RESUMO
BACKGROUND: Peptide based vaccines may suffer from limited stability and inefficient delivery to professional antigen-presenting cells (APCs), such as dendritic cells (DCs). In order to overcome such limitations, several types of biodegradable nanoparticles (NPs) have been developed as carrier system for antigens. The present study describes for the first time the extensive biological characterization of cationic NPs made of poly (D,L-lactide-co-glycolide) (PLGA) and polyethylenimine (PLGA/PEI) as delivery system for protein/peptide antigens, with potential in therapeutic cancer vaccine development. RESULTS: Flow cytometry as well as confocal laser scanning microscopy (CLSM) showed that PLGA/PEI NPs are more readily taken up than PLGA NPs by both human CD14(+) monocytes and mouse Hepa 1-6 hepatoma cell line. No signs of toxicity were observed in either cellular setting. Sequential image acquisition by TEM showed an intracellular apical localization for PLGA NPs and a perinuclear localization for PLGA/PEI NPs. Both NPs showed a clathrin-dependent as well as a caveolin-dependent internalization pathway and, once in the cells, they formed multivesicular endosomes (MVE). Finally, an ex vivo priming experiment showed that PLGA/PEI NPs are comparable to PLGA NPs in delivering a non-self antigen (i.e., ovalbumin - OVA) to immature dendritic cells (imDCs), which matured and induced autologous naïve CD4(+) T cells to differentiate to memory (i.e., central memory and effector memory) cells. Such a differentiation was associated with a Th1 phenotype suggesting a downstream activation and amplification of a CD8(+) T cell cytotoxic response. The same OVA antigen in a soluble form was unable to induce maturation of DCs, indicating that both NP formulations provided an intrinsic adjuvanting effect combined to efficient antigen delivery. CONCLUSIONS: Our study represents the first report on side-by-side comparison of PLGA and PLGA/PEI NPs as strategy for protein antigen delivery. PLGA/PEI NPs are superior for cellular uptake and antigen delivery as compared to PLGA NPs. Such an evidence suggests their great potential value for vaccine development, including therapeutic cancer vaccines.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Ácido Láctico/farmacologia , Polietilenoimina/farmacologia , Ácido Poliglicólico/farmacologia , Animais , Antígenos/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Clatrina/metabolismo , Humanos , Memória Imunológica/imunologia , Camundongos , Microscopia Confocal , Corpos Multivesiculares/metabolismo , Nanopartículas , Ovalbumina/imunologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Linfócitos T Citotóxicos/imunologia , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Through the application of next generation sequencing, in synergy with conventional cloning of DOP-PCR fragments, two double-stranded RNA (dsRNA) molecules of about 1.5 kbp in size were isolated from leaf tissue of a Japanese persimmon (accession SSPI) from Apulia (southern Italy) showing veinlets necrosis. High-throughput sequencing allowed whole genome sequence assembly, yielding a 1,577 and a 1,491 bp contigs identified as dsRNA-1 and dsRNA-2 of a previously undescribed virus, provisionally named as Persimmon cryptic virus (PeCV). In silico analysis showed that both dsRNA fragments were monocistronic and comprised the RNA-dependent RNA polymerase (RdRp) and the capsid protein (CP) genes, respectively. Phylogenetic reconstruction revealed a close relationship of these dsRNAs with those of cryptoviruses described in woody and herbaceous hosts, recently gathered in genus Deltapartitivirus. Virus-specific primers for RT-PCR, designed in the CP cistron, detected viral RNAs also in symptomless persimmon trees sampled from the same geographical area of SSPI, thus proving that PeCV infection may be fairly common and presumably latent.
