Novel Hepatitis B Virus Capsid Assembly Modulator Induces Potent Antiviral Responses In Vitro and in Humanized Mice.

Hepatitis B virus (HBV) affects an estimated 250 million chronic carriers worldwide. Though several vaccines exist, they are ineffective for those already infected. HBV persists due to the formation of covalently closed circular DNA (cccDNA)-the viral minichromosome-in the nucleus of hepatocytes. Current nucleoside analogs and interferon therapies rarely clear cccDNA, requiring lifelong treatment. Our group identified GLP-26, a novel glyoxamide derivative that alters HBV nucleocapsid assembly and prevents viral DNA replication. GLP-26 exhibited single-digit nanomolar anti-HBV activity, inhibition of HBV e antigen (HBeAg) secretion, and reduced cccDNA amplification, in addition to showing a promising preclinical profile. Strikingly, long term combination treatment with entecavir in a humanized mouse model induced a decrease in viral loads and viral antigens that was sustained for up to 12 weeks after treatment cessation.

Use of Expression Profiles of HBV-DNA Integrated Into Genomes of Hepatocellular Carcinoma Cells to Select T Cells for Immunotherapy.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is often associated with hepatitis B virus (HBV) infection. Cells of most HBV-related HCCs contain HBV-DNA fragments that do not encode entire HBV antigens. We investigated whether these integrated HBV-DNA fragments encode epitopes that are recognized by T cells and whether their presence in HCCs can be used to select HBV-specific T-cell receptors (TCRs) for immunotherapy. METHODS: HCC cells negative for HBV antigens, based on immunohistochemistry, were analyzed for the presence of HBV messenger RNAs (mRNAs) by real-time polymerase chain reaction, sequencing, and Nanostring approaches. We tested the ability of HBV mRNA-positive HCC cells to generate epitopes that are recognized by T cells using HBV-specific T cells and TCR-like antibodies. We then analyzed HBV gene expression profiles of primary HCCs and metastases from 2 patients with HCC recurrence after liver transplantation. Using the HBV-transcript profiles, we selected, from a library of TCRs previously characterized from patients with self-limited HBV infection, the TCR specific for the HBV epitope encoded by the detected HBV mRNA. Autologous T cells were engineered to express the selected TCRs, through electroporation of mRNA into cells, and these TCR T cells were adoptively transferred to the patients in increasing numbers (1 × 104-10 × 106 TCR+ T cells/kg) weekly for 112 days or 1 year. We monitored patients' liver function, serum levels of cytokines, and standard blood parameters. Antitumor efficacy was assessed based on serum levels of alpha fetoprotein and computed tomography of metastases. RESULTS: HCC cells that did not express whole HBV antigens contained short HBV mRNAs, which encode epitopes that are recognized by and activate HBV-specific T cells. Autologous T cells engineered to express TCRs specific for epitopes expressed from HBV-DNA in patients' metastases were given to 2 patients without notable adverse events. The cells did not affect liver function over a 1-year period. In 1 patient, 5 of 6 pulmonary metastases decreased in volume during the 1-year period of T-cell administration. CONCLUSIONS: HCC cells contain short segments of integrated HBV-DNA that encodes epitopes that are recognized by and activate T cells. HBV transcriptomes of these cells could be used to engineer T cells for personalized immunotherapy. This approach might be used to treat a wider population of patients with HBV-associated HCC.

Hepatitis B variants among Egyptian patients undergoing hemodialysis.

Hepatitis B virus (HBV) infection is a serious nosocomial infection that affects patients undergoing hemodialysis (HD). However, certain HBV variants are not detected by routine serological tests in Egyptian dialysis units because of mutations that change important viral antigens (Ags). Of note, these mutations can result in the appearance of different HBV variants with different clinical manifestations. Thus, the present study aimed to assess different clinical forms of HBV infections and viral genotypes among patients undergoing HD in the Ismailia governorate of Egypt. To this end, serum samples were collected from 150 patients undergoing HD and screened for HBV-DNA using a nested PCR technique. Positive samples were then screened for HBV serological markers (hepatitis B core antibody [HBcAb], hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B e antigen and hepatitis B e antibody) using ELISA and the HBV viral load quantitated by qPCR. HBV genotypes were detected by direct sequencing of the partial surface (S) gene. The most common clinical form of HBV infection in our study cohort was overt HBV infection (10%); followed by seropositive occult hepatitis B infection (7.3%), most of whom had an isolated HBcAb. The least common form was the precore mutant (1.3%). All HBV isolates were genotype D. This study reveals the importance of HBcAb and PCR in screening for HBV, especially for detection of occult hepatitis B infection.

