National Engineering Research Center of Immunological Products & Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third Military Medical University, Chongqing, PR China.
Department of Critical Care Medicine, Children's Hospital of Chongqing Medical University, Chongqing, PR China.
PLoS Pathog. 2021 Jul 21;17(7):e1009752. doi: 10.1371/journal.ppat.1009752. eCollection 2021 Jul.
Highly immunogenic exotoxins are used as carrier proteins because they efficiently improve the immunogenicity of polysaccharides. However, their efficiency with protein antigens remains unclear. In the current study, the candidate antigen PA0833 from Pseudomonas aeruginosa was fused to the α-hemolysin mutant HlaH35A from Staphylococcus aureus to form a HlaH35A-PA0833 fusion protein (HPF). Immunization with HPF resulted in increased PA0833-specific antibody titers, higher protective efficacy, and decreased bacterial burden and pro-inflammatory cytokine secretion compared with PA0833 immunization alone. Using fluorescently labeled antigens to track antigen uptake and delivery, we found that HlaH35A fusion significantly improved antigen uptake in injected muscles and antigen delivery to draining lymph nodes. Both in vivo and in vitro studies demonstrated that the increased antigen uptake after immunization with HPF was mainly due to monocyte- and macrophage-dependent macropinocytosis, which was probably the result of HPF binding to ADAM10, the Hla host receptor. Furthermore, a transcriptome analysis showed that several immune signaling pathways were activated by HPF, shedding light on the mechanism whereby HlaH35A fusion improves immunogenicity. Finally, the improvement in immunogenicity by HlaH35A fusion was also confirmed with two other antigens, GlnH from Klebsiella pneumoniae and the model antigen OVA, indicating that HlaH35A could serve as a universal carrier protein to improve the immunogenicity of protein antigens.
高度免疫原性的外毒素被用作载体蛋白，因为它们能有效地提高多糖的免疫原性。然而，它们与蛋白质抗原的效率仍不清楚。在本研究中，铜绿假单胞菌的候选抗原 PA0833 与金黄色葡萄球菌的α-溶血素突变体 HlaH35A 融合，形成 HlaH35A-PA0833 融合蛋白（HPF）。与单独免疫 PA0833 相比，免疫 HPF 导致 PA0833 特异性抗体滴度增加、保护效力提高、细菌负荷降低和促炎细胞因子分泌减少。使用荧光标记的抗原来追踪抗原摄取和递呈，我们发现 HlaH35A 融合显著改善了注射肌肉中的抗原摄取和向引流淋巴结的抗原递呈。体内和体外研究均表明，免疫 HPF 后抗原摄取的增加主要归因于单核细胞和巨噬细胞依赖的巨胞饮作用，这可能是由于 HPF 与 Hla 宿主受体 ADAM10 结合所致。此外，转录组分析显示，几种免疫信号通路被 HPF 激活，揭示了 HlaH35A 融合提高免疫原性的机制。最后，HlaH35A 融合对另外两种抗原，肺炎克雷伯菌的 GlnH 和模型抗原 OVA 的免疫原性的改善也得到了证实，表明 HlaH35A 可以作为一种通用载体蛋白来提高蛋白质抗原的免疫原性。
