Silicon or Calcium Doping Coordinates the Immunostimulatory Effects of Aluminum Oxyhydroxide Nanoadjuvants in Prophylactic Vaccines.

Aluminum salts still remain as the most popular adjuvants in marketed human prophylactic vaccines due to their capability to trigger humoral immune responses with a good safety record. However, insufficient induction of cellular immune responses limits their further applications. In this study, we prepare a library of silicon (Si)- or calcium (Ca)-doped aluminum oxyhydroxide (AlOOH) nanoadjuvants. They exhibit well-controlled physicochemical properties, and the dopants are homogeneously distributed in nanoadjuvants. By using Hepatitis B surface antigen (HBsAg) as the model antigen, doped AlOOH nanoadjuvants mediate higher antigen uptake and promote lysosome escape of HBsAg through lysosomal rupture induced by the dissolution of the dopant in the lysosomes in bone marrow-derived dendritic cells (BMDCs). Additionally, doped nanoadjuvants trigger higher antigen accumulation and immune cell activation in draining lymph nodes. In HBsAg and varicella-zoster virus glycoprotein E (gE) vaccination models, doped nanoadjuvants induce high IgG titer, activations of CD4+ and CD8+ T cells, cytotoxic T lymphocytes, and generations of effector memory T cells. Doping of aluminum salt-based adjuvants with biological safety profiles and immunostimulating capability is a potential strategy to mediate robust humoral and cellular immunity. It potentiates the applications of engineered adjuvants in the development of vaccines with coordinated immune responses.

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.

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.

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.

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.

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.

Molecular Evaluation of Oral Immunogenicity of Hepatitis B Antigen Delivered by Hydrogel Microparticles.

The development of oral vaccine formulation is crucial to facilitate an effective mass immunization program for various vaccine-preventable diseases. In this work, the efficacy of hepatitis B antigen delivered by bacterial nanocellulose/poly(acrylic acid) composite hydrogel microparticles (MPs) as oral vaccine carriers was assessed to induce both local and systemic immunity. Optimal pH-responsive swelling, mucoadhesiveness, protein drug loading, and drug permeability were characterized by MPs formulated with minimal irradiation doses and acrylic acid concentration. The composite hydrogel materials of bacterial nanocellulose and poly(acrylic acid) showed significantly greater antigen release in simulated intestinal fluid while ensuring the integrity of antigen. In in vivo study, mice orally vaccinated with antigen-loaded hydrogel MPs showed enhanced vaccine immunogenicity with significantly higher secretion of mucosal immunoglobulin A, compared to intramuscular vaccinated control. The splenocytes from the same group demonstrated lymphoproliferation and significant increased secretion of interleukin-2 cytokines upon stimulation with hepatitis B antigen. Expression of CD69 in CD4+ T lymphocytes and CD19+ B lymphocytes in splenocytes from mice orally vaccinated with antigen-loaded hydrogel MPs was comparable to that of the intramuscular vaccinated control, indicating early activation of lymphocytes elicited by our oral vaccine formulation in just two doses. These results demonstrated the potential of antigen-loaded hydrogel MPs as an oral vaccination method for hepatitis B.

PreS1 peptide-functionalized gold nanostructures with SERRS tags for efficient liver cancer cell targeting.

Early detection is the most effective mean of improving prognosis for many fatal diseases such as cancer. In this context, the Surface Enhanced Resonance Raman Scattering (SERRS) technique is being proposed as alternative to fluorescent methods in detection of biomarkers, because SERRS nanostructures are bright as fluorescent tags but more stable and clearly detectable using the narrow Raman "fingerprints" of a suitable reporter. Here we show that biocompatible SERRS active gold nanostructures, functionalized with an engineered PreS1 peptide (AuNP@PEG-PreS1), detect the presence of the SerpinB3 antigen overexpressed on liver tumor cells, a biomarker of the onset of liver cell carcinomatous transformation. A proper engineering of the targeting unit, linked to the nanostructure by a polymer chain, affords a sensitivity and specificity larger than 80%, at subnanomolar concentrations. Taking into account the high sensitivity of SERRS and that SB3 overexpression is an early event in liver cell carcinomatous transformation, AuNP@PEG-PreS1 nanostructures could be used in routine diagnostic activities, to improve the accuracy of HCC detection in particular in patients with chronic liver diseases.

