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.

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.

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.

Carbonyl J acid derivatives block protein priming of hepadnaviral P protein and DNA-dependent DNA synthesis activity of hepadnaviral nucleocapsids.

Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploiting in vitro priming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 µM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives.

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.

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.

Hepatitis B virus (HBV) surface antigen interacts with and promotes cyclophilin a secretion: possible link to pathogenesis of HBV infection.

Cyclophilin A (CypA), predominantly located intracellularly, is a multifunctional protein. We previously reported decreased CypA levels in hepatocytes of transgenic mice expressing hepatitis B virus (HBV) surface antigen (HBsAg). In this study, we found that expression of HBV small surface protein (SHBs) in human hepatoma cell lines specifically triggered CypA secretion, whereas SHBs added extracellularly to culture medium did not. Moreover, CypA secretion was not promoted by the expression of a secretion deficient SHBs mutant, suggesting a close association between secretion of CypA and SHBs. Interaction between CypA and SHBs was observed by using coimmunoprecipitation and glutathione S-transferase pull-down assays. Hydrodynamic injection of the SHBs expression construct into C57BL/6J mice resulted in increased serum CypA levels and ALT/AST levels, as well as the infiltration of inflammatory cells surrounding SHBs-positive hepatocytes. The inflammatory response and serum ALT/AST level were reduced when the chemotactic effect of CypA was inhibited by cyclosporine and anti-CD147 antibody. Furthermore, higher serum CypA levels were detected in chronic hepatitis B patients than in healthy individuals. In HBV patients who had received liver transplantation, serum CypA levels declined dramatically after the loss of HBsAg as a consequence of liver transplantation. Taken together, these results indicate that expression and secretion of SHBs can promote CypA secretion, which may contribute to the pathogenesis of HBV infection.

Transcriptional analysis of immune-related genes in dendritic cells from hepatitis B surface antigen (HBsAg)-positive transgenic mice and regulation of Fc gamma receptor IIB by HBsAg-anti-HBs complex.

To study the gene expression profiles in dendritic cells (DCs) from a hepatitis B surface antigen (HBsAg) positive host, transcriptional analysis of bone marrow -derived DCs from a lineage of HBsAg transgenic mice (#59) was compared to DCs from normal mice. Among the immune-related genes, 12 were up-regulated, and 14 were down-regulated in transgenic mice relative to those of normal mice. The up-regulated genes include genes encoding immunoglobulin, histocompatibility 2 (K region), and several complement component genes, while the down-regulated genes include the TAP1 (transporters associated with antigen processing gene-1), interferon induced gene (Ifi203), chemokine (C-X-C) ligands and leukocyte-immunoglobulin-like genes, Lck-interacting transmembrane adaptor genes and histocompatibility 2 (Q region and T region). Since an immunogenic complex containing HBsAg-anti-HBs has been used as a therapeutic vaccine for clinical trial in chronic hepatitis B patients, DCs from #59 were incubated with immunogenic complex compared to those incubated with HBsAg alone. The immune-related six genes up-regulated with immunogenic complex treatment were Fcgr2b, Cxcl2, Fth1, Clec4n, Lilrb4, and Dbh, with Fcgr2b (Fc gamma receptor IIB) being the highest up-regulated gene. Interestingly, levels of Fcgr2b were found up-regulated in patients with chronic hepatitis B undergoing immunogenic complex immunization, which returned to baseline when immunization was discontinued. In conclusion, by transcriptional analysis, immunogenic complex induced up-regulation of Fcgr2b expression both in dendritic cells from an HBsAg transgenic mouse model and peripheral B cells from patients with chronic hepatitis B, which indicates that Fcgr2b is one of the key molecules up-regulated by immunogenic complex.

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.

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.

A double-spliced defective hepatitis B virus genome derived from hepatocellular carcinoma tissue enhanced replication of full-length virus.

