靶向防龋DNA疫苗增强粘膜免疫的分子机制研究
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摘要
研究目的：
本课题组构建的靶向免疫防龋DNA疫苗经过多年的发展,在实验室研究中显示出较强的粘膜免疫效果和防龋保护作用。但是关于靶向免疫防龋DNA疫苗如何增强免疫反应的机制,尚有待进一步研究。本实验的研究目的是探讨靶向防龋DNA疫苗加强粘膜免疫的效果的生物学机制,为今后更好的促进靶向防龋DNA疫苗研究和应用提供的深入的分子机制研究基础。
研究方法：
本实验研究首先构建编码小鼠细胞毒性T淋巴细胞抗原4(cytotoxic T lymphocyte antigen 4, CTLA4)胞外区、人IgG1铰链区与Fc段、变形链球菌(Streptococcus mutans) PAc区和GLU区的靶向防龋质粒(pmGJA-P),构建编码人CD5分子引导序列、人IgGl铰链区与Fc段、PAc区和GLU区的非靶向防龋质粒做为对照(pCDA-P).然后,将BALB/c小鼠分成三组,每组20只,分别经过鼻腔滴注途径给予靶向防龋疫苗(给予pmGJA-P鼻腔滴注),非靶向防龋疫苗(给予pCDA-P滴注)和空质粒组(给予PCI滴注,阴性对照组)。比较各种质粒在各试验组诱导的抗体反应,抗原提呈细胞的形成,淋巴细胞增殖反应。此外,比较不同组小鼠头颈部淋巴结里树突状细胞(Dendritic cells, DCs)的表型变化,并应用抗原提呈细胞功能芯片分析不同实验组的DCs细胞在给予疫苗免疫前后基因的变化,实时定量PCR验证芯片分析的结果。
实验结果：
1特异性抗体反应：
本研究检测了14天和28天时各组的唾液和血清中抗PAc和抗GLU抗体水平。结果显示靶向疫苗组唾液中两种抗体水平均明显高于其他组。另外,靶向疫苗组血清中的抗PAc和抗GLU抗体水平也明显高于其他组。非靶向组的唾液和血清中抗PAc和抗GLU抗体水平则显著高于空质粒组。
2抗原提呈细胞：
通过运用抗原特异性的ELISPOT assay,本实验分析了从头颈部淋巴结和脾脏分离的单核细胞产生抗原提呈细胞的能力。从靶向疫苗组和非靶向疫苗组的头颈部淋巴结和脾脏分离的单核细胞产生特异性抗原提呈细胞的数目均显著的多于空质粒组。靶向疫苗组的头颈部淋巴结和脾脏分离的单核细胞形成抗原提呈细胞的数目则高于非靶向疫苗组。
3淋巴细胞增值实验
从靶向疫苗组和非靶向疫苗组的头颈部淋巴结分离的淋巴结细胞悬液的淋巴细胞增殖能力要明显高于空质粒组,两个使用疫苗的组的脾细胞淋巴细胞增殖能力也要明显高于空质粒组。
4细胞因子：
给予靶向和非靶向疫苗的两组,其头颈部淋巴结和脾脏分离的细胞悬液经抗原刺激之后,产生的分泌的IFN-γ和IL-4细胞要明显多于空质粒组。靶向和非靶向疫苗的两组比较,分泌IFN-γ淋巴细胞的数目无显著性差异。但是,无论头颈部淋巴结还是脾脏分离的细胞悬液,靶向疫苗组分泌IL-4的细胞的数目要明显多于非靶向疫苗组。
从两个疫苗组的小鼠头颈部和脾脏分离的细胞悬液经过刺激后,其IFN-γ,IL-4和IL-5的mRNA表达水平明显高于空质粒组。IFN-γ在两个疫苗组的表达水平无明显差异。但是IL-4和IL-5 mRNA的表达水平,则是靶向疫苗组明显高于非靶向疫苗组。这些结果表明靶向和非靶向组都产生了Th 1/Th2混合型的免疫反应。靶向疫苗组产生更高的Th2型的细胞因子表达,相比非靶向组加强了Th2型的免疫反应。
5头颈部淋巴结和脾脏分离的树突状细胞的表型分析：
从头颈部淋巴结和脾脏中分离的单核细胞的中CD11c阳性的DCs在靶向疫苗组要明显多于非靶向组。此外,在靶向组CD11c阳性的细胞的MHCⅡ、CD80和CD86表达更高。而非靶向疫苗组与空质粒组比较,其CD11c阳性细胞的数目,MHCⅡ、CD80和CD86的表达均无明显差异。
6芯片分析
有8个基因达到我们选定候选基因的标准,所有候选基因又通过Real-timePCR进行验证,其中有7个候选基因的表达得到Real-time PCR的证实。这些差异基因涉及DCs的成熟,抗原摄取、提呈,生存。
实验结论：
我们结果显示,靶向防龋疫苗可能通过提高抗原呈递的效果,促进DC细胞的成熟,并由此提高DCs对抗原的摄取和提呈能力,促进DCs的生存,进而加强DCs对T、B细胞的激活,加强粘膜免疫。
Aims:
The strategy to construct anti-caries DNA vaccine with targeting to cytotoxic T lymphocyte antigen 4 (CTLA-4) which binds to B7 molecule expressed on the surfaces of antigen-presenting cells(APC), is proved to be an effective way to enhancement of immune response and protective effect compared with untargeted DNA vaccine. But the mechanism of CTLA-4 fusion anticaries DNA vaccine to increase the antigen immune reponse and protection for caries challenge is still unclear.