Glucan Particles Are a Powerful Adjuvant for the HBsAg, Favoring Antiviral Immunity.

The lack of vaccine adjuvants that are able to induce robust T cell responses fosters the search for more powerful options. Pathogen-like particles are a promising approach. The adjuvant activity of pathogen-like particles is highly influenced by size and surface composition. This study aimed to evaluate the adjuvant potential of two different ß-glucan-based particles, blend chitosan/ß-glucan particles (ChiGluPs), which are positively charged and have mean size of 1276 nm, and neutral yeast-derived glucan particles (GPs), with a mean size of 3 µm. Additionally, chitosan particles (ChiPs) were used to understand the effect of ß-glucan addition (ChiGluPs). Mouse spleen cells responded through the production of either TNF-α or RANTES, following in vitro stimulation with particles containing either ß-glucan (ChiGluPs and GPs) or chitosan (ChiGluPs and ChiPs). Human monocytes responded to all particles through TNF-α secretion. Subcutaneous vaccination of mice with the hepatitis B surface antigen (HBsAg) showed increased serum IgG for all particles compared to HBsAg alone (435-, 4500-, or 2500-fold increase for either ChiPs, ChiGluPs, or GPs). Interestingly, only GPs elicited the secretion of HBsAg-specific Th1, Th2, Th9, Th17, Th22, and Treg-related cytokines. This study demonstrates, for the first time, that GPs can have a significant role against the hepatitis B virus by favoring antiviral immunity.

HBs antigen and mannose loading on the surface of iron oxide nanoparticles in order to immuno-targeting: fabrication, characterization, cellular and humoral immunoassay.

Mannosylation of nanovaccine is an appropriate strategy for targeting the mannose receptors on DCs. Here, HBsAg and mannose loaded on the surface of iron oxide nanoparticles to increases HBsAg vaccine potency. Nanoparticles are made by co-precipitation method and bonded to the HBsAg and mannose by chemical bonding. The physicochemical properties of nano-vaccines, their toxicity and antigenicity were determined. The synthesized nano-vaccine showed spherical shape with a mean particle size of 60 nm, a zeta potential of -44 mV, an antigen-binding efficiency of around 100% and for mannose 78%. In vitro release of nanoparticles exhibited about 30% at the first day and about 60% until the third day. SDSPAGE analysis confirmed structural integrity of HBsAg loaded on nanoparticles. The HBsAg-loaded LCMNP and MLCMNP nanoparticles had no toxic effects on HEK293 cell line. The quantification of the intracellular Fe by ICP-OES as a criterion of nano-vaccine uptake revealed mannose intensify uptake of MLCMNP. In addition, mannose in the structure of MLCMNP improved IL-6, TNF-α and IFN-γ (>16 fold) cytokines genes expression by macrophage/dendritic cells after exposure in 12 h. Immunization of experimental mice (subcutaneously, two times with 2-week intervals) with 5 µg of HBsAg loaded on MLCMNP nanoparticles increased specific total IgG and IgG2a/IgG1 ratio. In addition, TNF-α, IL-12, IL-2 and IL-4 cytokines in mannosylated nano-vaccine increased versus nano-vaccine group while lymphocyte proliferation and IFN-γ responses in the targeted nano-vaccine group show a tiny increase versus the nano-vaccine group. The results show that mannosylated nano-vaccine promotes higher level of cellular and humoural immune responses against HBsAg nano-vaccine.

Comparative purification and characterization of hepatitis B virus-like particles produced by recombinant vaccinia viruses in human hepatoma cells and human primary hepatocytes.

