Data_Sheet_1_Comparison of the immune effects of the Chlamydia abortus MOMP antigen displayed in different parts of bacterial ghosts.docx

Bacterial ghosts (BGs) are promising vaccine platforms owing to their high adjuvant properties and delivery efficiency. Heterologous antigens can be anchored to different parts of BGs using genetic engineering strategies to prepare vaccines. However, several key issues need to be resolved, including the efficient preparation of BGs and determining the optimal anchoring position of exogenous antigens in the BGs. Here, we prepared an efficient temperature-controlled lysis system using lysis gene E of phage PhiX174 and used the major outer membrane protein (MOMP) of Chlamydia abortus (C. abortus) as a model antigen to explore the optimal display location of exogenous antigens in BGs. We demonstrated that the constructed recombinant temperature-controlled lysis plasmid can still stably inhibit E gene expression at 37°C, and the lysis efficiency of E. coli can reach above 99.9%. Four recombinant MOMP Escherichia coli (E. coli) ghost vaccines were constructed using different anchor sequences. These vaccines all induced strong specific antibody responses and secrete high levels of IFN-γ in immunized mice and significantly increased the clearance of C. abortus in a mouse infection model. Notably, the strongest immune effect was observed when MOMP was displayed on the surface of E. coli ghosts (rECG-InpN-M), which resulted in the clearance of C. abortus in mice 6 days earlier than that with the recombinant MOMP vaccine. Altogether, we constructed an efficient BG temperature-controlled lysis system and provided a feasible strategy for developing a BG delivery platform with enhanced immune effects.

细菌幽灵（Bacterial Ghosts，BGs）凭借其优异的佐剂特性与递送效率，成为极具潜力的疫苗载体平台。可通过基因工程策略将异源抗原锚定至BGs的不同部位以制备疫苗。然而，仍有若干关键问题亟待解决，包括BGs的高效制备，以及确定外源抗原在BGs中的最优锚定位置。本研究利用噬菌体PhiX174的裂解基因E构建了高效温控裂解系统，并以流产衣原体（Chlamydia abortus，C. abortus）的主要外膜蛋白（MOMP）作为模式抗原，探究外源抗原在BGs中的最优展示位置。实验结果表明，所构建的重组温控裂解质粒仍可在37℃下稳定抑制E基因的表达，大肠杆菌（Escherichia coli，E. coli）的裂解效率可达99.9%以上。本研究通过不同锚定序列构建了4种重组MOMP大肠杆菌（E. coli）幽灵疫苗。上述疫苗均可在免疫小鼠体内诱导产生强烈的特异性抗体应答，并分泌高水平的干扰素-γ（IFN-γ），且在小鼠感染模型中显著提升了流产衣原体的清除效果。值得注意的是，当MOMP展示于大肠杆菌幽灵（rECG-InpN-M）表面时，免疫效果最为显著，其小鼠体内流产衣原体的清除时间较重组MOMP疫苗提前了6天。综上，本研究构建了高效的BG温控裂解系统，并为开发免疫效果更优的BG递送平台提供了可行策略。