Methods:
We constructed targeted anti-caries DNA vaccine encoding Streptococcus mutans antigens fused to murine CTLA4,untargeted anticaries DNA vaccine encoding Streptococcus mutans antigens fused to human CD5. Mice were given targeted, untargeted anti-caries DNA vaccine and empty plasmids pCI. Then, immune response, the numbers of AFC in CLNs and lymphcytes proliferation were determined. DCs in immune effector tissues were tested by FACS.The gene expression changes of DCs in different groups were tested by gene array. The candidate genes which were selected form gene array were confirmed by Real-time PCR.
Results
1. Ag-specific antibody responses
The salivary anti-PAc and anti-GLU IgA antibody level of Group pmGJA-P were both significantly higher than that of Group pCDA-P at day 14 and 28.The anti-PAc and anti-GLU IgG levels in serum samples of mice vaccinated with pmGJA-P showed significantly higher than those of mice vaccinated with pCDA-P at day 14 and 28.
Mice given pmGJA-P displayed increasing numbers of PAc and GLU specific IgA AFCs in CLNs. Elevated numbers of anti-PAc and anti-GLU IgG AFCs were detected in spleen of the group immunized with pmGJA-P compared with group with pCDA-P. Mice immunized with pCI as a control group did not exhibit any anti-Ag AFCs.
2. Lymphocytes proliferative and cytokine responses
Both splenic and CLNs lymphocytes from mice treated with pmGJA-P and pCDA-P showed significantly difference proliferative responses than pCI group. There was no significantly difference between Group pmGJA-P and pCDA-P.
Mice immunized pmGJA-P and pCDA-P had higher numbers of PAc and GLU specific IFN-y and IL-4 producing cells in their CLNS and spleens as compared with control groups. The numbers of PAc and GLU specific IFN-y producing cells of CLNs and spleens from mice immunised pmGJA-P were no significantly different in comparison to mice given pCDA-P. Spleens and CLNs from pmGJA-P immunised mice contained elevated numbers of Ag specific IL-4 producing cells had significantly greater numbers of cytokine-secreting than mice given pCDA-P.
CLNs or spleen from the mice given pmGJA-P exhibited significantly higher levels of IL-4 and IL-5 mRNA expression when compared with mice given pCDA-P. PAc or GLU stimulated lymphocytes from the spleen and CLNs of mice given pmGJA-P or pCDA-P contained significantly increased levels of IFN-y and IL-4 mRNA when compared with mice immunized nasally with pCI. In addition, IFN-γmRNA level was no significant different observed in mice given pmGJA-P and pCDA-P.
3. DCs subtype in mucosal tissues test by FACS
Our results observed major increases in numbers of CD11C+DCs in CLNs and spleen of mice given pmGJA-P when compared with mice given pCDA-P or pCI. Further, higher levels of MHC II, CD80, and CD86 were expressed by CD11C+DCs from mice given pmGJA-P compared with mice given pCDA-P or pCI.There was no significant difference in numbers of CD11c+DCs and costimulator molecule expression on CD11c+DCs between mice immunized pCDA-P and pCI.
4. Gene expression difference
We defined a selective group of 8 genes that were significantly different expression for at least in one group mice given pmGJA-P or pCDA-P compared with mice given empty plasmids. In selected 8 genes, 7 genes were conformed by Real-time PCR.These genes were involved in DCs mature, Ag uptake, presentation, survival.
Conclusion
The machenism of targeted CTLA-4 fusion DNA to enhance immunity may relate to increasing capture of antigen to stimulate DCs mature,migaration,antigen processing,and survival.
