at room
emperature. The amount of bound antibodies was then detectedith a peroxidase-conjugated goat anti-rat IgG (1:5000, Pierce,SA) or peroxidase-conjugated goat anti-rat SIgA (1:5000, Bethyl,SA) diluted in the blocking buffer. The action was followed bydding an ophenylenediamine substrate (Sigma, USA) with H2O2.fter incubating at 37 ◦C for 30 min, the reaction was stopped withM H2SO4. Optical density (OD) readings were taken and recordedt 490 nm (OD490). The final OD was defined as the differenceetween OD490 and the absorbance of the sham control (no sam-le added). The final result was counted according to the standardurve.
.7. Statistical treatment of data
Statistical analysis of the specific antibody levels and cariescores were performed with SPSS 10.0 software (SPSS, Inc., Chicago,
C. Liu et al. / Vaccine 26 (2008) 6685–6689 6687
Fig. 2. Serum IgG antibody levels of rats immunized with various treatments.Groups of rats were immunized with a DNA vaccine as follows: intramuscular injec-tion of pGJA-P/VAX (pGJA-P/i.m.); intramuscular injection of pVAX1 (pVAX1/i.m.);intranasal administration of pGJA-P/VAX (pGJA-P/i.n.); intranasal administration ofpVAX1 (pVAX1/i.n.). Serum and saliva samples were collected biweekly for ELISA.Data are expressed as the mean ± S.D. values. Symbols for statistical significance: (↑)b#
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Fig. 3. Salivary IgA antibody levels of rats immunized with various treatments.Groups of rats were immunized with a DNA vaccine as follows: intramuscular injec-tion of pGJA-P/VAX (pGJA-P/i.m.); intramuscular injection of pVAX1 (pVAX1/i.m.);intranasal administration of pGJA-P/VAX (pGJA-P/i.n.); intranasal administration ofpVAX1 (pVAX1/i.n.). Serum and saliva samples were collected biweekly for ELISA.Data are expressed as the means of the percent anti-PAc or anti-Glu SIgA antibodypndA
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efore immunization; *significantly different from the group pVAX1/i.m. (P < 0.05);significantly different from the group pVAX1/i.n. (P < 0.05). (A) Serum anti-PAc IgGntibody levels. (B) Serum anti-Glu IgG antibody levels.
L, USA). Differences between experimental and control groupsere determined by one-way analysis of variance (ANOVA) and
onsidered significant if P values were <0.05.
. Results
.1. Serum IgG responses in rats
After boosting, the mean specific serum IgG antibody level ofhe pGJA-P/VAX–bupivacaine (i.m.) group was higher than that ofhe pGJA-P/VAX–bupivacaine (i.n.) group at weeks 2, 4, 10, 12, 14,6, 18, and 20 (P < 0.01). At all tested time points, the specific serumgG levels of test groups were significantly higher than those of con-
rol groups (P < 0.01). There was no significant difference betweenhe pGJA-P/VAX and pVAX1 groups before treatment (P > 0.05). Asxpected, the specific serum IgG antibody response decreased inhe control group (Fig. 2 A and B).
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er total IgA ± S.D. values. Symbols for statistical significance: (↑) before immu-ization; *significantly different from the group pVAX1/i.m. (P < 0.05); #significantlyifferent from the group pVAX1/i.n. (P < 0.05). (A) Anti-PAc SIgA antibody levels. (B)nti-Glu SIgA antibody levels.
.2. Salivary SIgA responses in rats
After boosting, both the mean salivary anti-PAc and anti-lu SIgA antibody level of the pGJA-P/VAX–bupivacaine (i.n.)roup were higher than that of the pGJA-P/VAX–bupivacainei.m.) group after week 6 (P < 0.05). At all tested time points,he specific salivary SIgA levels of rats vaccinated with pGJA-/VAX–bupivacaine were significantly higher than that of ratsaccinated with pVAX1–bupivacaine (i.m.) (P < 0.05). While, at allested time points except at week 2, the specific salivary SIgA lev-ls of rats with pGJA-P/VAX–bupivacaine were significantly higherhan rats vaccinated with pVAX1–bupivacaine (i.n.) (P < 0.01). There
as no significant difference between the pGJA-P/VAX and pVAX1
roups before treatment (P > 0.05). As expected, the specific salivaryIgA antibody response decreased in the control group (Fig. 3A and).
