Occult HBV infection (OBI) is a threat for the safety of blood-supply, and has been associated with the onset of HBV-related hepatocellular carcinoma and lymphomagenesis. Nevertheless, genetic markers in HBsAg (particularly in D-genotype, the most common in Europe) significantly associated with OBI in vivo are missing. Thus, the goal of this study is to define: (i) prevalence and clinical profile of OBI among blood-donors; (ii) HBsAg-mutations associated with OBI; (iii) their impact on HBsAg-detection. OBI was searched among 422,278 blood-donors screened by Nucleic-Acid-Testing. Following Taormina-OBI-definition, 26 (0.006%) OBI-patients were identified. Despite viremia <50 IU/ml, HBsAg-sequences were obtained for 25/26 patients (24/25 genotype-D). OBI-associated mutations were identified by comparing OBI-HBsAg with that of 82 chronically-infected (genotype-D) patients as control. Twenty HBsAg-mutations significantly correlated for the first time with OBI. By structural analysis, they localized in the major HBV B-cell-epitope, and in HBsAg-capsid interaction region. 14/24 OBI-patients (58.8%) carried in median 3 such mutations (IQR:2.0–6.0) against 0 in chronically-infected patients. By co-variation analysis, correlations were observed for R122P + S167L (phi = 0.68, P = 0.01), T116N + S143L (phi = 0.53, P = 0.03), and Y100S + S143L (phi = 0.67, p < 0.001).
Mutants (obtained by site-directed mutagenesis) carrying T116N, T116N + S143L, R122P, R122P + Q101R, or R122P + S167L strongly decreased HBsAg-reactivity (54.9 ± 22.6S/CO, 31.2 ± 12.0S/CO, 6.1 ± 2.4S/CO, 3.0 ± 1.0S/CO and 3.9 ± 1.3S/CO, respectively) compared to wild-type (306.8 ± 64.1S/CO). Even more, Y100S and Y100S + S143L supernatants show no detectable-HBsAg (experiments in quadruplicate).
In conclusions, unique HBsAg-mutations in genotype-D, different than those described in genotypes B/C (rarely found in western countries), tightly correlate with OBI, and strongly affect HBsAg-detection. By altering HBV-antigenicity and/or viral-particle maturation, they may affect full-reliability of universal diagnostic-assays for HBsAg-detection.