Versatile performance edges of HBsAg Next assay in diagnosis and therapeutic monitoring of HBV infection.

BACKGROUND: HBsAg Next assay (HBsAgNx) claims improved detection of HBsAg. The aim was to investigate its performance in ascertaining HBsAg loss, ability to detect HBsAg in various phases of HBV infection, specificity and its amenability to in-house neutralization. METHODS: Analytical sensitivity was investigated using NIBSC standard (3rd WHO-IS). For clinical performance, out of 91,962 samples tested for HBsAg (Qual-II), 512 samples consisting of 170 cases with evidence of HBsAg loss during treatment (n = 116) and without treatment (n = 54), acute-hepatitis B (n = 90) and acute exacerbation of chronic-hepatitis B (n = 41), acute-hepatitis A (n = 24) and acute-hepatitis E (n = 9) positive, HIV-1 positive (n = 20), non-HBV, HAV and HEV related acute-hepatitis (n = 81) and HBsAg prozone (n = 14) as well as in-house neutralization (n = 63) were included. RESULTS: The calculated limit of detection (LOD) was 0.004 IU/mL. Of the 170 patients with apparent HBsAg loss, 18/116 (15.5%) among treated and 15/54 (27.7%) with spontaneous clearance were positive in HBsAgNx (p < 0.0001). Additionally, it detected HBsAg in 12/95 (12.6%) and 6/34 (17.6%) patients who were HBV DNA negative in treatment experienced and spontaneous clearance groups respectively (p < 0.001). The specificity of HBsAgNx was comparable to HBsAg Qual-II. The signal-intensity of HBsAgNx was significantly higher than HBsAg Qual-II across various phases of HBV infection and prozone samples. CONCLUSION: HBsAgNx significantly enhanced the accuracy of HBsAg detection without compromising the specificity in ascertaining HBsAg loss. The performance was superior in various phases of HBV infection including samples that exhibited prozone effect. Furthermore, it is amenable to cost-effective in-house neutralization to confirm low HBsAg levels.

Quantitative assessment of LPS-HBsAg interaction by introducing a novel application of immunoaffinity chromatography.

Lipopolysaccharide (LPS), as a stubborn contamination, should be monitored and kept in an acceptable level during the pharmaceutical production process. Recombinant hepatitis B surface antigen (r-HBsAg) is one of the recombinant biological products, which is probable to suffer from extrinsic endotoxin due to its long and complex production process. This research aims to assess the potential interaction between LPS and r-HBsAg by recruiting immunoaffinity chromatography (IAC) as a novel tool to quantify the interaction. Molecular modeling was performed on the HBsAg molecule to theoretically predict its potential binding and interaction sites. Then dynamic light scattering (DLS) analysis was implemented on HBsAg, LPS, and mixtures of them to reveal the interaction. The virus-like particle (VLP) structure of HBsAg and the ribbon-like structure of LPS were visualized by transmission electron microscopy (TEM). Finally, the interaction was quantified by applying various LPS/HBsAg ratios ranging from 1.67 to 120 EU/dose in the IAC. Consequently, the LPS/HBsAg ratios in the eluate were measured from 1.67 to a maximum of 92.5 EU/dose. The results indicated that 77 to 100% of total LPS interacted with HBsAg by an inverse relationship to the incubated LPS concentration. The findings implied that the introduced procedure is remarkably practical in the quantification of LPS interaction with a target recombinant protein.

Release of hepatitis B virions is positively regulated by glucose-regulated protein 78 through direct interaction with preS1.

