基于明尼苏达沙门氏菌Re595脂多糖低毒改造的外膜囊泡佐剂活性研究

Adjuvant activity of outer membrane vesicles from Salmonella minnesota Re595 with lipopolysaccharides modified for attenuating toxicity

摘要:

【目的】外膜囊泡(outer membrane vesicles, OMVs)是由革兰氏阴性菌和某些革兰氏阳性菌生成和分泌的球形双层膜结构，含有丰富的细菌表面抗原，因此在疫苗相关领域具有重要的研究价值。脂多糖(lipopolysaccharide, LPS)作为OMVs的主要结构，在诱导免疫活性的同时，也成为影响OMVs安全性的主要因素。因此，通过基因工程改造细菌LPS以生产安全高效的OMVs，是促进其生产应用的有效途径之一。【方法】以O抗原和大部分核心抗原缺失的明尼苏达沙门氏菌(Salmonella minnesota) Re595为研究对象，通过缺失酰基链编码基因msbB和导入新凶手弗朗西丝氏菌(Francisella novicida)磷酸酶基因lpxE，减少类脂A上的酰基链和磷酸基团，从而获得低毒LPS；提取亲本株与改造株LPS与OMVs，比较其刺激免疫细胞的炎性反应，以OMVs作为免疫增强剂制备口蹄疫灭活疫苗，评价免疫活性。【结果】Re595经LPS低毒改造成功降低该菌OMVs的内毒素活性和炎性反应，进一步评价亲本株Re595与低毒改造株的OMVs免疫活性发现，LPS低毒改造虽降低了OMVs的炎性反应，但其免疫增强活性显著提高。【结论】本研究说明LPS低毒改造后可降低细菌OMVs毒性，并提高其免疫增强活性，该结果将为OMVs作为免疫佐剂的应用提供理论依据。

关键词:外膜囊泡;免疫佐剂;脂多糖;内毒素活性;基因编辑

Abstract:

[Objective] Outer membrane vesicles (OMVs) are spherical bilayer membrane structures secreted by Gram-negative and some Gram-positive bacteria. OMVs contain abundant surface antigens and are of great research significance in vaccine development. However, the presence of lipopolysaccharides (LPS), which is the primary component of OMVs, arouses safety concern. Therefore, genetically modifying bacterial LPS to produce safe and efficient OMVs is a viable approach to enhance the production and application of OMVs. [Methods] We modified Salmonella minnesota Re595 with O antigen and most core antigen deletions by deleting the acyl chain coding gene msbB and inserting the phosphatase coding gene lpxE from Francisella novicida to reduce acyl chains and phosphate groups on lipid A, thus obtaining less toxic LPS. LPS and OMVs were extracted from the starting strain and modified strain, and their pro-inflammatory activities were compared between the two strains. In addition, inactivated foot-and-mouth disease virus vaccines were prepared with OMVs to assess the immune adjuvant activity of OMVs. [Results] The modification of LPS reduced the endotoxin activity and pro-inflammatory responses while significantly increasing the immune adjuvant activity of OMVs. [Conclusion] This study demonstrates that the modification of LPS can attenuate the toxicity and enhance the immune adjuvant activity of bacterial OMVs. These findings provide a theoretical foundation for utilizing OMVs as immune adjuvants in the future.

Key words:outer membrane vesicles;immune adjuvant;lipopolysaccharide;endotoxin activity;gene editing