本课题组构建的靶向免疫防龋DNA疫苗经过多年的发展,在实验室研究中显示出较强的粘膜免疫效果和防龋保护作用。但是关于靶向免疫防龋DNA疫苗如何增强免疫反应的机制,尚有待进一步研究。本实验的研究目的是探讨靶向防龋DNA疫苗加强粘膜免疫的效果的生物学机制,为今后更好的促进靶向防龋DNA疫苗研究和应用提供的深入的分子机制研究基础。
研究方法：
本实验研究首先构建编码小鼠细胞毒性T淋巴细胞抗原4(cytotoxic T lymphocyte antigen 4, CTLA4)胞外区、人IgG1铰链区与Fc段、变形链球菌(Streptococcus mutans) PAc区和GLU区的靶向防龋质粒(pmGJA-P),构建编码人CD5分子引导序列、人IgGl铰链区与Fc段、PAc区和GLU区的非靶向防龋质粒做为对照(pCDA-P).然后,将BALB/c小鼠分成三组,每组20只,分别经过鼻腔滴注途径给予靶向防龋疫苗(给予pmGJA-P鼻腔滴注),非靶向防龋疫苗(给予pCDA-P滴注)和空质粒组(给予PCI滴注,阴性对照组)。比较各种质粒在各试验组诱导的抗体反应,抗原提呈细胞的形成,淋巴细胞增殖反应。此外,比较不同组小鼠头颈部淋巴结里树突状细胞(Dendritic cells, DCs)的表型变化,并应用抗原提呈细胞功能芯片分析不同实验组的DCs细胞在给予疫苗免疫前后基因的变化,实时定量PCR验证芯片分析的结果。
实验结果：
1特异性抗体反应：
本研究检测了14天和28天时各组的唾液和血清中抗PAc和抗GLU抗体水平。结果显示靶向疫苗组唾液中两种抗体水平均明显高于其他组。另外,靶向疫苗组血清中的抗PAc和抗GLU抗体水平也明显高于其他组。非靶向组的唾液和血清中抗PAc和抗GLU抗体水平则显著高于空质粒组。
2抗原提呈细胞：
通过运用抗原特异性的ELISPOT assay,本实验分析了从头颈部淋巴结和脾脏分离的单核细胞产生抗原提呈细胞的能力。从靶向疫苗组和非靶向疫苗组的头颈部淋巴结和脾脏分离的单核细胞产生特异性抗原提呈细胞的数目均显著的多于空质粒组。靶向疫苗组的头颈部淋巴结和脾脏分离的单核细胞形成抗原提呈细胞的数目则高于非靶向疫苗组。
3淋巴细胞增值实验
从靶向疫苗组和非靶向疫苗组的头颈部淋巴结分离的淋巴结细胞悬液的淋巴细胞增殖能力要明显高于空质粒组,两个使用疫苗的组的脾细胞淋巴细胞增殖能力也要明显高于空质粒组。
4细胞因子：
给予靶向和非靶向疫苗的两组,其头颈部淋巴结和脾脏分离的细胞悬液经抗原刺激之后,产生的分泌的IFN-γ和IL-4细胞要明显多于空质粒组。靶向和非靶向疫苗的两组比较,分泌IFN-γ淋巴细胞的数目无显著性差异。但是,无论头颈部淋巴结还是脾脏分离的细胞悬液,靶向疫苗组分泌IL-4的细胞的数目要明显多于非靶向疫苗组。
从两个疫苗组的小鼠头颈部和脾脏分离的细胞悬液经过刺激后,其IFN-γ,IL-4和IL-5的mRNA表达水平明显高于空质粒组。IFN-γ在两个疫苗组的表达水平无明显差异。但是IL-4和IL-5 mRNA的表达水平,则是靶向疫苗组明显高于非靶向疫苗组。这些结果表明靶向和非靶向组都产生了Th 1/Th2混合型的免疫反应。靶向疫苗组产生更高的Th2型的细胞因子表达,相比非靶向组加强了Th2型的免疫反应。
5头颈部淋巴结和脾脏分离的树突状细胞的表型分析：
从头颈部淋巴结和脾脏中分离的单核细胞的中CD11c阳性的DCs在靶向疫苗组要明显多于非靶向组。此外,在靶向组CD11c阳性的细胞的MHCⅡ、CD80和CD86表达更高。而非靶向疫苗组与空质粒组比较,其CD11c阳性细胞的数目,MHCⅡ、CD80和CD86的表达均无明显差异。
6芯片分析
有8个基因达到我们选定候选基因的标准,所有候选基因又通过Real-timePCR进行验证,其中有7个候选基因的表达得到Real-time PCR的证实。这些差异基因涉及DCs的成熟,抗原摄取、提呈,生存。
实验结论：
我们结果显示,靶向防龋疫苗可能通过提高抗原呈递的效果,促进DC细胞的成熟,并由此提高DCs对抗原的摄取和提呈能力,促进DCs的生存,进而加强DCs对T、B细胞的激活,加强粘膜免疫。
Aims:
The strategy to construct anti-caries DNA vaccine with targeting to cytotoxic T lymphocyte antigen 4 (CTLA-4) which binds to B7 molecule expressed on the surfaces of antigen-presenting cells(APC), is proved to be an effective way to enhancement of immune response and protective effect compared with untargeted DNA vaccine. But the mechanism of CTLA-4 fusion anticaries DNA vaccine to increase the antigen immune reponse and protection for caries challenge is still unclear.
