Immunogenicity of Wild Type and Mutant Hepatitis B Surface Antigen Virus-like Particles (VLPs) in Mice with Pre-Existing Immunity against the Wild Type Vector.

Virus-like particles (VLPs), composed of the small hepatitis B virus surface antigen (HBsAgS), are the antigenic components of the hepatitis B virus (HBV) vaccine and represent the backbones for a chimeric anti-malaria vaccine and various vaccine candidates. Biological vectors have to face pre-existing anti-vector immune responses due to previous immune exposure. Vector recognition after natural infections or vaccinations can result in unwarranted outcomes, with compromising effects on clinical outcomes. In order to evaluate the impact of a pre-existing anti-HBsAgS immune response, we developed mutant VLPs composed of subunits with reduced HBsAgS-specific antigenicity. The insertion of a Plasmodium falciparum circumsporozoite protein (CSP)-derived epitope as a read-out allowed the assessment of wild type (wt) and mutant VLPs in the context of a pre-existing immune response. Mutant and wt VLP platforms with a CSP-epitope insert are immunogenic and have the ability to generate anti-CSP antibody responses in both naïve BALB/c mice and mice with a pre-existing anti-HBsAgS immune response, but with superior anti-CSP responses in mice with a pre-existing immunity. The data indicate that previous HBsAgS exposure facilitates enhanced antibody responses against foreign epitopes delivered by the HBsAgS platform, and, in this context, the state of immune sensitization alters the outcome of subsequent vaccinations.

Role of anti-HBs in functional cure of HBeAg+ chronic hepatitis B patients infected with HBV genotype A.

BACKGROUND & AIMS: HBsAg-specific antibody responses are difficult to detect during chronic hepatitis B infection (CHB) and are often overlooked. The aim of this study was to examine whether anti-HBs may be involved in functional cure (FC) by profiling anti-HBs responses in patients with CHB using a panel of specific assays. METHODS: Longitudinal serum samples were obtained from 25 patients with CHB who were infected with HBV genotype A and were undergoing nucleos(t)ide analogue (NA) treatment: 14 achieved FC while 11 remained infected (non-FC). Anti-HBs immune complexes (HBsAg-IC), FcγRIIIa dimer binding, epitope specificity and neutralisation efficacy were measured. RESULTS: HBsAg-IC peaks were detected prior to HBsAg loss in 10/14 FC patients. These HBsAg-IC peaks overlapped with either an alanine aminotransferase (ALT) flare (8/10 patients), or a rise in ALT (2/10 patients). HBsAg-IC peaks were detected in 7/11 non-FC patients, but were not associated with an ALT flare. FCγRIIIa binding was detected in 9/14 FC patients, independent from detection of overlapping HBsAg-IC/ALT peaks. FC patients had stable HBsAg epitope occupancy across the study, whereas non-FC patients had a reduction in HBsAg epitope occupancy within the first 12-24 weeks of NA treatment. Convalescent sera from FC patients recognised more HBsAg epitopes and neutralised HBV infection more potently than anti-HBs derived from vaccinees. Neutralisation potency appeared to increase post-HBsAg loss in 4/5 FC patients examined. CONCLUSIONS: Using these assays, we confirm that anti-HBs responses are present and fluctuate over time in this cohort of patients with HBeAg+ CHB, who were infected with HBV genotype A and treated with NAs. Key anti-HBs profiles associated with either FC or failure to achieve FC were also identified, suggesting a role for anti-HBs responses in FC. LAY SUMMARY: Using a panel of assays to characterise hepatitis B surface antibody (anti-HBs) responses in a group of patients with chronic hepatitis B, we identified anti-HBs profiles associated with either functional cure, or failure to achieve functional cure. Functional cure was associated with immune complex peaks which overlapped with alanine aminotransferase flares. Conversely, in those who did not achieve functional cure, immune complex peaks were present, but were not associated with alanine aminotransferase flares, and a decline in anti-HBs diversity was observed early during treatment.

