RESUMO
Hepatitis B virus (HBV) affects an estimated 250 million chronic carriers worldwide. Though several vaccines exist, they are ineffective for those already infected. HBV persists due to the formation of covalently closed circular DNA (cccDNA)-the viral minichromosome-in the nucleus of hepatocytes. Current nucleoside analogs and interferon therapies rarely clear cccDNA, requiring lifelong treatment. Our group identified GLP-26, a novel glyoxamide derivative that alters HBV nucleocapsid assembly and prevents viral DNA replication. GLP-26 exhibited single-digit nanomolar anti-HBV activity, inhibition of HBV e antigen (HBeAg) secretion, and reduced cccDNA amplification, in addition to showing a promising preclinical profile. Strikingly, long term combination treatment with entecavir in a humanized mouse model induced a decrease in viral loads and viral antigens that was sustained for up to 12 weeks after treatment cessation.
Assuntos
Antivirais/farmacologia , Capsídeo/química , Vacinas contra Hepatite B/farmacologia , Vírus da Hepatite B/química , Animais , Antivirais/química , Capsídeo/imunologia , DNA Circular/genética , DNA Circular/metabolismo , Cães , Guanina/análogos & derivados , Hepatite B/tratamento farmacológico , Antígenos da Hepatite B/química , Antígenos da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/química , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/metabolismo , Hepatócitos/virologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Nucleocapsídeo/efeitos dos fármacos , Ratos , Montagem de VírusRESUMO
OBJECTIVE: The incredibly serious problem of Hepatitis B is virus-related chronic liver disease. The conventional preventive treatment of Hepatitis B requires booster dose, which requires repeated administration of the vaccine to the subject. Thus, there is a need to develop a formulation which can eliminate the need of frequent dosing and enhance patient's acquiescence. To prepare single dose nanovaccine of HBsAg by utilizing central composite design for optimization and interaction of independent variables effects on measured response. METHODS: Nanovaccines were characterized for particle size, morphology, integrity, internalization, proliferation response and haemocompatibility. Nanoparticles at single and multiple doses were compared with booster dose of alum-HBsAg vaccine and measure the immunological marker and cytokine (interleukin-2 and interferon-Y) levels by ELISA techniques in BALB/c mice. RESULTS: The designed nanoparticles were found to have nanometric size, high entrapment efficiency and retained antigen integrity. Nanoparticles showed maximum proliferation and efficiently internalized by lymph and spleen cell without eliciting significant toxicity and haemocampatible. CONCLUSION: The comparable data of anti-HBsAg titre between nanovaccine and alum adsorbed HBsAg demonstrated that single dose of nanoparticles is sufficient for production of immunoglobulin plus cytokine levels, maintain immunogenicity at longer period of time and eliminate the booster dose. Nanovaccines trigger immune responses and showing adjuvant properties.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Antígenos da Hepatite B/administração & dosagem , Imunização/métodos , Ácido Láctico/administração & dosagem , Nanopartículas/administração & dosagem , Ácido Poliglicólico/administração & dosagem , Adjuvantes Imunológicos/química , Compostos de Alúmen/química , Animais , Proliferação de Células/efeitos dos fármacos , Composição de Medicamentos , Liberação Controlada de Fármacos , Eritrócitos/efeitos dos fármacos , Feminino , Hemólise/efeitos dos fármacos , Antígenos da Hepatite B/química , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Interferon gama/imunologia , Interleucina-2/imunologia , Ácido Láctico/química , Linfócitos/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Agregação Plaquetária/efeitos dos fármacos , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
