Hepatitis B Virus (HBV) Subviral Particles as Protective Vaccines and Vaccine Platforms

Hepatitis B remains one of the major global health problems more than 40 years after the identification of human hepatitis B virus (HBV) as the causative agent. A critical turning point in combating this virus was the development of a preventative vaccine composed of the HBV surface (envelope) protein (HBsAg) to reduce the risk of new infections. The isolation of HBsAg sub-viral particles (SVPs) from the blood of asymptomatic HBV carriers as antigens for the first-generation vaccines, followed by the development of recombinant HBsAg SVPs produced in yeast as the antigenic components of the second-generation vaccines, represent landmark advancements in biotechnology and medicine. The ability of the HBsAg SVPs to accept and present foreign antigenic sequences provides the basis of a chimeric particulate delivery platform, and resulted in the development of a vaccine against malaria (RTS,S/AS01, MosquirixTM), and various preclinical vaccine candidates to overcome infectious diseases for which there are no effective vaccines. Biomedical modifications of the HBsAg subunits allowed the identification of strategies to enhance the HBsAg SVP immunogenicity to build potent vaccines for preventative and possibly therapeutic applications. The review provides an overview of the formation and assembly of the HBsAg SVPs and highlights the utilization of the particles in key effective vaccines.

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A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes

Viruses ◽

10.3390/v12090928 ◽

2020 ◽

Vol 12 (9) ◽

pp. 928 ◽

Cited By ~ 1

Author(s):

Eunji Jo ◽

Jaewon Yang ◽

Alexander Koenig ◽

Seung Yoon ◽

Marc Windisch

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Preparation Method ◽

Cost Effective ◽

Viral Dna ◽

Dna Preparation ◽

Hbv Replication ◽

Viral Particles ◽

B Virus ◽

Total Dna

Hepatitis B virus (HBV) is a para-retrovirus that reverse transcribes its pregenomic RNA into relaxed circular DNA inside viral nucleocapsids. The number of HBV genomes produced in vitro is typically quantified using commercial silica-membrane-based nucleic acid purification kits to isolate total DNA followed by HBV-specific quantitative PCR (qPCR). However, despite the convenience of commercial kits, this procedure is costly and time-consuming due to multiple centrifugation steps, which produce unnecessary waste. Here, we report a rapid, cost-effective, and environmentally friendly total DNA preparation method. The assay is based on the simple incubation of detergent and proteinase K with cells or cell-free supernatants to permeabilize cells and disrupt viral particles. After heat inactivation and subsequent centrifugation to clear the lysates, DNA samples are directly subjected to qPCR to quantify HBV genomes. As a proof of concept, the assay was developed in 12-well plates to assess intra- and extracellular HBV genome equivalents (GEqs) of stably viral-replicating cell lines (e.g., HepAD38) and HBV-infected HepG2-NTCP cells, both treated with lamivudine (LMV), an HBV replication inhibitor. Viral DNA was also prepared from the serum of patients chronically infected with HBV. To validate the assay, a representative commercial DNA isolation kit was used side-by-side to isolate intra- and extracellular HBV DNA. Both methods yielded comparable amounts of HBV GEqs with comparable LMV 50% efficient concentration (EC50) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC50 values were comparable to those obtained in 12-well plates. In addition, the calculated coefficient of variation, Z’ values, and assay window demonstrated high reproducibility and quality. We devised a novel, robust, reproducible, high-throughput microtiter plate DNA preparation method suitable for quantifying HBV GEqs by qPCR analysis. This strategy enables rapid and convenient quantitative analysis of multiple viral DNA samples in parallel to investigate intracellular HBV replication and the secretion of DNA-containing viral particles.