Characterization of Hepatitis B Precore/Core-Related Antigens.

Current therapies rarely cure chronic hepatitis B virus (HBV) infection due to the persistence of the viral episome, the covalently closed circular DNA (cccDNA), in hepatocytes. The hepatitis B virus core-related antigen (HBcrAg), a mixture of the viral precore/core gene products, has emerged as one potential marker to monitor the levels and activities of intrahepatic cccDNA. In this study, a comprehensive characterization of precore/core gene products revealed that HBcrAg components included the classical hepatitis B virus core antigen (HBc) and e antigen (HBeAg) and, additionally, the precore-related antigen, PreC, retaining the N-terminal signal peptide. Both HBeAg and PreC antigens displayed heterogeneous proteolytic processing at their C termini resulting in multiple species, which varied with viral genotypes. HBeAg was the predominant form of HBcrAg in HBeAg-positive patients. Positive correlations were found between HBcrAg and PreC, between HBcrAg and HBeAg, and between PreC and HBeAg but not between HBcrAg and HBc. Serum HBeAg and PreC shared similar buoyant density and size distributions, and both displayed density and size heterogeneity. HBc, but not HBeAg or PreC antigen, was found as the main component of capsids in DNA-containing or empty virions. Neither HBeAg nor PreC protein was able to form capsids in cells or in vitro under physiological conditions. In conclusion, our study provides important new quantitative information on levels of each component of precore/core gene products as well as their biochemical and biophysical characteristics, implying that each component may have distinct functions and applications in reflecting intrahepatic viral activities.IMPORTANCE Chronic hepatitis B virus (HBV) infection afflicts approximately 257 million people, who are at high risk of progressing to chronic liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. Current therapies rarely achieve cure of HBV infection due to the persistence of the HBV episome, the covalently closed circular DNA (cccDNA), in the nuclei of infected hepatocytes. Peripheral markers of cccDNA levels and transcriptional activities are urgently required to guide antiviral therapy and drug development. Serum hepatitis B core-related antigen (HBcrAg) is one such emerging peripheral marker. We have characterized the components of HBcrAg in HBV-infected patients as well as in cell cultures. Our results provide important new quantitative information on levels of each HBcrAg component, as well as their biochemical and biophysical characteristics. Our findings suggest that each HBcrAg component may have distinct functions and applications in reflecting intrahepatic viral activities.

Glycoengineered hepatitis B virus-like particles with enhanced immunogenicity.

Virus-like particles (VLP) represent biological platforms for the development of novel products such as vaccines and delivery platforms for foreign antigenic sequences. VLPs composed of the small surface antigen (HBsAgS) derived from the hepatitis B virus (HBV) are the immunogenic components of a licensed, preventative vaccine, which contains aluminum hydroxide as adjuvant. Herein, we report that glycoengineering of N-glycosylated HBsAgS to generate hyper-glycosylated VLPs display an enhanced immunogenicity relative to the wild type (WT) HBsAgS VLPs when expressed in FreeStyle HEK 293F cells. Comparative mass spectrometry-based N-glycan profiling, gel electrophoresis, and immunoassays demonstrated that WT and hyper-glycosylated HBsAgS VLPs contain the same type and distribution of N-glycan structures, but the latter shows a higher glycan abundance per protein mass. The antigenic integrity of the modified VLPs was also shown to be retained. To assess whether hyper-glycosylated VLPs induce an enhanced immune response in the presence of the adjuvant aluminum hydroxide, the anti-HBV surface antigen (anti-HBsAgS) antibody response was monitored in BALB/c mice, subcutaneously injected with different VLP derivatives. In the absence and presence of adjuvant, hyper-glycosylated VLPs showed an enhanced immunogenicity compared to WT VLPs. The ability of hyper-glycosylated VLPs to promote potent anti-HBsAgS immune responses compared to VLPs with a native N-glycan level as well as non-glycosylated, yeast-derived HBsAgS VLPs opens exciting avenues for generating more efficacious VLP-based vaccines against hepatitis B and improved HBsAgS VLP carrier platforms using glycoengineering.