BACKGROUND & AIMS: All known hepatitis B virus (HBV) genotypes occur in humans and hominoid Old World non-human primates (NHPs). The divergent woolly monkey HBV (WMHBV) forms another orthohepadnavirus species. The evolutionary origins of HBV are unclear. METHODS: We analysed sera from 124 Brazilian monkeys collected during 2012-2016 for hepadnaviruses using molecular and serological tools, and conducted evolutionary analyses. RESULTS: We identified a novel orthohepadnavirus species in capuchin monkeys (capuchin monkey hepatitis B virus [CMHBV]). We found CMHBV-specific antibodies in five animals and high CMHBV concentrations in one animal. Non-inflammatory, probably chronic infection was consistent with an intact preCore domain, low genetic variability, core deletions in deep sequencing, and no elevated liver enzymes. Cross-reactivity of antisera against surface antigens suggested antigenic relatedness of HBV, CMHBV, and WMHBV. Infection-determining CMHBV surface peptides bound to the human HBV receptor (human sodium taurocholate co-transporting polypeptide), but preferentially interacted with the capuchin monkey receptor homologue. CMHBV and WMHBV pseudotypes infected human hepatoma cells via the human sodium taurocholate co-transporting polypeptide, and were poorly neutralised by HBV vaccine-derived antibodies, suggesting that cross-species infections may be possible. Ancestral state reconstructions and sequence distance comparisons associated HBV with humans, whereas primate hepadnaviruses as a whole were projected to NHP ancestors. Co-phylogenetic analyses yielded evidence for co-speciation of hepadnaviruses and New World NHP. Bayesian hypothesis testing yielded strong support for an association of the HBV stem lineage with hominoid ancestors. Neither CMHBV nor WMHBV was likely the ancestor of the divergent human HBV genotypes F/H found in American natives. CONCLUSIONS: Our data suggest ancestral co-speciation of hepadnaviruses and NHP, and an Old World origin of the divergent HBV genotypes F/H. The identification of a novel primate hepadnavirus offers new perspectives for urgently needed animal models of chronic hepatitis B. LAY SUMMARY: The origins of HBV are unclear. The new orthohepadnavirus species from Brazilian capuchin monkeys resembled HBV in elicited infection patterns and could infect human liver cells using the same receptor as HBV. Evolutionary analyses suggested that primate HBV-related viruses might have emerged in African ancestors of New World monkeys millions of years ago. HBV was associated with hominoid primates, including humans and apes, suggesting evolutionary origins of HBV before the formation of modern humans. HBV genotypes found in American natives were divergent from those found in American monkeys, and likely introduced along prehistoric human migration. Our results elucidate the evolutionary origins and dispersal of primate HBV, identify a new orthohepadnavirus reservoir, and enable new perspectives for animal models of hepatitis B.
Assuntos
Cebus/virologia , Evolução Molecular , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Orthohepadnavirus/genética , Orthohepadnavirus/isolamento & purificação , Sequência de Aminoácidos , Animais , Teorema de Bayes , Brasil , Especiação Genética , Genoma Viral , Hepatite B/veterinária , Hepatite B/virologia , Antígenos da Hepatite B/química , Antígenos da Hepatite B/genética , Antígenos da Hepatite B/imunologia , Vírus da Hepatite B/classificação , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Modelos Genéticos , Doenças dos Macacos/virologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Orthohepadnavirus/classificação , Filogenia , Primatas/virologia , Receptores Virais/fisiologia , Simportadores/fisiologia , Internalização do VírusRESUMO
Presently available marketed alum adsorbed hepatitis B vaccine used for prophylactic immunization, can effectively elicit humoral immunity but is poor inducer of cell-mediated immunity (CMI). Besides, conventional alum-adjuvant vaccines require multiple injections to achieve long-lasting protective immune responses. Therefore, as a result of insufficient immunization, infections are still the leading killer among diseases. The present investigation was therefore, aimed at developing "single-shot" HBsAg adsorbed microspheres of poly (DL)-lactide-co-glycolide (PLGA) (L/G 50:50 and 75:25) and their capability to stimulate the cell mediated immune response against hepatitis B surface antigen. These microspheres were characterized in vitro for their size, shape polydispersity index, percentage HBsAg adsorption efficiency and in vitro release profile. The immune-stimulating activities were also studied following subcutaneous injection of HBsAg adsorbed PLGA microspheres (single-dose on day 0) and compared with alum adsorbed vaccines (two-doses on 0 and 28 days) in Balb/c mice. Specific cell-mediated immune responses such as lymphocyte transformation assay (stimulation-index) including release of interferon-gamma (IFN-γ), interleukin-2 (IL-2) and nitric-oxide were determined. Cellular responses in case of alum adsorb HBsAg vaccine was very low. These studies demonstrate the potential of cationic polymeric microspheres based vaccine in stimulating cell mediated immune response along with humoral response against hepatitis B.