In this study, we investigated the mechanism of hepatitis B virus (HBV)-enveloped particle release. Specifically, we used preS1 as a bait protein to screen host proteins using mass spectroscopy, with the results of immunofluorescence, western blot, co-immunoprecipitation, isothermal titration calorimetry, and pull-down assays identifying glucose-regulated protein (GRP)78 as a specific target for preS1 binding. We employed transcriptome sequencing, enzyme-linked immunosorbent assays, and particle gel assays to investigate the mechanism of GRP78-mediated positive regulation of HBV-enveloped particle release. Additionally, we performed phage-display, surface plasmon resonance, and molecular-docking assays to assess peptides inhibiting enveloped-particle release. We found that HBV upregulated GRP78 expression in liver cell lines and the serum of patients with chronic hepatitis B. Furthermore, GRP78 promoted the release of HBV-enveloped particles in vitro and in vivo within an HBV transgenic mouse model. Moreover, we identified interactions of preS1 peptides with GRP78 via hydrogen bonding and hydrophobic interactions, which effectively inhibited its interaction with HBV-enveloped particles and their subsequent release. These findings provide novel insights regarding HBV virion release, and demonstrated that GRP78 interacted with preS1 to positively regulate the release of HBV-enveloped particles, suggesting GRP78 as a potential therapeutic target for inhibiting HBV infection.

[Neutralization of hepatitis B virus with vaccine-escape mutations by novel hepatitis B vaccine with large-HBs antigen].

Although the current hepatitis B (HB) vaccine comprising yeast-derived small hepatitis B surface antigen (HBsAg) is potent and safe and used worldwide, specific concerns should not be ignored, such as the attenuated prophylaxis against hepatitis B virus (HBV) infection with specific amino acid polymorphisms, called vaccine-escape mutations (VEMs). We investigated a novel HB vaccine consisting of large-HBsAg that covers the shortcomings of the current HB vaccine in a nonhuman primate model. The yeast-derived large-HBsAg was mixed with the adjuvant and used to immunize rhesus macaques, and the induction of antibodies to HBsAg was compared with that of the current HB vaccine. The current HB vaccine predominantly induced antibodies to small-HBsAg, whereas immunization with the large-HBsAg vaccine mainly induced antibodies to the preS1 region. Although the antibodies induced by the current HB vaccine could not prevent infection of HBV with VEMs, the large-HBsAg vaccine-induced antibodies neutralized infection of HBV with VEMs at levels similar to those of the wild type. The HBV genotypes that exhibited attenuated neutralization by induced antibodies differed between these vaccines. In conclusion, the novel HB vaccine consisting of large-HBsAg was revealed to be useful to compensate for shortcomings of the current HB vaccine. The combined use of these HB vaccines may be able to induce antibodies that can neutralize HBV strains with VEMs or multiple HBV genotypes.

Binding of Hepatitis B Virus Pre-S1 Domain-Derived Synthetic Myristoylated Peptide to Scavenger Receptor Class B Type 1 with Differential Properties from Sodium Taurocholate Cotransporting Polypeptide.

(1) Background: The myristoylated pre-S1 peptide (Myr47) synthesized to mimic pre-S1 domain (2-48) in large (L) surface protein of hepatitis B virus (HBV) prevents HBV infection to hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP). We previously demonstrated that yeast-derived nanoparticles containing L protein (bio-nanocapsules: BNCs) bind scavenger receptor class B type 1 (SR-B1). In this study, we examined the binding of Mry47 to SR-B1. (2) Methods: The binding and endocytosis of fluorescence-labeled Myr47 to SR-B1 (and its mutants)-green fluorescence protein (GFP) fusion proteins expressed in HEK293T cells were analyzed using flow cytometry and laser scanning microscopy (LSM). Various ligand-binding properties were compared between SR-B1-GFP and NTCP-GFP. Furthermore, the binding of biotinylated Myr47 to SR-B1-GFP expressed on HEK293T cells was analyzed via pull-down assays using a crosslinker and streptavidin-conjugated beads. (3) Conclusions: SR-B1 bound not only Myr47 but also its myristoylated analog and BNCs, but failed to bind a peptide without myristoylation. However, NTCP only bound Myr47 among the ligands tested. Studies using SR-B1 mutants suggested that both BNCs and Myr47 bind to similar sites of SR-B1. Crosslinking studies indicated that Myr47 binds preferentially SR-B1 multimer than monomer in both HEK293T and HepG2 cells.

