RESUMEN
The available HBV vaccines based on the HBV surface protein are manufactured in yeasts and demonstrate excellent prophylactic but no therapeutic activity and are thus ineffective against chronic HBV infection. Five different HBV core proteins (HBc)-full length and C-terminally truncated-were used for the insertion of the short, preS1,aa 20-47 and long, preS1phil, aa 12-60 + 89-119 fragments. Modified virus-like particles (VLPs) were compared for their biotechnological and immunological properties. The expression level of HBc-preS1 proteins was high for all investigated proteins, allowing us to obtain 10-20 mg of purified VLPs from a gram of biomass with the combination of gel filtration and ion-exchange chromatography to reach approximately 90% purity of target proteins. The immunogenicity of chimeric VLPs was tested in BALB/c mice, showing a high anti-preS1 response and substantial T-cell proliferation after stimulation with HBc protein. Targeted incorporation of oligonucleotide ODN 1668 in modified HBc-preS1 VLPs was demonstrated.
RESUMEN
BACKGROUND: The lack of a universal influenza vaccine is a global health problem. Interest is now focused on structurally conserved protein domains capable of eliciting protection against a broad range of influenza virus strains. The long alpha helix (LAH) is an attractive vaccine component since it is one of the most conserved influenza hemagglutinin (HA) stalk regions. For an improved immune response, the LAH domain from H3N2 strain has been incorporated into virus-like particles (VLPs) derived from hepatitis B virus core protein (HBc) using recently developed tandem core technology. RESULTS: Fermentation conditions for recombinant HBc-LAH were established in yeast Pichia pastoris and a rapid and efficient purification method for chimeric VLPs was developed to match the requirements for industrial scale-up. Purified VLPs induced strong antibody responses against both group 1 and group 2 HA proteins in mice. CONCLUSION: Our results indicate that the tandem core technology is a useful tool for incorporation of highly hydrophobic LAH domain into HBc VLPs. Chimeric VLPs can be successfully produced in bioreactor using yeast expression system. Immunologic data indicate that HBc VLPs carrying the LAH antigen represent a promising universal influenza vaccine component.
Asunto(s)
Hemaglutininas Virales/aislamiento & purificación , Antígenos del Núcleo de la Hepatitis B/genética , Vacunas contra la Influenza/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Virión/aislamiento & purificación , Animales , Anticuerpos Antivirales , Femenino , Hemaglutininas Virales/genética , Hemaglutininas Virales/inmunología , Hemaglutininas Virales/metabolismo , Subtipo H3N2 del Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/metabolismo , Ratones , Ratones Endogámicos BALB C , Pichia/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Virión/genética , Virión/inmunología , Virión/metabolismoRESUMEN
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.
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Cápside , Fagos ARN , Vacunas de Partículas Similares a Virus/química , Secuencia de Aminoácidos , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Leviviridae/clasificación , Leviviridae/genética , Conformación Molecular , Nanotecnología/métodos , Vacunas de Partículas Similares a Virus/inmunologíaRESUMEN
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.
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Vectores Genéticos , Virus de la Hepatitis B/genética , Vacunas de Partículas Similares a Virus/inmunología , Proteínas del Núcleo Viral/genética , Secuencia de Aminoácidos , Animales , Epítopos/genética , Epítopos/inmunología , Femenino , Variación Genética , Anticuerpos contra la Hepatitis B/sangre , Anticuerpos contra la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Vacunas de Partículas Similares a Virus/genética , Proteínas del Núcleo Viral/químicaRESUMEN
Virus-like particles (VLPs) are multisubunit self-assembly competent protein structures with identical or highly related overall structure to their corresponding native viruses. To construct a new filamentous VLP carrier, the coat protein (CP) gene from potato virus M (PVM) was amplified from infected potato plants, cloned, and expressed in Escherichia coli cells. As demonstrated by electron microscopy analysis, the PVM CP self-assembles into filamentous PVM-like particles, which are mostly 100-300 nm in length. Adding short Gly-Ser peptide at the C-terminus of the PVM, CP formed short VLPs, whereas peptide and protein A Z-domain fusions at the CP N-terminus retained its ability to form typical PVM VLPs. The PVM-derived VLP carrier accommodates up to 78 amino acid-long foreign sequences on its surface and can be produced in technologically significant amounts. PVM-like particles are stable at physiological conditions and also, apparently do not become disassembled in high salt and high pH solutions as well as in the presence of EDTA or reducing agents. Despite partial proteolytic processing of doubled Z-domain fused to PVM VLPs, the rabbit IgGs specifically bind to the particles, which demonstrates the functional activity and surface location of the Z-domain in the PVM VLP structure. Therefore, PVM VLPs may be recognized as powerful structural blocks for new human-made nanomaterials.
