Comparative analysis of HBV basic core promoter/pre-core gene mutations and viral quasispecies diversity in HIV/HBV co-infected and HBV mono-infected patients.

Human immunodeficiency virus (HIV)/hepatitis B virus (HBV) co-infection accelerates the progression of HBV-related liver diseases. HBV basic core promoter (BCP)/pre-core (preC) gene mutations may be one of the most important risk factors. In this study, a total of 230 patients were recruited, and 199 patients whose HBV BCP/preC gene were successfully amplified and sequenced, including 99 HIV/HBV co-infected and 100 HBV mono-infected patients. Next-generation sequencing was used for detection of BCP/preC mutations which were then compared in patients with different HBV genotypes and different HBeAg statuses, and 1% and 20% cutoff values were defined to evaluate the mutations. HBV quasispecies diversity was also compared in HIV/HBV co-infected and HBV mono-infected patients. Among the patients infected with HBV genotype C and HBeAg-negative status, the frequency of A1762T/G1764A double mutations was significantly lower in HIV/HBV co-infected patients than in HBV mono-infected patients (53.3% vs. 100.0%, P = 0.008) regardless of the 1% or 20% cutoff value level. However, A1762T/G1764A double mutations did not differ in the other groups (P >0.05). Viral quasispecies diversity was lower in HIV/HBV co-infected patients than in HBV mono-infected patients (P Keywords: human immunodeficiency virus, hepatitis B virus; mutations; viral quasispecies; next-generation sequencing.

Distinct T-cell receptor profiles associated with hepatitis B e antigen seroconversion during entecavir treatment.

BACKGROUND & AIMS: T-cell receptor (TCR) repertoire is ambiguously changed in chronic hepatitis B (CHB) patients during antivirus therapy. We tried to assess TCR repertoire dynamics and its clinical significance upon HBeAg seroconversion in CHB patients. METHODS: Twenty CHB patients undergoing 1-year entecavir (ETV) treatment were enrolled, including 10 complete response (CR) vs 10 non-complete response (NCR) patients based on HBeAg seroconversion at week 48. The TCRß complementarity-determining region 3 (CDR3) of peripheral CD4+ and CD8+ T cells at weeks 0, 12 and 48 was analyzed by unbiased high-throughput sequencing. The TCR repertoire profiles and their correlations with serological parameters were analyzed. RESULTS: The diversity of TCRß repertoires was decreasing in CR patients but increasing in NCR patients. The distribution pattern of TCR repertoires stratified according to clonotype frequencies changed in the opposite direction between CR and NCR patients. Narrow amounts of newly appearing clonotypes in CR patients experienced a more intensive and robust expansion and this phenomenon could occur as early as week 12 for the CD4+ subset but later at week 48 for the CD8+ subset. There existed some CR-exclusive clonotypes with a relatively low but increasing frequency at week 48. The number of unique TCRß clonotypes was positively correlated with the ALT or HBV DNA level in CR patients but showed no or negative correlation in NCR patients. CONCLUSION: Distinct TCR profiles contribute to predicting HBeAg seroconversion in CHB patients during ETV treatment and certain TCRß CDR3 motif may be utilized for CHB immunotherapy in the future.

Naturally occurring antiviral drug resistance in HIV patients who are mono-infected or co-infected with HBV or HCV in China.

Drug resistance mutations (DRMs) may reduce the efficacy of antiviral therapy. However, the studies focused on naturally occurring, pre-existing DRMs among co-infected patients in China are limited. To investigate DRMs prevalence in treatment-naïve human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) mono- and co-infected patients in China, a total of 570 patients were recruited for this study. DRMs sequences were amplified and successfully sequenced in 481 of these patients, who were grouped into three cohorts: (i) The HBV cohort included 100 HIV/HBV co-infected and 110 HBV mono-infected patients who were sequenced for HBV; (ii) The HCV cohort included 91 patients who were HIV/HCV co-infected and 72 who were HCV mono-infected for HCV sequencing; and (iii) The HIV cohort included 39 HIV mono-infected, 22 HIV/HCV, and 47 HIV/HBV co-infected patients for HIV sequencing. Next-generation sequencing and Sanger sequencing were used in this study. The results showed that in the HCV cohort, HCV genotypes 6a (P < 0.001) and 3b (P = 0.004) were more prevalent in HIV/HCV co-infected patients, however, the prevalence of HBV and HIV genotypes were similar within the HBV and HIV cohorts. HBV DRMs prevalence was significantly higher in HIV/HBV co-infected than HBV mono-infected patients (8.0% vs 0.9%, P = 0.015), whereas HCV and HIV DRMs did not differ within the HCV and HIV cohort (P > 0.05). This study revealed that HBV DRMs were more prevalent in HIV/HBV co-infected patients in China, while DRMs in HCV and HIV patients did not differ. Further dynamic surveillance of DRMs may be needed.

The dynamics and association of B and T cell receptor repertoires upon antibody response to hepatitis B vaccination in healthy adults.