Interleukin-21 receptor gene polymorphism is associated with hepatitis B virus-related hepatocellular carcinoma in Chinese patients.

BACKGROUND: We investigated the relationship between hepatitis B virus (HBV)-related pathogenesis and single nucleotide polymorphisms (SNPs) in interleukin-21 (IL-21)-JAK-STAT signaling pathway genes. METHODS: We used the high-resolution melting (HRM) method to genotype five SNPs (IL-21 rs2221903, IL-21 rs4833837, IL-21 receptor (IL-21R) rs2285452, JAK3 rs3008, and STAT3 rs1053023) in 546 HBV-infected patients and 353 healthy Chinese subjects. The HBV-infected patients were further divided into subgroups based on the HBV-related pathologies: chronic hepatitis B (CHB), HBV-related liver cirrhosis (LC), and HBV-related hepatocellular carcinoma (HCC). RESULTS: There were no significant differences in the genotype and allele distributions of the five SNPs between the HBV-infected patients and healthy subjects. The genotype and allele frequencies were similar in the two groups for IL-21 rs2221903 (A>G, P = 0.83 and 0.67), rs4833837 (A>G, P = 0.80 and 0.49), IL-21R rs2285452 (G>A, P = 0.25 and 0.68), STAT3 rs1053023 (A>G, P = 1.00 and 0.96), and JAK3 rs3008 (C>T, P = 0.32 and 0.54). However, patients with the IL-21R rs2285452 AA genotype were more susceptible to HBV-related HCC than those with the IL-21R rs2285452 GA/GG genotype (P = 0.03, OR = 3.27, 95% CI = 1.16-9.20). The serological marker model of "HBsAg+, HBeAg+, HBcAb+" was predominant among patients with HBV infection. However, there was no association between the genotype's distribution of the five SNPs and the serological marker models (P > 0.05). CONCLUSIONS: These findings demonstrate that the IL-21R rs2285452 AA genotype increases the risk of HBV-related HCC in Chinese patients.

A novel hepatitis B virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses.

BACKGROUND & AIMS: All known hepatitis B virus (HBV) genotypes occur in humans and hominoid Old World non-human primates (NHPs). The divergent woolly monkey HBV (WMHBV) forms another orthohepadnavirus species. The evolutionary origins of HBV are unclear. METHODS: We analysed sera from 124 Brazilian monkeys collected during 2012-2016 for hepadnaviruses using molecular and serological tools, and conducted evolutionary analyses. RESULTS: We identified a novel orthohepadnavirus species in capuchin monkeys (capuchin monkey hepatitis B virus [CMHBV]). We found CMHBV-specific antibodies in five animals and high CMHBV concentrations in one animal. Non-inflammatory, probably chronic infection was consistent with an intact preCore domain, low genetic variability, core deletions in deep sequencing, and no elevated liver enzymes. Cross-reactivity of antisera against surface antigens suggested antigenic relatedness of HBV, CMHBV, and WMHBV. Infection-determining CMHBV surface peptides bound to the human HBV receptor (human sodium taurocholate co-transporting polypeptide), but preferentially interacted with the capuchin monkey receptor homologue. CMHBV and WMHBV pseudotypes infected human hepatoma cells via the human sodium taurocholate co-transporting polypeptide, and were poorly neutralised by HBV vaccine-derived antibodies, suggesting that cross-species infections may be possible. Ancestral state reconstructions and sequence distance comparisons associated HBV with humans, whereas primate hepadnaviruses as a whole were projected to NHP ancestors. Co-phylogenetic analyses yielded evidence for co-speciation of hepadnaviruses and New World NHP. Bayesian hypothesis testing yielded strong support for an association of the HBV stem lineage with hominoid ancestors. Neither CMHBV nor WMHBV was likely the ancestor of the divergent human HBV genotypes F/H found in American natives. CONCLUSIONS: Our data suggest ancestral co-speciation of hepadnaviruses and NHP, and an Old World origin of the divergent HBV genotypes F/H. The identification of a novel primate hepadnavirus offers new perspectives for urgently needed animal models of chronic hepatitis B. LAY SUMMARY: The origins of HBV are unclear. The new orthohepadnavirus species from Brazilian capuchin monkeys resembled HBV in elicited infection patterns and could infect human liver cells using the same receptor as HBV. Evolutionary analyses suggested that primate HBV-related viruses might have emerged in African ancestors of New World monkeys millions of years ago. HBV was associated with hominoid primates, including humans and apes, suggesting evolutionary origins of HBV before the formation of modern humans. HBV genotypes found in American natives were divergent from those found in American monkeys, and likely introduced along prehistoric human migration. Our results elucidate the evolutionary origins and dispersal of primate HBV, identify a new orthohepadnavirus reservoir, and enable new perspectives for animal models of hepatitis B.

