Viral and host factors related to the clinical outcome of COVID-19.

In December 2019, coronavirus disease 2019 (COVID-19), which is caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in Wuhan (Hubei province, China)1; it soon spread across the world. In this ongoing pandemic, public health concerns and the urgent need for effective therapeutic measures require a deep understanding of the epidemiology, transmissibility and pathogenesis of COVID-19. Here we analysed clinical, molecular and immunological data from 326 patients with confirmed SARS-CoV-2 infection in Shanghai. The genomic sequences of SARS-CoV-2, assembled from 112 high-quality samples together with sequences in the Global Initiative on Sharing All Influenza Data (GISAID) dataset, showed a stable evolution and suggested that there were two major lineages with differential exposure history during the early phase of the outbreak in Wuhan. Nevertheless, they exhibited similar virulence and clinical outcomes. Lymphocytopenia, especially reduced CD4+ and CD8+ T cell counts upon hospital admission, was predictive of disease progression. High levels of interleukin (IL)-6 and IL-8 during treatment were observed in patients with severe or critical disease and correlated with decreased lymphocyte count. The determinants of disease severity seemed to stem mostly from host factors such as age and lymphocytopenia (and its associated cytokine storm), whereas viral genetic variation did not significantly affect outcomes.

Modulation of monocyte activity by hepatocellular MicroRNA delivery through HBsAg particles: Implications for pathobiology of chronic hepatitis B.

BACKGROUND AND AIMS: HBsAg serves as an important immune-modulatory factor in chronic hepatitis B. One aspect of such modulation may act through monocytes, which are the major Ag-presenting cells taking up HBsAg. There is evidence for the encapsulation of hepatocellular microRNAs (miRNAs) by HBsAg particles, while its pathobiological significance is unclear. Here, we characterized the miRNA profile in patients with chronic hepatitis B and probed their association with liver inflammation. APPROACHES AND RESULTS: We collected plasma from patients that are treatment-naive with chronic hepatitis B (n = 110) and quantified total/HBsAg-enveloped miRNAs by qRT-PCR and plasma cytokines by ELISA. The biological effects of HBsAg-delivered miRNAs in monocytes were evaluated using multiple approaches. The clinical significance of candidate miRNAs and cytokines was corroborated in patients with HBV-associated advanced liver diseases. The plasma miRNA profile showed 2 major clusters, one significantly associated with HBsAg titer and the other correlated with liver inflammation. Among HBsAg-carried miRNAs, miR-939 displayed the most significant correlation with IL-8. Mechanistically, miR-939 in subviral particles enters monocytes and significantly augments IL-8 production through the mitogen-activated protein kinase (MAPK) p38 signaling pathway. Finally, the findings that miR-939 positively correlated with IL-8 level and inflammation/fibrosis stage in the cohort of HBV-associated advanced liver diseases support its causative role in the progression of liver diseases. CONCLUSIONS: HBsAg particles carry hepatocellular miRNAs, including miR-939, which enter monocytes and alter their functional status, such as IL-8 secretion. Our findings demonstrate that the HBsAg-miR-939-IL-8 axis may play a crucial role in HBV-induced hepatic necro-inflammation and the progression of advanced liver diseases.

Chimeric antigen receptors of HBV envelope proteins inhibit hepatitis B surface antigen secretion.

OBJECTIVES: Chronic hepatitis B (CHB) caused by HBV infection greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. Hepatitis B surface antigen (HBsAg) plays critical roles in the pathogenesis of CHB. HBsAg loss is the key indicator for cure of CHB, but is rarely achieved by current approved anti-HBV drugs. Therefore, novel anti-HBV strategies are urgently needed to achieve sustained HBsAg loss. DESIGN: We developed multiple chimeric antigen receptors (CARs) based on single-chain variable fragments (scFvs, namely MA18/7-scFv and G12-scFv), respectively, targeting HBV large and small envelope proteins. Their impacts on HBsAg secretion and HBV infection, and the underlying mechanisms, were extensively investigated using various cell culture models and HBV mouse models. RESULTS: After secretory signal peptide mediated translocation into endoplasmic reticulum (ER) and secretory pathway, MA18/7-scFv and CARs blocked HBV infection and virion secretion. G12-scFv preferentially inhibited virion secretion, while both its CAR formats and crystallisable fragment (Fc)-attached versions blocked HBsAg secretion. G12-scFv and G12-CAR arrested HBV envelope proteins mainly in ER and potently inhibited HBV budding. Furthermore, G12-scFv-Fc and G12-CAR-Fc strongly suppressed serum HBsAg up to 130-fold in HBV mouse models. The inhibitory effect lasted for at least 8 weeks when delivered by an adeno-associated virus vector. CONCLUSION: CARs possess direct antiviral activity, besides the well-known application in T-cell therapy. Fc attached G12-scFv and G12-CARs could provide a novel approach for reducing circulating HBsAg.

