乙肝疫苗高免疫原性相关基因的研究

Immunisation with hepatitis B vaccine is the most effective means of preventing acute infection by hepatitis B virus. However, the duration of protection remains unclear. It has been reported that the titre of anti-HBs induced by the vaccine determines the duration of protection. In many cases, when scientists studied the association of host factors with the response to the vaccine, they tended to focus on response or non-response, rather than the difference between those who maintain a high titre of anti-HBs for a long time and those whose anti-HBs became negative soon. Longan county, Guangxi was one of the first research areas to evaluate the effectiveness of hepatitis B vaccine in our country. It has the details of immunization..The proposed study will select subjects from Longan to establish a l:1 case-control study. All of the cases and controls responded with protective levels of anti-HBs when immunized 28 years ago. Cases will be those whose anti-HBs titres remained above 100 mIU/ml for 28 years after immunization. Controls will be those with anti-HBs titres below 10 mIU/ml but who remain negative for HBsAg. A genome-wide association study will be performed to search for genes that are strongly related to the response to the hepatitis B vaccine..The results will not only reveal the factors influencing the duration of high titre of anti-HBs, which may be helpful in improving current vaccines and their immune-protection and lead to life-long protection, but also will be helpful in resolving low and non-responses.

乙肝疫苗免疫是预防乙肝病毒新感染最有效的手段，但免疫保护期长短尚无定论。有报道表明，免疫产生抗体滴度的高低影响免疫保护期的长短。长期以来人们在探讨宿主因素与乙肝疫苗免疫应答关系时，主要集中在探索应答与不应答的原因，忽视了常规免疫都产生保护性抗体人群中，为何有些能长时间维持高抗体滴度、有些抗体很快变阴性。广西隆安县是我国最早开展乙肝疫苗免疫效果观察的现场，有完整的免疫史记录。本项目将按1:1匹配病例对照研究原理，在隆安选择1987年出生、常规免疫都产生保护性抗体且目前抗体滴度仍≧100 mIU/ml者为病例，滴度﹤10mIU/ml但HBsAg仍为阴性者为对照，用全基因组关联研究方法，探索乙肝疫苗高免疫原性相关基因，研究结果不仅能阐明正常免疫应答人群高抗体滴度维持时间长、短的原因，从而为改善现有疫苗、提高其免疫保护效果以达到终身免疫提供科学依据，而且有助于解决低免疫应答或不应答问题。

本研究旨在阐明乙肝疫苗免疫后高抗体组和低抗体组在基因上的差异，探索乙肝疫苗免疫原性相关基因，阐明正常免疫应答人群高抗体滴度维持时间长、短的原因，从而为改善现有疫苗、提高其免疫保护效果以达到终身免疫提供科学依据。本研究按 1:1 匹配病例对照研究的原理，在广西隆安县选择219对病例和对照样本，选择1987年出生、常规免疫都产生保护性抗体且目前抗体滴度仍≧100 mIU/ml者 为病例，滴度﹤10mIU/ml但HBsAg仍为阴性者为对照。用芯InfiniumCore-24v1-2_A1 进行全基因组SNP扫描并采用 PLINK 1.90 进行基于三种模型的单位点关联分析。全基因组关联分析（GWAS）发现有46个SNP 与抗体水平显著相关。46个SNP位点应用MassARRAY SNP分型检测平台在231对高、低抗体样本中进行验证，结果分析发现位于一号染色体非编码区的3个SNP位点（rs7037200、rs17628432、rs11699316）在高、低抗体组中差异显著（P<0.05）。随机挑选50对高低抗体样本用定量PCR方法进行3个SNP关联基因 （PNPLA7、ALPK1、ENTPD6，分别与炎症反应、先天免疫和巨噬细胞的活化相关）的mRNA 表达检测，发现这3个基因的mRNA表达在高低抗体组中均差异不显著（P>0.05）。这可能由于样本在快递运输过程中干冰耗尽，使得部分样本提取的RNA质量较差，影响了mRNA检测结果的准确性，后续我们会进行进一步的验证和分析。本研究从基因组水平发现了3个乙肝疫苗高免疫原性相关的SNP，初步的检测结果表明这3个SNP关联基因的mRNA表达在高低抗体组中差异不显著，后续我们将进行更加深入的研究，阐明这3个SNP及其关联基因影响乙肝疫苗抗体水平的机制。