换位免疫对DNA和病毒载体疫苗免疫原性的改善作用及其机制研究

Improving the immunogenicities of DNA and recombinant viral vector vaccines is the key step for accelerating their translation from bench to bedside. Our previous work showed that changing the injection site during repeated inoculation (Successive Site Translocating Inoculation, SSTI) could significantly improve the specific immune responses elicited by both DNA and recombinant vaccinia vaccines. Preliminary mechanistic study suggested that SSTI improved the immunogenicity of DNA vaccine by avoiding the inhibition of in vivo antigen expression mediated by specific antibodies in the limbs that have been repeatedly inoculated. To completely characterize this strategy and to clarify the underlying mechanisms, in this project, we will first observe the impacts of SSTI on specific T cell function and antibody affinity, and investigate its improving effect on vaccine-induced protection in mice. Then, by employing recombinant Tiantan vaccinia as a model vaccine, we will elaborate how the immunogenicity of viral vector vaccine is enhanced by SSTI. Finally, we will delineate the source of specific antibodies in muscles that have been repeatedly inoculated with DNA or recombinant vaccinia vaccines，which will provide clues for further optimizing the SSTI strategy. Taken together, our study will not only provide a new way to improve the immunogenicities of both DNA and viral vector vaccines, but also help to deepen the understanding of the mechanisms about how these vaccines work.

寻求安全有效的免疫原性增强手段是促进DNA疫苗和重组病毒载体疫苗向临床应用转化的关键。我们在前期研究中发现，与常规的固定部位接种相比，在重复接种过程中改变肌肉注射部位（换位免疫）可显著增强DNA疫苗和重组痘病毒疫苗的免疫原性。初步机制探索提示：换位免疫可使DNA疫苗避免由特异性抗体介导的体内抗原表达抑制。为进一步阐明换位免疫策略的作用特点与机制，本课题将通过小鼠实验首先观察换位免疫对抗原特异性T细胞功能及特异性抗体亲和力的影响，并阐明其对疫苗保护效果的改善作用；随后，以重组天坛株痘病毒疫苗为模型，解析换位免疫策略增强病毒载体疫苗免疫原性的机制；最后，利用多色流式及组织原位检测技术，分析固定接种部位肌肉组织中特异性抗体的来源，为进一步优化换位免疫策略提供线索。以上工作不仅能为改善DNA疫苗和重组病毒载体疫苗的免疫原性提供一条新的途径，还将有助于深化对这两类疫苗免疫应答机理的认识。

以核酸疫苗和重组病毒载体疫苗为代表的新型疫苗是当前疫苗研究的热点。我们在前期研究中发现，相比于常规的固定部位肌注接种，在重复免疫时更换接种部位（换位免疫）可以显著提高DNA疫苗、重组痘病毒疫苗以及DNA初免-重组痘病毒加强策略所诱发的免疫反应强度。初步的机制探索发现，换位免疫能够避免接种部位特异性抗体应答对体内抗原表达的抑制，从而改善DNA疫苗的免疫原性。为了进一步解析换位免疫对特异性免疫应答的影响和机制，本课题以DNA疫苗和重组痘病毒疫苗为主要研究模型，通过小鼠实验，我们首先证明了换位免疫不仅能够提高DNA疫苗与重组痘病毒疫苗所激发的细胞免疫反应强度，还可以提高抗原特异性T细胞应答的质量；随后，分别通过豚鼠和小鼠实验，证实了换位免疫策略能够显著提高特异性抗体的应答强度，并且发现细胞因子佐剂（IL-21）能够显著提高换位免疫策略所诱发的特异性抗体的亲合力。接下来，为了阐明换位免疫策略的作用机制，我们进一步观察了疫苗接种次数和骨骼肌神经支配对特异性免疫应答的影响，发现特异性抗体亲合力峰值出现的时间显著滞后于抗体滴度峰值；并且发现骨骼肌神经支配对肌肉注射疫苗的接种效果具有显著影响。最后，我们运用RNA测序技术分析了疫苗接种部位肌肉组织中的抗体分泌细胞的表型和功能特征，初步证明了这些定居型抗体分泌细胞具有与骨髓浆细胞不同的表面分子标记和抗体分泌特征。综上所述，本课题通过多轮动物实验，系统阐述了换位免疫策略的作用特点和影响因素，证实了组织定居型抗体分泌细胞的存在，对未来的疫苗研发和免疫规划制定具有潜在参考价值。