RESUMO
RNA phages are often used as prototypes for modern recombinant virus-like particle (VLP) technologies. Icosahedral RNA phage VLPs can be formed from coat proteins (CPs) and are efficiently produced in bacteria and yeast. Both genetic fusion and chemical coupling have been successfully used for the production of numerous chimeras based on RNA phage VLPs. In this review, we describe advances in RNA phage VLP technology along with the history of the Leviviridae family, including its taxonomical organization, genomic structure, and important role in the development of molecular biology. Comparative 3D structures of different RNA phage VLPs are used to explain the level of VLP tolerance to foreign elements displayed on VLP surfaces. We also summarize data that demonstrate the ability of CPs to tolerate different organic (peptides, oligonucleotides, and carbohydrates) and inorganic (metal ions) compounds either chemically coupled or noncovalently added to the outer and/or inner surfaces of VLPs. Finally, we present lists of nanotechnological RNA phage VLP applications, such as experimental vaccines constructed by genetic fusion and chemical coupling methodologies, nanocontainers for targeted drug delivery, and bioimaging tools.
Assuntos
Capsídeo , Fagos RNA , Vacinas de Partículas Semelhantes a Vírus/química , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Leviviridae/classificação , Leviviridae/genética , Conformação Molecular , Nanotecnologia/métodos , Vacinas de Partículas Semelhantes a Vírus/imunologiaRESUMO
A vaccine protecting against all influenza strains is a long-sought goal, particularly for emerging pandemics. As previously shown, vaccines based on the highly conserved extracellular domain of M2 (M2e) may protect against all influenza A strains. Here, we demonstrate that M2e-specific monoclonal antibodies (mAbs) protect mice from a lethal influenza infection. To be protective, antibodies had to be able to bind to Fc receptors and fix complement. Furthermore, mAbs of IgG2c isotype were protective in mice, while antibodies of identical specificity, but of the IgG1 isotype, failed to prevent disease. These findings readily translated into vaccine design. A vaccine targeting M2 in the absence of a toll-like receptor (TLR) 7 ligand primarily induced IgG1, whilst the same vaccine linked to a TLR7 ligand yielded high levels of IgG2c antibodies. Although both vaccines protected mice from a lethal challenge, mice treated with the vaccine containing a TLR7 ligand showed significantly lower morbidity. In accordance with these findings, vaccination of TLR7(-/-) mice with a vaccine containing a TLR7 ligand did not result in protection from a lethal challenge. Hence, the innate immune system is required to direct isotype switching toward the more protective IgG2a/c antibodies.
Assuntos
Anticorpos Antivirais/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Glicoproteínas de Membrana/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Switching de Imunoglobulina/efeitos dos fármacos , Switching de Imunoglobulina/genética , Imunoglobulina G/sangue , Imunoglobulina G/genética , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Vacinas contra Influenza/farmacologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Pandemias , Transdução de Sinais/genética , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , VacinaçãoRESUMO
BACKGROUND: Subviral particles of hepatitis B virus (HBV) composed of L protein deletion variants with the 48 N-terminal amino acids of preS joined to the N-terminus of S protein (1-48preS/S) induced broadly neutralizing antibodies after immunization of mice with a Semliki Forest virus vector. A practical limitation for use as vaccine is the suboptimal secretion of such particles. The role of the N-terminal preS myristoylation in the cellular retention of full-length L protein is described controversially in the literature and the relation of these data to the truncated L protein was unknown. Thus, we studied the effect of preS myristoylation signal suppression on 1-48preS/S secretion efficiency, glycosylation and subcellular distribution. FINDINGS: The findings are that 1-48preS/S is secreted, and that removal of the N-terminal myristoylation signal in its G2A variant reduced secretion slightly, but significantly. The glycosylation pattern of 1-48preS/S was not affected by the removal of the myristoylation signal (G2A mutant) but was different than natural L protein, whereby N4 of the preS and N3 of the S domain were ectopically glycosylated. This suggested cotranslational translocation of 1-48preS in contrast to natural L protein. The 1-48preS/S bearing a myristoylation signal was localized in a compact, perinuclear pattern with strong colocalization of preS and S epitopes, while the non-myristoylated mutants demonstrated a dispersed, granular cytoplasmic distribution with weaker colocalization. CONCLUSIONS: The large deletion in 1-48preS/S in presence of the myristoylation site facilitated formation and secretion of protein particles with neutralizing preS1 epitopes at their surface and could be a useful feature for future hepatitis B vaccines.
