新型冠状病毒T细胞表位多肽不同免疫途径及佐剂对免疫应答的影响及长效免疫研究  

Effects of Different Immunization Routes and Adjuvants on Immune Response and Long-term Immunity of SARS-CoV-2 Derived T-cell Epitopic Peptides

作　　者：宋洋[1] 胡宏俏 郭宏 时雨晴 黄星湖 赵建楠 江洁 曹蕾[1] 张勇[1] 张燕[1] 许文波[1] SONG Yang;HU Hongqiao;GUO Hong;SHI Yuqing;HUANG Xinghu;ZHAO Jiannan;JIANG Jie;CAO Lei;ZHANG Yong;ZHANG Yan;XU Wenbo(National Institute for Viral Diseases Control and Prevention,China CDC,NHC Key Laboratory of Medical Virology and Viral Diseases,WHO WPRO Regional Polio Reference Laboratory,WHO WPRO Regional Reference Measles/Rubella Laboratory,Beijing 102206,China)

机构地区：[1]国家卫生健康委员会医学病毒和病毒病重点实验室,中国疾病预防控制中心病毒病预防控制所,世界卫生组织西太平洋区脊髓灰质炎参比实验室,世界卫生组织西太平洋地区麻疹/风疹参比实验室,北京102206

出　　处：《病毒学报》2023年第3期631-643,共13页Chinese Journal of Virology

基　　金：国家重点研发计划(项目号:2022YFE0202800),题目:新型冠状病毒通用治疗性多肽疫苗的研究。

摘　　要：很多研究表明T细胞免疫在抗新型冠状病毒(SARS-CoV-2)感染和预防重症死亡中起到关键作用。T细胞识别由抗原递呈细胞(Antigen-Presenting Cell,APC)递呈的与主要组织相容性复合体(Major Histocompatibility Complex,MHC)结合的短肽,因此新冠T细胞多肽疫苗成为研究重点之一。本研究利用三条C57BL/6J小鼠MHC II类分子限制性的新冠T细胞表位多肽分别结合目前已经在临床上市使用的佐剂:铝佐剂,5’cytosine-phosphateguanine 3’oligonucleotide(CpG-OND,以下简称CpG)佐剂以及铝加CpG佐剂制备成小鼠新冠T细胞多肽疫苗,通过皮下、肌肉、滴鼻(CpG佐剂组别)三种给药方式两次免疫C57BL/6J小鼠,在二免后14 d和6个月(CpG佐剂组别)后利用荧光免疫斑点实验(FluoroSpot)评价疫苗刺激小鼠脾脏淋巴细胞产生的辅助型T细胞1(T helper 1 cell,Th1)应答细胞因子—干扰素γ(Interferon-γ, IFN-γ)、肿瘤坏死因子α(Tumor Necrosis Factor α, TNF-α)和白细胞介素2(Interleukin 2, IL-2)以及Th2应答细胞因子—IL-4、IL-10和IL-5的水平情况。实验结果显示,不管使用哪种佐剂,皮下免疫产生了更好的效果;皮下和肌肉免疫使用CpG佐剂产生的细胞因子水平良好但应答水平较AL加CpG佐剂比更向Th1极化,而滴鼻免疫的Th1/Th2应答水平更为平衡。结合AL加CpG佐剂的皮下免疫组小鼠分泌了高水平且Th1/2平衡的细胞因子。结合CpG佐剂的小鼠二免后6个月IFN-γ分泌水平降低,但仍能产生较多的斑点,且滴鼻免疫组小鼠IFN-γ并没有出现显著降低。本研究结果提示T细胞多肽疫苗结合AL加CpG佐剂通过皮下免疫途径产生更好的细胞免疫应答,结合单独CpG佐剂的滴鼻免疫方式同样产生了较好的T细胞免疫应答和长效T细胞免疫,二者同时也维持了良好的Th1/Th2应答平衡。本研究对新冠T细胞多肽进行了免疫途径和佐剂的条件摸索,具有一定参考价值。Many studies have indicated that T cell immunity plays a key role in preventing SARS-CoV-2 infection and COVID-19 severe illnesses and deaths.It also has broad-spectrum effects against variants of concern(VOCs).T cells recognize short peptides that bind to major histocompatibility complex(MHC)presented by antigen-presenting cells(APCs).In that case,T-cell peptide vaccines have become one of the research priorities.In this study,three SARS-CoV-2 derived T-cell epitopic peptides that bind to MHC class II molecules of C57BL/6J mice were used.Each peptide were mixed with the dose of 30μg per mouse in combination with adjuvants currently in clinical marketing-aluminum adjuvant,CpG-OND adjuvant,and aluminum plus CpG-OND adjuvant-to prepare a SARS-CoV-2 T-cell peptide vaccine for C57BL/6J mouse.C57BL/6J mice were immunized twice by subcutaneous,intramuscular,or intranasal(CpG-OND adjuvant group only)and administrated on days O and 21.Splenic lymphocytes were isolated from mice on day 35 and 6 months after the second immunization(CpG-OND adjuvant group only),and the levels of Thl-response cytokines-interferon gamma(IFN-),tumor necrosis factor alpha(TNF-α),and interleukin-2(IL-2),and Th2 response cytokines-IL-4,IL-10,and IL-5 produced in vaccine-stimulated mice were evaluated using fluorescent immunospot assay(FluoroSpot).Results indicated that subcutaneous immunization produced a better immune effect than intramuscular immunization regardless of which adjuvant was used.AL adjuvant alone produced very low levels of cytokines that were biased to Th2 responses.CpG adjuvant alone produced good levels of cytokines comparable to AL plus CpG adjuvant,while the response levels were more biased to Thl than AL plus CpG adjuvant at subcutaneous and intramuscular injection.The Th1/Th2 response levels were more balanced in intranasal immunization.Mice immunized subcutaneously with combined AL plus CpG adjuvant secreted high levels of Thl/2 balanced cytokines.Mice treated with CpG adjuvant showed decreased levels of IFN-secretion 6

关 键 词：新型冠状病毒 细胞免疫 多肽 免疫途径 CpG佐剂 长效免疫 

分 类 号：R392.9[医药卫生—免疫学] R373.1[医药卫生—基础医学]