[Hepatitis B Virus Infection: Current Management and Prevention].

Hepatitis B virus (HBV) is transmitted mainly via percutaneous or permucosal exposure to HBV- containing body fluids. Because HB vaccination is effective and safe, it is recommended for all children, ado- lescents, and all unvaccinated adults at risk of HBV infection (individuals with occupational risk, immunosup- pressed individuals, and sexually active individuals). In Japan, universal HB vaccination will be introduced for all infants in October 2016. In Japan, the prevalence of HBV genotype A, which is frequently found in North America, northwestern Europe, India, and Africa, has been increasing as a sexually transmitted infection. HBV reactivation under anticancer chemotherapy and immunosuppressive therapy is well-known as a seri- ous complication in HBV-resolved patients. Monthly monitoring of HBV-DNA is recommended for prevent- ing HBV reactivation-related hepatitis among HBV-resolved patients with non-Hodgkin B-cell lymphoma under steroid plus rituximab chemotherapy. Recently, a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for HBsAg detection by Lumipulse HBsAg-HQ was reported as the latest clinical application. Although the sensitivity of this assay (≥5 mIU/mL) is 10-fold higher than the conventional assay, it is still lower than that of the HBV-DNA assay. The useful HBsAg-HQ will be applied for detecting occult HBV infection and HBV reactivation. The aim of treatment for chronic HBV infection is to reduce the risk of complications, including cirrhosis and hepatocellular carcinoma (HCC). Pegylated interferon alfa and nucleoside/nucleotide analogues (NAs) are the current treatments for chronic HBV infection. NAs have improved the outcomes of patients with cirrhosis and HCC, and decreased the incidence of acute liver failure. [Review].

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection.

Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T- and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.

Antihepatitis B virus activity of a protein-enriched fraction from housefly (Musca domestica) in a stable HBV-producing cell line.

Hepatitis B virus (HBV) infection remains a major public health problem. Although several vaccines and therapeutic strategies are currently being implemented to combat HBV virus, effective antiviral therapy against HBV infection has not been fully developed. Alternative strategies and new drugs to combat this disease are urged. Insects and insect derivatives are a large and unexploited source of potentially useful compounds for modern medicine. In the present study, we investigated the first anti-HBV activity of a protein-enriched fraction (PE) from the larvae of the housefly (Musca domestica) in a stable HBV-producing cell line. HBsAg and HBeAg in the culture medium were measured by enzyme-linked immunosorbent assay. HBV-DNA was quantified by fluorescent quantification PCR. HBV core protein was assayed by immunofluorescent staining. Results indicate PE treatment inhibited both HBsAg, HBeAg secretion, and HBV-DNA replication. Furthermore, PE could also suppress HBV core protein expression. PE could be a potential candidate for the development of a novel and effective drug for the treatment of HBV infection.

Recombinant Semliki Forest virus vectors encoding hepatitis B virus small surface and pre-S1 antigens induce broadly reactive neutralizing antibodies.

Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV-genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1-48 of the pre-S1 domain, fused to S (pre-S1.1-48/S). The antigen loop in S protein and the selected pre-S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 10(7) rSFV particles and 10(8) rSFV particles 3 weeks later. Antibodies induced by rSFV encoding S proteins reacted preferentially with subtype determinants of yeast-derived S antigen but equally well with patient-derived S antigen. Immunization with rSFV encoding pre-S1.1-48/S resulted in formation of pre-S1- and S-specific immunoglobulin G (IgG), while immunization with the isogenic mutant without S start codon induced pre-S1 antibodies only. Neutralizing antibodies were determined by mixing with plasma-derived HBV/ayw2 and subsequent inoculation of susceptible primary hepatocyte cultures from Tupaia belangeri. S/adw2 antisera neutralized HBV/ayw2 as effectively as antisera raised with S/ayw2. The pre-S1 antibodies also completely neutralized HBV infectivity. The IgG1/IgG2a ratios ranged from 0.28 to 0.88 in the four immunized groups and were lowest for the pre-S1.1-48/S vector, indicating the strongest Th1 response. This vector type may induce subtype-independent and S-escape-resistant neutralizing antibodies against HBV.