Assuntos
Antígenos Virais/imunologia , Deleção de Genes , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Processamento de Proteína Pós-Traducional , Proteínas do Envelope Viral/genética , Animais , Anticorpos Neutralizantes/sangue , Antígenos Virais/genética , Antígenos Virais/metabolismo , Vetores Genéticos , Anticorpos Anti-Hepatite B/sangue , Vacinas contra Hepatite B/administração & dosagem , Vacinas contra Hepatite B/genética , Vírus da Hepatite B/genética , Camundongos , Testes de Neutralização , Vírus da Floresta de Semliki/genéticaRESUMO
Virus-like particle (VLP) technology is a promising approach for the construction of novel vaccines, diagnostic tools, and gene therapy vectors. Initially, VLPs were primarily derived from non-enveloped icosahedral or helical viruses and proved to be viable vaccine candidates due to their effective presentation of epitopes in a native conformation. VLP technology has also been used to prepare chimeric VLPs decorated with genetically fused or chemically coupled epitope stretches selected from immunologically defined target proteins. However, structural constraints associated with the rigid geometrical architecture of icosahedral or helical VLPs pose challenges for the expression and presentation of large epitopes. Complex VLPs derived from non-symmetric enveloped viruses are increasingly being used to incorporate large epitopes and even full-length foreign proteins. Pleomorphic VLPs derived from influenza or other enveloped viruses can accommodate multiple full-length and/or chimeric proteins that can be rationally designed for multifunctional purposes, including multivalent vaccines. Therefore, a second generation of VLP carriers is represented by complex particles reconstructed from natural or chimeric structural proteins derived from complex enveloped viruses. Further development of safe and efficient VLP nanotechnology may require a rational combination of both approaches.
Assuntos
Biotecnologia/métodos , Portadores de Fármacos , Engenharia Genética/métodos , Tecnologia Farmacêutica/métodos , Virossomos/genética , Animais , Epitopos/genética , Epitopos/imunologia , Humanos , Vírus/genéticaRESUMO
Rheumatoid arthritis (RA) is closely associated with HLA-DRB1 alleles that code a five-amino acid sequence motif in positions 70-74 of the HLA-DRbeta-chain, called the shared epitope (SE). The mechanistic basis of SE-RA association is unknown. We recently found that the SE functions as an allele-specific signal-transducing ligand that activates an NO-mediated pathway in other cells. To better understand the role of the SE in the immune system, we examined its effect on T cell polarization in mice. In CD11c(+)CD8(+) dendritic cells (DCs), the SE inhibited the enzymatic activity of indoleamine 2,3 dioxygenase, a key enzyme in immune tolerance and T cell regulation, whereas in CD11c(+)CD8(-) DCs, the ligand activated robust production of IL-6. When SE-activated DCs were cocultured with CD4(+) T cells, the differentiation of Foxp3(+) T regulatory cells was suppressed, whereas Th17 cells were expanded. The polarizing effects could be seen with SE(+) synthetic peptides, but even more so when the SE was in its natural tridimensional conformation as part of HLA-DR tetrameric proteins. In vivo administration of the SE ligand resulted in a greater abundance of Th17 cells in the draining lymph nodes and increased IL-17 production by splenocytes. Thus, we conclude that the SE acts as a potent immune-stimulatory ligand that can polarize T cell differentiation toward Th17 cells, a T cell subset that was recently implicated in the pathogenesis of autoimmune diseases, including RA.
