Single-cell RNA-sequencing of virus-specific cellular immune responses in chronic hepatitis B patients.

Chronic hepatitis B (CHB) is a major global health challenge. CHB can be controlled by antivirals but a therapeutic cure is lacking. CHB is characterized by limited HBV-specific T cell reactivity and functionality and expression of inhibitory receptors. The mechanisms driving these T cell phenotypes are only partially understood. Here, we created a single-cell RNA-sequencing dataset of HBV immune responses in patients to contribute to a better understanding of the dysregulated immunity. Blood samples of a well-defined cohort of 21 CHB and 10 healthy controls, including a subset of 5 matched liver biopsies, were collected. scRNA-seq data of total immune cells (55,825) plus sorted HBV-specific (1,963), non-naive (32,773) and PD1+ T cells (96,631) was generated using the 10X Genomics platform (186,123 cells) or the full-length Smart-seq2 protocol (1,069 cells). The shared transcript count matrices of single-cells serve as a valuable resource describing transcriptional changes underlying dysfunctional HBV-related T cell responses in blood and liver tissue and offers the opportunity to identify targets or biomarkers for HBV-related immune exhaustion.

Cambios en las características epidemiológicas y en el pronóstico de la hepatitis crónica B en personas que viven con VIH / Epidemiological characteristic and prognosis changes in chronic hepatitis B in people living with HIV

Introducción: La vacunación, los avances en el tratamiento frente al virus de la hepatitis B (VHB) y los cambios epidemiológicos producidos en España en las últimas décadas han podido modificar las características y el pronóstico de la hepatitis crónica B (HCB) en personas que viven con VIH (PVIH). Métodos: Estudio observacional retrospectivo donde se incluyeron PVIH-HCB en seguimiento en una unidad de referencia madrileña hasta el año 2019. Se comparó la incidencia y las características epidemiológicas y clínicas según el momento del diagnóstico (antes del año 2000 y posteriormente en periodos de cinco años). Además, se realizó un estudio longitudinal retrospectivo evaluando la tasa de mortalidad, descompensación hepática y factores asociados. Resultados: De 5.452 PVIH, 160 presentaban HCB en el momento basal (prevalencia 2,92%, IC 95%: 2,5-3,4), 85,6% hombres, edad mediana al diagnóstico 32,1 (27-37,2) años. La incidencia (2,4/100 pacientes-año) no varió en los diferentes periodos. Los pacientes diagnosticados antes del 2000 (n = 87) comparados con los diagnosticados entre 2015-2019 (n = 11) con mayor frecuencia eran nativos españoles (90,8 vs. 18,2%), habían consumido drogas intravenosas (55,2 vs. 0), tenían antecedentes de hepatitis C (40 vs. 9,1%) y delta (30,4 vs. 0) y mayor afectación hepática (24,1% cirróticos vs. 0). Tras un seguimiento de 20,4 años, 23 pacientes murieron (7,1/1.000 pacientes-año) y 19 presentaron descompensación hepática (4,9/1.000 pacientes-año), todos diagnosticados antes del año 2010. La mortalidad se asoció con mayor fibrosis hepática basal estimada por Fibroscan® (HR 1,06; IC 95%: 1,03-1,09). Conclusión: Las PVIH-HCB con diagnóstico previo al año 2000 son más frecuentemente de nacionalidad española, infectadas por vía parenteral y con mayor prevalencia de otras coinfecciones. Los pacientes diagnosticados antes del 2010 tienen peor pronóstico condicionado por presentar mayor grado de fibrosis hepática.(AU)

Introduction: Due to hepatitis B virus (HBV) treatment and vaccination during the last decades in Spain, epidemiological and prognosis of chronic hepatitis B (CHB) may have changed. Methods: Retrospective review of CHB–HIV coinfected patients in a single reference center in Madrid until year 2019. We compared incidence, epidemiological and clinical characteristics according diagnosis period (before 2000, 2000–2004, 2005–2009, 2010–2014, 2015–2019). A retrospective longitudinal study was done to assess mortality, related risk factors and hepatic decompensation. Results: Out of 5452 PLHIV, 160 had CHB (prevalence 2.92%; 95% CI: 2.5–3.4), 85.6% were men, median age 32.1 (27–37.2). Incidence rate did not change over the years (2.4/100 patients-year). PLHIV with CHB diagnosed before year 2000 (n = 87) compared with those diagnosed between 2015 and 2019 (n = 11) were more often native-Spanish (90.8% vs. 18.2%), had infected using intravenous drugs (55.2% vs. 0), were coinfected with hepatitis C (40% vs. 9.1%) or hepatitis delta virus (30.4% vs. 0) and had more severe liver disease (cirrhosis 24.1% vs. 0). After a median follow-up of 20.4 years, 23 patients died (7.1/1000 patients-year) and 19 had liver decompensation (4.9/1000 patients-year). All deaths and liver decompensation occurred in patients diagnosed before year 2010. Mortality was associated with higher liver fibrosis in Fibroscan® (HR 1.06, 95% CI: 1.03–1.09). Conclusion: The epidemiology of CHB in PLHIV in our cohort is changing with less native Spanish, more sexually transmitted cases and less coinfection with other hepatotropic virus. Patients diagnosed before 2010 have worst prognosis related to higher grades of liver fibrosis.(AU)