This study describes the comparative expression and purification of hepatitis B surface antigen (HBsAg) particles produced upon infection of human primary hepatocytes and human hepatoma cell lines (HuH-7 and HepG2) with recombinant vaccinia viruses. The highest levels of HBsAg expression were found in HuH-7 hepatoma cells following infection with recombinant vaccinia viruses, which contain the S gene under control of a 7.5 k-promoter. Four different methods for purification of the HBsAg particles were examined: isopycnic ultracentrifugation, sucrose cushion sedimentation, isocratic column gel filtration, and binding to anti-HBs-coated microparticles. The highest degree of purity of HBsAg particles was reached by the method based on anti-HBs-coated microparticles. The resulting product was >98% pure. Biochemical analysis and characterization of purified HBsAg particles were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and electron microscopy. The HBsAg, purified from human hepatoma cell lines and from human primary hepatocytes, consisted of both the non-glycosylated (p25) and the glycosylated (gp27) form and assembled into typical 22-nm particles, and thus may be of great interest and importance for research, diagnostics, and medical treatments.

Polymeric nanoengineered HBsAg DNA vaccine designed in combination with ß­glucan.

Antigen-specific immune responses following DNA vaccination are hard to achieve, owing to the difficulty to mediate efficient gene delivery. This study proposed the use of PDMAEMA:PßAE/DNA polyplexes (Pol) as the vehicle of a pDNA vaccine encoding the hepatitis B surface antigen (HBsAg), with these Pol designed in combination with a soluble (Glu) or a particulate (GPs) form of ß­glucan. ß­Glucans are recognized adjuvants that activate immune cells, a good strategy to improve transfection efficiency and vaccine efficacy. Results showed that Pol produced at a 19:1 polymer:DNA (+/-) charge ratio were positively charged (+41 mV), had a mean size of 180 nm and presented high stability under different storage conditions. These polyplexes resulted in enhanced transfection activity than the positive control, showing even higher luciferase gene expression in the presence of GPs (COS-7 and RAW 264.7 cell lines). Additionally, no alterations in hemolysis and plasma coagulation time of human blood were found in the non-cytotoxic working range. Mice vaccination studies (pCMV-S), resulted in a seroconversion rate of 40%, regardless of the additional ß­glucan adjuvants. This work showed the potential of this nanosystem together with GPs to enhance in vitro transfection capacity and to be further studied as a DNA vaccination platform.

Generation and Characterization of a Neutralizing Human Monoclonal Antibody to Hepatitis B Virus PreS1 from a Phage-Displayed Human Synthetic Fab Library.

The hepatitis B virus (HBV) envelope contains small (S), middle (M), and large (L) proteins. PreS1 of the L protein contains a receptor-binding motif crucial for HBV infection. This motif is highly conserved among 10 HBV genotypes (A-J), making it a potential target for the prevention of HBV infection. In this study, we successfully generated a neutralizing human monoclonal antibody (mAb), 1A8 (IgG1), that recognizes the receptor-binding motif of preS1 using a phage-displayed human synthetic Fab library. Analysis of the antigen-binding activity of 1A8 for different genotypes indicated that it can specifically bind to the preS1 of major HBV genotypes (A-D). Based on Bio-Layer interferometry, the affinity (KD) of 1A8 for the preS1 of genotype C was 3.55 nM. 1A8 immunoprecipitated the hepatitis B virions of genotypes C and D. In an in vitro neutralization assay using HepG2 cells overexpressing the cellular receptor sodium taurocholate cotransporting polypeptide, 1A8 effectively neutralized HBV infection with genotype D. Taken together, the results suggest that 1A8 may neutralize the four HBV genotypes. Considering that genotypes A-D are most prevalent, 1A8 may be a neutralizing human mAb with promising potential in the prevention and treatment of HBV infection.

HDV Can Constrain HBV Genetic Evolution in HBsAg: Implications for the Identification of Innovative Pharmacological Targets.

