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.

A 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments.

Single-chain variable fragments (scFvs), composed of variable domains of heavy and light chains of an antibody joined by a linker, share antigen binding capacity with their parental antibody. Due to intrinsically low solubility and stability, only two Escherichia coli-produced scFvs have been approved for therapy. Here we report that a 33-residue peptide, termed P17 tag, increases the solubility of multiple scFvs produced in Escherichia coli SHuffle strain by up to 11.6 fold. Hydrophilic sequence, especially charged residues, but not the predicted α-helical secondary structure of P17 tag, contribute to the solubility enhancement. Notably, the P17 tag elevates the thermostability of scFv as efficiently as intra-domain disulfide bonds. Moreover, a P17-tagged scFv targeting hepatitis B virus surface proteins shows over two-fold higher antigen-binding affinity and virus-neutralizing activity than the untagged version. These data strongly suggest a type I intramolecular chaperone-like activity of the P17 tag. Hence, the P17 tag could benefit the research, production, and application of scFv.

Use of oral anticoagulants and its associated factors among nonvalvular atrial fibrillation patients with new-onset acute ischemic stroke: A report from the China Atrial Fibrillation Registry study.

BACKGROUND: The adherence of oral anticoagulant (OAC) therapy among nonvalvular atrial fibrillation (NVAF) patients with acute ischemic stroke (AIS) in China during recent years was unclear, and the possible factors that influenced the initiation and persistent use of OAC were needed to be explored. METHODS: A total of 1085 NVAF patients, who experienced new-onset and nonfatal AIS from August 2011 to December 2020 during follow-ups in the China Atrial Fibrillation Registry (China-AF), were enrolled. Information including patients' demographic characteristics, medical history, medication usage, which were collected before and after the index stroke, were used in the analysis. RESULTS: OAC was initiated in 40% (434/1085) NVAF patients within 3 months after new-onset AIS. High-reimbursement-rate insurance coverage (odds ratio [OR]: 1.51, 95% confidence interval [CI]: 1.03-2.22, p = .036), 3-month-peri-stroke AF episodes (OR: 2.63, 95% CI: 1.88-3.69, p < .001), and pre-stroke OAC usage (OR: 8.92, 95% CI: 6.01-13.23, p < .001), were positively associated with initiation of OAC within 3 months after new-onset AIS, while age (OR: 0.98, 95% CI: 0.96-1.00, p = .024), female (OR: 0.63, 95% CI: 0.44-0.90, p = .012) and higher modified HASBLED score (OR: 0.45, 95% CI: 0.37-0.55, p < .001) were negatively associated with it. Among 3-month-post-stroke OAC users, history of radiofrequency ablation (hazard ratio: 1.65, 95% CI: 1.16-2.35; p = .006) was positively associated with non-persistence of OAC usage. CONCLUSIONS: In China, the proportion of NVAF patients who initiated OAC therapy since new-onset AIS was still low. More efforts are needed on improving patients' adherence to anticoagulant therapy.

Expression Level of Small Envelope Protein in Addition to Sequence Divergence inside Its Major Hydrophilic Region Contributes to More Efficient Surface Antigen Secretion by Hepatitis B Virus Subgenotype D2 than Subgenotype A2.

Hepatitis B surface antigen (HBsAg) promotes persistent hepatitis B virus (HBV) infection. It primarily corresponds to small (S) envelope protein secreted as subviral particles. We previously found that genotype D clones expressed less S protein than genotype A clones but showed higher extracellular/intracellular ratio of HBsAg suggesting more efficient secretion. The current study aimed to characterize the underlying mechanism(s) by comparing a subgenotype A2 clone (geno5.4) with a subgenotype D2 clone (geno1.2). Five types of full-length or subgenomic constructs were transfected to Huh7 cells at different dosage. HBsAg was quantified by enzyme linked immunosorbent assay while envelope proteins were detected by Western blot. We found that ratio of extracellular/intracellular HBsAg decreased at increasing amounts of DNA transfected. Conflicting findings from two types of subgenomic construct confirmed stronger secretion inhibitory effect of the genotype D-derived large envelope protein. Chimeric constructs followed by site-directed mutagenesis revealed geno1.2 specific V118/T127 and F161/A168 in the S protein as promoting and inhibitory of HBsAg secretion, respectively. In conclusion, more efficient HBsAg secretion by subgenotype D2 than subgenotype A2 is attributed to lower level of S protein expression in addition to V118 and T127 in S protein, although its F161 and A168 sequences rather reduce HBsAg secretion.