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Carlavirus/genética , Genoma Viral , Nanopartículas/virología , Vacunas de Partículas Similares a Virus/química , Animales , Carlavirus/aislamiento & purificación , Carlavirus/fisiología , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Inmunoglobulina G/sangre , Inmunoglobulina G/química , Conejos , Solanum tuberosum/virología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vacunas de Partículas Similares a Virus/aislamiento & purificación , Ensamble de VirusRESUMEN
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.
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Álcalis/química , Antígenos del Núcleo de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Virión/inmunología , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Terapia Genética/métodos , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos del Núcleo de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Aminoácido , Espectrofotometría Ultravioleta , Virión/genética , Virión/metabolismo , Ensamble de Virus/genética , Ensamble de Virus/inmunologíaRESUMEN
Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic and therapeutic vaccines. In the present study, we have compared the adjuvanting potential of commercially available Silica nanoparticles (initial particles size of 10-20 nm) with that of aluminium hydroxide, or Alum, as well as that of complete and incomplete Freund's adjuvants for the immunisation of BALB/c mice with virus-like particles (VLPs) formed by recombinant full-length Hepatitis B virus core (HBc) protein. The induction of B-cell and T-cell responses was studied after immunisation. Silica nanoparticles were able to adsorb maximally 40% of the added HBc, whereas the adsorption capacity of Alum exceeded 90% at the same VLPs/adjuvant ratio. Both Silica and Alum formed large complexes with HBc VLPs that sedimented rapidly after formulation, as detected by dynamic light scattering, spectrophotometry, and electron microscopy. Both Silica and Alum augmented the humoral response against HBc VLPs to the high anti-HBc level in the case of intraperitoneal immunisation, whereas in subcutaneous immunisation, the Silica-adjuvanted anti-HBc level even exceeded the level adjuvanted by Alum. The adjuvanting of HBc VLPs by Silica resulted in the same typical IgG2a/IgG1 ratios as in the case of the adjuvanting by Alum. The combination of Silica with monophosphoryl lipid A (MPL) led to the same enhancement of the HBc-specific T-cell induction as in the case of the Alum and MPL combination. These findings demonstrate that Silica is not a weaker putative adjuvant than Alum for induction of B-cell and T-cell responses against recombinant HBc VLPs. This finding may have an essential impact on the development of the set of Silica-adjuvanted vaccines based on a long list of HBc-derived virus-like particles as the biological component.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos del Núcleo de la Hepatitis B/farmacología , Vacunas contra Hepatitis B/farmacología , Virus de la Hepatitis B/inmunología , Hepatitis B/prevención & control , Dióxido de Silicio/farmacología , Adyuvantes Inmunológicos/química , Compuestos de Alumbre/farmacología , Animales , Femenino , Adyuvante de Freund/inmunología , Adyuvante de Freund/farmacología , Hepatitis B/inmunología , Antígenos del Núcleo de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Inmunidad Humoral/efectos de los fármacos , Inmunización , Lípido A/análogos & derivados , Lípido A/inmunología , Lípido A/farmacología , Lípidos/inmunología , Lípidos/farmacología , Ratones Endogámicos BALB C , Nanopartículas/química , Dióxido de Silicio/química , Dióxido de Silicio/inmunologíaRESUMEN
The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber- or opal-suppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11-16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIII-induced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine.
Asunto(s)
Fagos ARN/genética , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/metabolismo , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Virus del Nilo Occidental/inmunologíaRESUMEN
Virus-like particles (VLPs) generated by heterologous expression of viral structural genes have become powerful tools in vaccine development. Recently, we and others have reported on the assembly of VLPs of the RNA bacteriophages MS2, Qß, and GA in yeast. Here, we investigate the formation of VLPs of five additional phages in the yeasts Saccharomyces cerevisiae and Pichia pastoris, namely, the coliphages SP and fr, Acinetobacter phage AP205, Pseudomonas phage PP7, and Caulobacter phage φCb5. In all cases except SP, particle formation was detected, although VLP outcome varied from 0.2 to 8 mg from 1 g of wet cells. We have found that phage φCb5 VLPs easily dissociate into coat protein dimers when applied to strong anion exchangers. Upon salt removal and the addition of nucleic acid or its mimics and calcium ions, the dimers re-assemble into VLPs with high efficiency. A variety of compounds, including RNA, DNA, and gold nanoparticles can be packaged inside φCb5 VLPs. The ease with which phage φCb5 coat protein dimers can be purified in high quantities and re-assembled into VLPs makes them attractive for downstream applications including the internal packaging of nanomaterials and the chemical coupling of peptides of interest on the surface.