Chronic HBV + HDV infection is associated with greater risk of liver fibrosis, earlier hepatic decompensation, and liver cirrhosis hepatocellular carcinoma compared to HBV mono-infection. However, to-date no direct anti-HDV drugs are available in clinical practice. Here, we identified conserved and variable regions in HBsAg and HDAg domains in HBV + HDV infection, a critical finding for the design of innovative therapeutic agents. The extent of amino-acid variability was measured by Shannon-Entropy (Sn) in HBsAg genotype-d sequences from 31 HBV + HDV infected and 62 HBV mono-infected patients (comparable for demographics and virological-parameters), and in 47 HDAg genotype-1 sequences. Positions with Sn = 0 were defined as conserved. The percentage of conserved HBsAg-positions was significantly higher in HBV + HDV infection than HBV mono-infection (p = 0.001). Results were confirmed after stratification for HBeAg-status and patients' age. A Sn = 0 at specific positions in the C-terminus HBsAg were correlated with higher HDV-RNA, suggesting that conservation of these positions can preserve HDV-fitness. Conversely, HDAg was characterized by a lower percentage of conserved-residues than HBsAg (p < 0.001), indicating higher functional plasticity. Furthermore, specific HDAg-mutations were significantly correlated with higher HDV-RNA, suggesting a role in conferring HDV replicative-advantage. Among HDAg-domains, only the virus-assembly signal exhibited a high genetic conservation (75% of conserved-residues). In conclusion, HDV can constrain HBsAg genetic evolution to preserve its fitness. The identification of conserved regions in HDAg poses the basis for designing innovative targets against HDV-infection.

Interaction of S17 Antibody with the Functional Binding Region of the Hepatitis B Virus Pre-S2 Epitope.

To understand the mechanism for inhibition of hepatitis B virus (HBV) infection is important. In this study, single-chain variable fragment (scFv) antibodies were generated and directed to the pre-S2 epitope of HBV surface antigen (HBsAg). These human scFvs were isolated from a person with history of HBV infection by phage display technology. An evaluation of panning efficiency revealed that the eluted phage titer was increased, indicating that specific clones were enriched after panning. Selected scFvs were characterized with the recombinant HBsAg through Western blotting and enzyme-linked immunosorbent assay to confirm the binding ability. Flow cytometry analysis and immunocytochemical staining revealed that one scFv, S17, could recognize endogenous HBsAg expressed on the HepG2215 cell membrane. Moreover, the binding affinity of scFv S17 to the pre-S2 epitope was determined to be 4.2 × 10-8 M. Two ion interactions were observed as the major driving forces for scFv S17 interacting with pre-S2 by performing a rational molecular docking analysis. This study provides insights into the structural basis to understand the interactions between an antibody and the pre-S2 epitope. The functional scFv format can potentially be used in future immunotherapeutic applications.

A virus-like particle of the hepatitis B virus preS antigen elicits robust neutralizing antibodies and T cell responses in mice.

The preS antigen of hepatitis B virus (HBV) corresponds to the N-terminal polypeptide in the large (L) antigen in addition to the small (S) antigen. The virus-like particle (VLP) of the S antigen is widely used as a vaccine to protect the population from HBV infection. The presence of the S antigen and its antibodies in patient blood has been used as markers to monitor hepatitis B. However, there is very limited knowledge about the preS antigen. We generated a preS VLP that is formed by a chimeric protein between preS and hemagglutinin (HA), and the matrix protein M1 of influenza virus. The HBV preS antigen is displayed on the surface of preS VLP. Asn112 and Ser98 of preS in VLP were found to be glycosylated and O-glycosylation of Ser98 has not been reported previously. The preS VLP shows a significantly higher immunogenicity than recombinant preS, eliciting robust anti-preS neutralizing antibodies. In addition, preS VLP is also capable of stimulating preS-specific CD8+ and CD4+ T cell responses in Balb/c mice and HBV transgenic mice. Furthermore, preS VLP immunization provided protection against hydrodynamic transfection of HBV DNA in mice. The data clearly suggest that this novel preS VLP could elicit robust immune responses to the HBV antigen, and can be potentially developed into prophylactic and therapeutic vaccines.