Assuntos
Artrite Reumatoide/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Epitopos/fisiologia , Antígenos HLA-DR/fisiologia , Sequência de Aminoácidos , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Polaridade Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/patologia , Epitopos/metabolismo , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/biossíntese , Inibidores do Crescimento/fisiologia , Cadeias HLA-DRB1 , Humanos , Interleucina-17/biossíntese , Ligantes , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Dados de Sequência Molecular , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
BACKGROUND: Escape mutations potentially allow viruses to avoid detection and clearance by the host immune system and may represent a mechanism through which infections may persist in some patients. The association of the mutations in the HBcAg gene with Hepatitis B asymptomatic carriers (ASC) has not been studied adequately. The current study was aimed to investigate HBcAg18-27 CTL epitope mutations in ASC patients in the South-Eastern region of Iran. METHODS: 100 ASC patients were selected for this study and screened for HLA-A2 using flow cytometry. HBV-DNA was extracted from the HLA-A2 positive patients and the HBc gene was amplified using PCR. Direct double sequencing was performed to analyse mutations in the HBc gene of HBV isolates from patients with ASC. RESULTS: Overall, 25 (25%) of individuals were HLA-A2 positive. Direct double sequencing indicated no mutations in the HBcAg18-27 epitope. However, four mutations within the T helper and three mutations within the B cell epitopes of ASC patients were identified. CONCLUSIONS: The lack of mutations within the HBcAg18-27 epitope suggests that the antigenicity of this region is not altered in HBV isolates of our patients and therefore antigen presentation would occur normally to the patient's immune system through HLA-A2. However, in the course of this study we revealed some novel mutations within the T helper and B cell epitopes that may affect the efficiencies of immune response of ASC patients against these novel HBV epitopes.
Assuntos
Infecções Assintomáticas , Linfócitos B/imunologia , Tolerância Central/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Hepatite B/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Sequência de Aminoácidos , Anticorpos Antivirais , Linfócitos B/citologia , Sequência de Bases , Tolerância Central/genética , DNA Viral/análise , Feminino , Antígenos do Núcleo do Vírus da Hepatite B/química , Interações Hospedeiro-Patógeno , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Linfócitos T Auxiliares-Indutores/citologiaRESUMO
BACKGROUNDS & AIMS: The N-terminally myristoylated preS1 domain of the large hepatitis B surface protein (LHBs) mediates specific attachment of hepatitis B virus (HBV) to hepatocytes. Its B-cell epitopes leading to neutralization of infectivity are not yet characterized. METHODS: We inserted C- and N-terminal preS1 peptides into the most immunogenic region of HBV core particles, therewith immunized Balb/c mice and determined binding properties and neutralization potential of resulting antibodies in vitro. RESULTS: The particles with preS1 inserts were highly immunogenic and the corresponding anti-preS antibodies strongly bound to HBV particles from chronic carriers infected with different HBV genotypes A-F. However, antibodies binding to the C-terminal part of preS1 did not neutralize HBV infectivity for susceptible hepatocyte cultures. In contrast, antibodies elicited by the complete N-terminal attachment site of preS1(2-48) strongly neutralized with an IC50<3µg/ml of total immunoglobulin. Interestingly, antibodies against the very N-terminal part of preS1(1-21) could not neutralize infectivity although this sequence contains the most conserved and essential part of the attachment site. These antibodies reacted well with non-myristoylated preS1 peptides but only weakly with myristoylated preS1 peptides, natural HBsAg or HBV. CONCLUSIONS: N-terminal myristic acid obviously favors a topology of LHBs that makes the most essential part of the preS1 attachment site inaccessible for neutralizing antibodies, whereas antibodies to neighbouring sequences neutralized very well. Thus, addition of the preS1(2-48) peptide in a highly immunogenic form to the current hepatitis B vaccine may improve protective immunity and reduce selection of escape mutations.