Assuntos
Portadores de Fármacos/química , Antígenos da Hepatite B/imunologia , Vacinas contra Hepatite B/administração & dosagem , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Ácido Láctico/química , Ácido Poliglicólico/química , Animais , Cátions , Estabilidade de Medicamentos , Eletroforese em Gel de Poliacrilamida , Antígenos da Hepatite B/administração & dosagem , Antígenos da Hepatite B/química , Injeções Subcutâneas , Interferon gama/sangue , Interferon gama/imunologia , Interleucina-2/sangue , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Microesferas , Peso Molecular , Óxido Nítrico/sangue , Óxido Nítrico/imunologia , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Propriedades de Superfície , Tecnologia FarmacêuticaRESUMO
Hepatitis B virus envelope is mainly composed of three forms of the same protein expressed from different start codons of the same open reading frame. The smaller form named S protein corresponds to the C-terminal common region and represents about 80% of the envelope proteins. It is mainly referred as hepatitis B virus surface antigen (HBsAg). Over expressed in the host cell, this protein can be produced as spherical and tubular self-organized particles. Highly immunogenic, these particles are used in licensed hepatitis B vaccines. In this study we have combined transmission electron microscopy and atomic force microscopy to determine the shape and size of HBsAg particles produced from the yeast Hansenula polymorpha. Tapping mode atomic force microscopy in liquid allows structural details of the surface to be delineated with a resolution in the nanometer range. Particles were decorated by closely packed spike-like structures protruding from particle surface. Protrusions appeared uniformly distributed at the surface and an average number of 75 protrusions per particle were calculated. Importantly, we demonstrated that proteins mainly contribute to the topography of the protrusions.
Assuntos
Antígenos da Hepatite B/química , Microscopia de Força Atômica/métodos , Nanotecnologia/métodos , Ditiotreitol/metabolismo , Antígenos da Hepatite B/genética , Antígenos da Hepatite B/metabolismo , Microscopia Eletrônica , Pichia/genéticaRESUMO
5S is a mouse monoclonal IgG1 that binds to the 'a' epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K(D) = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.
Assuntos
Antígenos de Superfície/química , Antígenos de Superfície/imunologia , Antígenos da Hepatite B/química , Antígenos da Hepatite B/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Engenharia de Proteínas/métodos , Antígenos de Superfície/genética , Antígenos da Hepatite B/genética , Humanos , Fragmentos Fab das Imunoglobulinas/genéticaRESUMO
BACKGROUND: Despite their ease of use, lateral flow immunoassays (LFIAs) often suffer from poor quantitative discrimination and low analytical sensitivity. We explored the use of a novel class of europium chelate-loaded silica nanoparticles as labels to overcome these limitations. METHODS: Antibodies were covalently conjugated onto europium chelate-loaded silica nanoparticles with dextran as a linker. The resulting conjugates were used as labels in LFIA for detection of hepatitis B surface antigen (HBsAg). We performed quantification with a digital camera and Adobe Photoshop software. We also used 286 clinical samples to compare the proposed method with a quantitative ELISA. RESULTS: A detection limit of 0.03 microg/L was achieved, which was 100 times lower than the colloidal gold-based LFIAs and lower than ELISA. A precise quantitative dose-response curve was obtained, and the linear measurement range was 0.05-3.13 microg/L, within which the CVs were 2.3%-10.4%. Regression analysis of LFIA on ELISA results gave: log (LFIA) = -0.14 log (ELISA) + 1.03 microg/L with r = 0.99 for the quantification of HBsAg in 35 positive serum samples. Complete agreement was observed for the qualitative comparison of 286 clinical samples assayed with LFIA and ELISA. CONCLUSIONS: Europium chelate-loaded silica nanoparticle labels have great potential to improve LFIAs, making them useful not only for simple screening applications but also for more sensitive and quantitative immunoassays.
