Implications of newly-added N-glycosylation mutation of hepatitis B virus s-gene in patients with coexistence of HBsAg and antiHBs / 解放军医学杂志

ABSTRACT

Objective To analyze the characteristics of newly added N-glycosylation mutation in major hydrophilic region (MHR) of HBV S gene in patients with coexistence of HBsAg and antiHBs, and reveal the generation mechanism and clinical implications of the coexistence. Methods HBV S genes from 284 patients with HBsAg+antiHBs and 314 patients with single HBsAg were amplified respectively for sequence analysis. A chronic hepatitis B (CHB) patient with HBsAg+antiHBs in MHR was found to harbor a novel double N-glycosylation mutation and selected for further study. Recombinant vectors harboring the novel mutant or control PreS/S genes were constructed and transfected in HepG2 cells respectively for phenotypic analysis, and the effects of the mutations on HBV duplication and antigenicity were investigated. Results The detection rate of MHR N-glycosylation mutation was significantly higher in HBsAg+antiHBs group than in single HBsAg group (11.3% vs. 2.9%, P<0.01, respectively). In HBsAg+antiHBs cohort, the proportion of hepatocellular carcinoma (HCC) patients accounted for 46.9%(15/32) in patients with N-glycosylation mutation at the time of testing; by contrast, the number was 22.6%(57/252) in patients with non-N-glycosylation mutation (P<0.01). N-glycosylation mutational pattern of the novel strain was s116-118TST → NST+s131-133TSM → NST concomitant with sP120 deletion+G145D mutation. The novel mutants accounted for 98.0%, 2.0% and 2.5%, respectively, of viral clones in three sequential serum samples. Mutants with single N-glycosylation mutation s130-132GTS→NSS without sP120 deletion+G145D were detected in sample 2, accounting for 17.6% of viral clones. Compared to the wild-type, the novel mutant had an increase of 31% in replication capacity, but a decrease of 99% in HBsAg level. Immunofluorescence showed that elimination of the two additional N-glycosylation mutations only partly restored HBsAg detection by antiHBs, suggesting that sP120 deletion+G145D mutation also attenuated HBsAg antigenicity. Conclusions Additional N-glycosylation mutation in MHR of HBV S gene is associated with coexisting HBsAg+antiHBs, and the two parameters together might be a better risk factor for HCC occurrence. Combination of two additional N-glycosylation mutation, sP120 deletion and sG145D mutation may co-play a role in silence of HBsAg antigenicity.