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RESEARCH REPORT |
1 The Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School & Hospital of Stomatology, Wuhan University, Luoyu Road 237, 430079 Wuhan, Hubei, China; and
2 now at Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China;
* corresponding author, kqyywjtx{at}public.wh.hb.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: DNA vaccines • Streptococcus mutans • dental caries
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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1 gene, the glucan-binding domain of the Streptococcus mutans (S. mutans) gtfB gene, and the A-P fragment of the S. mutans pac gene into the pCI vector—greatly accelerated and increased the antibody responses in mice compared with those generated by a fusion DNA construct pGLUA-P, which contains only the glucan-binding domain of the S. mutans gtfB gene and the A-P fragment of the S. mutans pac gene (Guo et al., 2004; Xu et al., 2005). pCI contains a late SV40 polyadenylation signal and an ampicillin resistance gene, which may cause chromosomal integration into the human genome and elicit allergic responses. To avoid these problems, we replaced the pCI vector with pVAX1, the only vector authorized by the FDA in clinical trials, and constructed a new targeted anti-caries DNA vaccine, pGJA-P/VAX (Jia et al., 2005, 2006). Salivary secretory IgA (SIgA)—the product of the common mucosal immune system, which consists of inductive sites where antigens are encountered, endocytosed, and presented to B- and T-cells and effector sites where antibodies, mainly SIgA, are produced—is considered to play a critical role in caries defense (Russell et al., 1999). Exposing antigens to the inductive sites at the gut, nasal cavity, bronchus, or rectum can generate SIgA not only in the region of induction, but also in remote locations. Compared with other mucosal delivery routes, the intranasal delivery is more convenient and acceptable. Moreover, lower doses of antigens are needed, because intranasal immunization does not expose antigens to low pH and a broad range of secreted degradative enzymes (Vajdy and O’Hagan, 2001). Our previous study showed that intranasal immunization with an anti-caries DNA vaccine, pCIA-P, elicited much higher specific SIgA responses compared with the intragastric and intrarectal routes. Correspondingly, rats immunized with pCIA-P via the intranasal route displayed the fewest caries lesions (Jia et al., 2004).
It is now clear that the nasal-associated lymphoid tissue, a paired lymph cell aggregate localized bilaterally on the posterior side of the palate, is the SIgA-inductive site following intranasal immunization in mice (Asanuma et al., 1997). Specialized epithelial cells called ’microfold cells’ overlying the nasal-associated lymphoid tissue facilitate the uptake of foreign antigens from the nasal cavity and transport them to the subepithelial antigen-presenting cells. Usually, DNA vaccination can induce long-term immune responses in animals, which is believed to be related to the persistence of plasmids in vivo. Studies have showed that plasmid DNA could be detected in injected muscle tissues for a long time (Ho et al., 1998); however, the presence of a plasmid in inoculated mucosal tissues is unclear. Whether the plasmid can exist in the nasal-associated lymphoid tissue after intranasal immunization deserves exploration.
In the present study, we observed the kinetics of the antibody responses generated following intranasal delivery of pGJA-P/VAX in mice and examined the persistence of the plasmid at both the nasal-associated lymphoid tissue and the draining lymph nodes.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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1 gene, the glucan-binding domain (GLU) (amino acid residues 1185–1475) of the gtfB gene from S. mutans GS-5, and the A-P fragment (amino acid residues 222–965) of the pac gene from S. mutans MT8148 was obtained and cloned into the NheI/NotI sites of pVAX1, to create the construct pGJA-P/VAX. For animal immunization, we prepared bupivacaine:DNA complexes by adding bupivacaine hydrochloride to the aqueous DNA solutions, using a fast-mixing method (Pachuk et al., 2000). The final bupivacaine and DNA concentrations were 0.25% and 1 µg/µL, respectively.