Analysis of carriers of hepatitis B virus from a tertiary referral hospital: does the viral load change during the natural course of infection?

This study was designed to determine the prevalence, forms of transmission, mutational profile and viral load at baseline of hepatitis B virus (HBV) carriers in Delhi. HBV surface antigen (HBsAg)-positive patients were enrolled and evaluated clinically for liver function, serological markers for hepatitis B and HBV DNA quantitation. Tests were carried out again 1 year later and the results were compared. Liver biopsy was carried out on all carriers with active viral replication. HBV DNA-positive samples were subjected to polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP) to screen mutations in the Precore, core, and the X-gene prior to sequencing analysis. Among the 100 patients examined, HBeAg was detected in 23% and 40% were HBV DNA-positive. Of the 40 HBV DNA-positive cases, 8 had precore/core mutations, [G1896A (10%), T2066A (12.5%), T2050C (10%), and G1888A (7.5%)]. No X gene mutants were detected. Reduction in viral load was higher in HBeAg-positive patients, as compared to HBeAg-negative patients, over 1 year. At follow-up, 2/8 HBV mutants corresponded with altered liver function and morphological changes suggestive of chronic hepatitis. One patient was re-designated as DNA-negative on follow-up and had wild-type virus infection with a relatively low viral load. The predominant route for HBV transmission was determined to be parenteral. Twenty percent of the HBV carriers were infected with precore and core mutant HBV. Although the clinical and biochemical profiles of these HBV carriers remained largely stable on follow-up, there was evidence of spontaneous reduction in the mean viral load over the 1-year study period.

Restoration of type I interferon signaling in intrahepatically primed CD8+ T cells promotes functional differentiation.

Hepatitis B virus-specific (HBV-specific) CD8+ T cells fail to acquire effector functions after priming in the liver, but the molecular basis for the dysfunction is poorly understood. By comparing the gene expression profile of intrahepatically primed, dysfunctional HBV-specific CD8+ T cells with that of systemically primed, functional effector counterparts, we found that the expression of interferon-stimulated genes (ISGs) is selectively suppressed in the dysfunctional CD8+ T cells. The ISG suppression was associated with impaired phosphorylation of STAT1 in response to IFN-α treatment. Importantly, a strong induction of type I interferons (IFN-Is) in the liver facilitated the functional differentiation of intrahepatically primed HBV-specific CD8+ T cells in association with the restoration of ISGs' expression in the T cells. These results suggest that intrahepatic priming suppresses IFN-I signaling in CD8+ T cells, which may contribute to the dysfunction. The data also suggest a therapeutic value of the robust induction of intrahepatic IFN-Is for the treatment of chronic HBV infection.

Affibody-displaying bionanocapsules for specific drug delivery to HER2-expressing cancer cells.

A novel HER2-targeted carrier was developed using bionanocapsules (BNCs). Bionanocapsules (BNCs) are 100-nm hollow nanoparticles composed of the L-protein of hepatitis B virus surface antigen. An affibody of HER2 was genetically displayed on the BNC surface (Z(HER2)-BNC). For the investigation of binding affinity, Z(HER2)-BNC was incubated with the cancer cell lines SK-BR-3 (HER2 positive), and MDA-MB-231 (HER2 negative). For analysis of HER2 targeting specificity, Z(HER2)-BNC or Z(WT)-BNC (without affibody) was incubated with both SK-BR-3 and MDA-MB-231 cells by time lapse and concentration. For the delivery of encapsulated molecules (calcein), fluorescence of Z(HER2)-BNC mixed with liposomes was also compared with that of Z(WT)-BNC and nude liposomes by incubation with SK-BR-3 cells. As a result, Z(HER2)-BNC-liposome complex demonstrated the delivery to HER2-expressing cells (SK-BR-3) with a high degree of specificity. This indicates that genetically engineered BNCs are promising carrier for cancer treatment.

Recognition of HBV antigens and HBV DNA by dendritic cells.

