Risk Stratification for Hepatitis B Virus Reactivation in Kidney Transplant Recipients With Resolved HBV Infection.

The prophylaxis strategy for hepatitis B virus (HBV) reactivation in kidney transplant recipients (KTRs) with resolved HBV infection remains unclear. In this hospital-based retrospective cohort study, consecutive KTRs with resolved HBV infection were screened from the years 2000 through 2020. After excluding confounding conditions, 212 and 45 patients were respectively recruited into Anti-HBs positive and Anti-HBs negative groups. Cumulative incidences of, and subdistribution hazard ratios (SHRs) for HBV reactivation were analyzed after adjusting the competing risk. During a median 8.3 (mean 8.4 ± 4.9) years of follow-up, the 10-year cumulative incidence of HBV reactivation was significantly higher in Anti-HBs negative group when compared to that in Anti-HBs positive group (15.2%, 95% CI: 3.6-26.7 vs. 1.3%, 95% CI: 0.0-3.0; p < 0.001). In multivariable regression analysis, absence of anti-HBs (SHR 14.2, 95% CI: 3.09-65.2; p < 0.001) and use of high-dose steroids, i.e., steroid dose ≥20 mg/day of prednisolone equivalent over 4 weeks (SHR 8.96, 95% CI: 1.05-76.2; p = 0.045) were independent risk factors related to HBV reactivation. Accordingly, the 10-year cumulative incidence of HBV reactivation occurring in patients with two, one and zero risk factors was 42.7% (95% CI: 0.0-87.1), 7.9% (95% CI: 1.2-14.7) and 0%, respectively (p < 0.001). In conclusion, the strategy of HBV antiviral prophylaxis may be defined according to the risk stratification.

Risk of hepatitis B virus reactivation during immunosuppressive treatment.

OBJECTIVE: In this study, we aimed to rate Hepatitis B Virus (HBV) reactivation, risk factors for reactivation and compare the efficacy of prophylactic antiviral therapy in patients who initiated immunosuppressive therapy. PATIENTS AND METHODS: A total of 177 patients with Chronic Hepatitis B or resolved HBV infection who had received immunosuppressive treatment were analyzed in this retrospective study. Demographic features, relevant liver tests, prophylactic treatment type, duration of treatment, transaminase levels and HBV serology and clinical conditions were recorded from all patients who received prophylactic treatment. RESULTS: Eleven reactivation occurred in all groups. The mean age of patients who developed reactivation was statistically significantly lower (p=0.049). Three (27.3%) of the patients were male and 8 (72.7%) were female (p=0.66). Eight (36.36%) of 22 HB surface antigen (HBsAg) positive patients developed reactivation, 3 (155%) of 155 HBsAg negative patients developed reactivation. HBsAg positivity was determined as a risk factor for reactivation (p<0.001). There was no significant difference neither in reactivation, nor in the type of antiviral treatment (p=0.2) according to anti-HBs serology (p=0.366). CONCLUSIONS: As a result, early age, baseline HBsAg positivity, moderate risk group, baseline HBV DNA positivity were associated with reactivation. Gender, immunosuppressive therapy type, preemptive antiviral therapy type, and anti-HBs titers were not associated with reactivation.

Patients treated with rituximab are poorly screened for hepatitis B infection: Data from a low-incidence country.