Immune response and treatment targets of chronic hepatitis B virus infection: innate and adaptive immunity.

Chronic hepatitis B virus (HBV) infection is a major global public health risk that threatens human life and health, although the number of vaccinated people has increased. The clinical outcome of HBV infection depends on the complex interplay between viral replication and the host immune response. Innate immunity plays an important role in the early stages of the disease but retains no long-term immune memory. However, HBV evades detection by the host innate immune system through stealth. Therefore, adaptive immunity involving T and B cells is crucial for controlling and clearing HBV infections that lead to liver inflammation and damage. The persistence of HBV leads to immune tolerance owing to immune cell dysfunction, T cell exhaustion, and an increase in suppressor cells and cytokines. Although significant progress has been made in HBV treatment in recent years, the balance between immune tolerance, immune activation, inflammation, and fibrosis in chronic hepatitis B remains unknown, making a functional cure difficult to achieve. Therefore, this review focuses on the important cells involved in the innate and adaptive immunity of chronic hepatitis B that target the host immune system and identifies treatment strategies.

Baseline Hepatitis B Virus Surface Antigen Titers in Childhood Predict the Risk of Advanced Liver Fibrosis in Adulthood.

BACKGROUND & AIMS: Hepatitis B virus (HBV) surface antigen (HBsAg) is a marker of both HBV covalently closed circular DNA and integrated HBV genome, whereas the HBV core-related antigen (HBcrAg) indicates the transcriptional activity of covalently closed circular DNA. This study examined the relationship between HBsAg and HBcrAg titers in childhood and advanced fibrosis in adulthood. METHODS: We recruited 214 initially hepatitis B e antigen-positive chronic HBV-infected patients who were followed for a total of 6371 person-years. None of the patients were co-infected with hepatitis C or D virus. Serum HBsAg and HBcrAg titers were assessed at 10 and 15 years of age. Transient elastography was performed at a mean final age of 38.21 years to identify advanced fibrosis. RESULTS: Patients with advanced fibrosis in adulthood had a higher rate of genotype C HBV infection and a higher HBsAg titer at 10 and 15 years of age (P = .003, P = .03, and P = .005, respectively). The HBcrAg titer was not correlated with advanced fibrosis (P > .05). Receiver operating characteristic curve analysis showed that HBsAg cutoffs of >4.23 and >4.44 log10 IU/mL at 10 and 15 years of age, respectively, best predicted advanced fibrosis in the fourth decade of life (P = .001 and P < .001, respectively). In a multivariate analysis, both an HBsAg titer >4.44 log10 IU/mL at 15 years of age and HBV genotype C were predictors of advanced fibrosis (odds ratios, 15.43 and 4.77; P = .01 and P = .02, respectively). CONCLUSIONS: HBsAg titers in childhood predict the progression to liver fibrosis in adulthood.

HBV transcription and translation persist despite viral suppression in HBV-HIV co-infected patients on antiretroviral therapy.

BACKGROUND AND AIMS: Liver injury may persist in patients with HBV receiving antiviral therapy who have ongoing transcription and translation. We sought to assess ongoing HBV transcription by serum HBV RNA, translation by serum hepatitis B core related antigen (HBcrAg), and their associations with hepatic HBsAg and HBcAg staining in patients coinfected with HBV and HIV. METHODS: This is a cross-sectional study of 110 adults coinfected with HBV and HIV who underwent clinical assessment and liver biopsy. Immunohistochemistry (IHC) was performed for HBsAg and HBcAg. Viral biomarkers included quantitative HBsAg, HBV RNA, and HBcrAg. RESULTS: Participants' median age was 49 years (male, 93%; Black, 51%; HBeAg+, 65%), with suppressed HBV DNA (79%) and undetectable HIV RNA (77%) on dually active antiretroviral therapy. Overall, HBV RNA and HBcrAg were quantifiable in 81% and 83%, respectively (96% and 100% in HBeAg+, respectively). HBcAg staining was detected in 60% and HBsAg in 79%. Higher HBV RNA was associated with higher HBcAg and HBsAg IHC grades (both p < 0.0001). The HBsAg membranous staining pattern was significantly associated with higher HBV-RNA and HBcrAg levels. CONCLUSION: HBcAg and HBsAg IHC staining persisted despite viral suppression, and IHC grades and staining patterns correlated with markers of transcription (HBV RNA) and translation (HBcrAg). These data indicate that apparent HBV suppression is associated with residual transcription and translation that could contribute to liver pathology. Additional antiviral strategies directed to HBV protein expression may be useful to ameliorate liver injury.