Assuntos
Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Precursores de Proteínas/química , Precursores de Proteínas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes , Sítios de Ligação , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Genótipo , Anticorpos Anti-Hepatite B , Antígenos de Superfície da Hepatite B/genética , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/genética , Hepatite B Crônica/imunologia , Hepatite B Crônica/prevenção & controle , Hepatite B Crônica/virologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ácido Mirístico/química , Ácido Mirístico/imunologia , Precursores de Proteínas/genética , Homologia de Sequência de AminoácidosRESUMO
Assembly and disassembly of viral capsids are essential steps in the viral life cycle. Studies on their kinetics are mostly performed in vitro, allowing application of biochemical, biophysical and visualizing techniques. In vivo kinetics are poorly understood and the transferability of the in vitro models to the cellular environment remains speculative. We analyzed capsid disassembly of the hepatitis B virus in digitonin-permeabilized cells which support nuclear capsid entry and subsequent genome release. Using gradient centrifugation, size exclusion chromatography and immune fluorescence microscopy of digitonin-permeabilized cells, we showed that capsids open and close reversibly. In the absence of RNA, capsid re-assembly slows down; the capsids remain disintegrated and enter the nucleus as protein dimers or irregular polymers. Upon the presence of cellular RNA, capsids re-assemble in the nucleus. We conclude that reversible genome release from hepatitis B virus capsids is a unique strategy different from that of other viruses, which employs irreversible capsid destruction for genome release. The results allowed us to propose a model of HBV genome release in which the unique environment of the nuclear pore favors HBV capsid disassembly reaction, while both cytoplasm and nucleus favor capsid assembly.
Assuntos
Capsídeo/metabolismo , Núcleo Celular/virologia , Vírus da Hepatite B/patogenicidade , Vírion/patogenicidade , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , DNA Viral/metabolismo , Eletroforese em Gel de Ágar , Escherichia coli/metabolismo , Escherichia coli/virologia , Vírus da Hepatite B/metabolismo , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Isótopos de Fósforo , Multimerização Proteica , RNA Viral/metabolismo , Vírion/metabolismo , Fenômenos Fisiológicos ViraisRESUMO
Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0mg per 1g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast.
Assuntos
Antígenos do Núcleo do Vírus da Hepatite B , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite B/genética , Hepatite B/imunologia , Pichia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus , Clonagem Molecular , Portadores de Fármacos , Fermentação , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/isolamento & purificação , Vírus da Hepatite B/química , Humanos , Fosforilação , Engenharia de Proteínas , Proteínas Recombinantes/química , Análise Espectral , Tecnologia Farmacêutica/métodos , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/ultraestruturaRESUMO
The mechanisms regulating systemic and mucosal IgA responses in the respiratory tract are incompletely understood. Using virus-like particles loaded with single-stranded RNA as a ligand for TLR7, we found that systemic vs mucosal IgA responses in mice were differently regulated. Systemic IgA responses following s.c. immunization were T cell independent and did not require TACI or TGFbeta, whereas mucosal IgA production was dependent on Th cells, TACI, and TGFbeta. Strikingly, both responses required TLR7 signaling, but systemic IgA depended upon TLR7 signaling directly to B cells whereas mucosal IgA required TLR7 signaling to lung dendritic cells and alveolar macrophages. Our data show that IgA switching is controlled differently according to the cell type receiving TLR signals. This knowledge should facilitate the development of IgA-inducing vaccines.
Assuntos
Células Dendríticas/imunologia , Imunoglobulina A/biossíntese , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Mucosa/imunologia , RNA/imunologia , Animais , Glicoproteínas de Membrana , Camundongos , Camundongos Knockout , Linfócitos T Auxiliares-Indutores , Receptor 7 Toll-Like , Fator de Crescimento Transformador beta , Proteína Transmembrana Ativadora e Interagente do CAMLRESUMO
BACKGROUND: Since its first appearance in the USA in 1999, West Nile virus (WNV) has spread in the Western hemisphere and continues to represent an important public health concern. In the absence of effective treatment, there is a medical need for the development of a safe and efficient vaccine. Live attenuated WNV vaccines have shown promise in preclinical and clinical studies but might carry inherent risks due to the possibility of reversion to more virulent forms. Subunit vaccines based on the large envelope (E) glycoprotein of WNV have therefore been explored as an alternative approach. Although these vaccines were shown to protect from disease in animal models, multiple injections and/or strong adjuvants were required to reach efficacy, underscoring the need for more immunogenic, yet safe DIII-based vaccines. RESULTS: We produced a conjugate vaccine against WNV consisting of recombinantly expressed domain III (DIII) of the E glycoprotein chemically cross-linked to virus-like particles derived from the recently discovered bacteriophage AP205. In contrast to isolated DIII protein, which required three administrations to induce detectable antibody titers in mice, high titers of DIII-specific antibodies were induced after a single injection of the conjugate vaccine. These antibodies were able to neutralize the virus in vitro and provided partial protection from a challenge with a lethal dose of WNV. Three injections of the vaccine induced high titers of virus-neutralizing antibodies, and completely protected mice from WNV infection. CONCLUSIONS: The immunogenicity of DIII can be strongly enhanced by conjugation to virus-like particles of the bacteriophage AP205. The superior immunogenicity of the conjugate vaccine with respect to other DIII-based subunit vaccines, its anticipated favourable safety profile and low production costs highlight its potential as an efficacious and cost-effective prophylaxis against WNV.