Predicting HBsAg clearance in genotype A chronic hepatitis B using HBsAg epitope profiling: A biomarker for functional cure.

BACKGROUND AND AIM: Functional cure is the major goal of chronic hepatitis B (CHB) therapy though few biomarkers predict this outcome. HBsAg epitope occupancy can be influenced by therapeutic and immune pressure. The aim of this study was to map the HBsAg epitope profiles during long-term nucleos(t)ide analogue therapy in patients with genotype A CHB, in the context of HBsAg loss (SL)/seroconversion. METHODS: We evaluated 25 genotype A CHB patients in the GS-US-174-0103 trial of HBeAg-positive CHB patients treated with tenofovir or adefovir for 4 years, 14 who achieved SL whilst 11 had no change. We epitope mapped the major domains of HBsAg to identify those patients with HBsAg clearance profile (CP) (loss of binding at both loops 1 and 2 epitopes of the 'a' determinant) vs non-clearance profile (no change in epitope recognition, or loss of epitope binding at one loop only), correlating this to on-treatment HBsAg responses. Complexed anti-HBs was also measured. RESULTS: Analysis of the HBsAg epitope profiles of the 25 patients at baseline identified no predictive correlation with SL. In contrast, analysis at week 48 and end of study (week 192) or prior to SL identified significant predictive associations between development of HBsAg CPs and outcome of functional cure. The detection of a CP also correlated with the development of an alanine aminotransferase flare and detection of anti-HBs complexed with HBsAg. CONCLUSION: The detection of HBsAg CPs by epitope mapping represents a novel viral biomarker, reflecting an emerging anti-HBs selection pressure prior to functional cure.

Hepatitis B virus-like particles expressing Plasmodium falciparum epitopes induce complement-fixing antibodies against the circumsporozoite protein.

The repetitive structure of compact virus-like particles (VLPs) provides high density displays of antigenic sequences, which trigger key parts of the immune system. The hepatitis B virus (HBV) and human papilloma virus (HPV) vaccines exploit the assembly competence of structural proteins, which are the effective immunogenic components of the prophylactic HBV and HPV vaccines, respectively. To optimize vaccine designs and to promote immune responses against protective epitopes, the "Asp-Ala-Asp-Pro" (NANP)-repeat from the Plasmodium falciparum circumsporozoite protein (CSP) was expressed within the exposed, main antigenic site of the small HBV envelope protein (HBsAgS); this differs from the RTS,S vaccine, in which CSP epitopes are fused to the N-terminus of HBsAgS. The chimeric HBsAgS proteins are assembly competent, produce VLPs, and provide a high antigenic density of the NANP repeat sequence. Chimeric VLPs with four or nine NANP-repeats (NANP4 and NANP9, respectively) were expressed in mammalian cells, the HBsAgS- and CSP-specific antigenicity of the VLPs was determined, and the immunogenicity of the VLPs assessed in relation to the induction of anti-HBsAgS and anti-CSP antibody responses. The chimeric VLPs induced high anti-CSP titres in BALB/c mice independent of the number of the NANP repeats. However, the number of NANP repeats influenced the activity of vaccine-induced antibodies measured by complement fixation to CSP, one of the proposed effector mechanisms for Plasmodium neutralization in vivo. Sera from mice immunized with VLPs containing nine NANP repeats performed better in the complement fixation assay than the group with four NANP repeats. The effect of the epitope-specific density on the antibody quality may instruct VLP platform designs to optimize immunological outcomes and vaccine efficacy.

Stop codons in the hepatitis B surface proteins are enriched during antiviral therapy and are associated with host cell apoptosis.