Guava Leaf Extract Attenuates Insulin Resistance via the PI3K/Akt Signaling Pathway in a Type 2 Diabetic Mouse Model.

BACKGROUND AND OBJECTIVE: Insulin resistance is well known to exhibit essential effects on the progression of diabetes mellitus (DM). Guava leaf was also reported to exhibit anti-diabetic effects including decreasing blood glucose. Therefore, this present study aims to explore the role guava leaf extract (GLE) plays in insulin resistance and its mechanism of action via the PI3K/Akt signaling pathway. METHODS: KK-Ay mice is a spontaneous genetic type 2 diabetes mouse model induced by feeding a high fat and high sugar diet. Mice were randomly assigned into three groups: diabetic mice (DM), DM + MET (diabetic mice treated with metformin) and DM + GLE (diabetic mice treated with GLE) groups. After 8 weeks of treatment, body weight and levels of fasting plasma glucose (FPG), fasting insulin and lipids in plasma were measured. Mice were sacrificed and mRNA and protein expression of insulin receptor substrate1 (IRS1), phosphatidylinositol 3-kinase (PI3K) and serine/threonine kinase protein B (Akt) in livers were measured. RESULTS: GLE markedly reduced body weight, FPG, fasting insulin and insulin resistance index but increased the insulin sensitivity index of diabetic KK-Ay mice. Moreover, GLE upregulated the expression of IRS-1, PI3K and Akt mRNAs in livers of diabetic KK-Ay mice. In addition, GLE also elevated IRS-1, PI3K, Akt, p-PI3K and p-Akt protein expression in their livers. The results of the DM + MET group were similar to those of the DM + GLE group. CONCLUSION: GLE plays anti-diabetic roles by ameliorating insulin resistance in KK-Ay diabetic mice and this is related to the activation of PI3K/Akt signaling pathway.

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.

Response to immune complex vaccine in chronic hepatitis B patients is associated with lower baseline level of serum IgG galactosylation.

The composition of glycan in immunoglobulin G (IgG) has shown to affect various diseases and can be regulated by drugs and preventive vaccination. A hepatitis B surface antigen (HBsAg)-hepatitis B immunoglobulin (HBIG) immune complex (YIC) therapeutic vaccine for chronic hepatitis B (CHB) patients has undergone clinical trials. To explore for markers of CHB, which could be associated with responsiveness to YIC therapeutic vaccine, serum IgG glycosylation in CHB patients was analyzed.Kinetic changes of serum galactosylated IgG in 53 hepatitis Be antigen (HBeAg)-positive CHB patients treated with YIC were monitored by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) analysis. Whole blood cytokines were assayed by cytokine binding assay kits. All samples were back assayed before treatment, during therapy and follow-up for 6 months from a previous completed clinical trial.During YIC treatment, 26 patients with lower IgG galactosylation level at baseline [galactosylation level (Gal-ratio) = -0.29, 0.18 (mean, SD)] showed sustained increase of serum galactosylated IgG, and responded to YIC treatment by HBeAg seroconversion. While those who did not respond to YIC treatment [Gal-ratio = -0.40, 0.15 (mean, SD)] failed to show similar changes. Furthermore, this kinetic increase of galactosylated IgG correlated with marked up-regulated IL-2 level, confirming that effective cellular immune responses have participated in responsiveness.For HBeAg-positive CHB patients lower serum IgG galactosylation level may serve as an indicator for selecting a suitable subpopulation of candidates for YIC therapeutic vaccination.

Immune Complex Vaccination.