Virus-Like Particles Containing the E2 Core Domain of Hepatitis C Virus Generate Broadly Neutralizing Antibodies in Guinea Pigs.

A vaccine to prevent hepatitis C virus (HCV) infection is urgently needed for use alongside direct-acting antiviral drugs to achieve elimination targets. We have previously shown that a soluble recombinant form of the glycoprotein E2 ectodomain (residues 384 to 661) that lacks three variable regions (Δ123) is able to elicit a higher titer of broadly neutralizing antibodies (bNAbs) than the parental form (receptor-binding domain [RBD]). In this study, we engineered a viral nanoparticle that displays HCV glycoprotein E2 on a duck hepatitis B virus (DHBV) small surface antigen (S) scaffold. Four variants of E2-S virus-like particles (VLPs) were constructed: Δ123-S, RBD-S, Δ123A7-S, and RBDA7-S; in the last two, 7 cysteines were replaced with alanines. While all four E2-S variant VLPs display E2 as a surface antigen, the Δ123A7-S and RBDA7-S VLPs were the most efficiently secreted from transfected mammalian cells and displayed epitopes recognized by cross-genotype broadly neutralizing monoclonal antibodies (bNMAbs). Both Δ123A7-S and RBDA7-S VLPs were immunogenic in guinea pigs, generating high titers of antibodies reactive to native E2 and able to prevent the interaction between E2 and the cellular receptor CD81. Four out of eight animals immunized with Δ123A7-S elicited neutralizing antibodies (NAbs), with three of those animals generating bNAbs against 7 genotypes. Immune serum generated by animals with NAbs mapped to major neutralization epitopes located at residues 412 to 420 (epitope I) and antigenic region 3. VLPs that display E2 glycoproteins represent a promising vaccine platform for HCV and could be adapted to large-scale manufacturing in yeast systems. IMPORTANCE There is currently no vaccine to prevent hepatitis C virus infection, which affects more than 71 million people globally and is a leading cause of progressive liver disease, including cirrhosis and cancer. Broadly neutralizing antibodies that recognize the E2 envelope glycoprotein can protect against heterologous viral infection and correlate with viral clearance in humans. However, broadly neutralizing antibodies are difficult to generate due to conformational flexibility of the E2 protein and epitope occlusion. Here, we show that a VLP vaccine using the duck hepatitis B virus S antigen fused to HCV glycoprotein E2 assembles into virus-like particles that display epitopes recognized by broadly neutralizing antibodies and elicit such antibodies in guinea pigs. This platform represents a novel HCV vaccine candidate amenable to large-scale manufacture at low cost.

Multiple epitopes of hepatitis B virus surface antigen targeted by human plasma-derived immunoglobulins coincide with clinically observed escape mutations.

Hepatitis B immune globulin (HBIG) is a human plasma-derived immunoglobulin G concentrate that contains a high titer of neutralizing antibodies (anti-HBs) to the hepatitis B virus (HBV) surface antigen (HBsAg). HBIG is known to be highly effective in treating HBV infections, however, a more systematic characterization of the antibody binding sites on HBsAg and their correlation with emerging "escape" mutations in HBsAg was lacking. By using anti-HBs antibodies from HBIG lots to screen random peptide phage display libraries, we identified five clusters of peptides that corresponded to five distinct anti-HBs binding sites on the HBsAg. Three sites, Site II (C121-C124), Site III (M133-P135), and Site IV (T140-G145), were mapped within the "a" determinant, while the two other sites, Site I (Q101-M103) and Site V (I152-S154), were outside the "a" determinant. We then tested in binding assays HBsAg peptides containing clinically relevant mutations previously reported within these sites, such as Y134S, P142S, and G145R, and observed a significant reduction in anti-HBs binding activity to the mutated sites, suggesting a mechanism the virus may use to avoid HBIG-mediated neutralization. The current HBIG treatment could be improved by supplementing it with site-specific neutralizing monoclonal antibodies that target these mutations for control of HBV infections.

