Application of dendritic cells in tumor immunotherapy and progress in the mechanism of anti-tumor effect of Astragalus polysaccharide (APS) modulating dendritic cells: a review.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that are essential in mediating the body's natural and adaptive immune responses. The body can regulate the function of DCs in various ways to enhance their antitumor effects. In the tumour microenvironment (TME), antigen-specific T cell responses are initiated through DC processing and delivery of tumour-associated antigens (TAAs); conversely, tumour cells inhibit DC recruitment by releasing metabolites, cytokines and other regulatory TME and function. Different subpopulations of DCs exist in tumour tissues, and their functions vary. Insight into DC subgroups in TME allows assessment of the effectiveness of tumour immunotherapy. Astragalus polysaccharide (APS) is the main component of the Chinese herb Astragalus membranaceus. The study found that the antitumor effects of APS are closely related to DCs. APS can promote the expression of surface molecules CD80 and CD86, promote the maturation of DCs, and activate CTL to exert antitumor effects. We reviewed the application of DCs in tumor immunotherapy and the mechanism of modulation of DCs by Astragalus polysaccharide to provide new directions and strategies for tumor therapy and new drug development.

Prevalence of HBsAg among reproductive age couples in Chongqing: A population-based, cross-sectional study.

Hepatitis B is a leading cause of death worldwide. Here, we performed a large, population-based, cross-sectional study in Chongqing, China from 2011 to 2016 to assess the prevalence of HBsAg among couples of reproductive age, to predict subsequent trends, and to provide evidence for the WHO goal of "the elimination of viral hepatitis as a public health threat by 2030". A total of 386,286 couples aged 20 to 49 years were enrolled in the study. Approximately 14.35% of couples were HBsAg positive, including 95.00% with discordant HBsAg positivity. HBsAg prevalence was higher in men than in women. Among different occupations, the two categories of "houseworker" (female 6.73%, male 9.99%) and "unemployed" (female 6.64%, male 9.94%) showed the highest HBsAg positivity. In different regions, the lowest prevalence appeared in southeastern Chongqing (female 4.87%, male 7.71%). In 2030, the HBsAg positivity rate is expected to be 2.79%, 7.27% and 5.13% in females, males, and the whole population, respectively. According to the trends, this rate would drop to less than 2% in 2034, 2078 and 2051. In conclusion, the HBsAg prevalence in Chongqing is still relatively high compared with that in other parts of western China, especially among reproductive-age men. HBsAg-positive couples should be taken as an important unit of care. Vaccination is necessary before pregnancy if no antibody is found. More attention should be given to people without stable jobs. HBsAg-positive rate will decrease perceptibly by 2030 and will reach the level of low in epidemic areas by 2050.

Profiling T cell receptor ß-chain in responders after immunization with recombinant hepatitis B vaccine.

BACKGROUND: T cells with edited T cell receptor ß-chain variable (TRBV) are involved in the immune response to recombinant hepatitis B surface antigen (rHBsAg) vaccine and the production of hepatitis B surface antibody (HBsAb). The immune repertoire (IR) profile and mechanism of vaccination positive responders (VPR) with rHBsAg are not fully understood. METHODS: The IR of six VPRs (HBsAb+, HbsAg-) with rHBsAg vaccination was established by the high throughput sequencing technique and bioinformatics analysis and compared with those in five vaccination negative responders (VNRs) (HbsAb-, HbsAg-) who were also inoculated with rHBsAg. The repertoire features of the BV, BJ and V (CDR3) J genes and immune diversity in peripheral blood mononuclear cells, respectively, were analyzed for each subject. RESULTS: There was no significant difference in sequencing amplification indices of each sample. However, TRBV15/BJ2-3 demonstrated significantly high expression levels in VPR compared to those in the VNR group (both p < 0.05). Further results showed that the BV15/BJ2-5 level was significantly increased for VPR compared to that of VNR group. Interestingly, the motif of CDR3 in TRBV15/BJ2-5 was mostly expressed as "GGETQ" or "GETQ". Additionally, there was no remarkable difference between the two groups of distribution with respect to the different clone expression levels of V (CDR3) J. CONCLUSIONS: The features of IR in the VPR and VNR will contribute to the exploration of the mechanism of the positive response to rHBsAg, and also contribute to development of optimized hepatitis B vaccine, in addition to providing a partial interpretation of the VNR who has a relatively low infection with HBV.

