RESUMEN
Hepatitis C virus (HCV) infection is a major worldwide problem. Chronic hepatitis C is recognized as one of the major causes of cirrhosis, hepatocellular carcinoma, and liver failure. Although new, directly acting antiviral therapies are suggested to overcome the low efficacy and adverse effects observed for the current standard of treatment, an effective vaccine would be the only way to certainly eradicate HCV infection. Recently, polyhydroxybutyrate beads produced by engineered Escherichia coli showed efficacy as a vaccine delivery system. Here, an endotoxin-free E. coli strain (ClearColi) was engineered to produce polyhydroxybutyrate beads displaying the core antigen on their surface (Beads-Core) and their immunogenicity was evaluated in BALB/c mice. Immunization with Beads-Core induced gamma interferon (IFN-γ) secretion and a functional T cell immune response against the HCV Core protein. With the aim to target broad T and B cell determinants described for HCV, Beads-Core mixed with HCV E1, E2, and NS3 recombinant proteins was also evaluated in BALB/c mice. Remarkably, only three immunization with Beads-Core+CoE1E2NS3/Alum (a mixture of 0.1 µg Co.120, 16.7 µg E1.340, 16.7 µg E2.680, and 10 µg NS3 adjuvanted in aluminum hydroxide [Alum]) induced a potent antibody response against E1 and E2 and a broad IFN-γ secretion and T cell response against Core and all coadministered antigens. This immunological response mediated protective immunity to viremia as assessed in a viral surrogate challenge model. Overall, it was shown that engineered biopolyester beads displaying foreign antigens are immunogenic and might present a particulate delivery system suitable for vaccination against HCV.
Asunto(s)
Sistemas de Liberación de Medicamentos , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/sangre , Hidroxibutiratos/administración & dosificación , Poliésteres/administración & dosificación , Linfocitos T/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Animales , Modelos Animales de Enfermedad , Escherichia coli/genética , Escherichia coli/metabolismo , Hepatitis C/prevención & control , Interferón gamma/metabolismo , Ingeniería Metabólica , Ratones Endogámicos BALB C , Resultado del Tratamiento , Viremia/prevención & controlRESUMEN
The taxonomic position of a chlorophenol-degrading bacterium, strain S37T, was investigated. The 16S rDNA sequence indicated that this strain belongs to the genus Sphingopyxis, exhibiting high sequence similarity to the 16S rDNA sequences of Sphingomonas alaskensis LMG 18877T (98.8%), Sphingopyxis macrogoltabida LMG 17324T (98.2%), Sphingopyxis terrae IFO 15098T (95%) and Sphingomonas adhaesiva GIFU 11458T (92%). These strains (except Sphingopyxis terrae IFO 15098T, which was not investigated) and the novel isolate accumulated polyhydroxyalkanoates consisting of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from glucose as carbon source. The G + C content of the DNA of strain S37T was 65.5 mol%. The major cellular fatty acids of this strain were octadecenoic acid (18 : 1omega7c), heptadecenoic acid (17 : 1omega6c) and hexadecanoic acid (16 : 0). The results of DNA-DNA hybridization experiments and its physiological characteristics clearly distinguished the novel isolate from all known Sphingopyxis species and indicated that the strain represents a novel Sphingopyxis species. Therefore, the species Sphingopyxis chilensis sp. nov. is proposed, with strain S37T (=LMG 20986T =DSM 14889T) as the type strain. The transfer of Sphingomonas alaskensis to the genus Sphingopyxis as Sphingopyxis alaskensis comb. nov. is also proposed.
Asunto(s)
Alphaproteobacteria/clasificación , Alphaproteobacteria/metabolismo , Clorofenoles/metabolismo , Ácidos Grasos/química , ARN Ribosómico 16S/análisis , Alphaproteobacteria/química , Alphaproteobacteria/genética , Alphaproteobacteria/aislamiento & purificación , Composición de Base , Biodegradación Ambiental , Ácidos Grasos/análisis , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Filogenia , ARN Ribosómico 16S/genética , Ácidos EsteáricosRESUMEN
Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for beta-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.