Assuntos
Quelantes/química , Európio/química , Imunoensaio/métodos , Nanopartículas/química , Dióxido de Silício/química , Anticorpos Monoclonais/química , Corantes Fluorescentes/química , Antígenos da Hepatite B/sangue , Antígenos da Hepatite B/química , Humanos , Análise de Regressão , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Software , Coloração e RotulagemRESUMO
Hepatitis B is a deadly disease, and is carried by 30% of the world's population. Antibodies are produced through a series of three manual vaccinations during infancy and childhood. However, the current needle vaccination not only induces pain in patients, but also can be inconvenient to administer. This is particularly true for the case of newborn babies. Intranasal vaccination is emerging as an alternative parenteral drug delivery method that facilitates drug delivery without causing pain. Chitosan, which is obtained through the deacetylation of chitin from crustacea, is a cationic polymer that is biodegradable, avirulent, and highly absorptive. In this study, ionic gelation between chitosan and TPP was conducted to synthesize chitosan nanoparticles with sizes of 200-400 nm and a surface potential of 55-60 mV, and which can be used as Hepatitis B vaccine carriers. Then, Hepatitis B antigen protein was impregnated to manufacture chitosan-recombinant gene vaccine protein (RGVP) nanoparticles. AFM, SEM, TEM, and STEM were used to analyze the manufactured nanoparticles, whose function as drug carriers and whose usefulness for intranasal vaccination were confirmed through in vivo tests with SD rats.
Assuntos
Quitosana/administração & dosagem , Sistemas de Liberação de Medicamentos , Anticorpos Anti-Hepatite B/sangue , Antígenos da Hepatite B/administração & dosagem , Vacinas contra Hepatite B/administração & dosagem , Hepatite B/prevenção & controle , Nanopartículas/administração & dosagem , Animais , Quitosana/química , Quitosana/metabolismo , Hepatite B/imunologia , Antígenos da Hepatite B/química , Antígenos da Hepatite B/genética , Antígenos da Hepatite B/imunologia , Vacinas contra Hepatite B/genética , Vacinas contra Hepatite B/imunologia , Masculino , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Polifosfatos/administração & dosagem , Polifosfatos/química , Ratos , Ratos Sprague-DawleyRESUMO
The nonstructural hepatitis B virus (HBV) protein HBx has an important role in HBV replication and in HBV-associated liver disease. Many activities have been linked to HBx expression; however, the molecular mechanisms underlying many of these activities are unknown. One proposed HBx function is the regulation of cytosolic calcium. We analyzed calcium levels in HepG2 cells that expressed HBx or replicating HBV, and we demonstrated that HBx, expressed in the absence of other HBV proteins or in the context of HBV replication, elevates cytosolic calcium. We linked this elevation of cytosolic calcium to the association of HBx with the mitochondrial permeability transition pore.
Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Regulação da Expressão Gênica , Vírus da Hepatite B/fisiologia , Mitocôndrias/metabolismo , Replicação Viral , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Ciclosporina/farmacologia , DNA Viral , Regulação Viral da Expressão Gênica , Genoma Viral , Antígenos da Hepatite B/química , Vírus da Hepatite B/metabolismo , Humanos , Neoplasias Hepáticas/virologia , Mitocôndrias/virologiaAssuntos
Capsídeo/fisiologia , Vírus da Hepatite B/química , Vírus da Hepatite B/fisiologia , Montagem de Vírus , Capsídeo/química , Capsídeo/imunologia , Antígenos da Hepatite B/química , Antígenos da Hepatite B/fisiologia , Antígenos do Núcleo do Vírus da Hepatite B/biossíntese , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Conformação ProteicaRESUMO
OBJECTIVE: To screen and identify the protein interacting with HBV antigen in hepatocytes. Then investigate the biological functions of hepatitis B virus antigen in the pathogenesis of hepatitis B and seek effective methods to prevent and treat it. METHODS: The yeast two-hybrid system-3 technique was used to construct HBV PreS2, HBeAg, HBcAg, HBxAg bait plasmids. The bait plasmids transformed the yeast AH109 and expressed themselves in it. After being identified by SDS-PAGE and Western blot, the AH109 yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium to form diploid yeast and was then plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for screening. Plasmids of blue colonies were extracted and transformed into Escherichia coli, then analyzed by DNA sequencing and bioinformatics. To further prove the interaction between HBV antigen and metallothionein, translation was performed by using reticulocyte lysate and coimmunoprecipitation was displayed in vitro. RESULTS: Genes coding for HBV antigen binding protein were successfully cloned and metallothionein was found in that protein. The interaction between HBeAg, HBcAg and HBxAg and metallothionein were further proved by coimmunoprecipitation in vitro. CONCLUSION: The interaction between HBV antigen and metallothionein indicates that metallothionein may participate in the pathogenesis of hepatitis B
Assuntos
Antígenos da Hepatite B/química , Hepatite B/metabolismo , Hepatócitos/metabolismo , Metalotioneína/química , Humanos , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-HíbridoRESUMO
Ligand activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been reported to induce growth inhibition and apoptosis in various cancers including hepatocellular carcinoma (HCC). However, the effect of hepatitis B virus X protein (HBx) on PPARgamma activation has not been characterized in hepatitis B virus (HBV)-associated HCC. Herein, we demonstrated that HBx counteracted growth inhibition caused by PPARgamma ligand in HBx-associated HCC cells. We found that HBx bound to DNA binding domain of PPARgamma and HBx/PPARgamma interaction blocked nuclear localization and binding to recognition site of PPARgamma. HBx significantly suppressed a PPARgamma-mediated transactivation. These results suggest that HBx modulates PPARgamma function through protein-protein interaction.
Assuntos
Receptores Citoplasmáticos e Nucleares/metabolismo , Transativadores/farmacologia , Fatores de Transcrição/metabolismo , Adenoviridae , Apoptose/efeitos dos fármacos , Sequência de Bases , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Primers do DNA , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde , Antígenos da Hepatite B/química , Antígenos da Hepatite B/farmacologia , Humanos , Ligantes , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Proteínas Luminescentes/genética , Transporte Proteico/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/química , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Transfecção , Células Tumorais Cultivadas , Proteínas Virais Reguladoras e AcessóriasRESUMO
The use of transgenic plants to express orally immunogenic protein antigens is an emerging strategy for vaccine biomanufacturing and delivery. This concept has particular suitability for developing countries. One factor that has limited the development of this technology is the relatively modest levels of accumulation of some antigenic proteins in plant tissues. We used fusion protein design to improve expression of the hepatitis B surface antigen (HBsAg) by attempting to mimic the process of HBsAg targeting to the endoplasmic reticulum of human liver cells during hepatitis B virus infection. We created a gene encoding a recombinant HBsAg modified to contain a plant signal peptide fused to its amino terminus. The signal peptide from soybean vegetative storage protein vspA (VSP alpha S) directed endoplasmic reticulum targeting of HBsAg in plant cells, but was not cleaved and resulted in enhanced VSP alpha S-HBsAg fusion accumulation. This product was more stable and presented the protective "a" antigenic determinant to significantly higher levels than unmodified native HBsAg expressed in plant cells. It also showed a greater extent of intermolecular disulfide bond formation and formation of virus-like particles. Moreover, VSP alpha S-HBsAg stimulated higher levels of serum IgG than native HBsAg when injected into mice. We conclude that HBsAg tolerates a polypeptide fusion at the amino terminus and that VSP alpha S-HBsAg is an improved antigen for plant-based expression of a subunit vaccine for hepatitis B virus.