Methods:
We constructed targeted anti-caries DNA vaccine encoding Streptococcus mutans antigens fused to murine CTLA4,untargeted anticaries DNA vaccine encoding Streptococcus mutans antigens fused to human CD5. Mice were given targeted, untargeted anti-caries DNA vaccine and empty plasmids pCI. Then, immune response, the numbers of AFC in CLNs and lymphcytes proliferation were determined. DCs in immune effector tissues were tested by FACS.The gene expression changes of DCs in different groups were tested by gene array. The candidate genes which were selected form gene array were confirmed by Real-time PCR.
Results
1. Ag-specific antibody responses
The salivary anti-PAc and anti-GLU IgA antibody level of Group pmGJA-P were both significantly higher than that of Group pCDA-P at day 14 and 28.The anti-PAc and anti-GLU IgG levels in serum samples of mice vaccinated with pmGJA-P showed significantly higher than those of mice vaccinated with pCDA-P at day 14 and 28.
Mice given pmGJA-P displayed increasing numbers of PAc and GLU specific IgA AFCs in CLNs. Elevated numbers of anti-PAc and anti-GLU IgG AFCs were detected in spleen of the group immunized with pmGJA-P compared with group with pCDA-P. Mice immunized with pCI as a control group did not exhibit any anti-Ag AFCs.
2. Lymphocytes proliferative and cytokine responses
Both splenic and CLNs lymphocytes from mice treated with pmGJA-P and pCDA-P showed significantly difference proliferative responses than pCI group. There was no significantly difference between Group pmGJA-P and pCDA-P.
Mice immunized pmGJA-P and pCDA-P had higher numbers of PAc and GLU specific IFN-y and IL-4 producing cells in their CLNS and spleens as compared with control groups. The numbers of PAc and GLU specific IFN-y producing cells of CLNs and spleens from mice immunised pmGJA-P were no significantly different in comparison to mice given pCDA-P. Spleens and CLNs from pmGJA-P immunised mice contained elevated numbers of Ag specific IL-4 producing cells had significantly greater numbers of cytokine-secreting than mice given pCDA-P.
CLNs or spleen from the mice given pmGJA-P exhibited significantly higher levels of IL-4 and IL-5 mRNA expression when compared with mice given pCDA-P. PAc or GLU stimulated lymphocytes from the spleen and CLNs of mice given pmGJA-P or pCDA-P contained significantly increased levels of IFN-y and IL-4 mRNA when compared with mice immunized nasally with pCI. In addition, IFN-γmRNA level was no significant different observed in mice given pmGJA-P and pCDA-P.
3. DCs subtype in mucosal tissues test by FACS
Our results observed major increases in numbers of CD11C+DCs in CLNs and spleen of mice given pmGJA-P when compared with mice given pCDA-P or pCI. Further, higher levels of MHC II, CD80, and CD86 were expressed by CD11C+DCs from mice given pmGJA-P compared with mice given pCDA-P or pCI.There was no significant difference in numbers of CD11c+DCs and costimulator molecule expression on CD11c+DCs between mice immunized pCDA-P and pCI.
4. Gene expression difference
We defined a selective group of 8 genes that were significantly different expression for at least in one group mice given pmGJA-P or pCDA-P compared with mice given empty plasmids. In selected 8 genes, 7 genes were conformed by Real-time PCR.These genes were involved in DCs mature, Ag uptake, presentation, survival.
Conclusion
The machenism of targeted CTLA-4 fusion DNA to enhance immunity may relate to increasing capture of antigen to stimulate DCs mature,migaration,antigen processing,and survival.
引文
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