Premature stop codons in the hepatitis B virus (HBV) surface protein can be associated with nucleos(t)ide analogue resistance due to overlap of the HBV surface and polymerase genes. The aim of this study was to determine the effect of the replication of three common surface stop codon variants on the hepatocyte. Cell lines were transfected with infectious HBV clones encoding surface stop codons rtM204I/sW196*, rtA181T/sW172*, rtV191I/sW182*, and a panel of substitutions in the surface proteins. HBsAg was measured by Western blotting. Proliferation and apoptosis were measured using flow cytometry. All three surface stop codon variants were defective in HBsAg secretion. Cells transfected with these variants were less proliferative and had higher levels of apoptosis than those transfected with variants that did not encode surface stop codons. The most cytopathic variant was rtM204I/sW196*. Replication of HBV encoding surface stop codons was toxic to the cell and promoted apoptosis, exacerbating disease progression.

In Vitro Studies Show that Sequence Variability Contributes to Marked Variation in Hepatitis B Virus Replication, Protein Expression, and Function Observed across Genotypes.

The hepatitis B virus (HBV) exists as 9 major genotypes (A to I), one minor strain (designated J) and multiple subtypes. Marked differences in HBV natural history, disease progression and treatment response are exhibited by many of these genotypes and subtypes. For example, HBV genotype C is associated with later hepatitis B e antigen (HBeAg) seroconversion and high rates of liver cancer compared to other HBV genotypes, whereas genotype A2 is rarely associated with HBeAg-negative disease or liver cancer. The reasons for these and other differences in HBV natural history are yet to be determined but could in part be due to sequence differences in the HBV genome that alter replicative capacity and/or gene expression. Direct comparative studies on HBV replication and protein expression have been limited to date due largely to the absence of infectious HBV cDNA clones for each of the HBV genotypes present in the same genetic arrangement. We have produced replication-competent infectious cDNA clones of the most common subtypes of genotypes A to D, namely, A2, B2, C2, D3, and the minor strain J, and compared their HBV replication phenotype using transient-transfection models. We identified striking differences in HBV replicative capacity as well as HBeAg and surface (HBsAg) protein expression across genotypes, which may in part be due to sequence variability in regulatory regions of the HBV genome. Functional analysis showed that sequence differences in the major upstream regulatory region across genotypes impacted promoter activity. IMPORTANCE: There have been very few studies directly comparing the replication phenotype of different HBV genotypes, for which there are marked differences in natural history and disease progression worldwide. We have generated replication-competent 1.3-mer cDNA clones of the major genotypes A2, B2, C2, and D3, as well as a recently identified strain J, and identified striking differences in replicative capacity and protein expression that may contribute to some of the observed differences in HBV natural history observed globally.

Modification of Asparagine-Linked Glycan Density for the Design of Hepatitis B Virus Virus-Like Particles with Enhanced Immunogenicity.