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Subviral Hepatitis B Virus Filaments, like Infectious Viral Particles, Are Released via Multivesicular Bodies

Journal of Virology ◽

10.1128/jvi.03109-15 ◽

2015 ◽

Vol 90 (7) ◽

pp. 3330-3341 ◽

Cited By ~ 37

Author(s):

Bingfu Jiang ◽

Kiyoshi Himmelsbach ◽

Huimei Ren ◽

Klaus Boller ◽

Eberhard Hildt

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Secretory Pathway ◽

Current Model ◽

Surface Protein ◽

Surface Proteins ◽

Multivesicular Bodies ◽

Viral Particles ◽

B Virus ◽

Subviral Particles

ABSTRACTIn addition to infectious viral particles, hepatitis B virus-replicating cells secrete large amounts of subviral particles assembled by the surface proteins, but lacking any capsid and genome. Subviral particles form spheres (22-nm particles) and filaments. Filaments contain a much larger amount of the large surface protein (LHBs) compared to spheres. Spheres are released via the constitutive secretory pathway, while viral particles are ESCRT-dependently released via multivesicular bodies (MVBs). The interaction of virions with the ESCRT machinery is mediated by α-taxilin that connects the viral surface protein LHBs with the ESCRT component tsg101. Since filaments in contrast to spheres contain a significant amount of LHBs, it is unclear whether filaments are released like spheres or like virions. To study the release of subviral particles in the absence of virion formation, a core-deficient HBV mutant was generated. Confocal microscopy, immune electron microscopy of ultrathin sections and isolation of MVBs revealed that filaments enter MVBs. Inhibition of MVB biogenesis by the small-molecule inhibitor U18666A or inhibition of ESCRT functionality by coexpression of transdominant negative mutants (Vps4A, Vps4B, and CHMP3) abolishes the release of filaments while the secretion of spheres is not affected. These data indicate that in contrast to spheres which are secreted via the secretory pathway, filaments are released via ESCRT/MVB pathway like infectious viral particles.IMPORTANCEThis study revises the current model describing the release of subviral particles by showing that in contrast to spheres, which are secreted via the secretory pathway, filaments are released via the ESCRT/MVB pathway like infectious viral particles. These data significantly contribute to a better understanding of the viral morphogenesis and might be helpful for the design of novel antiviral strategies.

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Cell Culture Derived Recombinant HBsAg is Highly Immunogenic and Protects Chimpanzees from Infection with Hepatitis B Virus

Nature Biotechnology ◽

10.1038/nbt0786-630 ◽

1986 ◽

Vol 4 (7) ◽

pp. 630-636 ◽

Cited By ~ 13

Author(s):

Eric J. Patzer ◽

Gerald R. Nakamura ◽

Robert D. Hershberg ◽

Timothy J. Gregory ◽

Craig Crowley ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis B Virus ◽

Hepatitis B ◽

B Virus ◽

Recombinant Hbsag

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Trans-complementation among naturally occurring deletion mutants of hepatitis B virus and integrated viral DNA for the production of viral particles with mutant genomes in hepatoma cell lines

Journal of General Virology ◽

10.1099/0022-1317-74-3-407 ◽

1993 ◽

Vol 74 (3) ◽

pp. 407-414 ◽

Cited By ~ 19

Author(s):

H. Okamoto ◽

Y. Wang ◽

T. Tanaka ◽

A. Machida ◽

Y. Miyakawa ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Cell Lines ◽

Hepatoma Cell ◽

Deletion Mutants ◽

Viral Dna ◽

Hepatoma Cell Lines ◽

Naturally Occurring ◽

Viral Particles ◽

B Virus

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Modification of the Hepatitis B Virus Envelope Protein Glycosylation Pattern Interferes with Secretion of Viral Particles, Infectivity, and Susceptibility to Neutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.01161-14 ◽

2014 ◽

Vol 88 (16) ◽

pp. 9049-9059 ◽

Cited By ~ 39

Author(s):

R. Julithe ◽

G. Abou-Jaoude ◽

C. Sureau

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Envelope Protein ◽

Neutralizing Antibodies ◽

Protein Glycosylation ◽

Glycosylation Pattern ◽

Virus Envelope ◽

Viral Particles ◽

B Virus ◽

Virus Envelope Protein

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Isolation and Purification of Recombinant HBsAg of Human Hepatitis B Virus from Silkworm Larvae

Chemistry of Natural Compounds ◽

10.1007/s10600-005-0213-9 ◽

2005 ◽

Vol 41 (5) ◽

pp. 583-587

Author(s):