HBsAg-specific CD8+ T cells as an indispensable trigger to induce murine hepatocellular carcinoma.

Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) is mediated by an inappropriate attack by HBV-specific T cells in patients. However, this immunopathogenic process has not been clarified because of the lack of a suitable animal model. Here, we used immunocompetent Fah-/- mice as the recipients in the adoptive transfer of HBsAg+ hepatocytes from HBs-Tg mice to replace the recipient hepatocytes (HBs-HepR). HBs-HepR mice exhibited persistent HBsAg expression with chronic hepatitis and eventually developed HCC with a prevalence of 100%. HBsAg-specific CD8+ T cells were generated and specifically and continuously induced hepatocyte apoptosis with progressive chronic inflammation, and the depletion of CD8+ T cells or their deficiency prevented HCC, which could then be reproduced by the transfer of HBsAg-specific CD8+ T cells. In summary, our results demonstrated that CD8+ T cells plays a critical role in HBsAg-driven inflammtion and HCC tumorigenesis.

Distinct disease features in chimpanzees infected with a precore HBV mutant associated with acute liver failure in humans.

Transmission to chimpanzees of a precore hepatitis B virus (HBV) mutant implicated in acute liver failure (ALF) in humans did not cause ALF nor the classic form of acute hepatitis B (AHB) seen upon infection with the wild-type HBV strain, but rather a severe AHB with distinct disease features. Here, we investigated the viral and host immunity factors responsible for the unusual severity of AHB associated with the precore HBV mutant in chimpanzees. Archived serial serum and liver specimens from two chimpanzees inoculated with a precore HBV mutant implicated in ALF and two chimpanzees inoculated with wild-type HBV were studied. We used phage-display library and next-generation sequencing (NGS) technologies to characterize the liver antibody response. The results obtained in severe AHB were compared with those in classic AHB and HBV-associated ALF in humans. Severe AHB was characterized by: (i) the highest alanine aminotransferase (ALT) peaks ever seen in HBV transmission studies with a significantly shorter incubation period, compared to classic AHB; (ii) earlier HBsAg clearance and anti-HBs seroconversion with transient or undetectable hepatitis B e antigen (HBeAg); (iii) limited inflammatory reaction relative to hepatocellular damage at the ALT peak with B-cell infiltration, albeit less extensive than in ALF; (iv) detection of intrahepatic germline antibodies against hepatitis B core antigen (HBcAg) by phage-display libraries in the earliest disease phase, as seen in ALF; (v) lack of intrahepatic IgM anti-HBcAg Fab, as seen in classic AHB, but at variance with ALF; and (vi) higher proportion of antibodies in germline configuration detected by NGS in the intrahepatic antibody repertoire compared to classic AHB, but lower than in ALF. This study identifies distinct outcome-specific features associated with severe AHB caused by a precore HBV mutant in chimpanzees, which bear closer resemblance to HBV ALF than to classic AHB. Our data suggest that precore HBV mutants carry an inherently higher pathogenicity that, in addition to specific host factors, may play a critical role in determining the severity of acute HBV disease.

A recombinant human immunoglobulin with coherent avidity to hepatitis B virus surface antigens of various viral genotypes and clinical mutants.

The hepatitis B virus (HBV) envelope is composed of a lipid bilayer and three glycoproteins, referred to as the large (L), middle (M), and small (S) hepatitis B virus surface antigens (HBsAg). S protein constitutes the major portion of the viral envelope and an even greater proportion of subviral particles (SVP) that circulate in the blood. Recombinant S proteins are currently used as a preventive vaccine, while plasma fractions isolated from vaccinated people, referred to as hepatitis B immune globulin (HBIG), are used for short-term prophylaxis. Here, we characterized a recombinant human IgG1 type anti-S antibody named Lenvervimab regarding its binding property to a variety of cloned S antigens. Immunochemical data showed an overall consistent avidity of the antibody to S antigens of most viral genotypes distributed worldwide. Further, antibody binding was not affected by the mutations in the antigenic 'a' determinant found in many clinical variants, including the immune escape mutant G145R. In addition, mutations in the S gene sequence that confer drug resistance to the viral polymerase did not interfere with the antibody binding. These results support for a preventive use of the antibody against HBV infection.