Interferon-inducible MX2 is a host restriction factor of hepatitis B virus replication.

BACKGROUND & AIMS: Non-cytolytic cure of HBV-infected hepatocytes by cytokines, including type I interferons (IFNs), is of importance for resolving acute and chronic infection. However, as IFNs stimulate hundreds of genes, those most relevant for HBV suppression remain largely unknown. Amongst them are the large myxovirus resistance (Mx) GTPases. Human MX1 (or MxA) is active against many RNA viruses, while MX2 (or MxB) was recently found to restrict HIV-1, HCV, and herpesviruses. Herein, we investigated the anti-HBV activity of MX2. METHODS: The potential anti-HBV activity of MX2 and functional variants were assessed in transfected and HBV-infected hepatoma cells and primary human hepatocytes, employing multiple assays to analyze the synthesis and decay of HBV nucleic acids. The specific roles of MX2 in IFN-α-driven inhibition of HBV transcription and replication were assessed by MX2-specific shRNA interference (RNAi). RESULTS: Both MX2 alone and IFN-α substantially inhibited HBV replication, due to significant deceleration of the synthesis and slight acceleration of the turnover of viral RNA. RNAi knockdown of MX2 significantly reduced the inhibitory effects of IFN-α. Strikingly, MX2 inhibited HBV infection by reducing covalently closed circular DNA (cccDNA), most likely by indirectly impairing the conversion of relaxed circular DNA to cccDNA rather than by destabilizing existing cccDNA. Various mutations affecting the GTPase activity and oligomerization status reduced MX2's anti-HBV activity. CONCLUSION: MX2 is an important IFN-α inducible effector that decreases HBV RNA levels but can also potently inhibit HBV infection by indirectly impairing cccDNA formation. MX2 likely has the potential for therapeutic applications aimed at curing HBV infection by eliminating cccDNA. LAY SUMMARY: This study shows that the protein MX2, which is induced by interferon-α, has important anti-hepatitis B virus (HBV) effector functions. MX2 can reduce the amount of covalently closed circular DNA, which is the form of DNA that HBV uses to maintain viral persistence within hepatocytes. MX2 also reduces HBV RNA levels by downregulating synthesis of viral RNA. MX2 likely represents a novel intrinsic HBV inhibitor that could have therapeutic potential, as well as being useful for improving our understanding of the complex biology of HBV and the antiviral mechanisms of interferon-α.

Quantitative HBcrAg and HBcAb versus HBsAg and HBV DNA in predicting liver fibrosis levels of chronic hepatitis B patients.

OBJECTIVE: To evaluate the performance of the quantitative markers of hepatitis B core-related antigen (HBcrAg) and anti-hepatitis B core antigen antibodies HbcAb versus hepatitis B surface antigen (HBsAg) and hepatitis B virus DNA (HBV DNA) in predicting liver fibrosis levels in chronic hepatitis B patients. METHODS: Two hundred and fifty hepatitis B e antigen (HBeAg)-positive and 245 HBeAg-negative patients were enrolled. With reference to the Scheuer standard, stage 2 or higher and stage 4 liver disease were defined as significant fibrosis and cirrhosis, respectively. A receiver operating characteristic (ROC) curve was used to evaluate the performance of the HBV markers investigated. RESULTS: The areas under the ROC curves (AUCs) of HBcrAg in predicting significant fibrosis and cirrhosis in HBeAg-positive patients (0.577 and 0.700) were both close to those of HBsAg (0.617 and 0.762) (both P> 0.05). In HBeAg-negative patients (0.797 and 0.837), they were both significantly greater than those of HBV DNA (0.723 and 0.738) (P=0.0090 and P=0.0079). The AUCs of HBcAb in predicting significant fibrosis and cirrhosis in HBeAg-positive patients (0.640 and 0.665) were both close to those of HBsAg. In HBeAg-negative patients (0.570 and 0.621), they were both significantly less than those of HBcrAg (P <0.0001 and P=0.0001). Specificity in predicting significant fibrosis and sensitivity in predicting cirrhosis in HBeAg-positive patients, using a single cut-off of HBsAg ≤5,000 IU/ml, were 76.5% and 72.7%, respectively. In HBeAg-negative patients, using a single cut-off of HBcrAg>80kU/ml, they were 85.9% and 81.3%, respectively. CONCLUSIONS: HBsAg has good performance in predicting liver fibrosis levels in HBeAg-positive and HBeAg-negative patients, and HBcrAg has very good performance in predicting liver fibrosis levels in HBeAg-negative patients.