High prevalence of tryptophan-truncated S quasispecies in treatment-naïve chronic hepatitis B patients.

Hepatitis B virus surface antigen (HBsAg) encoded by the S gene is highly expressed during the replication cycle of hepatitis B virus (HBV). However, the frequent usage of tryptophan in HBsAg, which leads to a high cost of biosynthesis, is inconsistent with the high expression level of this protein. Tryptophan-truncated mutation of HBsAg, that is, a tryptophan to stop codon mutation resulting in truncated HBsAg, might help to maintain its high expression with lower biosynthetic cost. We aimed to investigate the prevalence of tryptophan-truncated S quasispecies in treatment-naïve patients with chronic hepatitis B (CHB) by applying CirSeq as well as a site-by-site algorithm developed by us to identify variants at extremely low frequencies in the carboxyl terminus of HBsAg. A total of 730 mutations were identified in 27 patients with CHB, varying from seven to 56 mutations per sample. The number of synonymous mutations was much higher than that of nonsynonymous mutations in the reverse transcriptase (RT) coding region and vice versa in the S coding region, implying that the evolutionary constraints on the RT and S genes might be different. We showed that 25 (92.6 %) of 27 patients had at least one S-truncated mutation, most of which were derived from tryptophan, indicating a high prevalence of tryptophan-truncated S mutations in treatment-naïve patients with CHB. In terms of the RT gene, 21 (77.8 %) patients had pre-existing drug-resistant mutations, while no truncated mutations were detected. Our findings that tryptophan-truncated S quasispecies and drug-resistant RT mutants were highly prevalent in treatment-naïve patients with CHB provide new insights into the composition of the HBV population, which might help optimize the treatment and management of patients with CHB.

A novel piperazine derivative that targets hepatitis B surface antigen effectively inhibits tenofovir resistant hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is a global problem. The loss of hepatitis B surface antigen (HBsAg) in serum is a therapeutic end point. Prolonged therapy with nucleoside/nucleotide analogues targeting the HBV-polymerase may lead to resistance and rarely results in the loss of HBsAg. Therefore, inhibitors targeting HBsAg may have potential therapeutic applications. Here, we used computational virtual screening, docking, and molecular dynamics simulations to identify potential small molecule inhibitors against HBsAg. After screening a million molecules from ZINC database, we identified small molecules with potential anti-HBV activity. Subsequently, cytotoxicity profiles and anti-HBV activities of these small molecules were tested using a widely used cell culture model for HBV. We identified a small molecule (ZINC20451377) which binds to HBsAg with high affinity, with a KD of 65.3 nM, as determined by Surface Plasmon Resonance spectroscopy. Notably, the small molecule inhibited HBsAg production and hepatitis B virion secretion (10 µM) at low micromolar concentrations and was also efficacious against a HBV quadruple mutant (CYEI mutant) resistant to tenofovir. We conclude that this small molecule exhibits strong anti-HBV properties and merits further testing.

HBV Pre-S1-Derived Myristoylated Peptide (Myr47): Identification of the Inhibitory Activity on the Cellular Uptake of Lipid Nanoparticles.