Long-term Hepatitis B Surface Antigen Profile and Seroclearance after Severe Acute Flares of Chronic Hepatitis B.

Background/Aims: Hepatitis B surface antigen (HBsAg) seroclearance remains uncommon in chronic hepatitis B (CHB) infection. During acute flares of CHB (AFOCHB), alanine aminotransferase elevation reflects a mounting immune response toward viral clearance. We hypothesized that severe AFOCHB is associated with a greater quantitative HBsAg (qHBsAg) decline and HBsAg seroclearance rate. Methods: A total of 75 patients with severe AFOCHB with alanine aminotransferase 10× the upper limit of normal were matched to a control group by age and sex in a 1:2 ratio. qHBsAg levels were measured at the time of flare and annually (for both cases and controls) until the last follow-up. Results: The median follow-up times for patients with severe AFOCHB and controls were 8.8 and 10.5 years, respectively. The cumulative rate of HBsAg seroclearance was higher in the severe AFOCHB group than in the control group (11.8% vs 5.0%, p=0.04) despite the former group having a trend of a higher baseline median qHBsAg (3,127 IU/mL vs 1,178 IU/mL, p=0.076). Compared with the control group, the severe AFOCHB group had a greater annual qHBsAg reduction (-242.4 IU/mL/yr vs -47.3 IU/mL/yr, p=0.002). Increasing age (p=0.049), lower baseline qHBsAg (p=0.002), and severe AFOCHB (p=0.014) were independently associated with HBsAg seroclearance. However, the cumulative rate of hepatocellular carcinoma was significantly higher in the severe AFOCHB group than in the control group (15.8% vs 1.9%, p<0.001). Conclusions: Severe AFOCHB was associated with a greater incidence of HBsAg seroclearance and qHBsAg decline. However, it was associated with a higher incidence of hepatocellular carcinoma.

Combined baseline HBcrAg and end-of-treatment HBsAg predict HBV relapse after entecavir or tenofovir cessation.

BACKGROUND: For patients with chronic hepatitis B (CHB), the optimal stopping criteria for entecavir or tenofovir disoproxil fumarate treatment remain unclear. METHODS: This study recruited CHB patients with levels of hepatitis B surface antigen (HBsAg) <100 IU/mL at the end of treatment (EOT) from Kaohsiung (n = 190) and Linkou (n = 188) Chang Gung Memorial Hospitals for use as development and validation groups, respectively. RESULTS: In the development group, 108 patients with HBsAg ≤40 IU/mL were used for analysis of predictors of HBV relapse and HBsAg loss. Multivariate analysis showed that age, nucleos(t)ide analogue (NA)-experienced status, baseline hepatitis B core-related antigen (HBcrAg) and HBsAg at EOT were associated independently with virological and clinical relapse. An HBsAg level of 20 IU/mL at EOT was the best cut-off value for minimizing HBV relapse. Patients with EOT HBsAg ≤20 IU/mL had lower virological and clinical relapse rates and higher HBsAg loss rates than those with EOT HBsAg 21-40 IU/mL and HBsAg 41-100 IU/mL in the development and validation groups. The virological and clinical relapse rates were very low (5-year rates: 6.5% and 0%, respectively) and HBsAg loss rate was very high (5-year rate: 81.7%) in patients with a combination of baseline HBcrAg ≤4 log U/mL and EOT HBsAg ≤20 IU/mL in the development group. CONCLUSIONS: A combination of baseline HBcrAg ≤4 log U/mL and EOT HBsAg level ≤20 IU/mL might reduce the risk of HBV relapse and increase HBsAg loss rate, and might be helpful for off-NA follow-up strategy.

Interleukin-33 mediates both immune-related and non-immune-related inhibitory effects against hepatitis B virus.