BACKGROUND: Hepatitis B virus (HBV) is a hepatotropic, noncytopathic, DNA virus which can cause acute and chronic infection. Viral persistence is associated with a weak or absent specific immune responses to HBV, particularly the cellular immune response. Dendritic cells (DCs) are professional antigen-presenting cells with a unique T cell stimulatory aptitude that play a crucial role in the instruction of adaptive immune responses upon infection. An impaired function of DCs was suggested by recent studies to account for the T and B cell hyporesponsiveness in chronic HBV infection. This review summarizes recent insights into the recognition of HBV antigens by DCs. DATA SOURCES: Studies were identified by searching MEDLINE and/or PubMed for articles using the key words "hepatitis B virus (HBV)", "dendritic cells", "C-type lectins", "mannose receptor", "toll-like receptor", and "dendritic cell-specific intercellular-adhesion-molecule-3 grabbing nonintegrin (DC-SIGN)" up to December 2009. Additional papers were identified by a manual search of the references from the key articles. RESULTS: DCs play an important role in the progress of hepatitis B, especially in the recognition of HBV. There are three main ways of recognition of HBV antigens by DCs. First, HBV DNA can be recognized by DCs through toll-like receptor 9 (TLR9) which activates the NF-κB signal pathway and p38 MAPK to up-regulate the expression of interferon (IFN) regulatory factor 7 (IRF-7) in a manner independent of type I IFN signaling, resulting in secretion of type I IFN and inflammatory cytokines, and induction of DC maturation and the adaptive immune response. Second, HBc/HBeAg cannot be recognized by DCs, but DNA or ssRNA encapsulated within HBcAg can be internalized by DCs through TLRs. Third, HBsAg can be internalized by DCs through the mannose receptor, which lacks the ability to induce DC maturation without the assistance of DC-SIGN. Meanwhile, there is some cross-talk among the three mechanisms, which induces an effective anti-viral response or HBV persistence. CONCLUSIONS: On the basis of these recognition processes, methods have been used to enhance the efficacy of DC-based vaccine against HBV and have been useful in the clinical application of HBV vaccine therapy. But the interactions between HBV antigens/HBV DNA and DCs are not clear, and cross-talk between TLRs and various ligands makes HBV antigen recognition by DCs more complicated. More efforts should be made to define the mechanisms and develop effective vaccines and therapies.

Intratumoral heterogeneity and clonal evolution in liver cancer.

Clonal evolution of a tumor ecosystem depends on different selection pressures that are principally immune and treatment mediated. We integrate RNA-seq, DNA sequencing, TCR-seq and SNP array data across multiple regions of liver cancer specimens to map spatio-temporal interactions between cancer and immune cells. We investigate how these interactions reflect intra-tumor heterogeneity (ITH) by correlating regional neo-epitope and viral antigen burden with the regional adaptive immune response. Regional expression of passenger mutations dominantly recruits adaptive responses as opposed to hepatitis B virus and cancer-testis antigens. We detect different clonal expansion of the adaptive immune system in distant regions of the same tumor. An ITH-based gene signature improves single-biopsy patient survival predictions and an expression survey of 38,553 single cells across 7 regions of 2 patients further reveals heterogeneity in liver cancer. These data quantify transcriptomic ITH and how the different components of the HCC ecosystem interact during cancer evolution.

Novel evidence indicates the presence and replication of hepatitis B virus in breast cancer tissue.

As a member of the liver tropic virus family, hepatitis B virus (HBV) was thought to only infect and replicate within the liver. Sodium taurocholate co­transporting polypeptide (NTCP) has been identified as a functional cellular receptor and a major determinant of liver tropism and HBV entry level species specificity. In the present study, the Oncomine database was used to explore differences in NTCP expression among cancerous and normal tissues. The results revealed that NTCP was highly expressed in breast cancer (BC), which was subsequently verified in clinical samples. Furthermore, in the BC tissue of patients with chronic HBV, HBV antigens, viral DNA/RNA and specific viral particles were detected via immunohistochemistry, ELISA, western blotting, reverse transcription­quantitative PCR and electron microscopy. Different HBV biomarkers and Dane particles were detected in BC. Furthermore, high levels of HBV­specific RNAs, the characteristic signals of HBV replication, were also detected, indicating that HBV infects BC tissue by binding to NTCP and replicating within. Based on the data of the present study, BC tissue may represent a second location of HBV infection and replication in addition to the liver.