6688 C. Liu et al. / Vaccine 26 (
Fig. 4. The enamel, slight dentinal, moderate dentinal, and extensive dentinal cariesscores of rats immunized with various treatments. Groups of rats were immu-nized with a DNA vaccine as follows: (A) intramuscular injection of pGJA-P/VAX(pGJA-P/i.m.); (B) intramuscular injection of pVAX1 (pVAX1/i.m.); (C) intranasaladministration of pGJA-P/VAX (pGJA-P/i.n.); (D) intranasal administration of pVAX1(edg
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pVAX1/i.n.). The caries levels were scored by the Keyes’ method. The results arexpressed as mean ± S.D. values. Symbols for statistical significance: *significantlyifferent from the group pVAX1/i.m. (P < 0.05); #significantly different from theroup pVAX1/i.n. (P < 0.05).
.3. Caries protection
To compare the anti-caries ability of the DNA vaccine pGJA-/VAX in vivo, SPF Wistar rats infected with S. mutans Ingbritt wereacrificed 5 months after initial caries occurrence. The extensionnd depth of carious lesion involvement were scored as enamelE), slight dentinal (Ds), moderate dentinal (Dm), and extensiveentinal (Dx). The pGJA-P/VAX–bupivacaine (i.n.) group displayedignificantly fewer enamel and dentinal lesions than pGJA-/VAX–bupivacaine (i.m.) group (P < 0.05) and pVAX1–bupivacainei.n.) group (P < 0.01). However, there was no significant differencen dental caries lesions between the pGJAP/VAX–bupivacaine (i.m.)roup and the pVAX1–bupivacaine (i.m.) group (P > 0.05) (Fig. 4).
. Discussion
S. mutans is an oral facultative anaerobe which has been impli-ated as the primary cariogenic organism. Due to its extracellularrowth character, the anti-caries DNA vaccine works throughnducing the specific antibodies included IgG from gingival sulcusnd SIgA from saliva partially [2,15]. Several reports [16,17] demon-trated that specific IgG presumably might inhibit adherence of therganism to the tooth surface or inhibit colonization by interferenceith enzyme activity. In addition, through agglutination, anti-PAc
IgA can inhibit sucrose-independent and sucrose-dependent cari-genic bacteria attachment to the tooth surface and then eliminatehem. Anti-Glu SIgA can interfere with bacteria growth and controlental plaque formation through inhibiting glucanosyltransferaseGTFase) metabolic activity. In the present research, we foundhat specific SIgA antibody concentrations generated by pGJA-/VAX via intranasal immunization in rats were significantly higherhan those via intramuscular immunization at most time points.owever, for the specific IgG antibody, results were the opposite,hich is consistent with previous research results [10]. Further,
previous study showed that the fusion anti-caries DNA vaccineGJA-P/VAX–bupivacaine complex could significantly increase the
evels of specific anti-Pac or anti-Glu IgG and SIgA antibodies in rats,s well as significant protective effects in prophylactic studies. Here,hese specific antibodies were also detected before immunization,
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2008) 6685–6689
uggesting that at the beginning, S. mutans had provoked a cer-ain extent immune response through colonization. However, testroup antibody levels were not only much higher than the controlroups, but could also maintain for a long time post immunization.he possible reason of high immune response was that the DNA vac-ine antigenicity was different from the bacteria, which could alsotimulate and modulate immune responses [18]. Booster immu-ization might recall a strong specific memory immune responses,hich is associated with long-term T and B cell memory, to make
ntibodies forestall colonization at later time points [15,19]. There-ore, owing to its ability to properly switch immune status, andeinforce immune defence against infections, the vaccination doesave a certain effect on dental caries. Zhu et al. [20] demonstratedhat switching improper immune status and recalling a strong andarly specific memory immune response were so important in theontrol of M. tuberculosis.