Chronic infection by hepatitis B virus (HBV) is associated with high risks of liver fibrosis, cirrhosis and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocyte serves as transcription template. Neither natural resolution of acute infection nor current treatment options for chronic infection are believed to cause cccDNA clearance. We previously showed that injection of IL-33-expressing plasmid facilitated clearance of intrahepatic HBV DNA in a mouse model of HBV persistence. In this work, HBV-targeting therapeutic effects of IL-33 were further explored. Murine IL-33 delivered by recombinant adeno-associated virus (AAV-mIL-33) induced clearance of both serum HBV markers and intrahepatic HBV DNA in two mouse models of HBV persistence based on replicon plasmid and recombinant cccDNA (rcccDNA) respectively. Clearance was associated with serum ALT elevations and liver infiltrations by CD4+ and CD8+ T cells, indicating IL-33-induced cellular immune responses against HBV-harboring cells. Adoptive transfer of splenocytes from AAV-mIL-33-cured mice was indeed sufficient to engender similar clearance in recipient mice. In vitro, intracellular, instead of extracellular, IL-33 was mainly responsible for repressing viral transcription, protein production and genome replication in Huh7 cells transfected with HBV replicon or rcccDNA. IL-33 was shown to be recruited onto rcccDNA minichromosome accompanied by loss of transcriptional activation epigenetic marks. Finally, transfection of IL-33 into HBV-infected HepG2/NTCP cells resulted in reduced transcription, antigen expression and genome replication, suggesting repression of canonical cccDNA. These data demonstrated diverse inhibitory effects on HBV and HBV-infected cells mediated by IL-33, and suggest IL-33 as an interesting therapeutic candidate.

Toll-like receptor-mediated innate immunity orchestrates adaptive immune responses in HBV infection.

Chronic hepatitis B virus (HBV) infection remains to be a substantial global burden, especially for end-stage liver diseases. It is well accepted that HBV-specific T and B cells are essential for controlling HBV infection. Toll-like receptors (TLRs) represent one of the major first-line antiviral defenses through intracellular signaling pathways that induce antiviral inflammatory cytokines and interferons, thereby shaping adaptive immunity. However, HBV has evolved strategies to counter TLR responses by suppressing the expression of TLRs and blocking the downstream signaling pathways, thus limiting HBV-specific adaptive immunity and facilitating viral persistence. Recent studies have stated that stimulation of the TLR signaling pathway by different TLR agonists strengthens host innate immune responses and results in suppression of HBV replication. In this review, we will discuss how TLR-mediated responses shape HBV-specific adaptive immunity as demonstrated in different experimental models. This information may provide important insight for HBV functional cure based on TLR agonists as immunomodulators.

Toll-Like Receptor 7 Agonist RG7854 Mediates Therapeutic Efficacy and Seroconversion in Woodchucks With Chronic Hepatitis B.

Conventional treatment of chronic hepatitis B (CHB) is rarely curative due to the immunotolerant status of patients. RG7854 is an oral double prodrug of a toll-like receptor 7 (TLR7) agonist that is developed for the treatment of CHB. The therapeutic efficacy, host immune response, and safety of RG7854 were evaluated in the woodchuck model of CHB. Monotreatment with the two highest RG7854 doses and combination treatment with the highest RG7854 dose and entecavir (ETV) suppressed viral replication, led to loss of viral antigens, and induced seroconversion in responder woodchucks. Since viral suppression and high-titer antibodies persisted after treatment ended, this suggested that a sustained antiviral response (SVR) was induced by RG7854 in a subset of animals. The SVR rate, however, was comparable between both treatment regimens, suggesting that the addition of ETV did not enhance the therapeutic efficacy of RG7854 although it augmented the proliferation of blood cells in response to viral antigens and magnitude of antibody titers. The induction of interferon-stimulated genes in blood by RG7854/ETV combination treatment demonstrated on-target activation of TLR7. Together with the virus-specific blood cell proliferation and the transient elevations in liver enzymes and inflammation, this suggested that cytokine-mediated non-cytolytic and T-cell mediated cytolytic mechanisms contributed to the SVR, in addition to the virus-neutralizing effects by antibody-producing plasma cells. Both RG7854 regimens were not associated with treatment-limiting adverse effects but accompanied by dose-dependent, transient neutropenia and thrombocytopenia. The study concluded that finite, oral RG7854 treatment can induce a SVR in woodchucks that is based on the retrieval of antiviral innate and adaptive immune responses. This supports future investigation of the TLR7 agonist as an immunotherapeutic approach for achieving functional cure in patients with CHB.

Role of Innate Immune Regulatory Genes, FOXP3 and FOS in Chronic Hepatitis B Infection.

