Jiang Hao, Hu Yijun, Yang Mei, Liu Hao, Jiang Guangshui
Key Laboratory for Oral Biomedical Research of Shandong Province, School of Dentistry, Shandong University, Jinan, China.
Yuhuangding Hospital, Yantai, China.
Immunobiology. 2017 May;222(5):730-737. doi: 10.1016/j.imbio.2017.01.007. Epub 2017 Jan 25.
The strength of immune responses induced by DNA vaccine is closely associated with the expression level of cloned antigens available to the antigen presenting cells (APCs). To acquire a larger and more persistent amount of antigen, a dual-promoter, which could double the target antigen output through its expression both in prokaryotic and eukaryotic cells, was employed in the constructed anti-caries DNA vaccine with attenuated Salmonella as mucosal delivery vector in this study. Here, both CMV and nirB promoters were included in the plasmid that harbors the genes encoding the functional epitopes of two virulence factors of S. mutans, i.e. the saliva-binding region (SBR) of PAc and the glucan-binding region (GBR) of glucosyltransferase-I (GTF-I). Delivered by attenuated Salmonella Typhimurium strain SL3261, the anti-caries vaccine was administered intragastrointestinally to BALB/c mice for evaluation of the effectiveness of this immune regime. Specific anti-SBR and anti-GBR antibodies were detected in the serum and saliva of experimental animals by week 3 after immunization. These immune responses were further enhanced after a booster vaccination at week 16. However, in mice receiving Salmonella expressing SBR and GBR under the control of nirB alone these antibody responses were significantly (P<0.01) lower. The serum IgG subclass profiles suggested a Th1/Th2-mixed but Th2 biased immune response to the cloned antigens, which was further confirmed by a significant increase in the Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-10) cytokines in splenocytes of immunized mice upon stimulation with SBR or GBR. To further determine the protective efficacy of these responses, a challenge test with S. mutans strain UA159 was performed in mice after the second immunization. Following challenge, mice immunized with Salmonella expressing SBR and GBR under the control of the CMV-nirB promoter showed a significant (P<0.01) reduction in the number of S. mutans in the dental plaque compared to the empty vector-immunized or unimmunized mice, and the reduction was also significant at weeks 3-8 (P<0.05) post-challenge when compared with those receiving Salmonella clones with nirB promoter alone. These results provide evidence for the effectiveness of a dual-promoter strategy in the anti-caries DNA vaccine when employing attenuated Salmonella as delivering vehicle for mucosal immunization.
DNA疫苗诱导的免疫反应强度与抗原呈递细胞（APC）可利用的克隆抗原表达水平密切相关。为了获得更大且更持久的抗原量，本研究构建了以减毒沙门氏菌为黏膜递送载体的抗龋DNA疫苗，采用了一种双启动子，该启动子可通过在原核细胞和真核细胞中表达使靶抗原产量翻倍。在这里，CMV和nirB启动子都包含在质粒中，该质粒携带编码变形链球菌两种毒力因子功能表位的基因，即表面蛋白A（PAc）的唾液结合区域（SBR）和葡糖基转移酶-I（GTF-I）的葡聚糖结合区域（GBR）。通过减毒鼠伤寒沙门氏菌菌株SL3261递送，将抗龋疫苗经胃肠道给予BALB/c小鼠，以评估这种免疫方案的有效性。免疫后第3周，在实验动物的血清和唾液中检测到特异性抗SBR和抗GBR抗体。在第16周进行加强免疫后，这些免疫反应进一步增强。然而，在仅接受在nirB控制下表达SBR和GBR的沙门氏菌的小鼠中，这些抗体反应显著（P<0.01）降低。血清IgG亚类谱表明对克隆抗原的免疫反应是Th1/Th2混合但以Th2为主，在用SBR或GBR刺激后，免疫小鼠脾细胞中Th1（IFN-γ、IL-2）和Th2（IL-4、IL-10）细胞因子显著增加进一步证实了这一点。为了进一步确定这些反应的保护效力，在第二次免疫后对小鼠进行了变形链球菌菌株UA159的攻毒试验。攻毒后，与空载体免疫或未免疫的小鼠相比，用在CMV-nirB启动子控制下表达SBR和GBR的沙门氏菌免疫的小鼠牙菌斑中变形链球菌数量显著（P<0.01）减少，与仅接受nirB启动子的沙门氏菌克隆的小鼠相比，攻毒后第3 - 8周减少也显著（P<0.05）。这些结果为在以减毒沙门氏菌作为黏膜免疫递送载体的抗龋DNA疫苗中双启动子策略的有效性提供了证据。