Host genetic background affects the course of infection and treatment response in patients with chronic hepatitis B.

BACKGROUND: Hepatitis B virus (HBV) utilizes proteins encoded by the host to infect hepatocytes and replicate. Recently, several novel host factors have been identified and described as important to the HBV lifecycle. The influence of host genetic background on chronic hepatitis B (CHB) pathogenesis is still poorly understood. OBJECTIVES: Here, we aimed to investigate the association of NTCP, FXRα, HNF1α, HNF4α, and TDP2 genetic polymorphisms with the natural course of CHB and antiviral treatment response. STUDY DESIGN: We genotyped 18 single-nucleotide polymorphisms using MALDI-TOF mass spectrometry in 136 patients with CHB and 100 healthy individuals. We investigated associations of the selected polymorphisms with biochemical, serological and hepatic markers of disease progression and treatment response. RESULTS: No significant differences in genotypic or allelic distribution between CHB and control groups were observed. Within TDP2, rs3087943 variations were associated with treatment response, and rs1047782 modified the risk of advanced liver inflammation. Rs7154439 within NTCP was associated with HBeAg seroconversion after 48 weeks of nucleos(t)ide analogue treatment. HNF1α genotypes were associated with treatment response, liver damage and baseline HBeAg presence. HNF4α rs1800961 predicted PEG-IFNα treatment-induced HBsAg clearance in long-term follow up. CONCLUSIONS: This study indicates host genetic background relevance in the course of CHB and confirms the role of recently described genes for HBV infection. The obtained results might serve as a starting point for validation studies on the clinical application of selected genetic variants to predict individual risks of CHB-induced liver failure and treatment response.

Prediction and clinical implications of HBV reactivation in lymphoma patients with resolved HBV infection: focus on anti-HBs and anti-HBc antibody titers.

Hepatitis B virus (HBV) reactivation (HBV-R) and hepatitis related to HBV-R are well-recognized complications that occur in patients who have undergone cytotoxic chemotherapy or immunosuppressive therapy. The degree of HBV-R in this population varies from self-limited or asymptomatic hepatitis to acute liver failure, which may lead to life-threatening events. However, no established treatment or standard surveillance method exists for monitoring patients to predict the development of HBV-R during or after chemotherapy or immunosuppressive therapy, particularly regarding resolved HBV infection. Prophylactic antiviral agents and regular monitoring of HBV-DNA levels are known to be useful methods for preventing HBV-R; however, these methods require considerable financial resources, and such resources are limited in the endemic areas of HBV infection. Most patients with resolved HBV infection do not develop a hepatitis flare or self-limited HBV-R with only an increase in HBV DNA. However, some patients may develop HBV-R even 1 year or more after the last chemotherapy treatment. Therefore, predicting the development of HBV-R and its timing is difficult, and exploring markers that could help predict whether or when HBV reactivation occurs is necessary. In this review, we address the predictive risk factors for HBV-R in patients with resolved HBV infection, focusing on the ability of anti-HBs and anti-HBc to predict HBV-R. We conclude that the combination of anti-HBc and anti-HBs titers may be a reliable and useful predictor for managing HBV-R.

High Frequency of Either Altered Pre-Core StartCodon or Weakened Kozak Sequence in the CorePromoter Region in Hepatitis B Virus A1 Strainsfrom Rwanda.