Persistence of hepatitis B virus (HBV) infection leading to chronic infection and its sequalae is responsible for over half a million deaths worldwide. The reason for persistence of chronic hepatitis B (CHB) infection is still not clearly understood. An attempt was made to understand the role of immune regulatory genes in CHB in comparison to spontaneously cleared HBV infection. Relative gene expression of 26 genes involved in innate immunity were studied using Real-Time Polymerase Chain Reaction Array. A total of 679 subjects from three different geographical regions of Northeast India (Assam, Arunachal Pradesh, and Tripura) were included in this case-control study. The cases were subdivided into CHB cases with HBeAg(+)(72), CHB with HBeAg(-)(278), spontaneously cleared controls (88), and healthy controls (228). Overall, 28.3% of the subjects had previous exposure with HBV, while 28.6% had protective antibodies IgG/IgM against HBV. There was a statistically higher number of CHB in men (66.4%) compared to women (33.6%) (p = 0.0001). Proto-oncogene FOS has been found to be moderately upregulated in CHB with HBeAg +ve (2.3-fold) and significantly upregulated (4.1-fold upregulation) in hepatocellular carcinoma. Further, FOXP3 was found to be significantly upregulated (3.0-fold, p = 0.01) in CHB with HBeAg (+) compared to spontaneously cleared HBV infection. In conclusion, CHB with HBeAg positivity was found to have disrupted immune response with upregulation of FOS and FOXP3. Thus, early induction of HBeAg seroconversion with interferon-based therapy or oral nucleos(t)ide analogs along with FOS inhibitors can have important clinical implications in the management of CHB and preventing cirrhosis and HCC.

CpG-C ODN M362 as an immunoadjuvant for HBV therapeutic vaccine reverses the systemic tolerance against HBV.

Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide (CpG-C) motifs may provide potential adjuvants for the immunotherapeutic strategy against CHB, since CpG-C ODNs stimulate both B cell and dendritic cell (DC) activation. However, the efficacy of CpG-C ODN as an anti-HBV vaccine adjuvant remains unclear. In this study, we demonstrated that CpG M362 (CpG-C ODN) as an adjuvant in anti-HBV vaccine (cHBV-vaccine) successfully and safely eliminated the virus in HBV-carrier mice. The cHBV-vaccine enhanced DC maturation both in vivo and in vitro, overcame immune tolerance, and recovered exhausted T cells in HBV-carrier mice. Furthermore, the cHBV-vaccine elicited robust hepatic HBV-specific CD8+ and CD4+ T cell responses, with increased cellular proliferation and IFN-γ secretion. Additionally, the cHBV-vaccine invoked a long-lasting follicular CXCR5+ CD8+ T cell response following HBV re-challenge. Taken together, CpG M362 in combination with rHBVvac cleared persistent HBV and achieved long-term virological control, making it a promising candidate for treating CHB.

TLR2, as a Pathogen Recognition Receptor, Plays Critical Roles in Hepatitis B Outcome.

The immune system of active and inactive chronic hepatitis B, as prolonged forms of hepatitis B, is unable to eradicate hepatitis B virus (HBV) from the infected hepatocytes completely. Toll-like receptors (TLRs) play key roles in the viral recognition and promotion of appropriate immune responses. The molecules also participate in the alteration of the target cell functions and transformation. TLR2 is the unique molecule that makes either homodimer or heterodimer with TLR1 and 6 and shows variable roles against viral infections. Therefore, it has been hypothesized that TLR2 may participate in both immune response against HBV and induction of the virus-related hepatic complications. The studies confirm the hypothesis and revealed that TLR2 is not only one of the main molecules altering the course of HBV infection, but also plays key roles in induction of hepatocellular carcinoma (HCC) and liver cirrhosis. However, recent studies demonstrated that the molecule can fight against HCC and liver cirrhosis. Collectively, it appears that nutrition habits, TLR2 gene polymorphisms, gut microbiome, HBV antigens, and activation of other receptors may play key roles in the determination of TLR2 functions.

An age-structured model of hepatitis B viral infection highlights the potential of different therapeutic strategies.

Hepatitis B virus (HBV) is a global health threat, and its elimination by 2030 has been prioritised by the World Health Organisation. Here we present an age-structured model for the immune response to an HBV infection, which takes into account contributions from both cell-mediated and humoral immunity. The model has been validated using published patient data recorded during acute infection. It has been adapted to the scenarios of chronic infection, clearance of infection, and flare-ups via variation of the immune response parameters. The impacts of immune response exhaustion and non-infectious subviral particles on the immune response dynamics are analysed. A comparison of different treatment options in the context of this model reveals that drugs targeting aspects of the viral life cycle are more effective than exhaustion therapy, a form of therapy mitigating immune response exhaustion. Our results suggest that antiviral treatment is best started when viral load is declining rather than in a flare-up. The model suggests that a fast antibody production rate always leads to viral clearance, highlighting the promise of antibody therapies currently in clinical trials.