BACKGROUND & AIMS: Patients with chronic or resolved hepatitis B are at risk of hepatitis B reactivation (HBVr) when treated with high-risk immunosuppressive therapy such as rituximab. Therefore, international guidelines recommend HBV screening prior to rituximab treatment and subsequent antiviral prophylaxis among patients with a (resolved) infection. In this study, we evaluated the adherence to those recommendations. METHODS: This is a retrospective multicentre study including patients treated with rituximab between 2000-2021. Performance of correct screening was assessed, defined as the measurement of hepatitis B surface antigen (HBsAg) and hepatitis B core antibodies (anti-HBc). Next, initiation of antiviral prophylaxis and HBVr rate among patients with a chronic or resolved HBV infection was studied. RESULTS: We enrolled 3,176 patients of whom 1,448 (46%) were screened correctly. Screening rates differed significantly between academic and non-academic hospitals; respectively 65% vs 32% (p<0.001). In addition, screening rates differed across specialties and improved throughout the years; from 32% before 2012 to 75% after 2020 among academic prescribers, versus 1% to 60% among non-academic prescribers (both p<0.001). Antiviral prophylaxis was initiated in 58% vs 36% of the patients with a chronic or resolved HBV infection. Seven patients experienced HBVr, including one fatal liver decompensation. CONCLUSIONS: Many patients treated with rituximab were not correctly screened for HBV infection and antiviral prophylaxis was often not initiated. Although screening rates improved over time, rates remain suboptimal. With the increasing number of indications for rituximab and other immunosuppressive agents these findings could raise awareness among all medical specialties prescribing these agents.

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.

Prophylactic effect of tenofovir on viral reactivation in immunocompromised pregnant women living with hepatitis B virus.

The appropriate prophylaxis for hepatitis B virus reactivation (HBVr) during gestation for immunocompromised pregnant women has yet to be determined. The prophylactic efficacy and safety of tenofovir disoproxil fumarate (TDF) in hepatitis B surface antigen (HBsAg)-positive patients and the HBVr risk in hepatitis B core antibody (HBcAb)-positive patients during gestation were investigated. Eligible pregnant women were diagnosed with rheumatic diseases and were administered prednisone (≤10 mg daily) with permitted immunosuppressants at screening. HBsAg-positive participants were instructed to take TDF; those unwilling to take TDF were followed up as the control group. Propensity score matching was applied to control for differences in confounding factors between the HBcAb-positive and uninfected groups. Hepatopathy, maternal, pregnancy, and safety outcomes were documented as endpoints. A cohort of 1292 women was recruited from 2017 to 2020, including 58 HBsAg-positive patients (29 in each group). A total of 120 pairs in the HBcAb-positive and noninfection groups were analyzed. Among HBsAg-positive patients, 6 (20.7%) cases of hepatitis flare (hazard ratio [HR]: 7.44; 95% confidence interval [CI]: 1.50-36.89; p = 0.014) and 12 (41.4%) cases of HBVr (HR: 8.71; 95% CI: 2.80-27.17; p < 0.001) occurred in the control group, while 0 occurred in the TDF prophylaxis group. The HBV level at delivery was the lowest (1.6 log10 IU/ml) for those who received TDF during the pregestation period with a good safety profile. More adverse maternal outcomes were observed in the control group (odds ratio: 0.19, 95% CI: 0.05-0.77, p = 0.021), including one death from fulminant hepatitis and two cases of vertical transmission. No HBVr was recorded in HBcAb-positive participants. Among immunocompromised pregnant women, prophylactic TDF during pregestation was necessary for HBsAg-positive women, whereas regular monitoring was recommended for HBcAb-positive women.

Significance of anti-HBc serological status in primary immune thrombocytopenia.

The association of previous hepatitis B virus (HBV) exposure [hepatitis B surface antigen (HBsAg) negative, hepatitis B core antibody (anti-HBc/HBcAb) positive] with disease severity and decision on treatment option in primary immune thrombocytopenia (ITP) patients remains unclear. Data from 725 patients diagnosed with ITP were analyzed to elucidate the association between anti-HBc serological status and disease severity. Data from a published prospective study [high-dose dexamethasone (HD-DXM), HD-DXM plus recombinant human thrombopoietin, NCT01734044] and two retrospective studies (standard-dose and low-dose rituximab) were rearranged to evaluate the impact of anti-HBc serological status on the response and outcome to ITP-specific treatments and the risk of HBV reactivation related to these treatments. The prevalence of HBsAg- HBcAb+ and HBsAg- HBcAb- in ITP patients was 51·03% and 48·97% respectively. Compared to the HBsAg- HBcAb- group, patients in the HBsAg- HBcAb+ group had lower platelet count, higher bleeding score, and longer hospitalization (P = 0·002, 0·033, and 0·008 respectively). Moreover, the initial complete response rate of HBsAg- HBcAb+ patients was lower than that of HBsAg- HBcAb- patients (45·2% vs 59·8%, P = 0·027). In conclusion, previous HBV exposure was correlated with disease severity and hospitalization in ITP patients. Anti-HBc positivity may be considered as a predictor for poor response to ITP-specific treatments.