Assuntos
Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Febre do Nilo Ocidental/prevenção & controle , Vacinas contra o Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologia , Animais , Bacteriófagos/genética , Bacteriófagos/imunologia , Bacteriófagos/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/química , Vacinas Conjugadas/genética , Vacinas Conjugadas/imunologia , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/mortalidade , Vacinas contra o Vírus do Nilo Ocidental/administração & dosagem , Vacinas contra o Vírus do Nilo Ocidental/química , Vacinas contra o Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/química , Vírus do Nilo Ocidental/genéticaRESUMO
BACKGROUND: Hepatitis C core protein is an attractive target for HCV vaccine aimed to exterminate HCV infected cells. However, although highly immunogenic in natural infection, core appears to have low immunogenicity in experimental settings. We aimed to design an HCV vaccine prototype based on core, and devise immunization regimens that would lead to potent anti-core immune responses which circumvent the immunogenicity limitations earlier observed. METHODS: Plasmids encoding core with no translation initiation signal (pCMVcore); with Kozak sequence (pCMVcoreKozak); and with HCV IRES (pCMVcoreIRES) were designed and expressed in a variety of eukaryotic cells. Polyproteins corresponding to HCV 1b amino acids (aa) 1-98 and 1-173 were expressed in E. coli. C57BL/6 mice were immunized with four 25-microg doses of pCMVcoreKozak, or pCMV (I). BALB/c mice were immunized with 100 microg of either pCMVcore, or pCMVcoreKozak, or pCMVcoreIRES, or empty pCMV (II). Lastly, BALB/c mice were immunized with 20 microg of core aa 1-98 in prime and boost, or with 100 microg of pCMVcoreKozak in prime and 20 microg of core aa 1-98 in boost (III). Antibody response, [3H]-T-incorporation, and cytokine secretion by core/core peptide-stimulated splenocytes were assessed after each immunization. RESULTS: Plasmids differed in core-expression capacity: mouse fibroblasts transfected with pCMVcore, pCMVcoreIRES and pCMVcoreKozak expressed 0.22 +/- 0.18, 0.83 +/- 0.5, and 13 +/- 5 ng core per cell, respectively. Single immunization with highly expressing pCMVcoreKozak induced specific IFN-gamma and IL-2, and weak antibody response. Single immunization with plasmids directing low levels of core expression induced similar levels of cytokines, strong T-cell proliferation (pCMVcoreIRES), and antibodies in titer 103(pCMVcore). Boosting with pCMVcoreKozak induced low antibody response, core-specific T-cell proliferation and IFN-gamma secretion that subsided after the 3rd plasmid injection. The latter also led to a decrease in specific IL-2 secretion. The best was the heterologous pCMVcoreKozak prime/protein boost regiment that generated mixed Th1/Th2-cellular response with core-specific antibodies in titer >or= 3 x 10(3). CONCLUSION: Thus, administration of highly expressed HCV core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. Instead, the latter is induced by a heterologous DNA prime/protein boost regiment that circumvents the negative effects of intracellular core expression.
RESUMO
The icosahedral capsid of duck hepatitis B virus (DHBV) is formed by a single core protein species (DHBc). DHBc is much larger than HBc from human HBV, and no high-resolution structure is available. In an accompanying study (M. Nassal, I. Leifer, I. Wingert, K. Dallmeier, S. Prinz, and J. Vorreiter, J. Virol. 81:13218-13229, 2007), we used extensive mutagenesis to derive a structural model for DHBc. For independent validation, we here mapped the epitopes of seven anti-DHBc monoclonal antibodies. Using numerous recombinant DHBc proteins and authentic nucleocapsids from different avihepadnaviruses as test antigens, plus a panel of complementary assays, particle-specific and exposed plus buried linear epitopes were revealed. These data fully support key features of the model.