UNLABELLED: The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic components of the HBV vaccine and are platforms for the delivery of foreign antigenic sequences. To investigate structure-immunogenicity relationships for the design of improved immunization vectors, we have generated biochemically modified virus-like particles (VLPs) exhibiting glycoengineered HBsAgS. For the generation of hypoglycosylated VLPs, the wild-type (WT) HBsAgS N146 glycosylation site was converted to N146Q; for constructing hyperglycosylated VLPs, potential glycosylation sites were introduced in the HBsAgS external loop region at positions T116 and G130 in addition to the WT site. The introduced T116N and G130N sites were utilized as glycosylation anchors resulting in the formation of hyperglycosylated VLPs. Mass spectroscopic analyses showed that the hyperglycosylated VLPs carry the same types of glycans as WT VLPs, with minor variations regarding the degree of fucosylation, bisecting N-acetylglucosamines, and sialylation. Antigenic fingerprints for the WT and hypo- and hyperglycosylated VLPs using a panel of 19 anti-HBsAgS monoclonal antibodies revealed that 15 antibodies retained their ability to bind to the different VLP glyco-analogues, suggesting that the additional N-glycans did not shield extensively for the HBsAgS-specific antigenicity. Immunization studies with the different VLPs showed a strong correlation between N-glycan abundance and antibody titers. The T116N VLPs induced earlier and longer-lasting antibody responses than did the hypoglycosylated and WT VLPs. The ability of nonnative VLPs to promote immune responses possibly due to differences in their glycosylation-related interaction with cells of the innate immune system illustrates pathways for the design of immunogens for superior preventive applications. IMPORTANCE: The use of biochemically modified, nonnative immunogens represents an attractive strategy for the generation of modulated or enhanced immune responses possibly due to differences in their interaction with immune cells. We have generated virus-like particles (VLPs) composed of hepatitis B virus envelope proteins (HBsAgS) with additional N-glycosylation sites. Hyperglycosylated VLPs were synthesized and characterized, and the results demonstrated that they carry the same types of glycans as wild-type VLPs. Comparative immunization studies demonstrated that the VLPs with the highest N-glycan density induce earlier and longer-lasting antibody immune responses than do wild-type or hypoglycosylated VLPs, possibly allowing reduced numbers of vaccine injections. The ability to modulate the immunogenicity of an immunogen will provide opportunities to develop optimized vaccines and VLP delivery platforms for foreign antigenic sequences, possibly in synergy with the use of suitable adjuvanting compounds.

Hepatitis B precore protein: pathogenic potential and therapeutic promise.

Hepatitis B virus (HBV), a small and economically packaged double-stranded DNA virus, represents an enormous global health care burden. In spite of an effective vaccine, HBV is endemic in many countries. Chronic hepatitis B (CHB) results in the development of significant clinical outcomes such as liver disease and hepatocellular carcinoma (HCC), which are associated with high mortality rates. HBV is a non-cytopathic virus, with the host's immune response responsible for the associated liver damage. Indeed, HBV appears to be a master of manipulating and modulating the immune response to achieve persistent and chronic infection. The HBV precore protein or hepatitis B e antigen (HBeAg) is a key viral protein involved in these processes, for instance though the down-regulation of the innate immune response. The development of new therapies that target viral proteins, such as HBeAg, which regulates of the immune system, may offer a new wave of potential therapeutics to circumvent progression to CHB and liver disease.

In vitro replication phenotype of a novel (-1G) hepatitis B virus variant associated with HIV co-infection.

The -1G mutant HBV is more prevalent in individuals co-infected with HIV/HBV than in individuals infected with HBV alone and in some cases is the dominant virus in circulation. This mutant is created by the deletion of a dGMP (-1G) from the guanine rich homopolymer sequence located at nts 2,085-2,090 (numbering from EcoRI site as position 1) in the HBV core gene. This deletion causes a frameshift generating a premature stop codon at (64) Asn in the HBV core gene (codon 93 in the precore gene), that truncates the precore protein, precursor of the secreted hepatitis B "e" antigen (HBeAg), and the core protein which forms the viral nucleocapsid. However, the replication phenotype of the -1G mutant HBV is unknown. An in vitro cell culture model in which hepatoma cells were transiently transfected with infectious cDNAs was used to show that the -1G mutant HBV is incapable of autonomous replication and, as expected, replication was restored to wild-type (wt) levels by supplying HBV core protein in trans. Although the -1G mutation had no deleterious effect on intracellular HBV-DNA levels, high levels of -1G mutant HBV relative to wt HBV reduced virus secretion and HBeAg secretion relative to empty vector controls. Importantly, the -1G mutant HBV also caused intracellular retention of truncated precore protein in the endoplasmic reticulum (ER) and Golgi apparatus. Together, these effects may be contributing to the increased pathology observed in the setting of HIV/HBV co-infection.

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.

Bioinformatic analysis of the hepadnavirus e-antigen and its precursor identifies remarkable sequence conservation in all orthohepadnaviruses.