Assuntos
Diospyros/virologia , Genoma Viral , Vírus de Plantas/isolamento & purificação , Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/genética , RNA Viral/genética , Análise de Sequência de DNA , Proteínas do Capsídeo/genética , Clonagem Molecular , Análise por Conglomerados , Sequenciamento de Nucleotídeos em Larga Escala , Japão , Dados de Sequência Molecular , Filogenia , Folhas de Planta/virologia , Vírus de Plantas/classificação , Vírus de Plantas/genética , Vírus de RNA/classificação , Vírus de RNA/genética , RNA Polimerase Dependente de RNA/genética , Homologia de SequênciaRESUMO
Chloroplast genetic engineering has long been recognised as a powerful technology to produce recombinant proteins. To date, however, little attention has been given to the causes of pleiotropic effects reported, in some cases, as consequence of the expression of foreign proteins in transgenic plastids. In this study, we investigated the phenotypic alterations observed in transplastomic tobacco plants accumulating the Pr55(gag) polyprotein of human immunodeficiency virus (HIV-1). The expression of Pr55(gag) at high levels in the tobacco plastome leads to a lethal phenotype of seedlings grown in soil, severe impairment of plastid development and photosynthetic activity, with chloroplasts largely resembling undeveloped proplastids. These alterations are associated to the binding of Pr55(gag) to thylakoids. During particle assembly in HIV-1 infected human cells, the binding of Pr55(gag) to a specific lipid [phosphatidylinositol-(4-5) bisphosphate] in the plasma membrane is mediated by myristoylation at the amino-terminus and the so-called highly basic region (HBR). Surprisingly, the non-myristoylated Pr55(gag) expressed in tobacco plastids was likely able, through the HBR motif, to bind to nonphosphorous glycerogalactolipids or other classes of lipids present in plastidial membranes. Although secondary consequences of disturbed chloroplast biogenesis on expression of nuclear-encoded plastid proteins cannot be ruled out, results of proteomic analyses suggest that their altered accumulation could be due to retrograde control in which chloroplasts relay their status to the nucleus for fine-tuning of gene expression.
Assuntos
Nicotiana/genética , Plastídeos/genética , Precursores de Proteínas/genética , Plântula/genética , Animais , Células COS , Chlorocebus aethiops , Cloroplastos/genética , Cloroplastos/fisiologia , Ácidos Graxos Monoinsaturados/metabolismo , HIV-1/genética , Humanos , Membranas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plastídeos/metabolismo , Ligação Proteica , Precursores de Proteínas/metabolismo , Nicotiana/metabolismoRESUMO
Molecular features and genomic organization were determined for Citrus yellow vein clearing virus (CYVCV), the putative viral causal agent of yellow vein clearing disease of lemon trees, reported in Pakistan, India, and more recently in Turkey and China. CYVCV isolate Y1 from Adana, Turkey, was used for deep sequencing analysis of the virus-induced small RNA fractions and for mechanical and graft inoculation of herbaceous and citrus indicator plants. A polyclonal antiserum was developed from CYVCV-Y1 purified from Phaseolus vulgaris and used in western blot assays to characterize the coat protein of CYVCV-Y1 and determine its serological relationship with related viruses. Contigs assembled from the Illumina sequenced short reads were used to construct the whole genome of Citrus yellow vein clearing virus (CYVCV), consisting in a positive-sense RNA of 7,529 nucleotides and containing six predicted open reading frames. The CYVCV genome organization and size resembled that of flexiviruses, and search for sequence homologies revealed that Indian citrus ringspot virus (ICRSV) (Mandarivirus, Alphaflexiviridae) is the most closely related virus. However, CYVCV had an overall nucleotide sequence identity of ≈74% with ICRSV. Although the two viruses were similar with regard to genome organization, viral particles, and herbaceous host range, CYVCV caused different symptoms in citrus and was serologically distinct from ICRSV. Primer pairs were designed and used to detect the virus by conventional and quantitative reverse transcription-polymerase chain reaction on yellow vein clearing symptomatic field trees as well as graft- and mechanically inoculated host plants. Collectively, these data suggest that CYVCV is the causal agent of yellow vein clearing disease and represents a new species in the genus Mandarivirus.
Assuntos
Citrus/virologia , Flexiviridae/classificação , Flexiviridae/genética , Doenças das Plantas/virologia , Regulação Viral da Expressão Gênica , Genoma Viral , FilogeniaRESUMO
We have previously described the establishment and characterization of a stably transfected insect cell line for the constitutive and efficient expression of Pr55 HIV Gag proteins, which auto-assemble into enveloped Virus-Like Particles (VLPs) released into the cell culture supernatant. Such HIV-Gag VLPs have been shown to elicit a specific systemic humoral response in vivo, proving the appropriate antigenic presentation of the HIV Gag protein to the immune system. Here we describe the establishment of a stable double transfected insect cell line for the constitutive and reproducible production of Pr55Gag-VLPs expressing on their surface trimeric forms of HIV-1 envelope glycoproteins. The persistence of HIV coding genes has been verified in clonal resistant insect cells, the protein expression and conformation has been verified by Western blot analysis. The resulting HIV-VLPs have been visualized by standard transmission electron microscopy and their immunogenicity has been evaluated in vivo. This represents, to our knowledge, the first example of stable double transfected insect cell line for the constitutive production of enveloped HIV-Gag VLPs presenting trimeric HIV-gp140 on their surface.