Assuntos
Avihepadnavirus/química , Vírus da Hepatite B do Pato/química , Nucleocapsídeo/química , Proteínas do Core Viral/química , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Avihepadnavirus/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Vírus da Hepatite B do Pato/imunologia , Modelos Moleculares , Nucleocapsídeo/imunologia , Estrutura Terciária de Proteína , Proteínas do Core Viral/imunologiaRESUMO
Envelope protein E2 of human hepatitis C virus (HCV) is an attractive component of a prototype HCV vaccine. Delivered by DNA immunogens, E2 evokes specific immune response of Th1-type, failing to induce either considerable antibody production, or T-helper cell proliferation. We aimed at modulating the immunogenic performance of E2 gene by changing the mode of protein expression in eukaryotic cells. Plasmids were constructed encoding full-length E2 and nonstructural protein 1 (p7) fused to either 13 or 38 C-terminal amino acids (aa) of HCV E1 that contain second hydrophobic segment of E1 stop-transfer signal, or a complete E1 stop-transfer signal with duplicated second hydrophobic segment. Injected into BALB/c mice, E2/p7 genes induced potent antibody and T-helper cell response targeted against hypervariable region 1, aa 472-586 of E2, and a novel epitope at aa 774-796 of p7. Profile of cytokines secreted by proliferating mouse splenocytes stimulated in vitro with E2- and p7-derived peptides, indicated mixed Th1/Th2 type of immune response. Thus, the full-length E2 and p7 genes supplied in one cassette were both immunogenic. E2/p7 containing a complete E1 stop-transfer signal with prolonged membrane spanning domain was superior to the shorter E2/p7 version in terms of both antibody and cellular immunogenicity. Optimal performance of HCV E2 could thus be achieved without the aid of external/heterologous signals by easing, through modification of the E2 signal sequence, the release of E2 from the rough ER while retaining full-length E2 and p7 sequences. This finding may help to improve the Th2 performance of HCV envelope genes as prototype vaccines.
Assuntos
Genes Virais , Hepacivirus/imunologia , Sinais Direcionadores de Proteínas/fisiologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular Transformada , Transformação Celular Viral , Chlorocebus aethiops , Escherichia coli/genética , Variação Genética , Células HeLa , Hepacivirus/genética , Humanos , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmídeos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas Virais/imunologiaRESUMO
Recombinant bacteriophage Qbeta coat protein (CP), which has been proposed as a promising carrier of foreign epitopes via their incorporation either by gene engineering techniques or by chemical coupling, efficiently self-assembles into virus-like particles (VLPs) when expressed in Escherichia coli. Here, we demonstrate expression and self-assembly of Qbeta CP in yeast Saccharomyces cerevisiae and Pichia pastoris. Production reached 3-4 mg/1g of wet cells for S. cerevisiae and 4-6 mg for P. pastoris, which was about 15-20% and 20-30% of the E. coli expression level, respectively. Qbeta VLPs were easily purified by size-exclusion chromatography in both cases and contained nucleic acid, shown by native agarose gel electrophoresis. The obtained particles were highly immunogenic in mice and the resulting sera recognized both E. coli- and yeast-derived Qbeta VLPs equally well.