Hepatitis B virus (HBV) is endemic in Rwanda and is a major etiologic agent for chronic liver disease in the country. In a previous analysis of HBV strains from Rwanda, the S genes of most strains segregated into one single clade of subgenotype, A1. More than half (55%) of the anti-HBe positive individuals were viremic. In this study, 23 complete HBV genomes and the core promoter region (CP) from 18 additional strains were sequenced. Phylogenetic analysis of complete genomes confirmed that most Rwandan strain formed a single unique clade, within subgenotype A1. Strains from 17 of 22 (77%) anti-HBe positive HBV carriers had either mutated the precore start codon (9 strains with either CUG, ACG, UUG, or AAG) or mutations in the Kozak sequence preceding the pre-core start codon (8 strains). These mutually exclusive mutations were also identified in subgenotypes A1 (70/266; 26%), A2 (12/255; 5%), and A3 (26/49; 53%) sequences from the GenBank. The results showed that previous, rarely described HBV variants, expressing little or no HBeAg, are selected in anti-HBe positive subgenotype Al carriers from Rwanda and that mutations reducing HBeAg synthesis might be unique for a particular HBV clade, not just for a specific genotype or subgenotype.

New and Old Biomarkers for Diagnosis and Management of Chronic Hepatitis B Virus Infection.

Tests to detect the presence and activity of hepatitis B virus (HBV) are the cornerstones of diagnosis and management. Assays that detect or measure serum levels of HB surface antigen, HB surface antibody, and HB core antibody are used to identify patients with exposure to HBV, whereas other tests provide information on the level of virus replication, presence of specific variants, and presence of virus reservoirs. Newer diagnostic tests, used only in research settings so far, aim to quantify levels of intrahepatic HBV replication. Other tests have been developed to detect HBV infection in resource-limited settings. We review point-of-care tests (essential in global screening efforts), standard diagnostic tests used in routine clinical management, and newer tests that might be used in clinical trials of agents designed to cure HBV infection.

T follicular helper cells and antibody response to Hepatitis B virus vaccine in HIV-1 infected children receiving ART.

HBV vaccine has 95% efficacy in children to prevent HBV infection and related cancer. We conducted a prospective study in HIV-1 infected children receiving ART (n = 49) and controls (n = 63) to assess humoral and cellular responses to HBV vaccine provided with three doses under an accelerated schedule of 4 weeks apart. At 1 month post-vaccination all children, except 4 HIV-1 infected, displayed protective antibody (ab) titers to HBV vaccine; ab titers were lower in infected children (P < 0.0001). Ab titers decreased (P < 0.0001) in both HIV-1 infected and control children at 6 months. The frequency of circulating Tfh (cTFh) cells was 20.3% for controls and 20.8% for infected children prior to vaccination and remained comparable post-vaccination. Cytokine expression by cTfh cells upon activation with HBV antigen was comparable in the two groups at baseline and 1 month post-vaccination. Higher plasma levels (P < 0.0001) of CXCL13 were found in infected children which correlated with cTfh cell frequency at baseline. In conclusion, a lower ab response to HBV vaccine was measured in HIV-1 infected children. The frequency and activation profile of cTfh cells was comparable in infected children and controls suggesting that cells other than Tfh cells are responsible for impaired ab response to HBV vaccine.

Construction and Characterization of an Anti-Hepatitis B Virus preS1 Humanized Antibody that Binds to the Essential Receptor Binding Site.

Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular carcinoma. With recent identification of HBV receptor, inhibition of virus entry has become a promising concept in the development of new antiviral drugs. To date, 10 HBV genotypes (A-J) have been defined. We previously generated two murine anti-preS1 monoclonal antibodies (mAbs), KR359 and KR127, that recognize amino acids (aa) 19-26 and 37-45, respectively, in the receptor binding site (aa 13-58, genotype C). Each mAb exhibited virus neutralizing activity in vitro, and a humanized version of KR127 effectively neutralized HBV infection in chimpanzees. In the present study, we constructed a humanized version (HzKR359-1) of KR359 whose antigen binding activity is 4.4-fold higher than that of KR359, as assessed by competitive ELISA, and produced recombinant preS1 antigens (aa 1-60) of different genotypes to investigate the binding capacities of HzKR359-1 and a humanized version (HzKR127-3.2) of KR127 to the 10 HBV genotypes. The results indicate that HzKR359-1 can bind to five genotypes (A, B, C, H, and J), and HzKR127-3.2 can also bind to five genotypes (A, C, D, G, and I). The combination of these two antibodies can bind to eight genotypes (A-D, G-J), and to genotype C additively. Considering that genotypes A-D are common, whereas genotypes E and F are occasionally represented in small patient population, the combination of these two antibodies might block the entry of most virus genotypes and thus broadly neutralize HBV infection.