Immunization of Mice
Four-week-old female BALB/c mice, 5 per group, were immunized with pGJA-P/VAX or pVAX1, respectively, by the intranasal route. A 50-µg quantity of the bupivacaine:DNA complex was applied in each nostril. The immunizations were given on days 0 and 14. Saliva and blood samples were collected prior to the primary immunization and then biweekly for 6 mos. Serum samples were obtained after centrifugation of blood collected from the retro-orbital plexus. Saliva samples were obtained after intraperitoneal injection of 5 µg of pilocarpine (Sigma Chemical Co., St. Louis, MO, USA) to stimulate salivation. All animal experiments performed in this study were approved by the Review Board of Hubei Medical Laboratory Animal Center.
Antibody Analysis
We used an enzyme-linked immunosorbent assay (ELISA) to determine the levels of specific antibodies in serum and saliva samples. Nintey-six-well flat-bottomed plates (Costar, Cambridge, MA, USA) were coated with 1 µg of rPAc (provided by Prof. Takahiko Oho) or GTF-I (Jia et al., 2003) (10 µg/mL) in carbonate buffer (pH 9.6). Non-specific binding sites were blocked with 3% bovine serum albumin (BSA) in PBS containing 0.05% Tween 20 (PBST) for 2 hrs at 37°C. Serially diluted sera or saliva were added in duplicate to individual wells and incubated at 37°C for 2 hrs. The bound antibodies were then detected with peroxidase-conjugated goat anti-mouse IgG (1:2500, Vector Labs, Inc., Burlingame, CA, USA) or peroxidase-conjugated goat anti-mouse IgA (1:1000, Sigma Chemical Co.) diluted in the blocking buffer, followed by the addition of O-phenylenediamine substrate with H2O2. After incubation at 37°C for 30 min, the reaction was stopped with 2 M H2SO4, and optical density at 490 nm (OD490) was recorded. The antibody titer was defined as the reciprocal of the highest dilution giving an OD490 of 0.1 above the control (adding 3% BSA/PBST instead of the serum or salivary samples after blocking).
Tissue Collection, Plasmid Extraction, and PCR Analysis
Four-week-old female BALB/c mice were randomly divided into 3 groups (n = 15) as follows: bupivacaine:pGJA-P/VAX complexes immunized group, bupivacaine:pVAX1 complexes immunized group, and the untreated group. The immunization procedure was the same as above. Five mice from each group were killed at 1 mo, 3 mos, and 6 mos, respectively, after the initial immunization. The draining cervical lymph nodes were then collected after an incision was made in the overlying skin along the midline of the cervix. For the nasal-associated lymphoid tissue collection, mice were killed and decapitated. The lower jaws, including the tongue. were then removed. After we separated the nose region from the rest of the head along the line of the eyeballs, we collected the nasal-associated lymphoid tissue by peeling away the palate. The excised tissues were minced and incubated in lysis buffer (20 mM Tris-HCl, 5 mM EDTA, 400 mM NaCl, 1% SDS) containing 400 µg/mL proteinase K at 55°C for 4 hrs. The mixture was then extracted once with phenol-chloroform and precipitated with ethanol. The DNA extracts were analyzed for the presence of plasmid pGJA-P/VAX by the PCR. Primers specific for the GLU region of S. mutans were as follows: sense primer, 5'-ATGGGCTATCAAGCCAAAGG-3'; antisense primer, 5'-AATCCGAACTCGTTCTCCAG-3'. PCR was carried out with the use of the Takara TaqTM (Takara Bio Inc., Otsu, Shiga, Japan), with 0.5 µL of the DNA extracts as templates. The amplification was performed for 40 cycles (45 sec at 94°C, 45 sec at 50.4°C, and 60 sec at 72°C). The products were analyzed by 1% agarose gel electrophoresis and DNA sequencing.
Statistical Analysis
Statistical analyses were performed with the use of SPSS10.0 software (SPSS Inc., Chicago, IL, USA). The differences in antibody levels between the test and control groups were determined by t test. A value of p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). These responses peaked at 12 wks and were approximately 3.5-fold greater than those in the control mice. A serum IgG anti-GTF-I response was also detected at 4 wks in mice treated with pGJA-P/VAX (Fig. 1B). The peak response was seen at 12 wks and was approximately 3.4-fold higher than that of the control mice.