E. Yu. Bachurina ◽

O. A. Likhoradova ◽

E. A. Zholdasova ◽

G. A. Piyakina ◽

Sh. S. Azimova

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Silkworm Larvae ◽

Isolation And Purification ◽

Human Hepatitis ◽

B Virus ◽

Recombinant Hbsag

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Carboxypeptidase D Is an Avian Hepatitis B Virus Receptor

Journal of Virology ◽

10.1128/jvi.73.10.8696-8702.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8696-8702 ◽

Cited By ~ 41

Author(s):

Shuping Tong ◽

Jisu Li ◽

Jack R. Wands

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Cell Lines ◽

Virus Binding ◽

Duck Hepatitis ◽

Viral Particles ◽

B Virus ◽

Carboxypeptidase D ◽

Immortalized Cell

ABSTRACT The receptor molecules for human and animal hepatitis B viruses have not been defined. Previous studies have described a 170 to 180 kDa molecule (p170 or gp180) that binds in vitro to the pre-S domain of the large envelope protein of duck hepatitis B virus (DHBV); cDNA cloning revealed the binding protein to be duck carboxypeptidase D (DCPD). In the present study, the DCPD cDNA was transfected into several nonpermissive human-, monkey-, and avian species-derived cell lines. Cells transfected with a plasmid encoding the full-length DCPD protein bound DHBV particles, whereas cells expressing truncated versions of DCPD protein that fail to bind the pre-S protein did not. The DHBV binding to DCPD-reconstituted cells was blocked by a monoclonal antibody that neutralizes DHBV infection of primary duck hepatocytes (PDH) and also by a pre-S peptide previously shown to inhibit DHBV infection of PDH. In addition to promoting virus binding, DCPD expression was associated with internalization of viral particles. The entry process was prevented by incubation of reconstituted cells with DHBV at 4°C and by the addition of energy-depleting agents known to block DHBV entry into PDH. These results demonstrated that DCPD is a DHBV receptor. However, the lack of complete viral replication in DCPD-reconstituted cells suggested that additional factors are required for postentry events in immortalized cell lines.

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Duck Hepatitis B Virus Requires Cholesterol for Endosomal Escape during Virus Entry

Journal of Virology ◽

10.1128/jvi.00422-08 ◽

2008 ◽

Vol 82 (21) ◽

pp. 10532-10542 ◽

Cited By ~ 16

Author(s):

Anneke Funk ◽

Mouna Mhamdi ◽

Heinz Hohenberg ◽

Jörg Heeren ◽

Rudolph Reimer ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Biological Membranes ◽

Endosomal Escape ◽

Cholesterol Depletion ◽

Virus Infectivity ◽

Duck Hepatitis B Virus ◽

Duck Hepatitis ◽

Viral Particles ◽

B Virus

ABSTRACT The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.

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Itinerary of Hepatitis B Viruses: Delineation of Restriction Points Critical for Infectious Entry

Journal of Virology ◽

10.1128/jvi.78.15.8289-8300.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 8289-8300 ◽

Cited By ~ 30

Author(s):

Anneke Funk ◽

Mouna Mhamdi ◽

Li Lin ◽

Hans Will ◽

Hüseyin Sirma

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Viral Entry ◽

Circular Dna ◽

Duck Hepatitis ◽

Viral Particles ◽

B Virus ◽

Number Of Binding Sites ◽

Infectious Entry ◽

First Time

ABSTRACT Little is known about cellular determinants essential for human hepatitis B virus infection. Using the duck hepatitis B virus as a model, we first established a sensitive binding assay for both virions and subviral particles and subsequently elucidated the characteristics of the early viral entry steps. The infection itinerary was found to initiate with the attachment of viral particles to a low number of binding sites on hepatocytes (about 104 per cell). Virus internalization was fully accomplished in less than 3 h but was then followed by a period of unprecedented length, about 14 h, until completion of nuclear import of the viral genome. Steps subsequent to virus entry depended on both intact microtubules and their dynamic turnover but not on actin cytoskeleton. Notably, cytoplasmic trafficking of viral particles and emergence of nuclear covalently closed circular DNA requires microtubules during entry only at and for specific time periods. Taken together, these data disclose for the first time a series of steps and their kinetics that are essential for the entry of hepatitis B viruses into hepatocytes and are different from those of any other virus reported so far.

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