乙肝疫苗长期免疫后NK细胞免疫记忆反应的作用及机制研究

Immunisation with hepatitis B vaccine is the most effective means of preventing infection by hepatitis B virus. However, the duration of protection remains unclear. Several studies aimed to detect and measure the HBV surface antigen (HBsAg)-specific T-cells and B-cells reactivity in vaccinees to show the presence of specific immune memory; however, the results were contradictory. Most studies found that T cell and B cell immunologic memory response against HBV infection would induced by HB revaccination and HBsAg stimulation even though the protective anti-HBs were not detectable. However, recent studies showed that about 10-25% vaccinees who were protective anti-HBs negative had lost their immunologic memory after long term HB vaccination despite the good initial HB vaccination at birth. Immune response was mainly mediated by memory B and T lymphocytes that are sensitized through an initial exposure to a specific antigen or vaccination. Recent studies demonstrated that NK cell possessed immunologic memory function and played an important role against HBV infection. However, the role and mechanism on immunologic memory of NK cell induced by long term vaccination of HB remained unclear. .In this study, a cross-sectional study will be performed. Participants born during 2002–2005 who had received a series of HB vaccinations at ages 0, 1, and 6 months will be enrolled. Serological HBV markers(HBsAg, anti-HBs, anti-HBc) are detected by MEIA, and participants who were HBsAg, anti-HBs, anti-HBc negative are revaccinated with 1 dozes of HB vaccine to evaluate the immunologic memory after long term HB vaccination. PBMCs are isolated from peripheral blood, and are cultured in vitro with inactivated HBsAg or negative control. The frequency of NK cells, molecule markers of memory NK cells, the expression of NK cells-specific cytokines and cytotoxicity production are compare in cells seeded with HBsAg or negative control. We aim to study the role and mechanism of immunologic memory response of NK cell after long term hepatitis B vaccination which will provide scientific evidence for the improvement of revaccination strategies to hepatitis B vaccine, and provide new method to develop a new type of last longer hepatitis B vaccine.

乙肝疫苗免疫是预防HBV感染的最有效措施，但保护期长短仍无定论。以往研究认为接种者即使失去保护性抗体，在加强免疫或遇HBsAg刺激后可产生免疫记忆反应，抵抗HBV感染。近年发现抗-HBs阴性的乙肝疫苗长期免疫者有10-24%已丧失免疫记忆。免疫记忆主要由B细胞和T细胞介导，最新研究表明NK细胞也有免疫记忆功能，在抗HBV感染中发挥重要作用，但乙肝疫苗免疫后NK细胞免疫记忆反应作用及机制尚未明确。本研究选取2002-2005出生并按程序接种乙肝疫苗的人群为研究对象，探讨乙肝疫苗长期免疫记忆反应；对抗-HBs阴性者的外周血分离PBMC，分别加入灭活HBsAg或阴性对照进行体外培养，比较两组NK细胞记忆相关分子、细胞因子、细胞毒性因子和特异性IFN-γ、TNF-α mRNA表达的差异，探讨乙肝疫苗长期免疫后NK细胞免疫记忆反应作用及机制，为完善乙肝疫苗加强免疫策略、研制更持久的乙肝疫苗提供新思路

乙型肝炎感染（以下简称乙肝）仍是全球严重的公共卫生问题，接种乙肝疫苗是预防和控制乙肝感染的有效途径，但是乙肝疫苗免疫记忆反应的保护性和保护时效仍无定论。乙肝疫苗免疫反应主要是基于T、B细胞介导的细胞和体液免疫，有关T、B细胞在乙肝疫苗发挥适应性免疫记忆反应已有较深入的论述。NK细胞作为天然免疫细胞，传统免疫学观点认为NK细胞不具有免疫记忆反应，可是近年来有研究表明NK细胞具有免疫记忆功能，可NK细胞是否在乙肝疫苗中发挥免疫记忆作用尚无相关研究。.本研究选取1980年代出生并已按0、1、6标准免疫程序接种3剂次乙肝疫苗的成年人（接种组）和出生时未接种乙肝疫苗且无补打过乙肝疫苗的成年人（未接种组）作为研究对象，并开展流行病学调查。采集接种乙肝疫苗前研究对象外周静脉血10ml，其中5ml采用MEIA法定量检测乙肝血清标志物，另外5ml使用乙肝表面抗原体外刺激研究对象外周血单个核细胞（PBMC）12小时后收获细胞，通过流式细胞术检测NK细胞记忆表型（NKp46、KLRG1、CD57、NKG2D）、细胞毒性（CD107a）和IFN-γ、颗粒酶B和穿孔素等细胞因子的表达和频率。再为研究对象接种1剂次乙肝疫苗，在随访的第14天和第28天采集研究对象外周血5ml并进行流式细胞术检测，检测的指标同体外实验。采用Flowjo软件分析流式细胞术数据，用Mann-Whitney U检验、Wilcoxon检验和Spearman等级检验评估相关细胞毒性、细胞因子频率和NK细胞免疫记忆反应的相关性。.本次研究发现疫苗接种组在抗原刺激以及补种1针乙肝疫苗后均发现mNKs的存在；而未接种组在接种1剂次乙肝疫苗后同样出现mNKs情况且由CD57、NKG2D和KLRG1的CD56dim细胞群介导此反应。结果显示使用抗原刺激和补种乙肝疫苗后研究对象外周血NK细胞表现出更明显的细胞脱颗粒作用和分泌更多相关细胞因子的情况。本次研究初步阐明乙肝疫苗有关NK细胞记忆表型，NK细胞免疫记忆反应，为进一部提高乙肝疫苗免疫后的长期保护效果，研制NK细胞免疫记忆反应的新型乙肝疫苗提供理论参考。