Correspondingly, compared to the control rats, pGJA-P/VAXmmunized (i.n.) rats showed higher levels of SIgA antibodies thatssociated with fewer caries lesions. This finding was consistentith another study that found cariogenic bacterial colonization
nhibition coincided with salivary antibody responses. Zhang [21]emonstrated that an anti-caries vaccine via i.n. could induceystemic immunity, as well as obtain a high level of specificIgA antibodies in saliva. However, in our study, the differencesn caries scores between the pGJA-P/VAX immunized (i.m.) andVAX1 immunized (i.m.) groups did not achieve statistical sig-ificance, which were inconsistent with previous results. Russell22] reported i.m. immunization induced specific IgG responses.s a result, caries scores were lower in immunized rats com-ared to the sham controls, though they were still higher thanats immunized via mucosal routes. There could be two reasonsor this phenomenon: one could be that the immune status isignificantly different in hosts that are infected or not infectedith S. mutans. Hence, bacteria products in challenged rats mightave influenced the vaccine to induce an acquired immune reac-ion [19]. Another possible reason is that although a specific IgGntibody could prevent caries by inhibiting a microbial invasion,he results might suggest that dental caries inhibition was mainlyue to SIgA response levels, rather than IgG response levels. It iselieved that the mucosal surface is the most important protec-ive barrier to the body. Secretory immunity is the key to defensegainst mucosal infections because it can inhibit initial pathogenolonization by providing SIgA antibody blocking activity on theucosal surface without causing tissue damage [23]. SIgA, which
s the immunoglobulin and predominant antibody class in thexternal secretions that bathe mucosal surfaces, plays a majorole in the entire immune system [24]. Therefore, by blockingdhesin–receptor interaction, SIgA antibodies can also be consid-red an important first line of defense against dental caries [22].
Repique et al. [25] suggested that the development of anmmunotherapeutic DNA vaccine was a promising but dauntingask that would require new strategies. In order to both increaselasmid uptake and limit degradation, various methods of for-ulating DNA for mucosal delivery have been proposed. In our
esearch group, bupivacaine was commonly used to coadministerith the plasmid in intranasal immunization and this delivery sys-
em could induce much higher protective antibody responses thanhe naked DNA [26]. In addition, as published, bupivacaine prein-ection into muscle, which could enhance DNA uptake and improveransfer efficiency [27], have been used in various plasmid DNA
onstruct immunizations, such as cytomegalovirus (CMV) and HIV28,29]. In the current work, the results indicate that a DNA vaccineith bupivacaine pre-treatment or co-administration, which couldrovoke effective immune response in rat models, has the potentialo be a novel class of gene therapy DNA delivery agents [13].
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C. Liu et al. / Vaccin
In conclusion, this was the first study to trace the antibodyesponse and caries scores after a caries challenge. The studyhowed that a targeted fusion anti-caries DNA vaccine, pGJA-/VAX–bupivacaine, could maintain long-term specific systemicgG and salivary SIgA responses in rats following intramuscularnd intranasal immunization. However, only caries scores of theGJA-P/VAX–bupivacaine immunized via i.n. group were signif-
cantly lower than that of the control group, which suggestedhat pGJA-P/VAX–bupivacaine (i.n.) could effectively control cariesevelopment to a certain extent. Therefore, the DNA vaccine pGJA-/VAX–bupivacaine (i.n.), beyond its prophylactic ability in ratithout caries, also displays a certain efficacy in rat with caries.owever, the vaccine should go hand in hand with any necessary
urgical treatment. Although the promising use of this DNA vac-ine to dental caries has been highlighted in this rat models, furthertudy must shed light on its effect in clinical research.
cknowledgements
We thank Prof. Takahiko Oho for providing the rPAc protein. Wehank Prof. Zhang for providing the Glu protein. This work was sup-orted by grant No. 30330660 from the Natural Science Foundationf China.
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