Hepatotoxicity associated with PD-1 blockade antibodies in cancer patients co-infected with hepatitis B virus.

The use of anti-programmed cell death-1 (PD-1) antibodies in treating malignancies is increasing; however, most registered clinical trials on anti-PD-1 antibodies exclude patients infected with hepatitis B virus (HBV). This retrospective study aimed to assess hepatotoxicity in cancer patients infected with HBV undergoing anti-PD1 antibody therapy and identify the associated risk factors. A total of 301 cancer patients positive for hepatitis B core antibodies (HbcAb) (negative or positive hepatitis B surface antigen [HBsAg]) who received PD-1 inhibitors were enrolled. The primary and secondary endpoints were the incidence rate of hepatotoxicity related to PD-1 inhibitor treatment, and risk factors associated with hepatic toxicity, respectively. Of the enrolled analyzed, 16.9% (n = 51) developed any grade and 4.7% (n = 14) developed grade 3-4 hepatotoxicity, respectively. Higher risk for any-grade hepatotoxicity development was associated with sero-positive HBsAg (OR = 6.30; P = 0.020), existence of liver involvement (OR = 2.10; P = 0.030), and detectable baseline HBV DNA levels (OR = 2.39; P = 0.012). Patients with prophylactic antiviral therapy decreased hazard for the incidence of grade 3-4 hepatotoxicity (OR = 0.10; P = 0.016). Our results suggested chronic (HBsAg-positive)/resolved (HBsAg-negative and HBcAb-positive) HBV-infected cancer patients are at an increased risk of hepatotoxicity following PD-1 inhibitor therapy. Cancer patients should be tested for HBsAg/HBcAb prior to the commencement of immune checkpoint inhibitor therapy. For patients with chronic/resolved HBV infection, ALT/AST and HBV DNA should be closely monitored during the whole immunotherapy period.

Risk of Hepatitis B Virus (HBV) Reactivation in Patients with Immune-Mediated Inflammatory Diseases Receiving Biologics: Focus on the Timing of Biologics after Anti-HBV Treatment.

Background/Aims: Anti-hepatitis B virus (HBV) therapy is required for patients with HBV infection receiving biologics because of the high risk of HBV reactivation. However, it is unclear when to start biologics after anti-HBV treatment. We investigated the risk of HBV reactivation according to the timing of biologics initiation after anti-HBV treatment in immune-mediated inflammatory disease (IMID) patients with HBV infection. Methods: We retrospectively evaluated the incidence of HBV reactivation in IMID patients who received biologics between July 2005 and April 2020. The patients were divided into two groups (within 1-week and after 1-week) according to the timing of biologics initiation after anti-HBV treatment. The cumulative probabilities and factors associated with HBV reactivation were evaluated. Results: A total of 60 hepatitis B surface antigen-positive patients with IMID received biologics (within 1-week group, n=23 [38%]; after 1-week group, n=37 [62%]). During a median follow-up of 34 months (interquartile range, 20 to 74 months), three patients (5%) developed HBV reactivation. In univariate analysis, the timing of biologics after anti-HBV treatment was not significantly associated with the risk of HBV reactivation (hazard ratio, 0.657; 95% confidence interval, 0.059 to 7.327; p=0.733). The cumulative probabilities of HBV reactivation did not significantly differ according to the timing of biologics (p=0.731). Conclusions: The risk of HBV reactivation was not significantly associated with the timing of biologics administration after anti-HBV treatment. Thus, biologics may be initiated early in patients with IMID undergoing treatment for HBV.