The Myr47 lipopeptide, consisting of hepatitis B virus (HBV) pre-S1 domain (myristoylated 2-48 peptide), is an effective commercialized anti-HBV drug that prevents the interaction of HBV with sodium taurocholate cotransporting polypeptide (NTCP) on human hepatocytes, an activity which requires both N-myristoylation residue and specific amino acid sequences. We recently reported that Myr47 reduces the cellular uptake of HBV surface antigen (HBsAg, subviral particle of HBV) in the absence of NTCP expression. In this study, we analyzed how Myr47 reduces the cellular uptake of lipid nanoparticles (including liposomes (LPs) and HBsAg) without NTCP expression. By using Myr47 mutants lacking the HBV infection inhibitory activity, they could reduce the cellular uptake of LPs in an N-myristoylation-dependent manner and an amino acid sequence-independent manner, not only in human liver-derived cells but also in human non-liver-derived cells. Moreover, Myr47 and its mutants could reduce the interaction of LPs with apolipoprotein E3 (ApoE3) in an N-myristoylation-dependent manner regardless of their amino acid sequences. From these results, lipopeptides are generally anchored by inserting their myristoyl residue into the lipid bilayer and can inhibit the interaction of LPs/HBsAg with apolipoprotein, thereby reducing the cellular uptake of LPs/HBsAg. Similarly, Myr47 would interact with HBV, inhibiting the uptake of HBV into human hepatic cells, while the inhibitory effect of Myr47 may be secondary to its ability to protect against HBV infection.

Performance of agarose and gigaporous chromatographic media as function of pore-to-adsorbate size ratio over wide span from ovalbumin to virus like particles.

Two commercially available agarose ion exchange media, DEAE-Capto and DEAE-Sepharose FF (DEAE-FF), and two gigaporous media DEAE -AP-120 nm and DEAE-AP-280 nm were evaluated for their applicability in adsorption of five proteins with large span of radius ranges from 2.9 nm to 14.1 nm, which include ovalbumin, bovine serum albumin (BSA), haptoglobin, thyroglobulin and hepatitis B surface antigen (HBsAg) virus like particle. The average pore radius of the four media was determined to be 6.9 nm, 18.5 nm, 59.4 nm and 139.3 nm, respectively, which was obtained by log normal distribution for DEAE-Capto and DEAE-FF and by bimodal Gaussian distribution for the two DEAE-AP media. The performance of these four media including phase ratio, static and dynamic binding capacity, and transport properties for the adsorption of these five model proteins as function of pore-to-adsorbate size ratio were investigated and compared. The best ratio of pore-to-adsorbate size was found dependent on the protein size. For protein with radius from 2.9 nm (ovalbumin) to 5.4 nm (BSA), the agarose media was superior to gigaporous media. Both the static and dynamic adsorption capacities reduced with the increase of pore size, and the highest values were obtained at the smallest pore-to-adsorbate size of about 2 times in this study, although the highest accessible surface area was obtained at pore-to-adsorbate size ratio about 16 to 20. For proteins with radius of 5.4 nm or larger than that, their adsorption capacities decreased firstly and then increased with the increase of ratio of pore-to-adsorbate size, and the highest values were obtained on the gigaporous media DEAE-AP-280 nm, which could provide faster diffusivity and larger accessible surface area. However, protein with radius of 14.1 nm (HBsAg) had much lower capacities compared to other proteins at the same pore-to-adsorbate size ratio, implying large protein needs greater pore-to-adsorbate size ratio to achieve a satisfactory capacity. For all the five tested proteins, the DEAE-Capto media having the smallest pore radius and branched dextran chains, was found superior to DEAE-FF in terms of both higher adsorption capacities and uptake kinetics, which suggested that the "chain delivery effect" took place on proteins over large size span from ovalbumin to HBsAg, though the effect on the larger proteins was much less significant than that on the smaller ones. Results from the present work provided more information on how do the relationships of pore size of chromatography media and adsorbate size interactively affect the chromatography behaviors, thus will provide general guidance for selection of suitable adsorbent for biologics of a given size.

Robust Immune Responses Elicited by a Hybrid Adjuvant Based on ß-Glucan Particles from Yeast for the Hepatitis B Vaccine.