Assuntos
Allolevivirus/metabolismo , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo , Vírion/imunologia , Vírion/metabolismo , Montagem de Vírus , Allolevivirus/genética , Animais , Clonagem Molecular , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Pichia/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Vírion/química , Vírion/genéticaRESUMO
The major immunodominant region (MIR) and N-terminus of the hepatitis B virus (HBV) core (HBc) protein were used to expose foreign insertions on the outer surface of HBc virus-like particles (VLPs). The additions to the HBc positively charged arginine-rich C-terminal (CT) domain are usually not exposed on the VLP surface. Here, we constructed a set of recombinant HBcG vectors in which CT arginine stretches were substituted by glycine residues. In contrast to natural HBc VLPs and recombinant HBc VLP variants carrying native CT domain, the HBcG VLPs demonstrated a lowered capability to pack bacterial RNA during expression in Escherichia coli cells. The C-terminal addition of a model foreign epitope from the HBV preS1 sequence to the HBcG vectors resulted in the exposure of the inserted epitope on the VLP surface, whereas the same preS1 sequences added to the native CT of the natural HBc protein remained buried within the HBc VLPs. Based on the immunisation of mice, the preS1 epitope added to the HBcG vectors as a part of preS1(20-47) and preS1phil sequences demonstrated remarkable immunogenicity. The same epitope added to the original C-terminus of the HBc protein did not induce a notable level of anti-preS1 antibodies. HBcG vectors may contribute to the further development of versatile HBc VLP-based vaccine and gene therapy applications.
Assuntos
Vetores Genéticos , Vírus da Hepatite B/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas do Core Viral/genética , Sequência de Aminoácidos , Animais , Epitopos/genética , Epitopos/imunologia , Feminino , Variação Genética , Anticorpos Anti-Hepatite B/sangue , Anticorpos Anti-Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas do Core Viral/químicaRESUMO
Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.
Assuntos
Álcalis/química , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vírion/imunologia , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Terapia Genética/métodos , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta , Vírion/genética , Vírion/metabolismo , Montagem de Vírus/genética , Montagem de Vírus/imunologiaRESUMO
Co-expression in Escherichia coli of wild-type (wt) hepatitis B virus core protein (HBc) and its naturally occurring variants with deletions at amino acid positions 77-93 or 86-93 leads to formation of mosaic particles, which consist of three dimer subunit compositions. These compositions are wt/variant HBc heterodimers and two types of homodimers, formed by wt HBc or the variant HBc themselves. Mosaic particles were found also when both HBc deletion variants 77-93 and 86-93 were co-expressed in E. coli. These findings are discussed in terms of their significance for hepatitis B virus pathogenesis and prospective use of mosaic particles in vaccine development.
Assuntos
Vírus da Hepatite B/química , Deleção de Sequência , Proteínas do Core Viral/genética , Clonagem Molecular , Dimerização , Variação Genética , Hepatite B/etiologia , Vacinas contra Hepatite B , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas do Core Viral/metabolismoRESUMO
In this study, we report the exact localization and substitutional characterization of a B-cell epitope domain at the N-terminus of the preS2 region of the hepatitis B surface antigen. A set of deletion variants containing preS2 sequences of different length was generated on the basis of frCP as a carrier. It was found after Western blot analysis that three monoclonal antibodies (MAbs) (2-11B1, 3-11C2, HB.OT10) recognized the linear preS2 sequence within the amino acid (aa) stretch 3-WNSTTFHQTLQDP-13. The importance of each aa residue of the epitope was proved by comparison of antibody binding to alanine-substituted peptides in both free-peptide and Pepscan variants.
Assuntos
Mapeamento de Epitopos , Epitopos de Linfócito B/análise , Antígenos de Superfície da Hepatite B/análise , Precursores de Proteínas/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Camundongos , Dados de Sequência Molecular , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes three structural proteins VP1, VP2, and VP3, where VP1 represents the major capsid protein of 384 amino acids. Screening of sera from HaPyV-infected papilloma-bearing and papilloma-free hamsters demonstrated the immunodominant features of all three capsid proteins. For both groups of hamsters in the C-terminal region of VP1 immunodominant B-cell epitopes were identified in the regions between amino acids 305 and 351 and amino acids 351 and 384. The high flexibility of the C-terminal region of VP1 was confirmed by the formation of chimeric virus-like particles based on the coat protein of the RNA bacteriophage fr which was previously found to tolerate only very short-sized foreign insertions. Phage fr coat protein-derived virus-like particles tolerated the N-terminal fusion of amino acids 333-384, 351-384, 351-374, and 364-384, respectively, of VP1. The induction of VP1-specific antibodies in rabbits and mice by immunization with chimeric virus-like particles harboring amino acids 333-384, 351-384, and 364-384, respectively, of VP1 suggested the immunodominant nature of the C-terminal region of VP1.