The Hepatitis B (HB) vaccine is efficacious in preventing hepatitis B virus infection. However, the association between antibody response to the HB vaccine and dynamic immune repertoire changes in different cell subsets remains unclear. Nine healthy participants were administered three doses of HB vaccine following the 0, 1, 6 month schedule. Peripheral CD4+ T, memory B (MB), naïve B (NB), and plasma cells (PCs) were sorted before vaccination and 7 days following each dose. The complementary determining region 3 of T-cell receptor ß (TCRß) chain and B-cell receptor (BCR) heavy chain (IgG, IgM, IgA) repertoires were analyzed by high-throughput sequencing. All nine participants elicited protective antibody titers to the vaccine at the end of immunization. Compared with the baseline, MB cells showed a significant increase in IgG usage and decreased IgM usage and repertoire diversity at the end of vaccination. TCRß diversity changes were highly correlated with those of the BCR in MB cells in participants with a faster and robust antibody responses. The percentage of shared clonotypes between NB and MB cells, and MB cells and PCs were much higher than that between NB cells and PCs. The more clonotypes sharing the faster and stronger antibody responses were observed after HB vaccination. These results suggest the integral involvement of MB cells in vaccine immunization. Interaction between CD4+ T and MB cells and B cell differentiation may improve antibody response to HB vaccine.

Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus.

Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector. To examine the efficacy of SaCas9 system on HBV genome destruction, we designed 5 guide RNAs (gRNAs) that targeted different HBV genotypes, 3 of which were shown to be effective. The SaCas9 system significantly reduced HBV antigen expression, as well as pgRNA and cccDNA levels, in Huh7, HepG2.2.15 and HepAD38 cells. The dual expression of gRNAs/SaCas9 in these cell lines resulted in more efficient HBV genome cleavage. In the mouse model, hydrodynamic injection of gRNA/SaCas9 plasmids resulted in significantly lower levels of HBV protein expression. We also delivered the SaCas9 system into mice with persistent HBV replication using an AAV vector. Both the AAV vector and the mRNA of Cas9 could be detected in the C3H mouse liver cells. Decreased hepatitis B surface antigen (HBsAg), HBV DNA and pgRNA levels were observed when a higher titer of AAV was injected, although this decrease was not significantly different from the control. In summary, the SaCas9 system accurately and efficiently targeted the HBV genome and inhibited HBV replication both in vitro and in vivo. The system was delivered by an AAV vector and maybe used as a novel therapeutic strategy against chronic HBV infection.

Dynamic Perturbations of CD4 and CD8 T Cell Receptor Repertoires in Chronic Hepatitis B Patients upon Oral Antiviral Therapy.

Long-term treatment with nucleos(t)ide analogs (NUCs) can improve the antiviral T cell response in chronic hepatitis B (CHB) patients. Whether and to what extent the T cell response is improved by NUCs in the early stage leading to hepatitis B e antigen (HBeAg) seroconversion remain to be clarified. A total of 22 CHB patients undergoing 2-year telbivudine-based therapy were enrolled, including 10 exhibiting a complete response (CR) and 12 exhibiting a non-complete response (NCR) according to HBeAg seroconversion at week 52. Peripheral CD4+ and CD8+ T cells were sorted at baseline, weeks 12, and 24. The T cell receptor ß chain (TCRß) complementarity-determining region 3 was analyzed by unbiased high-throughput sequencing. Compared with NCR group, patients in CR group had a much lower percentage of persistent clonotypes (P < 0.001) but remarkably higher percentages of new and expanded clonotypes (P < 0.05) between any two time points for both CD4 and CD8 subsets. The CD4 T cells exhibited a stronger response than CD8 population in the patients. The number of new and expanded clonotypes was inversely associated with the decline of viral antigen. In conclusion, NUC-based therapy induces a broad and vigorous T cell response with rapid decline of antigenemia during the early stage of treatment. A broad T cell expansion is crucial for HBeAg seroconversion. Our findings suggest that the potent suppression of hepatitis B virus replication by NUC monotherapy complemented with additional immunomodulatory strategies may increase the likelihood of a functional cure for CHB in the future.

High-throughput T cell receptor sequencing reveals distinct repertoires between tumor and adjacent non-tumor tissues in HBV-associated HCC.

T lymphocytes, which recognize antigen peptides through specific T cell receptors (TCRs), play an important role in the human adaptive immune response. TCR diversity is closely associated with host immune response and cancer prognosis. Although tumor-infiltrating T lymphocytes have implications for tumor prognosis, few studies have performed a detailed characterization of TCR diversity in both tumor and non-tumor tissues in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). Here, we performed high-throughput sequencing of the TCRß chain complementarity determining region 3 (CDR3) of liver-infiltrating T cells from 48 HBV-associated HCC patients. A significantly higher average number of CDR3 aa clonotypes (2259 vs. 1324, p < 0.001), and significantly higher TCR diversity (Gini coefficient, p < 0.001; Simpson index, p < 0.01; Shannon entropy, p < 0.001) were observed in tumor tissues compared with adjacent non-tumor tissues. The ratio of highly expanded clones (HECs) was significantly higher in non-tumor tissues than in tumor tissues when the HEC threshold was defined as 2% or greater (p < 0.05). Our analysis of the median Morisita-Horn index indicated weak TCR repertoire similarity between tumor and matched non-tumor tissues. The median number of shared clones in tumor tissue and matched non-tumor tissue from each patient was 360.5, representing 5.1-15.8% (10.6 ± 0.4%) of all clones in each patient. We observed extensive heterogeneity of T lymphocytes in tumors and higher HEC ratios in adjacent non-tumor tissues of HCC patients. The differential T cell repertoires in tumor and non-tumor tissues suggest a distinct T cell immune microenvironment in patients with HBV-associated HCC.