Interleukin-18 Plays a Positive Feedback Activity to Natural Killer-Like B Cells in Hepatitis B Virus-Associated Acute-on-Chronic Liver Failure.

Natural killer-like B (NKB) cells are a newly identified immune subset, which are separated from NK cells and B cells. NKB cells demonstrated immunoregulatory functions in elimination of microbial infection and inflammation through secretion of interleukin (IL)-12 and IL-18. However, the role of NKB cells in hepatitis B virus (HBV)-related diseases has not been reported. In this study, peripheral T cells, B cells, NK cells, and NKB cells in HBV-associated acute-on-chronic liver failure (ACLF), chronic hepatitis B (CHB), asymptomatic HBV carriers (AsC), and controls were investigated by flow cytometry. Plasma IL-12 and IL-18 levels were measured by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells from HBV-ACLF patients were stimulated with recombinant IL-12 or IL-18. Changes of immune cell percentage and nuclear factor-kappa B (NF-κB) phosphorylation were assessed. There were no statistical differences of T cell percentage, B cell percentage, or NK cell percentage among groups. NKB cell percentage within lymphocytes, and plasma IL-12 and IL-18 levels in HBV-ACLF patients were significantly elevated compared with in CHB, AsC, and controls. NKB cell percentage and IL-18, but not IL-12, had a better prognosis function for the 28-day survival status in HBV-ACLF patients. Recombinant IL-12 enhanced T cell and NK cell percentage, while only high concentrations (10 ng/mL) of IL-18 promoted NKB cell percentage in HBV-ACLF patients. High concentrations (10 ng/mL) of IL-18 induced NF-κB phosphorylation in NKB cells probably through suppression of IL-18 binding protein in HBV-ACLF patients. The current data indicated that elevated NKB cells and IL-18 might be important indicators for poor prognosis of HBV-ACLF patients. Increased IL-18 might play a positive feedback activity to NKB cells in HBV-ACLF patients.

[Clinical effect and influencing factors of pegylated interferon alfa-2a and entecavir monotherapy among children with HBeAg-positive chronic hepatitis B based on a real-world study].

Objective: To compare the efficacy, safety, and influencing factors among children with hepatitis B virus e antigen (HBeAg)-positive chronic hepatitis B (CHB) who received short-term therapy with pegylated interferon alfa-2a (Peg-IFNα-2a) or continuous therapy with entecavir (ETV). Methods: Quantitative data were compared using analysis of variance to compare the differences between groups. Enumeration data were compared by χ2 test (or Fisher's exact test). Univariate and multivariate logistic regressions were used to analyze the influencing factors. Results: Peg-IFNα-2a, ETV, and untreated group had HBsAg clearance rates of 46.2%, 5.3%, and 0 after 52 weeks of therapy, respectively. HBsAg clearance in the patients' group with Peg-IFNα-2a and ETV was all accompanied by anti-HBS positive conversion, and the difference was statistically significant (χ2=13.616, P=0.001). Peg-IFNα-2a group was followed-up for 104 weeks. Peg-IFNα-2a, ETV, and the untreated group had HBsAg clearance rates of 46.2%, 10.5%, and 0%, respectively, and the differences were statistically significant (χ2=11.056, P=0.004). Only one of the two children with HBsAg clearance in the ETV group had achieved anti-HBs antibodies, and the difference was statistically significant (χ2=13.616, P=0.001). Univariate and multivariate logistic regression analysis showed that HBsAg clearance was associated with age and antiviral therapy. During treatment, adverse events such as fever (n=4, 30.8%), rash (n=4, 30.8%), fatigue (n=1, 7.7%), leukopenia (n=7, 53.8%), arthritis (n=1, 7.7%), and alopecia (n=3, 23.1%) were observed in the Peg-IFNα-2a group, while none were observed in the ETV group. Conclusion: Peg-IFNα-2a antiviral therapy produced higher HBsAg clearance than ETV in five-year-old and younger children with HBeAg-positive CHB, while ETV had fewer adverse events and was safer than Peg-IFNα-2a.

[Sero-epidemiological characteristics of the hepatitis D virus infection among hepatitis B virus infected-patients at a single center in Xinjiang region].