[Hepatits B virus reactivation in patients receiving immunosuppressive therapy or chemotherapy and effective prophylactic management: a prospective observational study in a hospital over an 8-year period].

We investigated the development of hepatits B virus (HBV) reactivation in patients receiving immunosuppressive therapy or chemotherapy at our hospital for 8 years. Using the automatic checking system for HBV reactivation coded using medical information that has been in operation in our hospital since October 2012, we prospectively observed the occurrence status of HBV reactivation in immunosuppressive/chemotherapy cases for 8 years. HBV reactivation occurred in 31 of 1516 patients with HBV infection. It occurred annually between 1 and 7 cases in multiple clinical departments, and in 8 of 59 patients treated with rituximab, 10 of 653 patients treated with antineoplastic agents, 10 of 399 patients treated with steroids, and 3 of 212 patients treated with direct-acting antivirals. The cumulative incidence of HBV reactivation was 1.2%, 2.3%, and 3.4% at 1, 2, and 3 years, respectively. The results of Cox regression analysis showed that the incidence of HBV reactivation was significantly higher in patients who received rituximab (odds ratio:12.841) or steroid (hazard ratio:4.264) or those who tested positive for HBc antibody alone (hazard ratio:11.005). We observed the occurrence of HBV reactivation in HBV-infected patients treated with immunosuppressive therapy or chemotherapy. HBV reactivation by immunosuppressive therapy or chemotherapy still occurs, and further safety management and caution are required in the hospital.

Prevalence of hepatitis B virus infection and risk of reactivation in rheumatic population undergoing biological therapy.

OBJECTIVES: Hepatitis B (HBV) is a common comorbidity among rheumatic patients. The prevalence of HBV infection and the rate of reactivation remain unclear. The literature data suggested a higher risk in chronic than in past infection. Currently, the literature data are mostly focused on anti-TNF and rituximab. This retrospective observational study aimed to analyse the prevalence of HBV infection and the risk of viral reactivation in a population of rheumatic patients undergoing anti-TNF and non-anti-TNF agents. METHODS: We analysed 1216 rheumatic patients, treated with both csDMARDs and bDMARDs between 2006 and 2017. Serologic markers for HBV (HBsAg, anti-HBs, anti-HBc) were performed prior and during biologic treatment. Patients with chronic or resolved infection were monitored every 3 months. RESULTS: The prevalence of HBV in our cohort was 15.7% (chronic infection: 0.4%, resolved infection: 12.6%, anti-HBc positivity alone: 2.6%). 12 (6.2%) out of 191 HBV infected patients experienced a reactivation. All of them showed markers of past infection. One patient experienced HBV reactivation despite lamivudine. Only one patient experienced acute hepatitis, probably due to the interruption of immunosuppressors in anticipation of surgery, not preceded by any HBV prophylactic treatment. CONCLUSIONS: HBV reactivation is a rare event in patients treated with a bDMARD and it can also occur while taking lamivudine, not only in chronic carriers (as per the literature data) but also in inactive ones. Regular screening followed by prompt treatment can prevent symptoms or complications. Due to the risk of hepatitis following the immune reconstitution, an antiviral therapy should be considered in the case of sudden discontinuation of csDMARDs or bDMARD.

CD4+ T Cells Play a Critical Role in Microbiota-Maintained Anti-HBV Immunity in a Mouse Model.