Asunto(s)
Bacteriófagos/fisiología , Nanopartículas/química , Pichia/virología , Saccharomyces cerevisiae/virología , Vacunas de Partículas Similares a Virus/metabolismo , Ensamble de Virus , Secuencia de Aminoácidos , Antiportadores/metabolismo , Proteínas de la Cápside/metabolismo , Pichia/clasificación , ARN Viral/genéticaRESUMEN
An efficient pBR327- and Ptrp-based E. coli expression system was used to generate a large-scale library of virus like particles (VLP) formed by recombinant hepatitis B virus (HBV) core (HBc) protein derivatives. To construct the library, the gene of HBc protein of the genotype D/subtype ayw2 virus was gradually truncated from the 3`-end and twenty-two HBc variants (with truncation up to 139 aa) were expressed at high levels. The proteins were purified by salt precipitation and gel filtration. Background RNA binding was observed for VLPs formed by HBc1-149, which lacked all C-terminal Arg blocks, and the addition of three Arg residues (HBc1-152) only slightly increased RNA binding. The presence of two Arg blocks (proteins HBc1-162 and HBc1-163) resulted in approximately half of the typical level of RNA binding, and the presence of three blocks (protein HBc1-171) led to approximately 85% of the typical level of binding. Only a small increase in the level of RNA binding was found for the HBc1-175 VLPs, which contained all four Arg blocks but lacked the last 8 aa of the full-length HBc protein. VLPs containing high levels of RNA had higher antigenicity according to an ELISA with anti-HBc mAbs than the VLPs formed by HBc variants without C-terminal Arg blocks and lacking RNA. The results indicate that the VLPs were stabilised by nucleic acids. The immunogenicity in BALB/c mice was comparable for VLPs formed by different HBc proteins, but a clear switch from a Th1 response to a Th2 response occurred after the loss of encapsidated RNA. We did not observe significant differences in lymphocyte proliferation in vitro for the tested VLP variants; however, the loss of RNA encapsidation correlated with a decreased level of IFN-γ induction, which is a measure of the potential CTL activity of immunogens.
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Biblioteca de Genes , Antígenos del Núcleo de la Hepatitis B/genética , Virión/genética , Animales , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Ingeniería Genética/métodos , Antígenos del Núcleo de la Hepatitis B/metabolismo , Antígenos del Núcleo de la Hepatitis B/ultraestructura , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Three variants of the major rubella virus (RV) E1 protein virus-neutralizing epitope from position 214 to 285 were exposed on the hepatitis B virus (HBV) C-terminally truncated core (HBcΔ) in a virus-like particle (VLP) vector and were produced in Escherichia coli. All three chimeras demonstrated VLPs in bacterial cell lysates, but only HBcΔ-E1(245-285) demonstrated the correct VLP structure after purification. The other chimeras, HBcΔ-E1(214-285) and HBcΔ-E1(214-240), appeared after purification as non-VLP aggregates of 100 to 900 nm in diameter according to dynamic light scattering data. All three variants possessed the intrinsic antigenic activity of RV E1, since they were recognized by natural human anti-RV E1 antibodies and induced an anti-RV E1 response in mice. HBcΔ-E1(214-240) and HBcΔ-E1(245-285) can be regarded as prototypes for a putative RV vaccine because they were able to induce antibodies recognizing natural RV E1 protein in RV diagnostic kits.
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Epítopos/inmunología , Virus de la Hepatitis B/inmunología , Vacuna contra la Rubéola/inmunología , Vacunas de Partículas Similares a Virus/ultraestructura , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Epítopos/genética , Escherichia coli/genética , Femenino , Virus de la Hepatitis B/genética , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Vacuna contra la Rubéola/administración & dosificación , Vacuna contra la Rubéola/genética , Vacunas Sintéticas , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
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.