Exosomes Exploit the Virus Entry Machinery and Pathway To Transmit Alpha Interferon-Induced Antiviral Activity.

Alpha interferon (IFN-α) induces the transfer of resistance to hepatitis B virus (HBV) from liver nonparenchymal cells (LNPCs) to hepatocytes via exosomes. However, little is known about the entry machinery and pathway involved in the transmission of IFN-α-induced antiviral activity. In this study, we found that macrophage exosomes uniquely depend on T cell immunoglobulin and mucin receptor 1 (TIM-1), a hepatitis A virus (HAV) receptor, to enter hepatocytes for delivering IFN-α-induced anti-HBV activity. Moreover, two primary endocytic routes for virus infection, clathrin-mediated endocytosis (CME) and macropinocytosis, collaborate to permit exosome entry and anti-HBV activity transfer. Subsequently, lysobisphosphatidic acid (LBPA), an anionic lipid closely related to endosome penetration of virus, facilitates membrane fusion of exosomes in late endosomes/multivesicular bodies (LEs/MVBs) and the accompanying exosomal cargo uncoating. Together, our findings provide comprehensive insights into the transmission route of macrophage exosomes to efficiently deliver IFN-α-induced antiviral substances and highlight the similarities between the entry mechanisms of exosomes and virus.IMPORTANCE Our previous study showed that LNPC-derived exosomes could transmit IFN-α-induced antiviral activity to HBV replicating hepatocytes, but the concrete transmission mechanisms, which include exosome entry and exosomal cargo release, remain unclear. In this study, we found that virus entry machinery and pathway were also applied to exosome-mediated cell-to-cell antiviral activity transfer. Macrophage-derived exosomes distinctively exploit hepatitis A virus receptor for access to hepatocytes. Later, CME and macropinocytosis are utilized by exosomes, followed by exosome-endosome fusion for efficient transfer of IFN-α-induced anti-HBV activity. We believe that understanding the cellular entry pathway of exosomes will be beneficial to designing exosomes as efficient vehicles for antiviral therapy.

Quantitative Anti-HBc in Liver Pathological States in Patients with Chronic Hepatitis B Virus Infection.

BACKGROUND: Changes of hepatitis B core antigen antibody (anti-HBc) in liver pathological involvement in patients with chronic hepatitis B virus (HBV) infection have not been investigated in detail. This study aimed to explore evolving patterns of anti-HBc following liver pathological states and to investigate validities of anti-HBc for predicting liver pathological states. METHODS: 254 HBeAg-positive and 237 HBeAg-negative patients with chronic HBV infection were enrolled. Liver pathological diagnoses referred to Scheuer standard, and anti-HBc was measured using chemiluminescence microparticle immunoassay. RESULTS: Anti-HBc was significantly positively correlated with pathological grades and stages in both HBeAg-positive (r s = 0.312, P < 0.0001, and r s = 0.268, P < 0.0001) and HBeAg-negative (r s = 0.270, P < 0.0001, and r s = 0.147, P=0.0237) patients. The medians of anti-HBc in pathological grades of G1, G2, and G3 and stages of S1, S2, S3, and S4 in HBeAg-positive patients were all significantly lower than those in HBeAg-negative patients (all P < 0.005). The areas under receiver-operating characteristic curves (95% confidence interval) of anti-HBc for predicting pathological grades ≥G2 and ≥G3, and stages ≥S2 and =S4 in HBeAg-positive patients were 0.683 (0.622-0.740) and 0.662 (0.601-0.720), and 0.627 (0.564-0.687) and 0.683 (0.622-0.740), respectively, and in HBeAg-negative patients were 0.681 (0.618-0.740) and 0.702 (0.639-0.760), and 0.569 (0.503-0.633) and 0.630 (0.565-0.691), respectively. CONCLUSION: Following hepatic aggravation of necroinflammation and progression of fibrosis, anti-HBc increases gradually in HBeAg-positive patients and continues to increase gradually in HBeAg-negative patients, which is a useful but unsatisfactory marker for monitoring pathological states.