Assuntos
Técnicas Genéticas , Antígenos da Hepatite B/química , Antígenos da Hepatite B/metabolismo , Peptídeos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Centrifugação com Gradiente de Concentração , Eletroporação , Retículo Endoplasmático , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/química , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Plasmídeos/metabolismo , Testes de Precipitina , Proteínas Recombinantes de Fusão/metabolismo , Glycine max/metabolismo , Sacarose/farmacologia , Temperatura , Fatores de Tempo , NicotianaRESUMO
Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/fisiologia , Antígenos da Hepatite B/química , Antígenos da Hepatite B/fisiologia , Vírus da Hepatite B/química , Vírus da Hepatite B/fisiologia , Montagem de Vírus , Dimerização , Cinética , Conformação Proteica , Sais , Cloreto de Sódio/química , Relação Estrutura-Atividade , TermodinâmicaRESUMO
An estimated 400 million people are chronically infected with the hepatitis B virus (HBV). Chronic viral hepatitis infection incurs serious sequelae such as liver cirrhosis and hepatocellular carcinoma. Prevention and treatment, thus, represent an important target for public health. Preventive vaccines using HBsAg alone or combined with other antigens allow for the generation of neutralizing antibodies which effectively prevent infection in immunocompetent individuals. Cell-mediated immunological mechanisms are thought to be crucial in determining viral persistence or viral elimination. Therapeutic approaches aiming to shift cellular immunity towards viral elimination have been on the research agenda for many years. This paper summarizes pre-clinical and clinical results obtained with the use of immunogenic peptides formulated as vaccines to selectively boost cellular immune responses. Such vaccines are capable of generating cellular immune responses in animal models as well as in humans and represent an important step towards the development of a therapeutic vaccine against chronic hepatitis.
Assuntos
Antígenos da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Antígenos da Hepatite B/química , Vacinas contra Hepatite B/uso terapêutico , Hepatite B Crônica/prevenção & controle , Humanos , Imunidade Celular , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
The control of hepatitis B by vaccination is arguably one of medicine's greatest achievements in terms of protecting infants and adults at high risk of infection. Paradoxically, however, the existence of a large reservoir of chronically infected individuals will not diminish the risk of infection by those coming into close contact with such persons until universal infant immunisation is practised globally and vaccines are in place to ensure maximum efficacy in those with impaired immune responses, immunity is achieved with fewer doses, and immunisation as an adjunct to the antiviral treatment of chronic carriers is adopted. These imperatives have continued to stimulate research into vaccines based on chemically synthesised short peptides, and those systems best suited for their delivery. This review discusses the potential of synthetic peptide formulations as efficient inducers of both humoral and cellular immune responses against hepatitis B, and reviews recent advances in peptide delivery. Synthetic peptide and delivery systems technologies will, amongst others, be of paramount importance in the global fight for the eradication of hepatitis B in the 21st century.
Assuntos
Vacinas contra Hepatite B/imunologia , Hepatite B/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Hepatite B/prevenção & controle , Antígenos da Hepatite B/administração & dosagem , Antígenos da Hepatite B/química , Antígenos da Hepatite B/imunologia , Vacinas contra Hepatite B/administração & dosagem , Vacinas contra Hepatite B/química , Humanos , Peptídeos/administração & dosagem , Peptídeos/síntese química , Peptídeos/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/químicaRESUMO
Duck hepatitis B virus (DHBV) appears to lack a homologue of the X protein found in mammalian hepadnaviruses. By replacing stop codons in the corresponding region of the DHBV genome, a hypothetical protein which closely matches the hydrophilicity profile of X proteins can be predicted, despite limited sequence homology. We conclude that a full-length X protein was once a common feature of the hepadnaviruses, conserved in structure but not sequence.