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). The response reached a plateau at 10 wks and was 15-fold higher than that of the control group. A salivary IgA anti-GTF-I response was also detected at 6 wks post-prime in the pGJA-P/VAX-immunized group (Fig. 1D). It reached a plateau at 10 wks and was 23-fold higher than that of the salivary IgA anti-GTF-I level of the control group. The salivary IgA anti-PAc and anti-GTF-I responses were maintained 26 and 24 wks, respectively, after the initial immunization.
Persistence of pGJA-P/VAX in vivo
The presence of plasmid DNA in mice was detected by PCR. DNA extracts of normal mice were spiked with pGJA-P/VAX and used as positive controls. DNA extracts from pVAX1-treated animals were used as negative controls. The PCR analysis showed that pGJA-P/VAX could be detected in the nasal-associated lymphoid tissue and the draining cervical lymph nodes at all timepoints assessed (5/5 mice at 1 mo, 5/5 mice at 3 mos, and 4/5 mice at 6 mos) (see Fig. 2). An 870-bp PCR product was amplified. DNA sequencing confirmed that the nucleotide sequence of the amplified product was identical with that of pGJA-P/VAX. No PCR product was observed in either the nasal-associated lymphoid tissue or the draining cervical lymph nodes of the pVAX1-immunized mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The ability of the immune system to learn from and remember its first encounter with an antigen, to make a better secondary response, is described as ’immune memory’, which is the foundation of the practice of vaccination (Kurtz, 2004). Cells responsible for the improved protection are antigen-experienced T- and B-lymphocytes that can persist for long periods of time. Several mechanisms have been postulated to explain this persistence. The presence of small amounts of antigen sequestered on the surface of follicular dendritic cells in germinal centers (Mandel et al., 1981), which persist after the infection is resolved, could drive memory cell expansion. Periodic exposure may also provide antigen-driving rejuvenation of the memory cell population. However, in some cases, memory has been shown to be maintained by cross-reactive stimulations with environmental antigens (Beverley, 1990), or to be totally independent of antigens (Lau et al., 1994). In DNA immunization, memory cells could be generated during the initial period after inoculation, when expression levels of target protein are presumably the highest (Ho et al., 1998). It has been hypothesized that the immune memory may be maintained by the long-term persistence of plasmid DNA that expresses the target genes continually. Some studies showed that plasmid DNA could be detected in the injected muscle tissues for a long time (Wolff et al., 1992; Ho et al., 1998). But until now, whether plasmid DNA could be maintained in the inoculated mucosal tissues was unclear. Unlike undifferentiated myocytes, mucosal tissues have a more rapid cell turnover. Gene expression from plasmid DNA was transient in the mucosal tissues (Hazinski et al., 1991; McCluskie et al., 1998). In the present study, the intranasally delivered plasmid could still be detected at the inoculation site 6 mos after immunization. Furthermore, we detected the plasmid in the draining cervical lymph nodes at 6 mos. It seemed that the initial high levels of expression of target protein induced specific responses, and subsequent lower-level expression maintained them.
In summary, our study demonstrated that pGJA-P/VAX could induce long-time systemic and mucosal antibody responses following intranasal immunization in mice. Furthermore, a plasmid ’depot’ in both the nasal-associated lymphoid tissue and the draining cervical lymph nodes may act as a booster immunization to maintain the immune responses.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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authors contributing equally to the work 
Received November 28, 2005; Last revision June 14, 2006; Accepted June 21, 2006
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REFERENCES |
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days of immunization. (A) Serum IgG anti-PAc antibody levels. (B) Serum IgG anti-GTF-I antibody levels. (C) Salivary IgA anti-PAc antibody levels. (D) Salivary IgA anti-GTF-I antibody levels.