Antibody/antigen binding results in immune complexes (IC) that have a variety of regulatory functions. One important feature is the enhanced host immune activation against antigen contained in the complex. ICs play important roles at several critical steps that lead to B and T cell activation, including antigen targeting/retention, facilitated antigen uptake, antigen presenting cell activation and proper balancing of positive and negative stimulatory signals. In both poultry industry and clinical health care, ICs have been used as preventive and therapeutic vaccines. With our deepening understanding of antibody biology, particularly in light of new revelations of regulatory functions of Fc receptors, mechanistically more precise engineering has spearheaded tailored use of this tool for infection control and cancer therapy. IC-based treatment and prophylaxis have been tested to different extents in HBV, HIV and influenza viral infection control and are actively examined as an alternative treatment for several forms of tumor. As a part of this book series, this chapter aims to discuss the mechanistic aspects of IC signaling and their impact on immune cells. We give samples how this old technology has been used by practitioners over the last several decades and suggest potential paths for future development of IC-based immune therapy.

Circulating Hematopoietic Stem/Progenitor Cells are Associated with Coronary Stenoses in Patients with Coronary Heart Disease.

Inflammatory cells in atherosclerotic plaque exclusively originate from hematopoietic stem/progenitor cells (HSPCs). In this study, we investigated whether circulating HSPCs frequency related to coronary stenosis in patients with coronary heart disease (CHD). Coronary angiography was performed in 468 participants who were recruited at Cardiology Centre in LuHe Hospital from March 2016 to May 2017. Among these subjects, 344 underwent echocardiography. Mononuclear cells isolated from peripheral blood were stained with an antibody cocktail containing anti-human CD34, anti-human lineage, anti-human CD38, and anti-human CD45RA. Lineage-CD38-CD45RAdimCD34+HSPCs were quantified by flow cytometry. CHD was defined as coronary stenosis ≥50% and the extent of CHD was further categorised by coronary stenosis ≥70%. A p < 0.0031 was regarded statistically significant by the Bonferroni correction. Circulating HSPCs frequency was 1.8-fold higher in CHD patients than non-CHD participants (p = 0.047). Multivariate-adjusted logistic analysis demonstrated that HSPCs was the only marker that was associated with the odds ratio of having mild vs. severe coronary stenosis (2.08 (95% CI, 1.35-3.21), p = 0.0009). Left ventricular ejection fraction was inversely correlated with HSPCs frequency and CRP in CHD patients (p < 0.05 for both). In conclusion, HSPCs frequency in circulation is intimately related to coronary stenoses in CHD patients.

An E. coli-produced single-chain variable fragment (scFv) targeting hepatitis B virus surface protein potently inhibited virion secretion.

Hepatitis B virus (HBV) envelopes as well as empty subviral particles carry in their lipid membranes the small (S), middle (M), and large (L) surface proteins, collectively known as hepatitis B surface antigen (HBsAg). Due to their common S domain all three proteins share a surface-exposed hydrophilic antigenic loop (AGL) with a complex disulfide bridge-dependent structure. The AGL is critical for HBV infectivity and virion secretion, and thus represents a major target for neutralizing antibodies. Previously, a human monoclonal antibody (mAb) targeting a conformational epitope in the AGL, IgG12, exhibited 1000-fold higher neutralizing activity than hepatitis B immune globulin (HBIG). Here we designed a single-chain variable fragment (scFv) homolog of IgG12, G12-scFv, which could be efficiently produced in soluble form in the cytoplasm of E. coli SHuffle cells. Independent in vitro assays verified specific binding of G12-scFv to a conformational S epitope shared with IgG12. Despite 20-fold lower affinity, G12-scFv but not an irrelevant scFv potently neutralized HBV infection of susceptible hepatoma cells (IC50 = 1.8 nM). Strikingly, low concentrations of G12-scFv blocked virion secretion from HBV producing cells (IC50 = 1.25 nM) without disturbing intracellular viral replication, whereas extracellular HBsAg was reduced only at >100-fold higher though still nontoxic concentration. The inhibitory effects correlated with S binding specificity and presumably also G12-scFv internalization into cells. Together these data suggest G12-scFv as a highly specific yet easily accessible novel tool for basic, diagnostic, and possibly future therapeutic applications.

