HBsAg genetic elements critical for immune escape correlates with HBV reactivation upon immunosuppression

Background: HBV-reactivation is defined as an abrupt reappearance or rise of serum HBV-DNA in patients with resolved or inactive HBV infection (Hoofnagle, 2009). The role of HBsAg genetic diversity in this phenomenon is still anecdotic. Here, we investigate HBsAg genetic signatures underlying immunosuppression-driven HBV-reactivation. Methods: This study includes 93 HBsAg-sequences from 29 patients with HBV-reactivation triggered by immunosuppressive therapy, and 64 chronically HBV-infected drug-naïve patients as control (all genotype-D). HBsAg ultra-deep sequencing (UDPS) is also performed for 21/29 HBV-reactivating patients. Results: 55.2% of patients with HBV-reactivation is treated with rituximab for hematologic-malignancies, 24.1% with corticosteroids for auto-immune/inflammatory/neoplastic diseases, and 20.7% with other immunosuppressive-chemotherapeutics. 48.3% of patients experienced HBV-reactivation after completing immunosuppressive-therapy (range: 1–14 months). Among 9 HBV-reactivating patients despite lamivudine-prophylaxis, drugresistance is detected in 5 patients. 72.4% of HBV-reactivating patients (compared with ≤1.5% of controls, P < 0.001) carries specific HBsAg-mutations localized in HBsAg-regions relevant for HBV immune-control. Of the 13 HBsAg-mutations correlated with HBV-reactivation, 5/13 (T118KP120A-Y134H-S143L-D144E) reside in the a-determinant, and are known to hamper HBsAg-recognition by antibodies; 8/13 (C48G-V96A-M103I-L109I-S171F-L175S-G185E-V190A) are localized in Class-I/II-restrictedT-cell epitopes, playing a potential role in HBV-escape from T-mediated response. Furthermore, additional N-linked glycosylation-sites within the a-determinant are found in 24.1% of HBV-reactivating patients, compared with 0% of controls (P < 0.001); N-linked glycosylation can mask immunogenic-epitopes, abrogating HBsAg-recognition by antibodies. By UDPS-analysis, 38.1% of HBV-reactivating patients carries minority-mutations in a-determinant, including vaccineescape T131N-M133I-G145R (intra-patient prevalence:0.1–18.1%), confirming the “immune-escape” viral-phenotype characterizing HBV-reactivation. Conclusions: HBV-reactivation occurs in a wide variety of immunosuppressive clinical-settings, also after completing immunosuppressive-therapy, and is driven by a complex quasispecies carrying HBsAg-mutations with enhanced-capability to evade immune-response. This underlines the importance of a careful patient-monitoring in all immunosuppressive-settings at reactivation risk and of establishing a prompt and potent therapy in order to prevent HBV-related clinical complications.