Specific mutations in the C-terminus domain of HBV surface antigen significantly correlate with low level of serum HBV-DNA in patients with chronic HBV infection.

BACKGROUND: To define HBsAg-mutations correlated with different serum HBV-DNA levels in HBV chronically-infected drug-naive patients. METHODS: This study included 187 patients stratified into the following ranges of serum HBV-DNA:12-2000 IU/ml, 2000-100,000 IU/ml, and >100,000 IU/ml. HBsAg-mutations were associated with HBV-DNA levels by applying a Bayesian-Partitional-Model and Fisher-exact test. Mutant and wild-type HBV genotype-D genomes were expressed in Huh7 cells and HBsAg-production was determined in cell-supernatants at 3 days-post-transfection. RESULTS: Specific HBsAg-mutations (M197T,-S204N-Y206C/H-F220L) were significantly correlated with serum HBV-DNA <2000 IU/ml (posterior-probability>90%, P < 0.05). The presence of Y206C/H and/or F220L was also associated with lower median (IQR) HBsAg-levels and lower median (IQR) transaminases (for HBsAg:250[115-840] IU/ml for Y206C/H and/or F220L versus 4300[640-11,838] IU/ml for wild-type, P = 0.023; for ALT:28[21-40] IU/ml versus 53[34-90] IU/ml, P < 0.001). These mutations were localized in the HBsAg C-terminus, known to be involved in virion and/or HBsAg secretion. The co-occurrence of Y206C + F220L was found significant by cluster-analysis, (P = 0.02). In addition, in an in-vitro model Y206C + F220L determined a 2.8-3.3 fold-reduction of HBsAg-amount released in supernatants compared to single mutants and wt (Y206C + F220L = 5,679 IU/ml; Y206H = 16,305 IU/ml; F220L = 18,368 IU/ml; Y206C = 18,680 IU/ml; wt = 14,280 IU/ml, P < 0.05). CONCLUSIONS: Specific HBsAg-mutations (compartmentalized in the HBsAg C-terminus) correlated with low-serum HBV-DNA and HBsAg-levels. These findings can be important to understand mechanisms underlying low HBV replicative potential including the inactive-carrier state.

Novel HBsAg markers tightly correlate with occult HBV infection and strongly affect HBsAg detection.

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 <50IU/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.

Identification and structural characterization of novel genetic elements in the HIV-1 V3 loop regulating coreceptor usage.

BACKGROUND: The interaction between HIV-1 gp120 and CCR5 N terminus is critical for R5-virus entry and affects CCR5 antagonists' activity. Knowledge of how different genetic signatures of gp120 V3 domain effect the strength of this interaction is limited. METHODS: HIV-1 coreceptor usage was assessed in 251 patients using enhanced-sensitivity Trofile assay and V3 sequencing plus tropism prediction by Geno2pheno algorithm. Bayesian partitional model and recursive model selection have been used to define V3 genetic determinants correlated with different coreceptor usage. Gp120 interaction with CCR5 N terminus was evaluated by docking-analysis/molecular-dynamic simulations starting from the model described previously. RESULTS: Selected V3 genetic determinants (beyond known aminoacidic positions) significantly correlate with CCR5- or CXCR4-usage, and modulate gp120 affinity for CCR5 N terminus. This is the case for N5Y and N7K, absent in CCR5-using viruses and present in 4.5% and 6% of CXCR4-using viruses, respectively, and A19V, occurring in 2.6% of CCR5-using viruses and 22.0% of CXCR4-using viruses (P=10(-2) to 10(-7)). Their presence determines a decreased affinity for CCR5 N terminus even stronger than that observed in the presence of the well-known mutation S11R (N5Y: -6.60 Kcal/mol; N7K: -5.40 Kcal/mol; A19V: -5.60 Kcal/mol; S11R: -6.70 Kcal/mol; WT: -6.90 Kcal/mol). N7K significantly increases the distance between V3 position 7 and sulphotyrosine at CCR5 position 14 (crucial for binding to gp120; from 4.22 Å to 8.30 Å), thus abrogating the interaction between these two important residues. CONCLUSIONS: Key determinants for tropism within the V3 sequence, confirmed by structure- and by phenotypic-tropism, have been identified. This information can be used for a finer tuning of potential efficacy of CCR5-antagonists in clinical practice, and to provide molecular implications for design of new entry inhibitors.