The ability of the host to clear hepatitis B virus (HBV) is closely correlated to the establishment of commensal microbiota. However, how microbiota affects anti-HBV immunity is still unclear. Using a well-known hydrodynamical HBV transfection mouse model and treatment with antibiotics (Atb), we explored the change in adaptive immunity (CD4+ cells, germinal center B cells and anti-HBs Ab). In our setting, normal mice exhibited complete clearance of HBV within 6 weeks post-hydrodynamic injection (HDI) of HBV-containing plasmid, whereas Atb-treated mice lost this capacity, showing high serum level of hepatitis B surface antigen (HBsAg) without hepatitis B surface antibodies (anti-HBs), similar as what happened in Rag1-/- mice or CD4-/- mice, suggesting that microbiota may influence the function of CD4+ T cells. Furthermore, the numbers of splenic and hepatic effector CD4+ T cells (CD44hiCD62L-CD4+ T cells) both decreased with impaired function (IFN-γ synthesis), resulting in lower frequency of germinal center B cells and CD4+ follicular helper T cells, and impaired anti-HBs production. We further tried to find the bacterial species responsible for maintaining anti-HBV immunity, and found that each antibiotic alone could not significantly influence HBV clearance compared to antibiotic combination, suggesting that global commensal microbial load is critical for promoting HBV clearance. We also confirmed that TLRs (e.g., TLR2, 4, 9) are not major players in immune clearance of HBV using their agonists and knock-out mice. These results suggest that commensal microbiota play an important role in maintaining CD4+ T cell immunity against HBV infection.

A human monoclonal antibody against small envelope protein of hepatitis B virus with potent neutralization effect.

Hepatitis B virus (HBV) produces large (L), middle (M), and small (S) envelope proteins, alternatively referred to as hepatitis B surface antigen (HBsAg). Currently, yeast-derived S protein serves as the preventive vaccine, while hepatitis B immune globulin (HBIG) concentrated from pooled plasma of vaccine recipients is employed for post-exposure prophylaxis. However, only a small proportion of the antibodies in HBIG are HBV specific. In the present study, a human monoclonal anti-S antibody (G12) was developed, produced under GLP conditions, and subjected to a panel of functional assays. In vitro results demonstrated high affinity of G12 for the S protein (KD = 7.56 nM). It reacted with envelope proteins of all 7 HBV genotypes tested (A-F, H) by immunofluorescent staining, and more than 97% of HBsAg-positive patient serum samples by enzyme-linked immunosorbent assay. G12 recognized a conformational epitope, although the exact sequence remains unknown. Strikingly, G12 was at least 1,000-fold more potent than HBIG in neutralizing HBV infectivity in both HepaRG cell line and HepG2 cells reconstituted with the HBV receptor. In a transgenic mouse model of HBV persistence, a single peritoneal injection of G12 markedly diminished serum HBsAg titers in all 7 mice, which was sustained for the observation period of 144 d in mice with low pre-treatment levels. While the therapeutic potential of G12 warrants further investigation using a large number of animals, G12 is a potent neutralizing human monoclonal antibody and a promising candidate to replace or supplement HBIG in the prevention of HBV infection.

Enhanced humanization and affinity maturation of neutralizing anti-hepatitis B virus preS1 antibody based on antigen-antibody complex structure.

To improve a previously constructed broadly neutralizing hepatitis B virus (HBV)-specific preS1 humanized antibody (HzKR127), we further humanized it through specificity-determining residue (SDR) grafting. Moreover, we improved affinity by mutating two residues in heavy-chain complementarity-determining regions (CDR), on the basis of the crystal structure of the antigen-antibody complex. HzKR127-3.2 exhibited 2.5-fold higher affinity and enhanced virus-neutralizing activity compared to the original KR127 antibody and showed less immunogenic potential than HzKR127. Enhanced virus-neutralizing activity was achieved by the increased association rate, providing insights into engineering potent antibody therapeutics for HBV immunoprophylaxis. HzKR127-3.2 may be a good candidate for HBV immunoprophylaxis.

Inhibitors of hepatitis B virus attachment and entry.