A pre-S gene chip to detect pre-S deletions in hepatitis B virus large surface antigen as a predictive marker for hepatoma risk in chronic hepatitis B virus carriers.

BACKGROUND: Chronic hepatitis B virus (HBV) infection is an important cause of hepatocellular carcinoma (HCC) worldwide. The pre-S1 and -S2 mutant large HBV surface antigen (LHBS), in which the pre-S1 and -S2 regions of the LHBS gene are partially deleted, are highly associated with HBV-related HCC. METHODS: The pre-S region of the LHBS gene in two hundred and one HBV-positive serum samples was PCR-amplified and sequenced. A pre-S oligonucleotide gene chip was developed to efficiently detect pre-S deletions in chronic HBV carriers. Twenty serum samples from chronic HBV carriers were analyzed using the chip. RESULTS: The pre-S deletion rates were relatively low (7%) in the sera of patients with acute HBV infection. They gradually increased in periods of persistent HBV infection: pre-S mutation rates were 37% in chronic HBV carriers, and as high as 60% in HCC patients. The Pre-S Gene Chip offers a highly sensitive and specific method for pre-S deletion detection and is less expensive and more efficient (turnaround time 3 days) than DNA sequencing analysis. CONCLUSION: The pre-S1/2 mutants may emerge during the long-term persistence of the HBV genome in carriers and facilitate HCC development. Combined detection of pre-S mutations, other markers of HBV replication, and viral titers, offers a reliable predictive method for HCC risks in chronic HBV carriers.

Generation and characterization of high affinity humanized fab against hepatitis B surface antigen.

5S is a mouse monoclonal IgG1 that binds to the 'a' epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K(D) = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.

[A construct competent to support the replication of hepatitis B virus of genotype B].

OBJECTIVE: To construct a vector that is competent to support the replication of hepatitis B virus (HBV) of genotype B. METHODS: The HBV genome of genotype B was amplified by PCR and ligated into pBlueskript II KS(+) vector, the resulting plasmid was verified by enzyme digestion and DNA sequencing. After transfection of this plasmid into Huh7 cells, the HBsAg and HBeAg antigens in culture medium were quantified by ELISA, the transcripts and replication intermediates of HBV were detected by northern blot and southern blot respectively. On the other hand, the plasmid was hydrodynamically injected into BALB/cJ mice via tail vein. Then the HBV DNA in serum was quantified by real-time PCR, and HBcAg expression in liver tissue was detected by immunohistochemistry. RESULTS: After transfection of the plasmid into Huh7 cells, the HBsAg and HBeAg antigens were detected in the culture medium, the transcripts and replication intermediates of HBV were detected in the cells. High titer of HBV DNA was detected in the serum of hydrodynamic-injected mice. Immunostaining indicated that HBcAg was expressed in hepatocytes of injected mice. CONCLUSION: This construct is competent to support the replication of hepatitis B virus of genotype B.

Transmission of hepatitis B virus (HBV) genotypes among Japanese immigrants and natives in Bolivia.

Hepatitis B virus genotypes are associated with transmission pattern, virological and clinical features and outcome of the chronic infection course. HBV genotypes other than Genotype F (HBV/F) are considered a reflection of human migration into South America. A total of 487 individuals in Bolivia, including Japanese immigrants (n=287) and natives (n=200), were screened for HBV serological markers. Overall 22/487 (4.5%) of the subjects were positive for HBsAg, 217/487 (44.5%) for anti-HBc and 162/487 (33.3%) for anti-HBs. Genotypes were determinable in 22 cases by EIA, followed by sequencing and phylogenetic analysis in 17 cases. HBV genotype distribution in Japanese and Bolivians was HBV/F (4 and 8); HBV/C (5 and 3); and HBV/B (1 and 1), respectively. Phylogenetic analyses of nine complete and eight partial (HBsAg/pre-core/core region) genomes, revealed that HBV/F strains cluster with previously reported regional strains, whereas HBV/B and HBV/C strains belonged to Asian subgenotype B2 (Ba) and C2 (Ce), respectively. Japanese immigrants might have introduced HBV/B and HBV/C to natives in Bolivia, conversely, exposed to the indigenous HBV/F. This report provides evidence of an inter-communities transmission of HBV revealed by its genotypes. Further study is required to investigate peculiarities of the genotypes in different ethnic groups in Bolivia.