HBsAb阳性隐匿性乙型肝炎病毒T细胞及B细胞表位变异分析

Mutation analysis of T-cell and B-cell epitopes derived from HBV PreS-S protein in HBsAb positive occult hepatitis B virus infection

目的分析隐匿性乙型肝炎病毒(occult hepatitis B virus,OHBV)PreS-S基因T、B细胞表位变异情况,探讨隐匿性乙型肝炎病毒感染(occult hepatitis B virus infection,OBI)及HBsAb+OBI发生机制。方法利用巢式聚合酶链反应(nested PCR)扩增OBI样品PreS-S区并测序,确定基因型。生物信息学分析HBsAb+/HBsAb-以及不同基因型OBI毒株PreS-S基因T、B细胞表位变异情况,分析高频T细胞突变表位突变前后与人类白细胞抗原(human leukocyte antigen,HLA)亲和力变化以及高频B细胞突变表位突变后抗原特性的改变。结果85份OBI样品成功扩增出PreS-S基因21份,其中HBsAb+OBI_(B) 4份,HBsAb-OBI_(B) 6份,HBsAb+OBI_(C) 6份,HBsAb-OBI_(C) 5份。OBI毒株PreS-S区突变率高于野毒株,差异有显著的统计学意义(OBI_(B) vs.WT B:2.64%:0.66%,P<0.001;OBI_(C) vs.WT_(C):3.67%:1.19%,P<0.001)。OBI毒株蛋白免疫区突变率大于非蛋白免疫区,差异有显著的统计学意义(OBI_(B):3.57%:1.86%,P=0.005;OBI_(C):4.78%:2.65%,P=0.001)。OBI_(C)毒株蛋白免疫区及非蛋白免疫区突变率均大于OBI_(B)毒株,差异无统计学意义(蛋白免疫区:4.78%:3.57%,P=0.107;非蛋白免疫区:2.65%:1.86%,P=0.142)。HBsAb-OBI毒株T、B细胞表位突变率高于HBsAb+OBI毒株,差异无统计学意义(HBsAb-OBI_(B) vs.HBsAb+OBI_(B):4.17∶3.01,P=0.303;HBsAb-OBI_(C) vs.HBsAb+OBI_(C):5.65∶4.26,P=0.207)。4个T细胞表位高频突变(T47A/K、S174N、L175S、V177A)以及3个B细胞表位高频突变(G73S、K122R、I126M/T)的亲和力分析和抗原性分析显示多数T细胞表位突变前后与HLA分子亲和力变化不大,B细胞表位免疫原性变化不明显,某些位点突变后亲水性、表面可及性甚至优于野生毒株。结论OBI毒株PreS-S区突变率显著高于野毒株;OBI毒株蛋白免疫区突变率显著高于非蛋白免疫区;OBI_(C)变异活跃程度大于OBI_(B);各点突变可能在病毒复制过程中随机发生,未发现HBsAb压力或依赖性;PreS-S区关键位点突变或多表位共同突变可能在OBI形成中发挥重要作用。

ObjectiveTo analyze the mutation of T-cell and B-cell epitopes derived from HBV PreS-S protein in occult hepatitis B virus(OHBV)and investigate the biological mechanisms of occult hepatitis B virus infection(OBI)and HBsAb positive OBI.MethodsThe PreS-S region of OBI samples were amplified by nested PCR,the products were sequenced and HBV genotypes were determined.The mutations of T-cell and B-cell epitopes derived from HBV PreS-S protein were analyzed and compared among groups of HBV genotypes and the presence of HBsAb.The affinity of the high frequency of T-cell epitope substitutions were analyzed by SYF PEITHI,the changes of antigenic characteristics of high frequency of B-cell epitope substitutions were analyzed by Ab Designer,Expasy ProtParam tool,Epitope Prediction and Analysis Tools.ResultsThe PreS-S region of HBV was amplified in 21 samples,including 4 HBsAb+OBI_(B),6 HBsAb-OBI_(B),6 HBsAb+OBI_(C),5 HBsAb-OBI_(C).The mutation rates in PreS-S region of OBI were significantly higher than wild type HBV strains(OBI_(B) vs.WT B:2.64%:0.66%,P<0.001;OBI_(C) vs.WT_(C):3.67%:1.19%,P<0.001).The mutation rates of the immunoreactive area were significantly higher than non-immunoreactive area in OBI(OBI_(B):3.57%:1.86%,P=0.005;OBI_(C):4.78%:2.65%,P<0.001).The mutation rates of the immunoreactive and non-immunoreactive area in OBI_(C) were higher than OBI_(B),but there was no statistically significant difference(immunoreactive area:4.78%:3.57%,P=0.107;non-immunoreactive area:2.65%:1.86%,P=0.142).The mutation rates of T-cell and B-cell epitopes of HBsAb-OBI were higher than HBsAb+OBI,although there was no significant difference(HBsAb-OBI_(B) vs.HBsAb+OBI_(B):4.17∶3.01,P=0.303;HBsAb-OBI_(C) vs.HBsAb+OBI_(C):5.65∶4.26,P=0.207).The affinity analysis of 4 high frequency T-cell epitope substitutions,including T47A/K,S174N,L175S,V177A,showed that the changes of affinity of most mutation sites were not obvious;the antigenicity analysis of 3 high frequency B-cell epitope substitutions,including G73S,K122R,I126M/T,did not show noticeable changes and the hydrophilicity,surface accessibility of some mutation sites were even better than wild strain.ConclusionsThe mutation rates in PreS-S region of OBI were significantly higher than wild type HBV strains.The mutation rates of the immunoreactive area were higher than non-immunoreactive area in OBI.The variant activity of OBI_(C) was higher than OBI_(B).The mutations of OBI might occur randomly and were not selected by antibody pressure.Single epitope and multi-epitopes combinational mutations might be a reason for OBI.