From therapeutic antibodies to immune complex vaccines.

In recent years, therapeutic monoclonal antibodies have made impressive progress, providing great benefit by successfully treating malignant and chronic inflammatory diseases. Monoclonal antibodies with broadly neutralizing effects against specific antigens, or that target specific immune regulators, manifest therapeutic effects via their Fab fragment specificities. Subsequently therapeutic efficacy is mediated mostly by interactions of the Fc fragments of the antibodies with their receptors (FcR) displayed on cells of the immune system. These interactions can trigger a series of immunoregulatory responses, involving both innate and adaptive immune systems and including cross-presentation of antigens, activation of CD8 + T cells and CD4 + T cells, phagocytosis, complement-mediated antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The nature of the triggered effector functions of the antibodies is markedly affected by the glycosylation patterns of the Fc fragments. These can cause differences in the conformation of the heavy chains of antibodies, with resultant changes in antibody binding affinity and activation of the complement system. Studies of the Fc glycosylation profiles together with the associated Fc effector functions and FcR/CR interactions promoted interest and progress in engineering therapeutic antibodies. Furthermore, because antigen-antibody immune complexes (ICs) have shown similar actions, in addition to certain novel immunoregulatory mechanisms that also reshape immune responses, the properties of ICs are being explored in new approaches for prevention and therapy of diseases. In this review, both basic studies and experimental/clinical applications of ICs leading to the development of preventive and therapeutic vaccines are presented.

Analysis of immunological mechanisms exerted by HBsAg-HBIG therapeutic vaccine combined with Adefovir in chronic hepatitis B patients.

An HBsAg-HBIG therapeutic vaccine (Yeast-derived Immune Complexes, YIC) for chronic hepatitis B (CHB) patients has undergone a series of clinical trials. The HBeAg sero-conversion rate of YIC varied from 21.9% to 14% depending on the immunization protocols from 6 to 12 injections. To analyze the immunological mechanisms exerted by 6 injections of YIC, 44 CHB patients were separately immunized with YIC, alum as adjuvant control or normal saline as blank control, with add on of antiviral drug Adefovir in all groups. Kinetic increase in Th1 and Th2 cells CD4+ T cell sub-populations with association in decrease in Treg cells and increase of Tc1 and Tc17 cells in CD8+ T cells were observed in YIC immunized group. No such changes were found in the other groups. By multifunctional analysis of cytokine profiles, significant increase of IL-2 levels was observed, both in CD4+ and CD8+ T cells in the YIC immunized group, accompanied by increase in IFN-gamma and decrease of inhibitory factors (IL-10, TGF-ß and Foxp3) in CD4+ T cells. In the alum immunized group, slight increase of IL-10, TGF-ß and Foxp3 in CD4+ T cells was found after the second injection, but decreased after more injections, suggesting that alum induced early inflammatory responses to a certain extent. Similar patterns of responses of IL-17A and TNF-α in CD8+T cells were shown between YIC and the saline group. Results indicate that add on of Adefovir, did not affect host specific immune responses.

Immunoregulatory functions of immune complexes in vaccine and therapy.

Clinical and experimental preparations of IgG/soluble antigen complexes, as well as those formed following antibody therapy in vivo, are multifaceted immune regulators. These immune complexes (ICs) have been tested in humans and animal models, mostly in forms of experimental or clinical vaccination, for at least a century. With intensified research on Fcγ receptor-mediated immune modulation, as well as with immune complex-directed antigen processing, presentation, and inflammatory responses, there are renewed interests of using ICs in vaccines and immunotherapies. Currently, IC-based immune therapy has been broadly experimented in HBV and HIV viral infection control and antitumor treatments. However, mechanistic insights of IC-based treatments are relatively recent subjects of study; strong efforts are needed to establish links to connect laboratory findings with clinical practices. This review covers the history, mechanisms, and in vivo outcomes of this safe and effective therapeutic tool, with a clear aim to bridge laboratory findings with evolving clinical applications.

Results of a phase III clinical trial with an HBsAg-HBIG immunogenic complex therapeutic vaccine for chronic hepatitis B patients: experiences and findings.