In hepatitis B virus (HBV) replication cycle, pregenomic RNA undergoes splicing and the reverse transcribed defective genomes can be packaged and released. Various types of spliced defective HBV genomes have been isolated from the sera and liver tissues of viral hepatitis B patients. To explore the functions of a 2.2 kb double spliced HBV variant, a 3.2 kb full-length HBV isolate (#97-34) and its 2.2 kb double-spliced HBV variant (#AP-12) from the tumor tissue of a patient with hepatocellular carcinoma (HCC) were amplified and cloned. Sequencing results showed that #AP12 had deletions in pre-S2, part of pre-S1, S genes, part of the spacer, and part of the reverse transcriptase gene, while the X gene was intact. When this defective double-spliced genome and its full-length counterpart genome were co-transfected into HepG2 cells, the former was shown to enhance the replication of the latter, both by real-time PCR and Southern blotting. When a replication incompetent clone 97-34G1881A was used to co-transfect with #AP12, #AP12 DNA was increased, indicating that replication of the wild-type virus was not the only factor involved in this observation. However, the replication enhancing competency of #AP12 was shown to require an intact HBV X expression cassette. The double-spliced defective variant might contribute to persistent HBV replication in a subpopulation of HCC patients.

Interaction between SARS-CoV helicase and a multifunctional cellular protein (Ddx5) revealed by yeast and mammalian cell two-hybrid systems.

To reveal the putative cellular factors involved in SARS coronavirus replication, the helicase (Hel, nsp13) of SARS coronavirus was used to screen the cDNA library of rat pulmonary epithelial cells using the yeast two-hybrid system. Positively interacting proteins were further tested using a mammalian cell hybrid system and co-immunoprecipitation in the human A549 cell line, which has been shown to support SARS coronavirus replication. Out of the seven positive clones observed by yeast two-hybrid assay, only the Ddx5 (Asp-Glu-Ala-Asp box polypeptide 5) protein showed specific interaction with SARS-CoV helicase. When expression of DdX5 was knocked down by small interfering RNA (siRNA), SARS coronavirus replication was significantly inhibited in fetal rhesus kidney (FRhK-4) cells. Since Ddx5 is a multifunctional protein that plays important roles in transcriptional regulation, its interaction with SARS coronavirus helicase provides interesting clues for studying virus-host cell interactions in SARS-CoV infections.

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.

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.

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.

Mutational analysis of the "turn" of helix clamp motif of HIV-1 reverse transcriptase.

Helix clamp motifs of polymerases possessing the helix-turn-helix secondary structure are crucial for their polymerase activity by binding to the nucleic acid template/primer via the alpha helices. To study the functions of turn in helix clamp motif of human immunodeficiency virus (HIV)-1 RT, clones with turn mutants at rt271-274 of HIV-1 RT were generated and studied by one cycle infection assay. Mutants rtY271A and rtI274A almost lost their replication competency, while mutants rtA272P, rtA272S, and rtG273A retained comparable replication competency relative to wild type pseudotyped HIV-1. To study the mechanisms involved, RT proteins from rt271 to rt274 mutants were expressed and assayed for their RNA dependent DNA polymerase activity, DNA binding activity and processivity. Discordance between RT activity and viral replication efficiency of some turn mutants was observed, indicating that aside from RT, other steps in HIV replication could be affected by substitutions at the turn of helix clamp motif.

Mutational analysis revealed that conservation of hepatitis B virus reverse transcriptase residue 306 (rtP306) is crucial for encapsidation of pregenomic RNA.

Hepatitis B virus (HBV) is a DNA virus which replicates via reverse transcription. The structure and function of the reverse transcriptase play important roles in HBV replication. We have previously reported that when proline at residue 306 in HBV reverse transcriptase was substituted by other amino acids, most of the mutants showed decreased replicative competency. To explore the mechanisms for this decrease in replicative competency, constructs with substituted amino acid residues at rtP306 were used to transfect Huh-7 cells, and replication competencies, transcription levels and encapsidation efficiencies of the mutants and the parental viral strain were compared. Decreased replication competency was found with many of the mutants and confirmed by trans-complementation between each mutant and a replication-defective replicon. No change in transcriptional level was detected between all mutated constructs. The encapsidation competencies of these constructs were studied by assaying pregenomic RNAs in intracytoplamic core particles from transfected cells, which were normalized for the amount of HBV core protein by Western blotting using anti-core antibodies. Impaired encapsidation was found in several mutants substituted at residue 306, thereby demonstrating for the first time that conservation of proline at this residue is crucial for efficient encapsidation of pregenomic RNA.