Inhibition of virus entry has become a major concept in the development of new antiviral drugs. Entry inhibitors can either neutralize activities of viral surface proteins or target essential host factors such as (co)receptors. Due to its distinct tissue tropism and the highly specific viral and cellular factors involved in its entry, hepatitis B virus (HBV) is an ideal candidate for entry inhibition. Hepatitis B immunoglobulins neutralize infection by binding to the S-domain of HBV surface proteins and are used to prevent reinfection of the graft after liver transplantation. Novel S or preS-specific monoclonal antibodies are currently in development. The identification of sodium-taurocholate cotransporting polypeptide (NTCP) as a bona fide receptor has revealed a suitable target for HBV entry inhibition. NTCP receptor function is blocked by a variety of different agents including Myrcludex B, a synthetic N-acylated preS1-derived lipopeptide that inhibits HBV entry in vitro and in vivo with high efficacy. Current antiviral treatment for chronic HBV-infected patients focuses on the inhibition of the viral polymerase via nucleos(t)ide analogues (NA). Entry inhibitors in combination with NAs could block reinfection and shield naive hepatocytes that emerge from natural liver turnover, opening up new therapeutic options.

Intracellular-delivery of a single-chain antibody against hepatitis B core protein via cell-penetrating peptide inhibits hepatitis B virus replication in vitro.

Assembly of nucleocapsids is an attractive target for novel anti-hepatitis B virus (HBV) agents, and intracellular single-chain variable fragment (scFv) antibodies against HBV core (HBc) protein are a class of potential alternatives for this purpose; however, their application is limited by the lack of a suitable means of delivery. Owing to the favorable performance of cytoplasmic transduction peptide (CTP) in cargo delivery in hepatocytes, we purified an anti-HBc scFv fused to CTP using a previous screened sequence by a prokaryotic expression system and evaluated its efficacy in the inhibition of HBV in vitro. Our results showed that cytoplasmic translocation of the previous anti-HBc scFv was achieved by CTP in HepG2.2.15 cells. Immunoprecipitation analysis indicated the fusion protein anti-HBc scFv-CTP interacted with its target antigen HBc, and negligible cytotoxicity was observed. Moreover, the anti-HBc scFv-CTP interfered with nucleocapsid assembly and markedly reduced both the supernatant HBV DNA level and the intracellular DNA replication intermediates, with a 5.1 µM of half maximal effect concentration and a dose-dependent effect. In conclusion, this novel anti-HBc scFv fused to CTP demonstrated inhibitory activity of HBV replication in vitro and warrants further in vivo study.

Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody.

The Hepatitis B virus precore protein is processed in the endoplasmic reticulum (ER) into secreted hepatitis B e antigen (HBeAg), which acts as an immune tolerogen to establish chronic infection. Downregulation of secreted HBeAg should improve clinical outcome, as patients who effectively respond to current treatments (IFN-α) have significantly lower serum HBeAg levels. Here, we describe a novel reagent, a single variable domain (V(NAR)) of the shark immunoglobulin new antigen receptor (IgNAR) antibodies. V(NAR)s possess advantages in stability, size (~14 kDa) and cryptic epitope recognition compared to conventional antibodies. The V(NAR) domain displayed biologically useful affinity for recombinant and native HBeAg, and recognised a unique conformational epitope. To assess therapeutic potential in targeting intracellular precore protein to reduce secreted HBeAg, the V(NAR) was engineered for ER-targeted in vitro delivery to function as an intracellular antibody (intrabody). In vitro data from HBV/precore hepatocyte cell lines demonstrated effective intrabody regulation of precore/HBeAg.

Production, characterization and in vitro testing of HBcAg-specific VHH intrabodies.