Polarization of Monocytic Myeloid-Derived Suppressor Cells by Hepatitis B Surface Antigen Is Mediated via ERK/IL-6/STAT3 Signaling Feedback and Restrains the Activation of T Cells in Chronic Hepatitis B Virus Infection.

Chronic hepatitis B virus (HBV) infection is characterized by T cell tolerance to virus. Although inhibition of T cell responses by myeloid-derived suppressor cells (MDSCs) has been observed in patients with chronic hepatitis B (CHB), the mechanism for expansion of MDSCs remains ambiguous. In this study, a significant increased frequency of monocytic MDSCs (mMDSCs) was shown positively correlated to level of HBsAg in the patients with CHB. We further found hepatitis B surface Ag (HBsAg) efficiently promoted differentiation of mMDSCs in vitro, and monocytes in PBMCs performed as the progenitors. This required the activation of ERK/IL-6/STAT3 signaling feedback. Importantly, the mMDSCs polarized by HBsAg in vitro acquired the ability to suppress T cell activation. Additionally, treatment of all-trans retinoic acid, an MDSC-targeted drug, restored the proliferation and IFN-γ production by HBV-specific CD4(+) and CD8(+) T cells in PBMCs from patients with CHB and prevented increase of viral load in mouse model. In summary, HBsAg maintains HBV persistence and suppresses T cell responses by promoting differentiation of monocytes into mMDSCs. A therapy aimed at the abrogation of MDSCs may help to disrupt immune suppression in patients with CHB.

Low doses of CMV induce autoimmune-mediated and inflammatory responses in bile duct epithelia of regulatory T cell-depleted neonatal mice.

Recent studies have indicated that perinatal infection with cytomegalovirus (CMV) may promote bile duct damage in biliary atresia (BA) and that the decreased regulatory T cell (Treg) percentage associated with BA may further amplify the bile duct damage. Although a majority of BA patients have had previous CMV infections and lower percentages of Tregs, it is unknown whether an initial exposure to a low dose of CMV could induce exaggerated and progressive biliary injury. A Treg-depleted neonatal mouse was infected with low-dose CMV (LD-CMV) as a model to study BA patients. LD-CMV infection in Treg-depleted mice induced extensive inflammation in both the intrahepatic and extrahepatic bile ducts, accompanied with injury to and atresia of intrahepatic bile ducts and partial obstruction of the extrahepatic bile ducts. Serum total and direct bilirubin amounts were also elevated. Evidence for the involvement of cellular and humoral autoimmune responses in LD-CMV-infection of Treg-depleted mice was also obtained through detection of increased percentages of CD3 and CD8 mononuclear cells and serum autoantibodies reactive to bile duct epithelial proteins, one of which was identified as α-enolase. Depletion of Tregs that can lead to the decreased inhibition of aberrantly activated hepatic T-lymphocytes and generation of autoantibodies may lead to further injury. Increased hepatic expression of Th1-related genes (TNF-α), IFN-γ-activated genes (STAT-1) and Th1 cytokines (TNF-α, lymphotactin, IL-12p40 and MIP -1γ) were also identified. In conclusion, autoimmune-mediated and inflammatory responses induced by LD-CMV infection in Treg-depleted mice results in increased intrahepatic and extrahepatic bile duct injury and contributed to disease progression.

Hepatitis B virus surface antigen selectively inhibits TLR2 ligand-induced IL-12 production in monocytes/macrophages by interfering with JNK activation.

It is widely accepted that chronic hepatitis B virus (HBV) infection is the result of an ineffective antiviral immune response against HBV infection. Our previous study found that the hepatitis B surface Ag (HBsAg) was related to decreased cytokine production induced by the TLR2 ligand (Pam3csk4) in PBMCs from chronic hepatitis B patients. In this study, we further explored the mechanism involved in the inhibitory effect of HBsAg on the TLR2 signaling pathway. The results showed that both Pam3csk4-triggered IL-12p40 mRNA expression and IL-12 production in PMA-differentiated THP-1 macrophage were inhibited by HBsAg in a dose-dependent manner, but the production of IL-1ß, IL-6, IL-8, IL-10, and TNF-α was not influenced. The Pam3csk4-induced activation of NF-κB and MAPK signaling were further examined. The phosphorylation of JNK-1/2 and c-Jun was impaired in the presence of HBsAg, whereas the degradation of IκB-α, the nuclear translocation of p65, and the phosphorylation of p38 and ERK-1/2 were not affected. Moreover, the inhibition of JNK phosphorylation and IL-12 production in response to Pam3csk was observed in HBsAg-treated monocytes/macrophages (M/MΦs) from the healthy donors and the PBMCs and CD14-positive M/MΦs from chronic hepatitis B patients. Taken together, these results demonstrate that HBsAg selectively inhibits Pam3csk4- stimulated IL-12 production in M/MΦs by blocking the JNK-MAPK pathway and provide a mechanism by which HBV evades immunity and maintains its persistence.

Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance.

BACKGROUND: On March 30, a novel influenza A subtype H7N9 virus (A/H7N9) was detected in patients with severe respiratory disease in eastern China. Virological factors associated with a poor clinical outcome for this virus remain unclear. We quantified the viral load and analysed antiviral resistance mutations in specimens from patients with A/H7N9. METHODS: We studied 14 patients with A/H7N9 disease admitted to the Shanghai Public Health Clinical Centre (SPHCC), China, between April 4, and April 20, 2013, who were given antiviral treatment (oseltamivir or peramivir) for less than 2 days before admission. We investigated the viral load in throat, stool, serum, and urine specimens obtained sequentially from these patients. We also sequenced viral RNA from these specimens to study the mutations associated with resistance to neuraminidase inhibitors and their association with disease outcome. FINDINGS: All patients developed pneumonia, seven of them required mechanical ventilation, and three of them further deteriorated to become dependent on extracorporeal membrane oxygenation (ECMO), two of whom died. Antiviral treatment was associated with a reduction of viral load in throat swab specimens in 11 surviving patients. Three patients with persistently high viral load in the throat in spite of antiviral therapy became ECMO dependent. An Arg292Lys mutation in the virus neuraminidase (NA) gene known to confer resistance to both zanamivir and oseltamivir was identified in two of these patients, both also received corticosteroid treatment. In one of them, wild-type sequence Arg292 was noted 2 days after start of antiviral treatment, and the resistant mutant Lys292 dominated 9 days after start of treatment. INTERPRETATION: Reduction of viral load following antiviral treatment correlated with improved outcome. Emergence of NA Arg292Lys mutation in two patients who also received corticosteroid treatment led to treatment failure and a poor clinical outcome. The emergence of antiviral resistance in A/H7N9 viruses, especially in patients receiving corticosteroid therapy, is concerning, needs to be closely monitored, and considered in pandemic preparedness planning. FUNDING: National Megaprojects of China for Infectious Diseases, Shanghai Municipal Health and Family Planning Commission, the National Key Basic Research Program of China, Ministry of Science and Technology, and National Natural Science Foundation of China.

High-Resolution Genomic Profiling of a Genotype 3b Hepatitis C Virus from a Flare of an Occult Hepatitis Patient with Acute-on-Chronic Liver Failure.

Acute-on-chronic liver failure (ACLF) is defined as a syndrome of acutely decompensated cirrhosis in patients with chronic liver disease (CLD). Here we report an ACLF case caused by a flare of occult hepatitis C infection. This patient was infected with hepatitis C virus (HCV) more than a decade ago and hospitalized due to alcohol-associated CLD. Upon admission, the HCV RNA in the serum was negative and the anti-HCV antibody was positive, whereas the viral RNA in the plasma dramatically increased during hospitalization, which suggests an occult hepatitis C infection. Overlapped fragments encompassing the nearly whole HCV viral genome were amplified, cloned, and sequenced. Phylogenetic analysis indicated an HCV genotype 3b strain. Sanger sequencing to 10-fold coverage of the 9.4-kb nearly whole genome reveals high diversity of viral quasispecies, an indicator of chronic infection. Inherent resistance-associated substitutions (RASs) in the NS3 and NS5A but not in the NS5B regions were identified. The patient developed liver failure and accepted liver transplantation, followed by direct-acting antiviral (DAA) treatment. The hepatitis C was cured by the DAA treatment despite the existence of RASs. Thus, care should be taken for occult hepatitis C in patients with alcoholic cirrhosis. The analysis of viral genetic diversity may help to identify an occult hepatitis C virus infection and predict the efficacy of anti-viral treatment.

Inhibition of hepatitis B virus replication by cIAP2 involves accelerating the ubiquitin-proteasome-mediated destruction of polymerase.