The use of particulate adjuvants offers an interesting method for enhancing and modulating the immune responses elicited by vaccines. Aluminum salt (Alum) is one of the most important immune adjuvants approved by the Food and Drug Administration for use in humans because of its safety and efficacy, but it lacks the capacity to induce strong cellular and mucosal immune responses. In this study, we designed an antigen delivery system that combines aluminum salts with ß-glucan particles. The ß-glucan-aluminum particles (GP-Al) exhibited a highly uniform size of 2-4 µm and could highly specifically target antigen-presenting cells (APCs) and strongly induce dendritic cell (DC) maturation and cytokine secretion. In vivo studies showed that both WT mice and HBV-Tg mice immunized with hepatitis B surface antigen (HBsAg)-containing GP-Al displayed high anti-HBsAg IgG titers in the serum. Furthermore, in contrast to mice receiving the antigen alone, mice immunized with the particulate adjuvant exhibited IgG2a antibody titers and higher antigen-specific IFN-γ levels in splenocytes. In conclusion, we developed GP-Al microspheres to serve as a hepatitis B vaccine to enhance both humoral and cellular immune responses, representing a safe and promising system for antigen delivery.

A sandwich-type electrochemical immunosensor based on Au@Pd nanodendrite functionalized MoO2 nanosheet for highly sensitive detection of HBsAg.

In this work, a sandwich-type electrochemical immunosensor was fabricated to the effective detection of hepatitis B surface antigen (HBsAg). The designed electrochemical immunosensor was based on Au core and Pd shell nanodendrites loaded on amino functionalized molybdenum dioxide nanosheets (Au@Pd NDS/NH2-MoO2 NSs) as the secondary antibody (Ab2) label and silver nanoparticles were loaded by electrodeposited (D-Ag NPs) on the surface of electrode as the platform. Because of the synergistic effect and abundant catalytic activity sites provided by surface dendrite structure, Au@Pd NDs were more effective than single gold and palladium nanoparticles in catalytic reduction of hydrogen peroxide (H2O2). MoO2 had the good catalytic capacity for reduction of H2O2 and favourable electrical conductivity. Hence, the obtained Au@Pd NDS/NH2-MoO2 NSs were more effective than Au@Pd NDs and NH2-MoO2 NSs in catalytic reduction of hydrogen peroxide attribute to a synergistic effect. Also, Ag NPs with admirable electrical conductivity and biocompatibility were used as sensing platforms and primary antibodies (Ab1) carriers, which can accelerate the electron transfer and improve the sensitivity of the immunosensor. Here, the proposed electrochemical immunosensor offered a wide linear interval from 10 fg mL-1 to 100 ng mL-1 and the lower limit of detection of 3.3 fg mL-1 (S/N = 3) for detection of HBsAg under optimal experimental conditions. Furthermore, the accuracy of the actual serum sample analysis was satisfactory, which showed that the electrochemical immunosensor possessed a good application prospect in clinical detection.

Expression Level of Small Envelope Protein in Addition to Sequence Divergence inside Its Major Hydrophilic Region Contributes to More Efficient Surface Antigen Secretion by Hepatitis B Virus Subgenotype D2 than Subgenotype A2.

Hepatitis B surface antigen (HBsAg) promotes persistent hepatitis B virus (HBV) infection. It primarily corresponds to small (S) envelope protein secreted as subviral particles. We previously found that genotype D clones expressed less S protein than genotype A clones but showed higher extracellular/intracellular ratio of HBsAg suggesting more efficient secretion. The current study aimed to characterize the underlying mechanism(s) by comparing a subgenotype A2 clone (geno5.4) with a subgenotype D2 clone (geno1.2). Five types of full-length or subgenomic constructs were transfected to Huh7 cells at different dosage. HBsAg was quantified by enzyme linked immunosorbent assay while envelope proteins were detected by Western blot. We found that ratio of extracellular/intracellular HBsAg decreased at increasing amounts of DNA transfected. Conflicting findings from two types of subgenomic construct confirmed stronger secretion inhibitory effect of the genotype D-derived large envelope protein. Chimeric constructs followed by site-directed mutagenesis revealed geno1.2 specific V118/T127 and F161/A168 in the S protein as promoting and inhibitory of HBsAg secretion, respectively. In conclusion, more efficient HBsAg secretion by subgenotype D2 than subgenotype A2 is attributed to lower level of S protein expression in addition to V118 and T127 in S protein, although its F161 and A168 sequences rather reduce HBsAg secretion.

