【作者】 李安遥； 陆晓燕； 郭蓉芳； 郭文博； 杨鹏挥； 娄鹤； 陈杰； 黄恩善； 章荣华； 王汉波； 杨继鸿； 李振皓； 范骁辉；

【Author】 Anyao Li;Xiaoyan Lu;Rongfang Guo;Wenbo Guo;Penghui Yang;He Lou;Jie Chen;Enshan Huang;Ronghua Zhang;Hanbo Wang;Jihong Yang;Zhenhao Li;Xiaohui Fan;College of Pharmaceutical Sciences,Zhejiang University;State Key Laboratory of Chinese Medicine Modernization,Innovation Center of Yangtze River Delta,Zhejiang University;Zhejiang Provincial Center for Disease Control and Prevention;ShouXianGu Botanical Drug Institute;Zhejiang Key Laboratory of Biological Breeding and Exploitation of Edible and Medicinal Mushrooms;The Joint-laboratory of clinical multi-omics research between Zhejiang University and Ningbo Municipal Hospital of TCM,Ningbo Municipal Hospital of TCM;

【机构】 浙江大学药学院； 浙江大学长三角智慧绿洲创新中心中药现代化国家重点实验室； 浙江省疾病预防控制中心； 寿仙谷中药研究院； 浙江省食用及药用菌生物育种与开发利用重点实验室； 浙江大学与宁波市中医院联合临床多组学研究实验室；

【摘要】 新型冠状病毒肺炎(COVID-19)疫苗接种后保护力随时间下降,亟需开发有效佐剂以增强免疫应答。去壁灵芝孢子粉(RGLS)活性成分具有免疫调节潜力,但其辅助提升疫苗免疫应答的效果尚不明确。本研究旨在评估RGLS作为口服佐剂对COVID-19疫苗加强针的增效作用,并借助单细胞多组学技术探讨其机制。研究纳入195名已完成两剂灭活疫苗的志愿者,随机分为对照组与RGLS组。RGLS干预组在接种第三针CoronaVac疫苗同时口服RGLS (每日2次,每次2g),持续6个月。采用微量中和试验检测中和抗体水平,并通过单细胞RNA、V(D)J及ATAC测序分析外周血单核细胞免疫特征。结合UPLC-MS分析RGLS成分,并通过分子对接与细胞实验验证其活性。研究结果显示,RGLS在接种后90天显著提升中等基线免疫力人群的中和抗体抑制率。单细胞多组学分析表明,RGLS显著提高了髓系细胞比例,尤其是经典单核细胞(CM)亚群(CM_S100A与CM_HLAD),并增强了其与T细胞和B细胞之间的相互作用。在先天免疫方面,RGLS上调了与IFN-α信号通路的转录因子(如STAT1、STAT2、IRF7、IRF9)活性,并提升了IFN-α应答相关基因的染色质可及性。在适应性免疫方面,RGLS诱导了B细胞受体(BCR)和T细胞受体(TCR)的基因使用偏好与克隆扩增偏向,增强了其对病毒的识别与结合能力。此外,RGLS干预组血清IFN-α水平显著升高,且多个干扰素刺激基因(如ISG15、IFI44L、IFITM1)表达上调。更进一步,化学分析确定了RGLS中12种入血三萜类成分含量,分子对接显示其对IFN-α通路关键靶点(TLR7/8、STING等)具高亲和力;体外实验证实灵芝酸H可促进IFN-α分泌及其下游干扰素刺激基因(ISG15、IFI44L)表达。综上,RGLS作为一种口服疫苗佐剂,可通过增强IFN-α介导的先天与适应性免疫应答,有效提升COVID-19疫苗效果,为其临床应用提供了理论依据。更多还原

【Abstract】 The protective efficacy of COVID-19 vaccines declines over time,highlighting the need for effective adjuvants to enhance immune responses.Sporoderm-removed Ganoderma lucidum spore powder(RGLS) is rich in bioactive components and possesses immunomodulatory potential,yet its effect in assisting in enhancing the immune response to vaccines remains unclear.This study aimed to evaluate the boosting effect of RGLS as an oral adjuvant for the COVID-19 vaccine booster dose and to investigate its mechanism using single-cell multi-omics approaches.A total of 195 volunteers who had completed two doses of inactivated vaccine were enrolled and randomly assigned to a control group or an RGLS group.The RGLS intervention group received RGLS orally(2 g,twice daily) for six months along with the third dose of CoronaVac.Neutralizing antibody levels were assessed using a microneutralization assay,and peripheral blood mononuclear cells(PBMCs)were analyzed by single-cell RNA sequencing,V(D)J sequencing,and ATAC sequencing.RGLS components were characterized using UPLC-MS,and their bioactivity was validated through molecular docking and cellular assays.Results showed that RGLS significantly increased the neutralizing antibody inhibition rate in individuals with moderate baseline immunity at 90 days post-vaccination.Single-cell multi-omics analysis revealed that RGLS elevated the proportion of myeloid cells,particularly classical monocyte subsets(CM S100 A and CM HLA-D),and enhanced their interactions with T and B cells.In innate immunity,RGLS upregulated the activity of transcription factors(e.g.,STAT1,STAT2,IRF7,IRF9)involved in the IFN-a signaling pathway,and enhanced the chromatin accessibility of genes related to the IFN-a response.In adaptive immunity,RGLS induced biased gene usage and clonal expansion of B cell and T cell receptors,improving viral recognition and binding.Moreover,serum IFN-a levels and the expression of interferon-stimulated genes(e.g.,ISG15,IFI44 L,IFITMI) were significantly elevated in the RGLS group.Chemical analysis determined the contents of 12 kinds of blood-entry triterpenoid components in RGLS,and molecular docking predicted their high affinity for key targets in the IFN-a pathway(e.g.,TLR7/8,STING).In vitro experiments confirmed that ganoderic acid H,a key component,promoted IFN-a secretion and the expression of downstream genes(ISG15,IFI44 L).In summary,RGLS serves as an oral vaccine adjuvant that enhances COVID-19 vaccine efficacy by boosting IFN-a-mediated innate and adaptive immune responses,providing a theoretical foundation for its clinical application.更多还原