Cellular inhibitor of apoptosis protein 2 (cIAP2) is a potent suppressor of apoptotic cell death. We have shown previously that cIAP2 is involved in the tumor necrosis factor alpha (TNF-α)-induced anti-hepatitis B virus (HBV) response; however, the mechanism for this antiviral effect remains unclear. In the present study, we demonstrate that cIAP2 can significantly reduce the levels of HBV DNA replication intermediates but not the total viral RNA or core protein levels. Domain-mapping analysis revealed that the carboxy-terminal domains of cIAP2 were indispensable for this anti-HBV ability and that an E3 ligase-deficient mutant of cIAP2 (termed cIAP2*) completely lost its antiviral activity. We further identified HBV polymerase as the target of cIAP2. Overexpression of cIAP2 but not cIAP2* reduced polymerase protein levels, while cIAP2 knockdown increased polymerase expression. In addition, we observed that cIAP2 promoted the degradation of the viral polymerase through a proteasome-dependent pathway. Further experiments demonstrated that cIAP2 can bind to polymerase and promote its polyubiquitylation. Finally, we found that cIAP2 downregulated the encapsidation of HBV pregenomic RNA. Taken together, these data reveal a novel mechanism for the inhibition of HBV replication by cIAP2 via acceleration of the ubiquitin-proteasome-mediated decay of polymerase and reduction of the encapsidation of HBV pregenomic RNA, making this mechanism a novel strategy for HBV therapy.

Characterization of intrahepatic B cells in acute-on-chronic liver failure.

Background and objectives: Acute on chronic liver failure (ACLF) is characterized by the immunologic dissonance during the prolonged pathogenic development. Both abnormal innate immune response and adaptive T-cell response have been reported in patients with ACLF; however, less is known regarding B cells in ACLF pathogenesis. Previous reports were only based on immunophenotyping of peripheral blood samples. Here, we aim to dissect liver-infiltrating B-cell subpopulation in ACLF. Methods: Paired liver perfusate and peripheral blood were freshly collected from healthy living donors and recipients during liver transplantation. Liver tissues were obtained from patients with ACLF, cirrhosis, and healthy controls. Flow cytometry was used to characterize the phenotypic and functional alterations in intrahepatic and circulating B-cell populations from ACLF, cirrhosis, and healthy controls. The expression of CD19+ and CD138+ on liver tissues was examined by immunohistochemistry staining. Results: In this study, we first deciphered the intrahepatic B cells subsets of patients with ACLF. We found that the ACLF liver harbored reduced fraction of naïve B cells and elevated percentage of CD27+CD21- activated memory B cells (AM), CD27-CD21- atypical memory B cells (atMBC), CD27+IgD-IgM+(IgM+ memory B cells), and CD27+CD38++ plasma cells than cirrhosis and healthy controls. Moreover, these B subpopulations demonstrated enhanced activation and altered effector functions. Specifically, the ACLF liver was abundant in atMBC expressing higher CD11c and lower CD80 molecule, which was significantly correlated to alanine aminotransferase and aspartate aminotransferase. In addition, we found that intrahepatic CD27+CD38++plasma cells were preferentially accumulated in ACLF, which expressed more CD273 (PD-L2) and secreted higher granzyme B and IL-10. Finally, the enriched hepatic plasma B cells were in positive association with disease severity indices including alkaline phosphatase and gamma-glutamyl transferase. Conclusions: In this pilot study, we showed an intrahepatic B-cell landscape shaped by the ACLF liver environment, which was distinct from paired circulating B-cell subsets. The phenotypic and functional perturbation in atMBC and plasma cells highlighted the unique properties of infiltrating B cells during ACLF progression, thereby denoting the potential of B-cell intervention in ACLF therapy.

Monocytic MDSCs homing to thymus contribute to age-related CD8+ T cell tolerance of HBV.

Hepatitis B virus exposure in children usually develops into chronic hepatitis B (CHB). Although hepatitis B surface antigen (HBsAg)-specific CD8+ T cells contribute to resolve HBV infection, they are preferentially undetected in CHB patients. Moreover, the mechanism for this rarely detected HBsAg-specific CD8+ T cells remains unexplored. We herein found that the frequency of HBsAg-specific CD8+ T cells was inversely correlated with expansion of monocytic myeloid-derived suppressor cells (mMDSCs) in young rather than in adult CHB patients, and CCR9 was upregulated by HBsAg on mMDSCs via activation of ERK1/2 and IL-6. Sequentially, the interaction between CCL25 and CCR9 mediated thymic homing of mMDSCs, which caused the cross-presentation, transferring of peripheral HBsAg into the thymic medulla, and then promoted death of HBsAg-specific CD8+ thymocytes. In mice, adoptive transfer of mMDSCs selectively obliterated HBsAg-specific CD8+ T cells and facilitated persistence of HBV in a CCR9-dependent manner. Taken together, our results uncovered a novel mechanism for establishing specific CD8+ tolerance to HBsAg in chronic HBV infection.