Characterization of gene expression profiles in HBV-related liver fibrosis patients and identification of ITGBL1 as a key regulator of fibrogenesis.

Although hepatitis B virus (HBV) infection is the leading cause of liver fibrosis (LF), the mechanisms underlying liver fibrotic progression remain unclear. Here, we investigated the gene expression profiles of HBV-related LF patients. Whole genome expression arrays were used to detect gene expression in liver biopsy samples from chronically HBV infected patients. Through integrative data analysis, we identified several pathways and key genes involved in the initiation and exacerbation of liver fibrosis. Weight gene co-expression analysis revealed that integrin subunit ß-like 1 (ITGBL1) was a key regulator of fibrogenesis. Functional experiments demonstrated that ITGBL1 was an upstream regulator of LF via interactions with transforming growth factor ß1. In summary, we investigated the gene expression profiles of HBV-related LF patients and identified a key regulator ITGBL1. Our findings provide a foundation for future studies of gene functions and promote the development of novel antifibrotic therapies.

Effect of Dosage and Type of Hepatitis B Immunoglobulin on Hepatitis Antibody Levels in Liver Transplant Recipients.

BACKGROUND: The current study aimed to evaluate the effect of dosage and type (intramuscular [IM] vs intravenous [IV]) of hepatitis B immunoglobulin (HBIG) on hepatitis antibody level in liver transplant recipients. METHODS: Between September 2000 and August 2016, patients who underwent orthotropic liver transplantation for chronic liver failure or hepatocellular carcinoma secondary to chronic hepatitis B virus (HBV) were retrospectively reviewed from a prospectively maintained database. The analyses of risk factors for postoperative short- and long-term anti-hepatitis B surface antibody levels (as classified level I: 0 to 100 U; II: 100 to 500 U; III: 500 to 1000 U; IV: >1000 U) were performed based on demographic characteristics, hepatitis B envelope antigen, hepatitis B core antibody, HBV DNA, delta antigen, HBIG administration dosage during unhepatic phase (5000 or 10,000 I/U; IM or IV), and type of administration in post-transplant period. Patients who were followed for less than 12 months were excluded from long-term analysis. RESULTS: The mean follow-up of 58 orthotropic liver transplant patients was 72 (±45) months. No adverse events were observed during both IM and IV type of administration. Compared with IM type, IV administration was associated with a significantly higher HBV antibody level in the short term (for IM and IV: level I: 24% vs 6%; II: 49% vs 18%; III: 12% vs 35%; IV: 15% vs 41%, respectively, P = .007). In the long term, IV administration of hepatitis B immunoglobulin (HBIG) was reported as the sole factor causing higher antibody level (P = .002). Longer follow-up was associated with decreased levels of anti-hepatitis B surface antibody. CONCLUSION: IV HBIG administration in preoperative anhepatic phase and postoperative prophylaxis is associated with higher antibody level both the short and long term without any adverse event.

Wide dynamic range of surface-plasmon-resonance-based assay for hepatitis B surface antigen antibody optimal detection in comparison with ELISA.

Limit of detection (LOD), limit of quantification, and the dynamic range of detection of hepatitis B surface antigen antibody (anti-HBs) using a surface plasmon resonance (SPR) chip-based approach with Pichia pastoris-derived recombinant hepatitis B surface antigen (HBsAg) as recognition element were established through the scouting for optimal conditions for the improvement of immobilization efficiency and in the use of optimal regeneration buffer. Recombinant HBsAg was immobilized onto the sensor surface of a CM5 chip at a concentration of 150 mg/L in sodium acetate buffer at pH 4 with added 0.6% Triton X-100. A regeneration solution of 20 mM HCl was optimally found to effectively unbind analytes from the ligand, thus allowing for multiple screening cycles. A dynamic range of detection of ∼0.00098-0.25 mg/L was obtained, and a sevenfold higher LOD, as well as a twofold increase in coefficient of variance of the replicated results, was shown as compared with enzyme-linked immunosorbent assay (ELISA). Evaluation of the assay for specificity showed no cross-reactivity with other antibodies tested. The ability of SPR chip-based assay and ELISA to detect anti-HBs in human serum was comparable, indicating that the SPR chip-based assay with its multiple screening capacity has greater advantage over ELISA.