Assuntos
Expressão Gênica , Virossomos/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Animais , Western Blotting , Linhagem Celular , Insetos , Microscopia Eletrônica de Transmissão , Transfecção , Virossomos/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
A primary therapeutic goal in Alzheimer's disease (AD) is to reduce the quantity of amyloid ß protein (Aß) present in the brain. To develop an effective, safe system for vaccination against Alzheimer's disease, the plant virus Cucumber mosaic virus (CMV) was engineered genetically to express Aß-derived fragments that stimulate mainly humoral immune responses. Six chimeric constructs, bearing the Aß1-15 or the Aß4-15 sequence in positions 248, 392 or 529 of the CMV coat protein (CP) gene, were created. Viral products proved to be able to replicate in their natural host. However, only chimeric Aß1-15-CMVs were detected by Aß1-42 antiserum in Western blot analysis. Experimental evidence of Immunoelectron microscopy revealed a complete decoration of Aß1-15-CMV(248) and Aß1-15-CMV(392) following incubation with either anti-Aß1-15 or anti-Aß1-42 polyclonal antibodies. These two chimeric CMVs appear to be endowed with features making them possible candidates for vaccination against Alzheimer's disease.
Assuntos
Vacinas contra Alzheimer/biossíntese , Peptídeos beta-Amiloides/biossíntese , Cucumovirus/genética , Expressão Gênica , Vetores Genéticos , Doença de Alzheimer/prevenção & controle , Doença de Alzheimer/terapia , Vacinas contra Alzheimer/genética , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Humanos , Microscopia Imunoeletrônica , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Nicotiana , Cultura de VírusRESUMO
We have previously developed HIV-1 Pr55gag-based virus-like particles (HIV-VLPs) as presentation and delivery model using a transient Baculovirus expression system. Here we describe the establishment and characterization of stably transfected insect cell line for the constitutive and reproducible production of HIV-VLPs. The persistence of HIV gag coding gene has been verified in clonal resistant insect cells and the protein expression has been confirmed by Western blot analysis. The resulting HIV-VLPs have been evaluated by standard transmission electron microscopy and their immunogenicity has been evaluated in vivo. Our results demonstrate that this strategy is highly efficient for constitutive expression of conformational enveloped VLPs which can be employed as presentation and delivery system for pathogen as well as tumor-associated antigens. This represents, to our knowledge, the first example of stably transfected insect cell line for the constitutive production of VLPs.
Assuntos
Vacinas contra a AIDS/imunologia , Produtos do Gene gag/imunologia , HIV-1/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Formação de Anticorpos , Linhagem Celular , Escherichia coli/metabolismo , Anticorpos Anti-HIV/sangue , Insetos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , TransfecçãoRESUMO
The Cucumber mosaic virus (CMV) is a three-component isodiametric plant virus with an extremely wide host range, present worldwide. A pseudorecombinant form has been described, deriving from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA 1, 2 components of the CMV-D strain. The CP gene was then engineered to express one or two copies of a synthetic peptide derived from many hypervariable region 1 (HVR1) sequences of the Hepatitis C virus (HCV) envelope protein E2 (the so-called R9 mimotope). Study of the symptoms pattern displayed in tobacco by these chimeric CMV particles, together with determination of their structural characteristics, assessed by circular dichroism (CD) spectroscopy and electron microscopy, revealed a possible relationship between the biological behavior and the structural properties of virus components.