Evaluation of the in situ assay for HBV DNA: An observational real-world study in chronic hepatitis B.

ABSTRACT: The visualization of intrahepatic hepatitis B virus (HBV) DNA by in situ hybridization (ISH) has uncovered some interesting aspects of HBV life cycle at the single-cell level. In the current study, we intend to evaluate the reliability and robustness of this assay in the real-world clinical scenario and its relationship with currently available clinical biomarkers in chronic hepatitis B (CHB) patients.In this cross-sectional study, 94 CHB patients and 10 patients with non-HBV related liver diseases were enrolled. Liver biopsies and routine histopathology analysis were performed. Intrahepatic HBV DNA and viral antigens (HBsAg and HBcAg) were detected by ISH and immunohistochemistry (IHC), respectively. The basic biochemical and virological parameters such as alanine transaminase, serum HBV DNA, and serum HBsAg were measured.The HBV DNA-ISH assay showed 55.8% (53/94 cases) positive rate in CHB patients, no false positive was found in non-HBV related hepatitis. The IHC of HBsAg and HBcAg showed a positive rate of 94.7% (89/94 cases) and 19.5% (17/87 cases), respectively. Quantification of HBV DNA-ISH signal showed a significant correlation with serum HBV DNA (rs = 0.6223, P < .0001). In addition, the staining pattern of HBV DNA in situ in the context of collagen deposition informed the histopathological progression of chronic liver disease.The application of this ISH assay in evaluating intrahepatic viral replication in real-world CHB patients showed favorable performance. It can be a complementation to conventional liver histopathology examination and IHC detection of viral antigens. This methodology provides an intuitive assessment of virological and pathological state of CHB patients, and further supports clinical diagnosis and management.

Quantitative serum HBV markers in predicting phases of natural history of chronic HBV infection.

BACKGROUND: Clinical importance of commercially available quantitative HBV markers has not been fully investigated. OBJECTIVE: To choice and to evaluate clinically valuable HBV markers for predicting phases of natural history with chronic HBV infection. METHODS: 472 naïve patients with chronic HBV infection were enrolled, in which 21 and 220 were confirmed as HBeAg-positive inactive and active hepatitis (EPIH and EPAH), respectively, and 106 and 125 were confirmed as HBeAg-negative inactive and active hepatitis (ENIH and ENAH), respectively. HBsAg, HBcrAg and anti- HBc were measured using chemiluminescent immunoassay, and HBV DNA was measured using PCR-fluorescence probing assay. RESULTS: There were all statistical differences in medians of HBsAg, anti-HBc, HBcrAg and HBV DNA between EPIH and EPAH and between ENIH and ENAH (all P < 0.01). According to binary logistic stepwise regressions, HBsAg and anti-HBc were preferred variables for predicting EPAH, and HBcrAg and HBV DNA were preferred variables for predicting ENAH. Based on normalization for coefficients of preferred variables entering regression equations, a handy model of MEPAH for predicting EPAH and of MENAH for predicting ENAH was constructed, respectively. Area under receiver operating characteristic curves of MEPAH and MENAH for predicting EPAH and ENAH were 0.882 and 0.931, respectively. With standard of MEPAH ≤ 5.997 and MENAH > 10.535, sensitivity or specificity of which for predicting EPAH and ENAH were about 81.0 % and 87.0 %, respectively. CONCLUSION: HBsAg and anti-HBc for predicting EPAH and HBcrAg and HBV DNA for predicting ENAH are dependable markers; MEPAH for predicting EPAH and MENAH for predicting ENAH have very good performance.

Measles virus infection in adults induces production of IL-10 and is associated with increased CD4+ CD25+ regulatory T cells.

