Lu Lu, Zhai Lina, Ou Qikun, Sang Shuli, Cao Chen, Guan Yiyan, Mao Yunyun, Zhai Yanfang, Li Kai, Yu Rui, Wang Yanchun
Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, 20 Dongda Street, Beijing 100071, China.
Beijing International Science and Technology Cooperation Base for Antiviral Drugs, Beijing Key Laboratory of Environmental and Viral Oncology, College of Chemistry and Life Science, Beijing University of Technology, Beijing 100124, China.
Vaccines (Basel). 2025 May 22;13(6):552. doi: 10.3390/vaccines13060552.
Outer Membrane Vesicles (OMVs), nanosized particles derived from Gram-negative bacteria, are promising vaccine carriers due to innate immunogenicity and self-adjuvant properties. Yet the systematic evaluations of OMV-associated toxicity remain limited.
We developed a CRISPR/Cas9-engineered serovar Typhimurium mutant (Mut4_STM) to produce detoxified OMVs (Mut4_OMVs) with enhanced yield. Subcutaneous immunization of BALB/c mice with Mut4_OMVs to evaluate safety, and the adjuvant efficacy was also determined by injecting Mut4_OMVs with F1Vmut or PA_D4 antigens, mixing formulation, respectively.
Mut4_OMVs showed a 9-fold protein yield increase over wild-type OMVs. While all mice injected with wild-type OMVs died, 100% survival was observed in those receiving Mut4_OMVs. However, dose-dependent pathological alterations became evident in specific organs as the administration dose escalated, such as induced splenic extramedullary hematopoiesis and renal edema. Despite residual toxicity, 2-3 doses of 10 μg Mut4_OMVs elicited antigen-specific antibody titers comparable to aluminum adjuvant controls and superior T-cell immune responses.
While Mut4_OMVs retain potent adjuvant activity, their residual toxicity necessitates further biocompatibility optimization for safe vaccine applications.
外膜囊泡（OMV）是源自革兰氏阴性菌的纳米级颗粒，因其固有免疫原性和自我佐剂特性而成为有前景的疫苗载体。然而，对OMV相关毒性的系统评估仍然有限。
我们构建了一种经CRISPR/Cas9基因工程改造的鼠伤寒血清型突变株（Mut4_STM），以产生产量更高的解毒OMV（Mut4_OMV）。对BALB/c小鼠进行Mut4_OMV皮下免疫以评估安全性，还分别通过将Mut4_OMV与F1Vmut或PA_D4抗原混合制剂注射来确定其佐剂功效。
Mut4_OMV的蛋白质产量比野生型OMV增加了9倍。虽然所有注射野生型OMV的小鼠都死亡了，但接受Mut4_OMV的小鼠存活率为100%。然而，随着给药剂量的增加，特定器官中出现了剂量依赖性的病理改变，如诱导脾外造血和肾水肿。尽管存在残留毒性，但2 - 3剂10μg的Mut4_OMV引发的抗原特异性抗体滴度与铝佐剂对照组相当，且T细胞免疫反应更强。
虽然Mut4_OMV保留了强大的佐剂活性，但其残留毒性需要进一步优化生物相容性以实现安全的疫苗应用。