Objective: To investigate the sero-epidemiological characteristics of the hepatitis D virus (HDV) infection among hepatitis B virus (HBV)-infected patients in Xinjiang region. Methods: A single-center cross-sectional analysis method was used to select 264 cases of hepatitis B virus infection who were hospitalized in the Center for Infectious Diseases and Liver Diseases of the First Affiliated Hospital of Xinjiang Medical University from August 2021 to January 2022. All patients were tested for HDV Ag, HDV IgM, HDV IgG, and HDV RNA. The infection status of hepatitis D virus was analyzed by grouping according to their clinical type, HBV viral load, and HBsAg level. A paired t-test was used for data with measurement data conforming to normal distribution. A paired rank sum test was used for data that did not conform to normal distribution before and after treatment. Results: A total of 36 cases (13.64%) and 26 cases (9.85%) were positive for HDV serological markers and HDV RNA. According to clinical type grouping, the positive rates of HDV serum markers in patients with chronic hepatitis B, hepatitis B-related cirrhosis, liver cancer, and liver failure were 13.46%, 12.43%, and 20.83%, respectively, and there was no statistically significant difference among the three groups (χ2=0.86, P=0.649). The positive rates of HDV RNA were 11.54%, 8.11%, and 20.83%, respectively, and there was no statistically significant difference among the three groups (χ2=4.015, P=0.134). According to HBV viral load grouping, the positive rates of HDV serum markers among patients with viral loads <20, 20-2 000, and >2 000 IU/ml were 17.15%, 7.81%, and 6.67%, respectively, and the difference was not statistically significant among the three groups (χ2=4.846, P=0.089). The positive rates of HDV RNA were 9.47%, 10.94%, and 10%, respectively, and the difference was not statistically significant among the three groups (χ2=0.113, P=0.945). According to HBsAg level grouping, the positive rates of HDV serum markers in HBsAg<0.05, 0.05~250, and >250 IU/ml were 14.29%, 16.67%, and 10.85%, respectively, and there was no statistically significance between the three groups (χ2=1.745, P=0.418). The positive rates of HDV RNA were 4.76%, 8.77%, and 11.63%, respectively, and there was no statistically significant difference among the three groups (χ2=1.221, P=0.543). Clinical outcome, disease course, HBV DNA, serological markers of viral hepatitis, routine blood test, biochemical indicators, coagulation function, and other laboratory indicators were compared between HDV serum marker and/or nucleic acid positive and negative patients, and there was no statistically significant difference (P>0.05). Conclusion: The positive rate of HDV serological markers and HDV RNA is 13.64% and 9.85%, respectively, at a single center in the Xinjiang region, and there is still a high HDV infection rate among the HBV-infected patients with low levels of viral load and HBsAg.

Programmed death 1 expressing CD8+ CXCR5+ follicular T cells constitute effector rather than exhaustive phenotype in patients with chronic hepatitis B.

BACKGROUND AND AIMS: Classical CD8 T cells are implicated for protective and pathogenic roles in chronic hepatitis B (CHB) infection. Recently, a subset of CD8 T cells expressing C-X-C chemokine receptor type 5 (CXCR5) and exhibiting features of TFH cells has been identified during chronic viral infections. However, in CHB, knowledge of their roles is limited. APPROACH AND RESULTS: We characterized circulating CD8+ CXCR5+/- cells and investigated their association with clinical and viral factors. We found that CHB infection did not influence the overall frequencies of CD8+ CXCR5+ cells whereas CD8+ CXCR5- cells were increased. However, among CHB, CD8+ CXCR5+ cells were higher in patients with low HBsAg and HBV-DNA levels, patients who were HBeAg negative and had high fibrosis scores, and these cells exhibited a significant association with HBsAg and HBV-DNA reduction. Contrarily, CD8+ CXCR5- cells were expanded and positively correlated with patients having high HBsAg, HBV-DNA, and alanine aminotransferase levels. CD8+ CXCR5+ cells express costimulatory molecules ICOS, OX40, CD40 ligand, inhibitory molecule programmed death 1, transcription factors B-cell lymphoma (BCL)-2, BCL-6, and signal transducer and activator of transcription 3, and are enriched in effector and central memory phenotype. Moreover, these cells are heterogeneous in nature given that they constitute different subsets of cytotoxic follicular T cells (TCF), including TCF1, TCF2, TCF17, and TCF22. Despite expressing high PD-1, CD8+ CXCR5+ cells are activated, proliferating, secreting more IFN-γ, IL-21, and IL-22, and have better cytolytic potential than CD8+ CXCR5- cells, which were inhibited after PD-1/PD-L1 blockade. CD8+ CXCR5+ cells are efficient in helping B cells in terms of plasmablasts and plasma cell generation. CONCLUSIONS: In conclusion, CD8+ CXCR5+ cells are enriched in effector phenotypes, produce HBV-specific cytokines despite increased PD-1, and are associated with HBsAg and HBV-DNA reduction. These cells competently support B-cell function, required for viral clearance, which may serve as potential therapeutic targets for CHB.