BACKGROUND & AIMS: Even though various experimental therapeutic approaches for chronic hepatitis B infection have been reported, few of them have been verified by clinical trials. We have developed an antigen-antibody (HBsAg-HBIG) immunogenic complex therapeutic vaccine candidate with alum as adjuvant (YIC), aimed at breaking immune tolerance to HBV by modulating viral antigen processing and presentation. A double-blind, placebo-controlled, phase II B clinical trial of YIC has been reported previously, and herein we present the results of the phase III clinical trial of 450 patients. METHODS: Twelve doses of either YIC or alum alone as placebo were administered randomly to 450 CHB patients and they were followed for 24weeks after the completion of immunization. The primary end point was HBeAg seroconversion, and the secondary end points were decrease in viral load, improvement of liver function, and histology. RESULTS: In contrast to the previous phase II B trial using six doses of YIC and alum as placebo, six more injections of YIC or alum resulted in a decrease of the HBeAg seroconversion rate from 21.8% to 14.0% in the YIC group, but an increase from 9% to 21.9% in the alum group. Decrease in serum HBV DNA and normalization of liver function were similar in both groups (p>0.05). CONCLUSIONS: Overstimulation with YIC did not increase but decreased its efficacy due to immune fatigue in hosts. An appropriate immunization protocol should be explored and is crucial for therapeutic vaccination. Multiple injections of alum alone could have stimulated potent inflammatory and innate immune responses contributing to its therapeutic efficacy, and needs further investigation.

Hepatitis B virus genetic variants: biological properties and clinical implications.

Hepatitis B virus (HBV) causes a chronic infection in 350 million people worldwide and greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. The majority of chronic HBV carriers live in Asia. HBV can be divided into eight genotypes with unique geographic distributions. Mutations accumulate during chronic infection or in response to external pressure. Because HBV is an RNA-DNA virus the emergence of drug resistance and vaccine escape mutants has become an important clinical and public health concern. Here, we provide an overview of the molecular biology of the HBV life cycle and an evaluation of the changing role of hepatitis B e antigen (HBeAg) at different stages of infection. The impact of viral genotypes and mutations/deletions in the precore, core promoter, preS, and S gene on the establishment of chronic infection, development of fulminant hepatitis and liver cancer is discussed. Because HBV is prone to mutations, the biological properties of drug-resistant and vaccine escape mutants are also explored.

Erratum: H7N9 avian influenza virus - search and re-search.

[This corrects the article DOI: 10.1038/emi.2013.18.].

Responses to multiple injections with alum alone compared to injections with alum adsorbed to proteins in mice.

New effects and mechanisms of alum on innate immunity have emerged in recent years. A number of cellular and molecular mechanisms induced by aluminum adjuvant have been reported, while the role of NALP3 and inflammasome in the cellular pathway induced by alum is still controversial. The effect of injection of alum alone without vaccine antigen into human has not been reported so far. Recently, in a phase IIIa double-blinded placebo controlled clinical trial testing the therapeutic HBsAg-anti-HBs vaccine formulated with alum against chronic viral hepatitis B patients, the placebo group receiving alum only showed substantial therapeutic effects. To explore possible underlying therapeutic mechanisms, mice were treated either with multiple injections of alum alone or with alum adsorbed to proteins (HBsAg-anti-HBs). After 4 injections Gr1(+)/CD11b(+) cells in the spleen were increased in both alum alone and alum adsorbed in proteins groups. Increased Gr1(+)/CD11b(+) cells in spleens remained consistently high in the alum alone treated group, while Gr1(+)/CD11b(+)cells decreased in the alum adsorbed to proteins group after 6 injections. Both treatments triggered increased levels of TNF-alpha measured in the plasma, but only the alum alone treated mice showed increased levels of IL-10. Histology of the liver tissues revealed a higher number of spotty necrotic foci in the alum alone immunized group. Taken together, potent inflammatory responses were induced in the alum alone immunized mice, which suggests that the substantial therapeutic effects observed in chronic hepatitis B patients immunized with alum alone might be attributed to inflammatory responses.