RESUMO
The role of apoptosis in the persistence of hepatitis C virus (HCV) infection is controversial. Moreover, conflicting data on the modulation of this process by HCV proteins have been provided. We evaluated the susceptibility of peripheral lymphocytes from patients with chronic hepatitis C to apoptosis both spontaneous and after incubation with a chimeric Cucumber mosaic virus (CMV) carrying 180 copies of the synthetic R9 mimotope obtained from more than 200 hypervariable region-1 sequences of HCV. Resting T lymphocytes were found to be sensitized to apoptosis as a result of chronic HCV infection. The plant virus-derived vector R9-CMV displayed a strong pro-apoptotic effect associated with activation of both caspase-8 and -9, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. A parallel R9-CMV-mediated activation of endoplasmic reticulum-stress was suggested by the significant induction of BiP/GRP78, GADD153 and caspase-12. These data contribute to define the complex HCV/host interaction, and open new prospects for developing a plant-derived antigen-presenting system to strengthen host defences against persistent pathogens.
Assuntos
Apoptose , Caspases/metabolismo , Cucumovirus/genética , Estresse do Retículo Endoplasmático , Hepatite C Crônica/fisiopatologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Adulto , Idoso , Caspase 12/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Células Cultivadas , DNA Viral , Chaperona BiP do Retículo Endoplasmático , Epitopos , Feminino , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Mimetismo MolecularRESUMO
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
The Cucumber mosaic virus (CMV) is an isodiametric plant virus with an extremely wide host range, present worldwide. CMV chimeric particles (R9-CMV), engineered to express a 27-aa synthetic peptide derived from Hepatitis C virus (HCV), were demonstrated to be stable under simulated gastric and intestinal conditions. Then the possibility of inducing a humoral immune response in rabbits fed with R9-CMV infected lettuce plants was demonstrated, suggesting that this system could function as a confirming tool of a bioreactor for the production of a stable edible vaccine against HCV.
Assuntos
Cucumovirus/química , Hepacivirus/química , Hepatite C/prevenção & controle , Nanopartículas/química , Vacinas contra Hepatite Viral/química , Administração Oral , Sequência de Aminoácidos , Animais , Cucumovirus/genética , Epitopos/administração & dosagem , Epitopos/química , Epitopos/imunologia , Hepacivirus/genética , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Intestinos , Peptídeos/administração & dosagem , Peptídeos/síntese química , Peptídeos/imunologia , Coelhos , Vírus Reordenados/química , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Estômago , Vacinas de Plantas Comestíveis/administração & dosagem , Vacinas de Plantas Comestíveis/química , Vacinas de Plantas Comestíveis/imunologia , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/imunologia , Vírion/imunologiaRESUMO
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
Chimeric plant viruses are emerging as promising vectors for use in innovative vaccination strategies. In this context, cucumber mosaic virus (CMV) has proven to be a suitable carrier of the hepatitis C virus (HCV)-derived R9 mimotope. In the present work, a new chimeric CMV, expressing on its surface the HCV-derived R10 mimotope, was produced but lost the insert after the first passage on tobacco. A comparative analysis between R10- and R9-CMV properties indicated that R9-CMV stability was related to structural features typical of the foreign insert. Thus, in order to combine high virus viability with strong immuno-stimulating activity, we doubled R9 copies on each of the 180 coat protein (CP) subunits of CMV. One of the chimeras produced by this approach (2R9-CMV) was shown to systemically infect the host, stably maintaining both inserts. Notably, it was strongly recognized by sera of HCV-infected patients and, as compared with R9-CMV, displayed an enhanced ability to stimulate lymphocyte IFN-gamma production. The high immunogen levels achievable in plants or fruits infected with 2R9-CMV suggest that this chimeric form of CMV may be useful in the development of oral vaccines against HCV.
Assuntos
Proteínas do Capsídeo/imunologia , Cucumovirus/metabolismo , Epitopos/metabolismo , Vetores Genéticos/metabolismo , Hepacivirus/imunologia , Peptídeos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Vírus Reordenados/metabolismo , Vacinas contra Hepatite Viral/genética , Sequência de Aminoácidos , Especificidade de Anticorpos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Cucumovirus/genética , Cucumovirus/crescimento & desenvolvimento , Epitopos/química , Epitopos/genética , Hepatite C/imunologia , Humanos , Soros Imunes/imunologia , Interferon gama/biossíntese , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Vacinas contra Hepatite Viral/imunologiaRESUMO
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
Cucumber mosaic virus (CMV) is a three component isodiametric plant virus which is common worldwide and has an extremely wide host range. A pseudorecombinant was made, derived from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA1,2 components of the CMV-D strain. This system developed mild mosaic and vein clearing in Xanthi tobacco three weeks after inoculation. The CP gene was then engineered in three different positions, to encode a Hepatitis C virus (HCV) epitope. The selected peptide was the so-called R9 mimotope, a synthetic surrogate derived from a consensus profile of many hypervariable region 1 (HVR1) sequences of the putative HCV envelope protein E2. Serum samples from 60 patients with chronic hepatitis C displayed a significant immunoreactivity to crude plant extracts infected with the chimeric CMV. These results suggest that further investigation should be made into a possible vaccine function for the CMV-HCV mimotope system.
Assuntos
Cucumovirus/genética , Epitopos/imunologia , Antígenos da Hepatite C/imunologia , Proteínas do Envelope Viral/imunologia , Adulto , Idoso , Reações Cruzadas , Epitopos/química , Epitopos/genética , Feminino , Hepacivirus/imunologia , Antígenos da Hepatite C/química , Antígenos da Hepatite C/genética , Hepatite C Crônica/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênese Insercional , Doenças das Plantas/virologia , Nicotiana/virologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
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
An isolate of plum pox potyvirus from sweet cherry (PPV-SwC) in southern Italy was investigated. The isolate was mechanically or graft transmissible to different Prunus and Nicotiana spp. but not to Chenopodium spp. It was transmitted also by Aphis fabae and Myzus persicae in a nonpersistent manner. Restriction fragment length polymorphism analysis indicated and nucleotide sequencing confirmed that the isolate lacks AluI and RsaI sites in the C-terminal region of the coat protein (CP) gene. Western immunoblot results showed that the PPV-SwC CP has an electrophoretic mobility similar to that of strain PPV-D and faster than that of strain PPV-M. Double-antibody sandwich indirect enzyme-linked immunosorbent assay of the CP showed that PPV-SwC, although reacting with universal monoclonal antibodies to PPV, failed to react with antibodies specific to strains M and D. Results indicate that PPV-SwC is different from conventional strains of PPV but closely related to the sour cherry isolate of PPV from Moldova.