Immunomodulation and RNA interference alter hepatitis B virus-specific CD8 T-cell recognition of infected HepG2-NTCP.

BACKGROUND AND AIMS: CD8 T cells are essential in controlling HBV infection. Viral control is dependent on efficient recognition of HBV-infected hepatocytes by CD8 T cells, which can induce direct lysis of infected hepatocytes. In addition, CD8 T cells produce interferon (IFN)-γ, which mediates noncytopathic viral clearance. Innate immunomodulators and HBV-targeted RNA interference (RNAi) are being developed to treat chronic hepatitis B (CHB), but may modify HBV antigen presentation and impact CD8 T-cell recognition, in addition to their primary mechanisms of action. APPROACH AND RESULTS: HBV-infected HepG2-NTCP cells were treated with tenofovir disoproxil fumarate (TDF), Toll-like receptor (TLR) 7/8 agonists, TLR7/8 conditioned media (CM) collected from immune cells, or RNAi using short interfering RNAs. The effect of these treatments on antigen presentation was measured through coculture with CD8 T cells recognizing human leukocyte antigen-A0201 restricted epitopes, HBc18-27 or HBs183-191. Cytokine profiles of TLR7/8 CM were measured using a cytometric bead array. TDF reduced viral replication, but not CD8 T-cell recognition, of infected cells. Direct exposure of infected HepG2-NTCP to TLR7/8 agonists had no impact on T-cell recognition. Exposure of infected HepG2-NTCP to TLR7/8 CM enhanced HBV-specific CD8 T-cell recognition through type 1 interferon (IFN) and IFN-γ-dependent mechanisms. RNAi rapidly suppressed HBV-DNA, HBcAg, and HBsAg expression, impairing recognition by HBV-specific CD8 T cells. CONCLUSIONS: Immunomodulation and RNAi, but not nucleos(t)ide analogues, alter the recognition of infected HepG2-NTCP by HBV-specific CD8 T cells. Understanding these changes will inform combination treatments for CHB.

Incidence and prediction of HBsAg seroclearance in a prospective multi-ethnic HBeAg-negative chronic hepatitis B cohort.

BACKGROUND AND AIMS: Achieving HBsAg loss is an important landmark in the natural history of chronic hepatitis B (CHB). A more personalized approach to prediction of HBsAg loss is relevant in counseling patients. This study sought to develop and validate a prediction model for HBsAg loss based on quantitative HBsAg levels (qHBsAg) and other baseline characteristics. METHODS: The Hepatitis B Research Network (HBRN) is a prospective cohort including 1240 untreated HBeAg-negative patients (1150 adults, 90 children) with median follow-up of 5.5 years. Incidence rates of HBsAg loss and hepatitis B surface antibody (anti-HBs) acquisition were determined, and a predictor score of HBsAg loss using readily available variables was developed and externally validated. RESULTS: Crude incidence rates of HBsAg loss and anti-HBs acquisition were 1.6 and 1.1 per 100 person-years (PY); 67 achieved sustained HBsAg loss for an incidence rate of 1.2 per 100 PY. Increased HBsAg loss was significantly associated with older age, non-Asian race, HBV phenotype (inactive CHB vs. others), HBV genotype A, lower HBV-DNA levels, and lower and greater change in qHBsAg. The HBRN-SQuARe (sex,∆quantHBsAg, age, race) score predicted HBsAg loss over time with area under the receiver operating characteristic curve (AUROC) (95% CIs) at 1 and 3 years of 0.99 (95% CI: 0.987-1.00) and 0.95 (95% CI 0.91-1.00), respectively. In validation in another cohort of 1253 HBeAg-negative patients with median follow-up of 3.1 years, HBRN SQuARe predicted HBsAg loss at 1 and 3 years with AUROC values of 0.99 (0.98-1.00) and 0.88 (0.77-0.99), respectively. CONCLUSION: HBsAg loss in predominantly untreated patients with HBeAg-negative CHB can be accurately predicted over a 3-year horizon using a simple validated score (HBRN SQuARe). This prognostication tool can be used to support patient care and counseling.