Antibody-dependent and antibody-independent uptake of HBsAg across human leucocyte subsets is similar between individuals with chronic hepatitis B virus infection and healthy donors.

Maintaining detectable levels of antibodies to hepatitis B surface antigen (HBsAg) in serum after HBsAg sero-conversion is the key clinical endpoint indicative of recovery from infection with hepatitis B virus (HBV). As HBV-infected hepatocytes secrete HBsAg subviral particles in vast excess over HBV virions, detectable hepatitis B surface antibody (anti-HBs) titres imply complete elimination of HBV virions as well as HBsAg particles. Although intrahepatic phagocytes, for example Kupffer cells, are thought to mediate clearance of HBsAg via antibody (Ab)-dependent and Ab-independent mechanisms, the relative contributions of circulating phagocytic cell types to HBsAg elimination are poorly characterized. Understanding the role of various immune cell subsets in the clearance of HBsAg is important because Ab-dependent or Ab-independent phagocytic HBsAg uptake may modulate presentation of HBsAg-derived epitopes to antigen-specific T cells and hence plays a critical role in adaptive immunity against HBV. This study aims to characterize phagocytic leucocyte subsets capable of internalizing HBsAg immune complexes (HBsAg:IC) or un-complexed HBsAg particles in whole blood directly ex vivo. The data show that uptake of HBsAg:IC occurs most prominently in monocytes, B cells, dendritic cells and in neutrophils. In contrast, B cells, and to a lesser degree also monocytes, seem to be effective phagocytes for un-complexed HBsAg. Importantly, a similar pattern of phagocytic HBsAg uptake was observed in blood from chronic hepatitis B (CHB) patients compared to healthy controls, suggesting that phagocytosis-related cellular functions are not altered in the context of CHB.

Label Free Quantitative Immunoassay for Hepatitis B.

Complementary metal oxide semiconductor (CMOS) technology has already been proven in molecular diagnostics. The present research proved that CMOS image sensor based immunodetection is a suitable diagnostic system for hepatitis B virus antigen (HBV-Ag)-antibody (Ab) interaction. The Ag-Ab was interacted on InNP substrate which was analyzed by a CMOS image sensor by photon number variation. The photon passes through the protein adsorbed substrate and hits the sensor surface. The number of photons attained by the sensor depends on the Ag concentration, nanoparticles size and substrates thickness; therein substrate with higher concentrations of Ag were blocked more photons. The number of photons was detected by the sensor and converted into a digital number with the aid of an analog-to-digital-converter (ADC). A topographical study of AFM and fluorescence images have evaluated the morphological changes, height increment, surface roughness of protein treated and non-treated InNP substrates, to prove the efficiency of CMOS image sensor based immunodetection. Also, the study was compared with conventional ELISA method, to find the sensitivity of CMOS image sensor. Hence, the detection of HBV Ag-Ab interactions by CMOS image sensors is suitable for point-of-care diagnosis.

Characterization of C69R variant HBsAg: effect on binding to anti-HBs and the structure of virus-like particles.

Several variants of the major "a" determinant of the HBsAg, the main target of HBV neutralization by antibodies, have been described. However, mutations outside this region have not been as thoroughly investigated. During the genotyping of HBV from Tunisian patients with chronic hepatitis B, we identified a variant with a C69R substitution in the cytosolic loop of the S protein, resulting in a change in the hydrophobicity profile compared to the wild-type HBsAg. Wild-type and mutant HBsAgs were produced in Saccharomyces cerevisiae and recombinant proteins were tested for their ability to correctly self-assemble into virus-like particles (VLPs), and their ability to bind to HBs antibodies. The C69R substitution resulted in a decrease in binding to commercial anti-HBs antibodies, and although the variant appeared to assemble properly into VLPs, the average size of the particles was larger than that of the wild-type HBsAg. Prediction of the tertiary structure of the C69R mutant revealed a change in the first (aa 60-70) and the second loop (aa 110 to 120) compared to the wild-type protein. Furthermore, we showed by an isothermal titration calorimetry assay that the interaction between the wild-type HBsAg and the anti-HBs antibody was exothermic, whereas that with the mutant C69R was endothermic, indicating an effect on the binding affinity.

