RESUMO
A method for the large-scale production of a quadrivalent mammalian cell derived hepatitis C virus-like particles (HCV VLPs) is described. The HCV core E1 and E2 coding sequences of genotype 1a, 1b, 2a or 3a were co-expressed in Huh7 cell factories using a recombinant adenoviral expression system. The structural proteins self-assembled into VLPs that were purified from Huh7 cell lysates by iodixanol ultracentrifugation and Stirred cell ultrafiltration. Electron microscopy, revealed VLPs of the different genotypes that are morphologically similar. Our results show that it is possible to produce large quantities of individual HCV genotype VLPs with relative ease thus making this approach an alternative for the manufacture of a quadrivalent mammalian cell derived HCV VLP vaccine.
Assuntos
Hepacivirus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Proteínas Virais/metabolismo , Adenoviridae/genética , Linhagem Celular , Expressão Gênica , Vetores Genéticos , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Microscopia Eletrônica de Transmissão , Vacinas de Partículas Semelhantes a Vírus/ultraestrutura , Proteínas Virais/genéticaRESUMO
An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.
Assuntos
Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Linfócitos T/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Linhagem Celular , Células Cultivadas , Hepacivirus/genética , Hepatócitos/virologia , Humanos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Microscopia Eletrônica , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Proteínas do Core Viral/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Virossomos/genética , Virossomos/imunologia , Virossomos/metabolismo , Virossomos/ultraestruturaRESUMO
Plasmodium falciparum malaria remains a major public health hazard in sub-Saharan African children. While the factors that determine the variations in clinical outcome of a malaria have not been completely defined, both host and parasite factors, as well as the complex molecular interactions between them have been implicated. The cyto-adherent properties of the P. falciparum-infected red blood cells are considered as key properties in the pathogenesis of malaria and the polymorphisms of the host adhesion molecules could contribute to the severity of malaria. Clinical information and blood samples were collected from 223 children from Ibadan (south-west Nigeria), median age of 34.5 months, presenting with different clinical manifestations of malaria--clinically asymptomatic parasitism (ACP), acute uncomplicated malaria (UM) and severe malaria (SM)--as defined by WHO criteria. The polymorphisms of genes coding for four human adhesion molecules at six different loci (ICAM-1 exons 2, 4 and 6, E-selectin exon 2, CD36 exon 10, and PECAM exon 3) were studied. DNA samples were prepared for further genotyping of the six exons mentioned above by PCR-RFLPs using the appropriate restriction digests for each loci. The ICAM-1 exon 4 locus was monomorphic. All the other loci were at Hardy-Weinberg equilibrium (HWE). The E-selectin locus had very low heterozygosity (approximately 0.06) in contrast to the other loci under study (0.23-0.44). Once the data was further processed for covariates (age and parasite density) and taking as the reference category the ACP group, results show that in the presence of the G allele at the ICAM-1 exon 6 there is an increased risk (3.6 times) of severe malaria. As far as the T allele in the E-selectin exon is concerned, the number of sampled DNAs with the T allele within both the UM and SM categories is too low for drawing any relevant conclusion at this stage. In conclusion, these results suggest that genetic polymorphisms at host adhesion molecules loci are an important variable in the susceptibility to severe malaria. Further studies of host loci are needed to further delineate which polymorphisms are associated with severe malaria and increase our knowledge of the biology of host-parasite interactions.
Assuntos
Selectina E/genética , Molécula 1 de Adesão Intercelular/genética , Malária Falciparum/genética , Pré-Escolar , Feminino , Humanos , Malária Falciparum/sangue , Malária Falciparum/classificação , Masculino , Nigéria , Polimorfismo Genético , Índice de Gravidade de DoençaRESUMO
Genetic polymorphisms have been recognized as important determinants of gene expression. Three common single nucleotide polymorphisms have been identified in the promoter and 5' untranslated region of the vascular endothelial growth factor (VEGF) gene: -460 C --> T, -141 A --> C and +405 G --> C. As VEGF has been postulated to play a role in the pathogenesis of childhood steroid-sensitive nephrotic syndrome (SSNS), this study tested the hypothesis that VEGF genotype may be associated with susceptibility to SSNS. We examined the genotype frequencies of these polymorphisms in a total of 116 children with SSNS and 150 control subjects, using polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP). There were no statistically significant differences in any of the genotype frequencies between SSNS patients and controls. We conclude that VEGF -460, -141 and +405 genotypes are not associated with susceptibility to childhood SSNS.
Assuntos
Fatores de Crescimento Endotelial/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Linfocinas/genética , Síndrome Nefrótica/genética , Polimorfismo de Nucleotídeo Único , Regiões 5' não Traduzidas , Criança , Frequência do Gene , Predisposição Genética para Doença , Humanos , Síndrome Nefrótica/tratamento farmacológico , Regiões Promotoras Genéticas , Valores de Referência , Esteroides/farmacologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob-associated histidine-rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite-derived structures, known as Maurer's clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.
Assuntos
Eritrócitos/parasitologia , Peptídeos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Brefeldina A/farmacologia , Adesão Celular , Citosol/química , Eritrócitos/metabolismo , Eritrócitos/ultraestrutura , Complexo de Golgi/efeitos dos fármacos , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Substâncias Macromoleculares , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Fotoquímica , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Vacúolos/metabolismo , Vacúolos/parasitologiaRESUMO
Resistance to commonly used malaria drugs is spreading and new drugs are required urgently. The recent identification of a relict chloroplast (apicoplast) in malaria and related parasites offers numerous new targets for drug therapy using well-characterized compounds. The apicoplast contains a range of metabolic pathways and housekeeping processes that differ radically to those of the host thereby presenting ideal strategies for drug therapy. Indeed, many compounds targeting these plastid pathways are antimalarial and have favourable profiles based on extensive knowledge from their use as antibacterials.