Assuntos
Epitopos/química , Hepacivirus/genética , Recombinação Genética , Proteínas do Envelope Viral/química , Vírion , Dicroísmo Circular , Cucumovirus/química , Cucumovirus/genética , Cucumovirus/metabolismo , Cucumovirus/ultraestrutura , Epitopos/genética , Epitopos/metabolismo , Hepacivirus/química , Hepacivirus/metabolismo , Microscopia Eletrônica , Doenças das Plantas/virologia , Folhas de Planta/virologia , Nicotiana/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismo , Vírion/ultraestruturaRESUMO
Potato virus T (PVT), a member of an unassigned species in the family Flexiviridae, has a genome 6,539 nt in size with three ORFs coding for replication-associated proteins (185 kDa, ORF 1), movement protein (40 kDa, ORF 2) and coat protein (24 kDa, ORF 3), respectively. PVT differs from the type members of all genera of the family Flexiviridae with a 30K-type movement protein and is phylogenetically distant from all of these viruses, least so from grapevine virus A (GVA, genus Vitivirus), with which it groups in all trees. The viral genome resembles that of trichoviruses but is smaller and does not contain the 3' terminal fourth ORF found in some members of this genus. PTV may represent a new genus of plant viruses for which the provisional name of Andesvirus is proposed.
Assuntos
Flexiviridae/genética , Genoma Viral , Vírus de Plantas/genética , Solanum tuberosum/virologia , Sequência de Aminoácidos , Sequência de Bases , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Flexiviridae/classificação , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Proteínas do Movimento Viral em Plantas/química , Proteínas do Movimento Viral em Plantas/genética , Vírus de Plantas/classificação , RNA Viral/genéticaRESUMO
A vaccine against Hepatitis C virus (HCV) is urgently needed due to the unsatisfactory clinical response to current therapies. We evaluated the immunological properties of a chimeric Cucumber mosaic virus (CMV), a plant virus engineered to express on its surface a synthetic peptide derived from many HVR1 sequences of the HCV envelope protein E2 (R9 mimotope). Evidence was obtained that the chimeric R9-CMV elicits a specific humoral response in rabbits. Furthermore, in patients with chronic HCV infection, purified preparations of R9-CMV down-modulated the lymphocyte surface density of CD3 and CD8, and induced a significant release of interferon (IFN)-gamma, interleukin (IL)-12 p70 and IL-15 by lymphomonocyte cultures. Finally, an R9 mimotope-specific CD8 T-cell response, as assessed by intracellular IFN-gamma production, was achieved in the majority of the patients studied. Our results open up new prospects for the development of effective vaccines against HCV infection. Moreover, the wide edible host range of CMV makes the production of an edible vaccine conceivable.
Assuntos
Quimera/imunologia , Cucumovirus/genética , Cucumovirus/imunologia , Epitopos/imunologia , Hepacivirus/imunologia , Vacinas contra Hepatite Viral/imunologia , Adulto , Idoso , Animais , Células Cultivadas , Quimera/genética , Doença Crônica , Citocinas/metabolismo , Epitopos/genética , Feminino , Hepacivirus/genética , Hepatite C/genética , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Coelhos , Linfócitos T/metabolismo , Linfócitos T/virologiaRESUMO
Comparison of the predicted coat protein amino acid sequence of the 'sweet cherry' strain of plum pox potyvirus (PPV-SwC) with the corresponding regions of several other PPV strains indicated that the main differences are in the N-terminal region. Polyclonal antibodies were produced against a synthetic peptide corresponding to the 1-14 sequence of the N-terminal region of PPV-SwC coat protein. They specifically detected PPV-SwC in different immunochemical tests.
Assuntos
Anticorpos Antivirais/biossíntese , Proteínas do Capsídeo , Capsídeo/imunologia , Fragmentos de Peptídeos/imunologia , Potyvirus/imunologia , Potyvirus/isolamento & purificação , Sequência de Aminoácidos , Anticorpos Antivirais/imunologia , Western Blotting , Capsídeo/química , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Doenças das Plantas/virologia , Extratos Vegetais , Análise de Sequência , Especificidade da EspécieRESUMO
The supramolecular organisation of elastin and its soluble derivative alpha-elastin were studied by scanning and transmission electron microscopy. It was found a variety of different structures including filaments, fibrils, fibres, networks and dendritic, leaf-like forms. Self-similar patterns, extending for at least three orders of magnitude, were revealed, strongly suggesting the presence of fractal objects. The fractal dimension D was determined by using the box counting method.