Hepatitis B virus (HBV) infections represent a global health problem, since these account for 350 million chronic infections worldwide that result in 500,000-700,000 deaths each year. Control of viral replication and HBV-related disease and mortality are of utmost importance. Because the currently available antiviral therapies all have major limitations, new strategies to treat chronic HBV infection are eagerly awaited. Six single-domain antibodies (VHHs) targeting the core antigen of HBV (HBcAg) have been generated and three of these bound strongly to HBcAg of both subtype ayw and adw. These three VHHs were studied as intrabodies directed towards the nucleus or the cytoplasm of a hepatoma cell line that was co-transfected with HBV. A speckled staining of HBcAg was observed in the cytoplasm of cells transfected with nucleotropic VHH intrabodies. Moreover, an increased intracellular accumulation of hepatitis B e antigen (HBeAg) and a complete disappearance of intracellular HBcAg signal were observed with nuclear targeted HBcAg-specific VHHs. These results suggest that HBcAg-specific VHHs targeted to the nucleus affect HBcAg and HBeAg expression and trafficking in HBV-transfected hepatocytes.

Production of recombinant woodchuck IFNalpha and development of monoclonal antibodies.

Interferon alpha (IFNalpha) is the first line treatment for chronic hepatitis B and C. In order to test new IFNalpha delivery systems and investigate the function of this cytokine in the woodchuck model, the best animal model of chronic hepatitis B, we produced and purified recombinant woodchuck IFNalpha and used it to produce monoclonal antibodies. wIFNalpha5 was cloned in a prokaryotic expression system, expressed as His-tagged protein and then purified. The rwIFNalpha5 protein was found to induce STAT-3 phosphorylation, to enhance 2',5'-oligoadenylate synthetase mRNA levels and to possess a potent antiviral activity. Two monoclonal antibodies were obtained through immunization of rats with rwIFNalpha5. Both recognized rwIFNalpha5 in western blot analysis and one was able to neutralize the antiviral activity of the rwIFNalpha5 and lymphoblastoid IFNalpha preparations. Finally, a capture rwIFNalpha5 ELISA was developed using both antibodies. In summary, the tools generated in this study will allow different forms of IFNalpha delivery as well as different combination therapies in woodchuck hepatitis virus infection to be tested, thus providing useful information for the design of new strategies to treat chronic hepatitis B in humans.

Factors affecting the presentation of exogenous hepatitis B virus core antigen.

Hepatitis B virus core antigen (HBcAg) plays a critical role in terminating acute Hepatitis B virus infection and may be used as a potential vaccine candidate. The cell surface major histocompatibility complex (MHC) class 1 molecules are thought to be involved in the presentation of HBcAg. Surface MHC class 1 HLA A2 heavy chain (HC) and trimeric molecules were characterized on transfected Hela cells used as antigen presenting cells (APC) for the presentation of HBcAg. The results show that antibodies against HC HLA A2 and trimeric HLA-A2 molecules resulted in increased activation of HBcAg 18-27 minimal peptide specific cytotoxic T lymphocytes (CTLs), while the addition of exogenous beta2-microglobulin decreased the activation of HBcAg specific CTLs. Further, specific CD8+ T cells were activated only when Hela cells as APCs were primed with HBcAg (peptide, soluble or embedded on virosomes) at pH 6.5.

Broadly neutralizing anti-hepatitis B virus antibody reveals a complementarity determining region H3 lid-opening mechanism.

The humanized monoclonal antibody HzKR127 recognizes the preS1 domain of the human hepatitis B virus surface proteins with a broadly neutralizing activity in vivo. We present the crystal structures of HzKR127 Fab and its complex with a major epitope peptide. In the complex structure, the bound peptide forms a type IV beta-turn followed by 3(10) helical turn, the looped-out conformation of which provides a structural basis for broad neutralization. Upon peptide binding, the antibody undergoes a dramatic complementarity determining region H3 lid opening. To understand the structural implication of the virus neutralization, we carried out comprehensive alanine-scanning mutagenesis of all complementarity determining region residues in HzKR127 Fab. The functional mapping of the antigen-combining site demonstrates the specific roles of major binding determinants in antigen binding, contributing to the rational design for maximal humanization and affinity maturation of the antibody.