The risk of hepatitis B virus reactivation and the role of antiviral prophylaxis in hepatitis B surface antigen negative/hepatitis B core antibody positive patients with diffuse large B-cell lymphoma receiving rituximab-based chemotherapy.

The risk factors and the role of prophylactic antiviral therapy of hepatitis B virus (HBV) reactivation in patients with hepatitis B surface antigen (HBsAg) negative/hepatitis B core antibody (HBcAb) positive disease remain controversial. We reviewed 629 patients with diffuse large B-cell lymphoma (DLBCL). Among 629 patients, 150 of 246 patients with resolved HBV (HBsAg negative and HBcAb positive) were treated with rituximab-combined therapy. Among these 150 patients, none of 104 patients (0.0%) who were hepatitis B surface antibody (HBsAb) positive experienced HBV reactivation versus four of 46 patients (8.7%) who were HBsAb negative (p = 0.008). One of 113 patients (0.9%) with International Prognostic Index (IPI) 0-2 suffered HBV reactivation versus three of the remaining 37 patients (8.1%) with IPI 3-5 (p = 0.047). HBsAb and IPI are potential risk factors for HBV reactivation. The use of prophylactic agents may not be recommended for these patients until the occurrence of HBV reactivation.

Phosphoacceptors threonine 162 and serines 170 and 178 within the carboxyl-terminal RRRS/T motif of the hepatitis B virus core protein make multiple contributions to hepatitis B virus replication.

UNLABELLED: Phosphorylation of serines 157, 164, and 172 within the carboxyl-terminal SPRRR motif of the hepatitis B virus (HBV) core (C) protein modulates HBV replication at multiple stages. Threonine 162 and serines 170 and 178, located within the carboxyl-terminal conserved RRRS/T motif of HBV C protein, have been proposed to be protein kinase A phosphorylation sites. However, in vivo phosphorylation of these residues has never been observed, and their contribution to HBV replication remains unknown. In this study, [(32)P]orthophosphate labeling of cells expressing C proteins followed by immunoprecipitation with anti-HBc antibody revealed that threonine 162 and serines 170 and 178 are phosphoacceptor residues. A triple-alanine-substituted mutant, mimicking dephosphorylation of all three residues, drastically decreased pregenomic RNA (pgRNA) encapsidation, thereby decreasing HBV DNA synthesis. In contrast, a triple-glutamate-substituted mutant, mimicking phosphorylation of these residues, decreased DNA synthesis without significantly decreasing encapsidation. Neither triple mutant affected C protein expression or core particle assembly. Individual alanine substitution of threonine 162 significantly decreased minus-strand, plus-strand, and relaxed-circular DNA synthesis, demonstrating that this residue plays multiple roles in HBV DNA synthesis. Double-alanine substitution of serines 170 and 178 reduced HBV replication at multiple stages, indicating that these residues also contribute to HBV replication. Thus, in addition to serines 157, 164, and 172, threonine 162 and serines 170 and 178 of HBV C protein are also phosphorylated in cells, and phosphorylation and dephosphorylation of these residues play multiple roles in modulation of HBV replication. IMPORTANCE: Threonine 162, within the carboxyl-terminal end of the hepatitis B virus (HBV adw) core (C) protein, has long been ignored as a phosphoacceptor, even though it is highly conserved among mammalian hepadnaviruses and in the overlapping consensus RxxS/T, RRxS/T, and TP motifs. Here we show, for the first time, that in addition to the well-known phosphoacceptor serines 157, 164, and 172 in SPRRR motifs, threonine 162 and serines 170 and 178 in the RRRS/T motif are phosphorylated in cells. We also show that, like serines 157, 164, and 172, phosphorylated and dephosphorylated threonine 162 and serines 170 and 178 contribute to multiple steps of HBV replication, including pgRNA encapsidation, minus-strand and plus-strand DNA synthesis, and relaxed-circular DNA synthesis. Of these residues, threonine 162 is the most important. Furthermore, we show that phosphorylation of C protein is required for efficient completion of HBV replication.