A 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.

Lipopolysaccharide derived alginate coated Hepatitis B antigen loaded chitosan nanoparticles for oral mucosal immunization.

Mucosal administration of vaccine can produce a strong immune response. Antigens adhere to "M-cells", present at the intestinal mucosa and the M-cells produce immunity after actively transporting luminal antigens to the underlying immune cells. The objective of the present study was to prepare and characterize alginate coated chitosan nanoparticles (ACNPs) loaded with HBsAg as an antigen to produce immunity; additionally anchored with lipopolysaccharide (LPS) as an adjuvant. Ionic gelation method was used to prepare chitosan nanoparticles (CNPs) which were loaded with HBsAg and stabilized by alginate coating to protect from gastric environment. Results showed that the prepared LPS-HB-ACNPs were small and spherical with mean particle size 605.23 nm, polydispersity index 0.234 and Zeta potential -26.2 mV and could effectively protect antigen at GIT in acidic medium. HB-ANCPs were stable during storage at 4 ± 1 and 27 ± 2 °C. Anchoring with LPS showed increased immunity as compared to other formulations. Additionally, NPs elicited significant sIgA at mucosal secretions and IgG antibodies in systemic circulation. Thus, the prepared LPS anchored alginate coated chitosan NPs may be a promising approach as a vaccine delivery system for oral mucosal immunization.

A Label-Free Immunosensor Based on Graphene Oxide/Fe3O4/Prussian Blue Nanocomposites for the Electrochemical Determination of HBsAg.

In this article, a highly sensitive label-free immunosensor based on a graphene oxide (GO)/Fe3O4/Prussian blue (PB) nanocomposite modified electrode was developed for the determination of human hepatitis B surface antigen (HBsAg). In this electrochemical immunoassay system, PB was used as a redox probe, while GO/Fe3O4/PB nanocomposites and AuNPs were prepared and coated on screen-printed electrodes to enhance the detection sensitivity and to immobilize the hepatitis B surface antibody (HBsAb). The immunosensor was fabricated based on the principle that the decrease in peak currents of PB is proportional to the concentration of HBsAg captured on the modified immunosensor. The experimental results revealed that the immunosensor exhibited a sensitive response to HBsAg in the range of 0.5 pg·mL-1 to 200 ng·mL-1, and with a low detection limit of 0.166 pg·mL-1 (S/N = 3). Furthermore, the proposed immunosensor was used to detect several clinical serum samples with acceptable results, and it also showed good reproducibility, selectivity and stability, which may have a promising potential application in clinical immunoassays.

A Hyper-Glycosylation of HBV Surface Antigen Correlates with HBsAg-Negativity at Immunosuppression-Driven HBV Reactivation in Vivo and Hinders HBsAg Recognition in Vitro.

Immune-suppression driven Hepatitis B Virus (HBV)-reactivation poses serious concerns since it occurs in several clinical settings and can result in severe forms of hepatitis. Previous studies showed that HBV strains, circulating in patients with HBV-reactivation, are characterized by an enrichment of immune-escape mutations in HBV surface antigen (HBsAg). Here, we focused on specific immune-escape mutations associated with the acquisition of N-linked glycosylation sites in HBsAg (NLGSs). In particular, we investigated profiles of NLGSs in 47 patients with immunosuppression-driven HBV-reactivation and we evaluated their impact on HBsAg-antigenicity and HBV-replication in vitro. At HBV-reactivation, despite a median serum HBV-DNA of 6.7 [5.3-8.0] logIU/mL, 23.4% of patients remained HBsAg-negative. HBsAg-negativity at HBV-reactivation correlated with the presence of >1 additional NLGSs (p < 0.001). These NLGSs are located in the major hydrophilic region of HBsAg (known to be the target of antibodies) and resulted from the single mutation T115N, T117N, T123N, N114ins, and from the triple mutant S113N+T131N+M133T. In vitro, NLGSs strongly alter HBsAg antigenic properties and recognition by antibodies used in assays for HBsAg-quantification without affecting HBsAg-secretion and other parameters of HBV-replication. In conclusion, additional NLGSs correlate with HBsAg-negativity despite HBV-reactivation, and hamper HBsAg-antigenicity in vitro, supporting the role of NGSs in immune-escape and the importance of HBV-DNA for a proper diagnosis of HBV-reactivation.