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Antígenos Virales/inmunología , Eliminación de Gen , Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Procesamiento Proteico-Postraduccional , Proteínas del Envoltorio Viral/genética , Animales , Anticuerpos Neutralizantes/sangre , Antígenos Virales/genética , Antígenos Virales/metabolismo , Vectores Genéticos , Anticuerpos contra la Hepatitis B/sangre , Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/genética , Virus de la Hepatitis B/genética , Ratones , Pruebas de Neutralización , Virus de los Bosques Semliki/genéticaRESUMEN
Virus-like particles (VLPs) are created by the self-assembly of multiple copies of envelope and/or capsid proteins from many viruses, mimicking the conformation of a native virus. Such noninfectious nanostructures are mainly used as antigen-presenting platforms, especially in vaccine research; however, some of them recently were used as scaffolds in biotechnology to produce targeted nanoparticles for intracellular delivery. This study demonstrates the creation of fusion VLPs using hepatitis B core protein-based system maintaining a fibronectin-binding property from B. burgdorferi BBK32 protein, including the evidence of particles' transmission to BHK-21 target cells via caveolae/rafts endocythosis. These results make this construct to be an attractive model in development of HBc-based nanoparticles for cellular targeting applications and highlights the fragment of B. burgdorferi BBK32 as a novel cellular uptake-promoting peptide. FROM THE CLINICAL EDITOR: This paper discusses the nanotechnology-based application of self-assembling viral-like peptides (VLP-s) for targeted delivery using a hepatitis B core protein based system. Creating fusion VLPs may be an attractive model for cellular targeting applications.
Asunto(s)
Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/metabolismo , Fibronectinas/metabolismo , Nanopartículas , Secuencia de Bases , Cartilla de ADN , PlásmidosRESUMEN
Virus-like particle (VLP) technology represents a promising approach for the creation of efficient vaccines and materials for use in nanotechnological applications. For construction of a new carrier for foreign protein sequences, the coat protein (CP) gene from potato virus Y (PVY) was cloned and expressed in Escherichia coli cells. The PVY CP self-assembles into PVY-like particles, as demonstrated by electron microscopy analysis of purified VLP preparations. The PVY CP with an N-terminal insertion of a foreign epitope (preS1) or of a whole protein (rubredoxin) retains its ability to form filamentous particles, whereas adding a foreign sequence to the C-terminus of the PVY CP generates mostly unstructured protein aggregates. This new filamentous plant virus-derived VLP carrier accommodates a foreign protein sequence that is up to 71 amino acids in length on the VLP surface and can be produced in E. coli in preparative amounts. The PVY CP VLPs are stable in physiological conditions, but they are sensitive to EDTA, high salt, and extreme pH. The presence of the preS1 epitope decreases the stability of the chimeric PVY CP particles at elevated temperatures. Mice that are immunized with chimeric PVY CP particles carrying preS1 epitopes exhibit a strong anti-preS1 immune response, even in the absence of adjuvants.
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Presentación de Antígeno , Proteínas de la Cápside/metabolismo , Escherichia coli/genética , Antígenos de Superficie de la Hepatitis B/inmunología , Potyvirus/genética , Precursores de Proteínas/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Virión/genética , Animales , Biotecnología/métodos , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Clonación Molecular , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Escherichia coli/inmunología , Escherichia coli/metabolismo , Femenino , Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/metabolismo , Inmunización , Ratones , Ratones Endogámicos BALB C , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Virión/inmunología , Virión/metabolismoRESUMEN
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.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Hepatitis B/genética , Hepatitis B/inmunología , Pichia , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Vacunas de Partículas Similares a Virus , Clonación Molecular , Portadores de Fármacos , Fermentación , Hepatitis B/virología , Antígenos del Núcleo de la Hepatitis B/química , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos del Núcleo de la Hepatitis B/aislamiento & purificación , Virus de la Hepatitis B/química , Humanos , Fosforilación , Ingeniería de Proteínas , Proteínas Recombinantes/química , Análisis Espectral , Tecnología Farmacéutica/métodos , Vacunas de Partículas Similares a Virus/química , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/aislamiento & purificación , Vacunas de Partículas Similares a Virus/ultraestructuraRESUMEN
A multivalent vaccine candidate against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections was constructed on the basis of HBV core (HBc) virus-like particles (VLPs) as carriers. Chimeric VLPs that carried a virus-neutralizing HBV pre-S1 epitope corresponding to amino acids (aa) 20 to 47 in the major immunodominant region (MIR) and a highly conserved N-terminal HCV core epitope corresponding to aa 1 to 60 at the C terminus of the truncated HBcDelta protein (N-terminal aa 1 to 144 of full-length HBc) were produced in Escherichia coli cells and examined for their antigenicity and immunogenicity. The presence of two different foreign epitopes within the HBc molecule did not interfere with its VLP-forming ability, with the HBV pre-S1 epitope exposed on the surface and the HCV core epitope buried within the VLPs. After immunization of BALB/c mice, specific T-cell activation by both foreign epitopes and a high-titer antibody response against the pre-S1 epitope were found, whereas an antibody response against the HBc carrier was notably suppressed. Both inserted epitopes also induced a specific cytotoxic-T-lymphocyte (CTL) response, as shown by the gamma interferon (IFN-gamma) production profile.