Plant-expressed Hepatitis B core antigen virus-like particles: Characterization and investigation of their stability in simulated and pig gastro-intestinal fluids.

Virus-like particles (VLPs) are potential oral vaccine candidates, as their highly compact structure may allow them to withstand the harsh conditions of the gastro-intestinal (GI) environment. Hepatitis B core antigen (HBcAg) is an immunogenic protein that assembles into 30 or 34nm diameter VLPs. Here, the stabilities of both the HBcAg polypeptide itself and the three-dimensional structure of the VLPs upon exposure to in vitro and ex vivo simulated gastric and intestinal fluids were investigated. Plant-expressed HBcAg VLPs were efficiently purified by sucrose density gradient and characterized. The purified VLPs did not show major chemical or physical instability upon exposure to the low pH conditions typically found in the stomach; however, they completely agglomerated upon acidification and subsequent pH neutralization. The HBcAg polypeptide was highly digested upon exposure to pepsin in simulated gastric fluids. HBcAg appeared more stable in both simulated and ex vivo intestinal fluids, where despite a partial digestion of the HBcAg polypeptide, the VLPs maintained their most immunogenic epitopes and their particulate conformation. These results suggest that HBcAg VLPs are likely to be unstable in gastric fluids, yet if the gastric instability could be bypassed, they could maintain their particulate structure and immunogenicity in intestinal fluids.

Synthesis of stable isotopically labeled peptides with filter-assisted enzymatic labeling for the diagnosis of hepatitis B virus infection utilizing mass spectrometry-based proteomics strategy.

A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic 16O/18O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol-1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per µl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease.

Research progress of therapeutic vaccines for treating chronic hepatitis B.

Hepatitis B virus (HBV) is a member of Hepadnavirus family, which leads to chronic infection in around 5% of patients with a high risk of developing liver cirrhosis, liver failure, and hepatocellular carcinoma. 1 Despite the availability of prophylactic vaccines against hepatitis B for over 3 decades, there are still more than 2 billion people have been infected and 240 million of them were chronic. Antiviral therapies currently used in the treatment of CHB (chronic hepatitis B) infection include peg-interferon, standard α-interferon and nucleos/tide analogs (NAs), but none of them can provide sustained control of viral replication. As an alternative strategy, therapeutic vaccines for CHB patients have been widely studied and showed some promising efficacies in dozens of preclinical and clinical trials. In this article, we review current research progress in several types of therapeutic vaccines for CHB treatment, including protein-based vaccines, DNA-based vaccines, live vector-based vaccines, peptide-based vaccines and cell-based therapies. These researches may provide some clues for developing new treatments in CHB infection.

Characterization of the disassembly and reassembly of the HBV glycoprotein surface antigen, a pliable nanoparticle vaccine platform.

While nanoparticle vaccine technology is gaining interest due to the success of vaccines like those for the human papillomavirus that is based on viral capsid nanoparticles, little information is available on the disassembly and reassembly of viral surface glycoprotein-based nanoparticles. One such particle is the hepatitis B virus surface antigen (sAg) that exists as nanoparticles. Here we show, using biochemical analysis coupled with electron microscopy, that sAg nanoparticle disassembly requires both reducing agent to disrupt intermolecular disulfide bonds, and detergent to disrupt hydrophobic interactions that stabilize the nanoparticle. Particles were otherwise resistant to salt and urea, suggesting the driving mechanism of particle formation involves hydrophobic interactions. We reassembled isolated sAg protein into nanoparticles by detergent removal and reassembly resulted in a wider distribution of particle diameters. Knowledge of these driving forces of nanoparticle assembly and stability should facilitate construction of epitope-displaying nanoparticles that can be used as immunogens in vaccines.