Antigenic and physicochemical characterization of Hepatitis B surface protein under extreme temperature and pH conditions.

Hepatitis B virus causes acute and chronic infections in millions of people worldwide and, since 1982, a vaccine with 95% effectiveness has been available for immunization. The main component of the recombinant hepatitis B vaccine is the surface antigen protein (HBsAg). In this work, the effect of pH, ionic strength and temperature on the native state of the HBsAg antigen were studied by a combination of biophysical methods that included small angle X-ray scattering, synchrotron radiation circular dichroism, fluorescence and surface plasmon resonance spectroscopies, as well as in vivo and in vitro potency assays. The native conformation, morphology, radius of gyration, and antigenic properties of the HBsAg antigen demonstrate high stability to pH treatment, especially in the pH range employed in all stages of HBsAg vaccine production and storage. The HBsAg protein presents thermal melting point close to 56 °C, reaching a more unfolded state after crossing this point, but it only experiences loss of vaccine potency and antigenic properties at 100 °C. Interestingly, a 6-month storage period does not affect vaccine stability, and the results are similar when the protein is kept under refrigerated conditions or at room temperature (20 °C). At frozen temperatures, large aggregates (>200 nm) are formed and possibly cause loss of HBsAg content, but that does not affect the in vivo assay. Furthermore, HBsAg has a well-ordered secondary structure content that is not affected when the protein is formulated with silica SBA-15, targeting the oral delivery of the vaccine. The combined results from all the characterization techniques employed in this study showed the high stability of the antigen at different storage temperature and extreme values of pH. These findings are important for considering the delivery of HBsAg to the immune system via an oral vaccine.

The modulation of HBsAg level by sI126T is affected by additional amino acid substitutions in the S region of HBV.

The hepatitis B surface antigen (HBsAg) is a vital serum marker for hepatitis B virus (HBV) infection. Amino acid (AA) substitutions in small hepatitis B surface protein (SHBs) are known to affect HBsAg level. However, how the genetic backbones of SHBs sequences would affect the roles of a specific AA substitution on HBsAg level remains unclear. In this study, we found that sI126 had a very high substitution detection rate of 17.54% (40/228) in untreated chronic hepatitis B cohort with subgenotype C2 HBV infection. Among different substitution types at sI126, the sI126T (N = 28) was found to be associated with significantly lower serum HBsAg level. Clone sequencing revealed that sI126T-harboring SHBs sequences had varied genetic backbones with zero to nine additional AA substitutions. Thus, we constructed 24 HBsAg expression plasmids harboring sI126T without (plasmid 1, P1) or with (P2-P24) additional AA substitution(s) and studied them in the HepG2 cells. The HBsAg levels were determined by both ELISA and Western blot. In vitro experiments showed that P1 significantly reduced HBsAg level and its secretion (p < .05), however, P2-P24 showed various extracellular and intracellular HBsAg levels. No significant differences were detected among the HBsAg mRNA levels of nine representative mutant plasmids. Our findings suggest that the modulation of HBsAg level by sI126T is affected by additional AA substitution(s) in the S region of HBV. The effects of AA combination substitutions in SHBs sequences on HBsAg levels are worthwhile for more attentions in terms of HBV biology and its clinical application.