Despite steady progress in elimination of measles virus globally, measles infection still causes 500,000 annual deaths, mostly in developing countries where endemic measles strains still circulate. Many adults are infected every year in China, with symptoms more severe than those observed in children. In this study, we have used blood samples from adult measles patients in Shanghai and age-matched healthy controls to gain an understanding of the immune status of adult measles patients. IFN-alpha mRNA was reduced in patient PBMC compared with healthy controls. In contrast, gene expression and plasma production of IL-2, IL-10, and IFN-gamma were elevated in patient blood. A similar cytokine profile was observed at early times when cultured PBMC were infected with a clinical isolate of measles virus. In contrast to previous studies in pediatric patients, we did not find a reduction in total CD4(+) and CD8(+) T cells in patient PBMC. Interestingly, we found that CD4(+)CD25(+)CD127(low) regulatory T cells were significantly increased in patient PBMC compared with controls. Using intracellular cytokine staining we also show that the measles virus induces IL-10-producing CD14(+) and CD4(+)CD25(+) cells in PBMC. Our results show that adult measles patients in the acute phase of the disease have a mixed Th1/Th2 type response, accompanied with severe immunosuppression of both innate and adaptive responses including suppression of type I IFN. Both regulatory T cells and plasma IL-10 may contribute to the immunosuppression.

A novel recombinant cccDNA-based mouse model with long term maintenance of rcccDNA and antigenemia.

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is critical for viral persistence in vivo. The lack of reliable, characterized and convenient small animal models for studying cccDNA persistence has long been a bottleneck for basic and translational research on HBV cure. A mouse model that can maintain intrahepatic cccDNA is urgently needed. Through combining the Cre/loxP-mediated recombination and adeno-associated virus (AAV) vector delivery strategy, we establish a novel recombinant cccDNA (rcccDNA) mouse model. AAV-rcccDNA mice supported long-term maintenance of intrahepatic rcccDNA which could be easily detected by Southern blotting within 30 weeks after transduction. Quantitative PCR could detect the rcccDNA signal throughout the experiment duration (>51 weeks). Furthermore, rcccDNA supported persistent serum antigenemia (>72 weeks) and intrahepatic HBsAg and HBcAg expression (>51 weeks). Flow cytometry analysis and single-cell RNA sequencing showed that AAV-rcccDNA mice displayed a compromised CD8+ T cell response. Meanwhile, minimal intrahepatic inflammation and fibrosis were observed. Furthermore, three anti-HBV compounds, AKEX0007, a post-transcriptional inhibitor, Bay 41-4109, a capsid allosteric modulator, and Entecavir were assessed in this AAV-rcccDNA mouse model. The changes of viral markers by these drugs were consistent with their mode of action although neither of them diminished the level of rcccDNA. This mouse model recapitulated the immune tolerant state of HBV infection with long term maintenance of cccDNA and antigenemia, which will provide a suitable platform for studying cccDNA persistence and developing intervention strategies that would eventually break the tolerance and clear the virus.

Clinical relevance of the in situ assay for HBV DNA: a cross-sectional study in patients with chronic hepatitis B.

AIMS: The visualisation of HBV DNA in liver sections of patients with chronic hepatitis B (CHB) in our previous report uncovered a mosaic distribution of viral antigens and nucleic acids. Here we aim to further explore the clinical utility of the in situ hybridisation (ISH) assay for HBV DNA. METHOD: ISH of HBV DNA along with immunohistochemistry (IHC) of HBsAg, HBcAg and routine histopathology analysis was performed in 313 treatment-naive patients with CHB. Serum HBcrAg and HBcAb titre were also measured in addition to basic biochemical and virological parameters. RESULTS: The ISH of HBV DNA, HBsAg and HBcAg showed 95.2%, 97.1% and 42.8% positive rate, respectively. The staining pattern of HBV DNA differs significantly with that of HBsAg. Intrahepatic HBV DNA exhibited high-level of correlations with viral load, HBcrAg and HBsAg titre. In HBeAg-negative patients, higher intrahepatic HBV DNA is associated with histological evidence of liver inflammation and fibrosis, whereas no such trend was observed in HBeAg-positive patients. Finally, a triple staining protocol that combined the detection of HBV DNA, HBsAg and collagen fibre was developed to enable better evaluation of viral replication and antigen expression in the context of disease progression. CONCLUSIONS: The ISH assay for HBV DNA reflects the vigour of intrahepatic viral replication. It is complementary to the routine IHC assay for viral antigens and also related to the histopathological progression of liver diseases. The application of the HBV DNA ISH assay may help a better evaluation of virological and pathological condition of patients with CHB.