The hepatitis B e-antigen (HBeAg) is a non-particulate secretory protein expressed by all viruses within the family Hepadnaviridae. It is not essential for viral assembly or replication but is important for establishment of persistent infection in vivo. Although the exact mechanism(s) by which the HBeAg manifests chronicity are unclear, the HBeAg elicits both humoral and cell-mediated immunity, down-regulates the innate immune response to infection, as well as functioning as a T cell tolerogen and regulating the immune response to the intracellular nucleocapsid. A bioinformatics approach was used to show that the HBeAg and precursory genetic codes share remarkable sequence conservation in all mammalian-infecting hepadnaviruses, irrespective of host, genotype, or geographic origin. Whilst much of this sequence conservation was within key immunomodulatory epitopes, highest conservation was observed at the unique HBeAg N-terminus, suggesting this sequence in particular may play an important role in HBeAg function.

Display scaffolds: protein engineering for novel therapeutics.

Protein scaffolds represent a new generation of universal binding frameworks for use as future immunopharmaceuticals to complement the expanding repertoire of therapeutic monoclonal antibodies. Here, we review recent literature describing advances in protein scaffold development, including efforts to engineer the minimal immunoglobulin-based binding-domain and molecular library design. Several diverse protein folds are currently under development on the basis of modular construction, a strategy also observed in families of naturally evolved immune receptors. We describe potential therapeutic and intracellular applications where scaffold-specific features provide distinct advantages for targeting of non-conventional antigens and comment on the scientific progress and validation of several designed scaffolds in the voyage towards first-in-human trials.

Inclusion formation by ataxins -1, -2, -3, and -7.

The authors studied inclusion formation in vitro using transiently transfected PC12 cells, with epitope-tagged and untagged full-length and truncated wild-type and expanded ataxins -1, -2, -3, and -7. At 72 hours, no inclusions were seen with wild-type full-length or truncated ataxins -2, -3, or -7, and only one with ataxin-1. Truncation abolished nuclear localization of ataxins -1 and -7, and allowed nuclear entry of ataxin-2. Of the expanded ataxins, only -1 and -2 formed inclusions, and those of ataxin-2 were rare and exclusively cytoplasmic. Truncation resulted in inclusion formation by ataxins -3 and -7, increased ataxin-1 inclusions, and enabled formation of nuclear ataxin-2 inclusions. There was no recruitment of wild-type ataxin-1 to expanded ataxin-1 inclusions.

Different ataxin-2 antibodies display different immunoreactive profiles.

We have developed a monoclonal antibody (4A7) directed against the C-terminus of the ataxin-2 protein that is involved in the polyglutamine neurodegenerative disorder spinocerebellar ataxia type 2. Comparison with other ataxin-2 antibodies showed that 4A7 specifically recognized ataxin-2. In contrast, a previously reported ataxin-2 antibody (15F6) did not appear to recognize full-length ataxin-2 in our systems. Immunocytochemical and subcellular fractionation studies using 4A7 confirmed previous reports that ataxin-2 is localized to both the cytoplasm and the trans-Golgi network in rat PC12 cells and rat brain tissue. In contrast, 4A7 failed to label the trans-Golgi network in the three primate cell lines examined. Cytoplasmic ataxin-2 was not associated with mitochondria, lysosomes, endoplasmic reticulum, peroxisomes, proteasomes, clathrin-coated pits or vesicles, or F-actin. Ataxin-2 was found to be phosphorylated but not glycosylated, and exhibited an estimated half-life of not less than 21 h. Interestingly, another commercially available ataxin-2 antibody did not detect ataxin-2 localized to the trans-Golgi network. This antibody was also found to immunoprecipitate fewer proteins/protein partners than 4A7. Although cross-reactivity of the 4A7 antibody with other protein(s) cannot be ruled out, it appears likely that the interaction of ataxin-2 with other cell components is dependent on both the host cell type and its subsequent subcellular localization.