Assuntos
Genoma Viral , Antígenos da Hepatite B/genética , Vírus da Hepatite B do Pato/genética , Fases de Leitura Aberta , Transativadores/genética , Sequência de Aminoácidos , Animais , Patos , Antígenos da Hepatite B/química , Dados de Sequência Molecular , Biossíntese de Proteínas , Análise de Sequência de Proteína , Transativadores/química , Proteínas Virais Reguladoras e AcessóriasRESUMO
Hepatitis B virus (HBV) has a unique fourth open reading frame coding for a 16.5-kDa protein known as hepatitis B virus X protein (HBX). The importance of HBX in the life cycle of HBV has been well established, but the underlying molecular function of HBX remains controversial. We previously identified a proteasome subunit PSMA7 that interacts specifically with HBX in the Saccharomyces cerevisiae two-hybrid system. Here we demonstrate that PSMC1, an ATPase-like subunit of the 19 S proteasome component, also interacts with HBX and PSMA7. Analysis of the interacting domains among PSMA7, PSMC1, and HBX by deletion and site-directed mutagenesis suggested a mutually competitive structural relationship among these polypeptides. The competitive nature of these interactions is further demonstrated using a modified yeast two-hybrid dissociator system. The crucial HBX sequences involved in interaction with PSMA7 and PSMC1 are important for its function as a transcriptional coactivator. HBX, while functioning as a coactivator of AP-1 and acidic activator VP-16 in mammalian cells, had no effect on the transactivation function of their functional orthologs GCN4 and Gal4 in yeast. Overexpression of PSMC1 seemed to suppress the expression of various reporters in mammalian cells; this effect, however, was overcome by coexpression of HBX. In addition, HBX expression inhibited the cellular turnover of c-Jun and ubiquitin-Arg-beta-galactosidase, two well known substrates of the ubiquitin-proteasome pathway. Thus, interaction of HBX with the proteasome complex in metazoan cells may underlie the functional basis of proteasome as a cellular target of HBX.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Transativadores/química , Transativadores/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Clonagem Molecular , Antígenos da Hepatite B/química , Antígenos da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Cinética , Substâncias Macromoleculares , Modelos Moleculares , Fases de Leitura Aberta , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Biossíntese de Proteínas , Estrutura Quaternária de Proteína , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reticulócitos/metabolismo , Saccharomyces cerevisiae/genética , Transativadores/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
The existence of HBV as quasispecies is thought to be favoured by the infidelity of HBV RT, which would account for the emergence of the many natural mutants with point substitutions. RT infidelity may also underlie the hypermutation phenomenon. Indeed, the oft-reported point mutation in the preC gene that leads to failure of HBeAg synthesis may be driven by a hypermutation-related mechanism. The presence of mutants with deletions and insertions involving single nucleotides and oligonucleotides at specific positions in the genome, and of mutants with deletions of even longer stretches particularly in the C gene, suggests that other mutagenic mechanisms operate. Candidates include slippage during mispairing between template and progeny DNA strand, the action of cellular topoisomerase I, and gene splicing using alternative donor and acceptor sites. Natural substitutions, deletions or insertions involving the Cp/ENII locus in the X gene can significantly alter the extent of viral replicative activity. Similar mutations occurring at other locations of Cp/ENII, and at B-cell epitope sites of the S gene are associated with failure to detect serological markers of HBV infection. HBV variation can also arise from recombination between coinfecting strains. S gene mutations that become evident following HBIG administration and HBV vaccination are all point substitutions, as are mutations in functional RT domains of the P gene after treatment with viral RT-inhibitory drugs. Widespread and long-term use of prophylactic and therapeutic agents may potentially generate serologically occult HBV variants that might become difficult to eradicate.