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Immunogenicity and Persistence of a Targeted Anti-caries DNA Vaccine
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 Correspondence: * corresponding author, kqyywjtx{at}public.wh.hb.cn
We have previously reported that a targeted anti-caries DNA vaccine, pGJA-P, induced accelerated and increased antibody responses compared with a non-targeted anti-caries DNA vaccine. Recently, pGJA-P/VAX, a new targeted anti-caries DNA vaccine for human trials, was constructed by replacing the pCI vector used in the construction of pGJA-P with pVAX1, the only vector authorized by the US Food and Drug Administration in clinical trials. Here, we report on our exploration of the kinetics of the antibody responses generated following pGJA-P/VAX immunization and the persistence of pGJA-P/VAX at both the inoculation site and the draining lymph nodes. Intranasal vaccination of mice with pGJA-P/VAX induced strong antibody responses that lasted for more than 6 months. Furthermore, pGJA-P/VAX could still be detected at both the inoculation site and the draining cervical lymph nodes 6 months after immunization. Thus, the persistent immune responses are likely due to the DNA depot in the host, which acts as a booster immunization.
Key Words: DNA vaccines • Streptococcus mutans • dental caries
A DNA vaccine is a bacterial plasmid that is designed to express a gene for the antigen of interest in the cells of a host. Although rapid progress has been made in demonstrating the efficacy of DNA vaccines against numerous infectious diseases, autoimmune diseases, allergies, and cancers, poor immunogenicity is still a major problem in DNA vaccination. Cytotoxic T-lymphocyte-associated antigen 4 is a membrane-bound molecule located mainly on activated T-cells. Its extracellular V-domain is considered to be involved in mediating the binding to the B7 molecule on antigen-presenting cells, the potent initiators of immune responses (Parsons et al., 1996). By utilizing the interaction between cytotoxic T-lymphocyte-associated antigen 4 and B7, specific antigens can be targeted to antigen-presenting cells by fusion to cytotoxic T-lymphocyte-associated antigen 4. Our previous studies showed that a targeted anti-caries DNA vaccine, pGJA-P—which was constructed by cloning the signal peptide and extracellular regions of human cytotoxic T-lymphocyte-associated antigen 4 gene, the hinge and Fc regions of human Ig  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.
Plasmids pGJA-P/VAX was constructed as previously described (Jia et al., 2005, 2006). Briefly, pGJA-P was digested with the restriction endonucleases NheI and NotI. Then, the fragment containing the signal peptide and extracellular regions (amino acid residues 1–160) of the human cytotoxic T-lymphocyte-associated antigen 4 gene, the hinge and Fc regions (amino acid residues 1–216) of the human Ig 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
Antibody Analysis
Tissue Collection, Plasmid Extraction, and PCR Analysis
Statistical Analysis
Mouse Serum IgG Responses The kinetics of the serum IgG responses generated following intranasal delivery of pGJA-P/VAX was observed in this study. Serum IgG anti-PAc responses in mice treated with pGJA-P/VAX were detected at 4 wks after the initial immunization and continued throughout the experiment, compared with responses in the pVAX1 control mice (Fig. 1A  ). 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.
Mouse Salivary IgA Responses A salivary IgA anti-PAc response was detected at 6 wks post-prime in the pGJA-P/VAX-immunized group compared with the pVAX1 control group (Fig. 1C  ). 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
Compared with the previously used pCI vector, a different vector pVAX1 was used in this study. The major differences between these 2 vectors are as follows. First, the pCI vector contained the late SV40 polyadenylation signal, whereas the pVAX1 vector contained the bovine growth hormone polyadenylation signal. The different polyadenylation signals may cause differences in transcription termination and mRNA polyadenylation efficiency. Second, the kanamycin resistance gene was used in pVAX1 for selection in E. coli. Compared with the ampicillin resistance gene in pCI vector, the kanamycin resistance gene contained fewer unmethylated CpG motifs, which were unmethylated cytosine-guanine dinucleotides within a particular base context and are considered to be potent adjuvants for mucosal vaccination strategies (McCluskie and Davis, 1999). Recent studies showed that the immunomodulatory effects induced by CpG motifs vary from species to species, and the optimal CpG motif for humans is 5'TGTCGTT3'. Numbers of CpG motifs for DNA vaccines are also important. Studies showed that a DNA vaccine with 50 CpG-S motifs was less effective in inducing antibody responses against a hepatitis B surface antigen than that with 16 motifs (Krieg et al., 1998). Finally, pVAX1 is a 3.0-kb plasmid vector, whereas pCI is 4.0 kb. As we know, it is often more difficult for larger plasmids to enter the cells than for smaller ones, which may decrease the quantity of antigens expressed. Thus, we can see that many factors may influence the immunogenicity of DNA vaccines. In the present study, the immune responses generated with pGJA-P/VAX were not significantly different from those seen with pGJA-P. In a golden hamster study, we found that pGJA-P/VAX induced obvious serum and salivary antibody responses that showed no significant differences compared with those induced by pGJA-P. Moreover, no significant difference in the protective efficacy was observed between the pGJA-P/VAX- and pGJA-P-immunized groups in golden hamster models infected with S. mutans. 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.
We thank Prof. Takahiko Oho for providing the rPAc protein. This study was supported by grant No. 30330660 from the Natural Science Foundation of China.
 authors contributing equally to the work  Received for publication November 28, 2005. Revision received June 14, 2006. Accepted for publication June 21, 2006.
Journal of Dental Research, Vol. 85, No. 10,
915-918 (2006)
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ABSTRACT
INTRODUCTION
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.