Analysis of the factors influencing HBsAg serum levels in HBV chronic carriers

Background and Aims: HBsAg serum levels (HBsAgsl) decline during the natural course of HBV infection, being lower in the HBeAg-negative phase. Emergence of PreS variants could modify the ratio among PreS-1, PreS-2 and S proteins influencing HBsAg serum levels. Pre-S-region heterogeneity of circulating HBV-DNA quasispecies were characterized in 209 HBeAg-negative, genotype D carriers and correlated with HBsAg serum levels according to their virological and clinical profiles. Patients and Methods: HBV carriers (Male/Female 104/105; mean age 46,1+13y), followed-up prospectively for a mean period of 40 m (+28.1), were classified as Inactive carriers (IC; HBV-DNA< 2000); Active without significant liver disease (AC1; HBV-DNA>2000< 20000); Active with Chronic Hepatitis (CH; HBV-DNA≥20000); Cirrhotics (CI; HBV-DNA≥20000 IU/mL). PreS region HBV mutants (PreSmt) were characterized by direct sequencing (CEQ-2000-XL DS, Beckman; minor population detected = 20% total viremia) and correlated with HBsAg serum levels (Architect-i2000, Abbott) on the same samples. Results: PreS region was characterized in 204 (97.6%) sera and mixed PreS mutant/wild type HBV populations were found in 45 (22.5%): 1 (1.9%) IC, 1 (3.2%) AC1, 28 (34.0%) CH and 15 (38.9%) CI (p< 0.001). Mutations were: PreS1 ATG point mutations in 1 (0.5%, HBsAg: 1709 IU/ml); PreS2 ATG point mutations in 10 (4.8%, median HBsAg=2867, range: 749-11732); deletions involving MHC class I/II-restricted T-cell epitope ending at nt 57 of the PreS2 region in 18 (8.6%, median HBsAg=3213.5, range: 473-11227); deletions including a larger portion of PreS region in 15 (7.1%, median HBsAg=7170, range: 853-15770); simultaneous PreS1/PreS2 ATG point mutations in 1 (0.5%, HBsAg: 4594). Median HBsAg serum levels were lower (1445, range:0.06-82480) in 159 patients without PreS mutations than in 45 with PreS mutations (3930, range: 473-15770; Mann Whitney U test p< 0.001) but not significantly different within diagnostic categories. Multivariate analysis in 119 (Chronic and chirrotics) patients showed that lower LogHBsAg serum levels correlates with older age (p=0.007) and disease stage (p=0.036), but not with PreS mutations (p=0.905). Conclusions. Mixed PreS mutant/wild type HBV populations circulate in 1/3 of patients with significant liver disease (CH or CI), but rarely in AC1 and IC, therefore their low HBsAg serum levels do not depend on PreS mutations. PreS mutations is associated with age and disease stage. The most common mutations involve the MHC-class I/II restricted T cell epitope suggesting their selection by the host's immune response. All these findings support the hypothesis that HBsAg serum levels, reflecting the rate the transcriptionally active cccDNA and their quantification add further information to the monitoring of viral replication in the understanding of the complex equilibrium between the virus and the host’s immune response at the single carrier level.