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Epítopos/inmunología , Hepacivirus/inmunología , Antígenos del Núcleo de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Vacunas contra Hepatitis Viral/inmunología , Virión/inmunología , Animales , Citocinas/metabolismo , Epítopos/genética , Epítopos/metabolismo , Femenino , Hepacivirus/metabolismo , Anticuerpos contra la Hepatitis B/biosíntesis , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos del Núcleo de la Hepatitis B/metabolismo , Virus de la Hepatitis B/metabolismo , Inmunización , Inmunoglobulina G/sangre , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas contra Hepatitis Viral/genética , Virión/metabolismoRESUMEN
BACKGROUND: Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines. METHODOLOGY/PRINCIPAL FINDINGS: Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection. CONCLUSIONS/SIGNIFICANCE: AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.
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Epítopos/química , Virión/química , Animales , Clonación Molecular , Hormona Liberadora de Gonadotropina/química , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Péptidos/química , Estructura Terciaria de Proteína , Fagos ARN/metabolismo , Proteínas Recombinantes/química , Vacunación/instrumentación , Vacunación/métodosRESUMEN
The capsids of single-stranded RNA bacteriophages show remarkable structural similarity. In an attempt to test whether the coat protein (CP) from one bacteriophage could substitute for the CP of another and form mixed particles, we reassembled capsids in vitro from a mixture of different RNA phage CP dimers together with E. coli ribosomal RNA. Surprisingly, mixing CPs from phages belonging to groups I and II led to appearance of rod-like particles along with icosahedral spherical capsids, both containing a mixture of the two CPs. Rods and mixed spherical capsids containing host RNA were also obtained in vivo in bacteria expressing simultaneously fr and GA CPs. In a co-infection of the two phages, however, only authentic fr and GA virions were formed. Coat protein mutants in the FG loop were unable to assemble into rods, suggesting that these loops are involved in the formation of the aberrant particles.
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Proteínas de la Cápside/metabolismo , Colifagos/genética , Multimerización de Proteína , Fagos ARN/genética , Virión/metabolismo , Escherichia coli/genética , Modelos Moleculares , ARN Bacteriano/metabolismo , ARN Ribosómico/metabolismo , Virión/ultraestructuraRESUMEN
Vaccination of melanoma patients with tumor-specific antigens recognized by cytotoxic T lymphocytes (CTLs) may produce significant tumor regressions. Here, we suggest a novel type of tumor vaccines, with well-studied CTL epitopes presented on highly immunogenic virus-like particle (VLP) carriers. Cancer-germline gene MAGE-3 encodes for an antigenic nonapeptide (MAGE-3(168-176) peptide) that is recognized by CTLs on human leukocyte antigen (HLA)-A1 and HLA-B35 molecules. A set of recombinant genes encoding hepatitis B virus core protein carrying MAGE-3 epitope was constructed and expressed in Escherichia coli cells. Variants that led to formation of chimeric VLPs in vivo were purified and analyzed for their DNA binding properties in vitro. VLPs exhibiting the most pronounced nucleic acid binding affinity were selected and loaded either with single-stranded DNA oligodeoxynucleotides rich in nonmethylated CG motifs, or with longer double-stranded DNA fragments. Packaged DNA was protected, at least partially, against the action of bacterial DNase. Such highly purified chimeric VLPs with entrapped immunomodulatory sequences could possibly be used as antitumor vaccines.
Asunto(s)
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/uso terapéutico , Antígenos del Núcleo de la Hepatitis B/genética , Melanoma/terapia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Secuencia de Aminoácidos , Antígenos de Neoplasias/metabolismo , Secuencia de Bases , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Clonación Molecular , Islas de CpG , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Antígenos del Núcleo de la Hepatitis B/inmunología , Antígenos del Núcleo de la Hepatitis B/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T Citotóxicos , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéuticoRESUMEN
The preS1phil, a hydrophilic component of the hepatitis B virus (HBV) preS1 sequence, was exposed on the surface of three widely used virus-like particle (VLP) carriers by (i) insertion into the HI loop of the murine polyomavirus (MPyV) VP1, (ii) N-terminal addition to the hepatitis B surface (HBs) protein, and (iii) insertion into the major immunodominant region (MIR) of three hepatitis B core (HBc) vectors with different structure of their C-termini. Adjuvant-free immunisation of Balb/c mice demonstrated high preS1-specific antibody responses, but strong Th1 cell activation with efficient induction of IgG2a isotype antibodies was observed only in those VLPs, and namely in two of three HBc derivatives, which contained packaged RNA.