RESUMEN
Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. Between 15-70% of reported cases are fatal with no approved vaccine available. In the present study, the attenuated poxvirus vector, Modified Vaccinia virus Ankara, was used to develop a recombinant candidate vaccine expressing the CCHF virus nucleoprotein. Cellular and humoral immunogenicity was confirmed in 2 mouse strains, including type I interferon receptor knockout mice, which are susceptible to CCHF disease. Despite the immune responses generated post-immunisation, the vaccine failed to protect animals from lethal disease in a challenge model.
Asunto(s)
Anticuerpos Antivirales/sangre , Virus de la Fiebre Hemorrágica de Crimea-Congo/inmunología , Fiebre Hemorrágica de Crimea/prevención & control , Inmunogenicidad Vacunal/inmunología , Nucleoproteínas/inmunología , Vacunas Sintéticas/inmunología , Proteínas Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Embrión de Pollo , Chlorocebus aethiops , Cricetinae , Fiebre Hemorrágica de Crimea/inmunología , Humanos , Inmunización , Ratones , Ratones Noqueados , Receptor de Interferón alfa y beta/genética , Células Vero , Carga Viral/inmunologíaRESUMEN
The capacity of activated T cells to alter their cytokine expression profiles after migration into an effector site has not previously been defined. We addressed this issue by paired daughter analysis of a type 1-polarized CD8(+) effector T cell population freshly isolated from lung parenchyma of influenza virus-infected mice. Single T cells were activated to divide in vitro; individual daughter cells were then micromanipulated into secondary cultures with and without added IL-4 to assess their potential to express type 2 cytokine genes. The resultant subclones were analyzed for type 1 and 2 cytokine mRNAs at day 6-7. When the most activated (CD44(high)CD11a(high)) CD8(+) subpopulation from infected lung was compared with naive or resting (CD44(low)CD11a(low)) CD8(+) cells from infected lung and from normal lymph nodes (LNs), both clonogenicity and plasticity of the cytokine response were highest in the LN population and lowest in the activated lung population, correlating inversely with effector function. Multipotential cells were nevertheless detected among clonogenic CD44(high)CD11a(high) lung cells at 30-50% of the frequency in normal LNs. The data indicate that activated CD8(+) T cells can retain the ability to proliferate and express new cytokine genes in response to local stimuli after recruitment to an effector site.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Activación de Linfocitos/inmunología , Animales , Células Clonales , Citocinas/genética , Femenino , Receptores de Hialuranos/inmunología , Interferón gamma/genética , Interleucina-10/inmunología , Interleucina-4/inmunología , Interleucina-4/farmacología , Pulmón/inmunología , Pulmón/virología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/virología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Fenotipo , ARN Mensajero/metabolismoRESUMEN
The serine proteinase inhibitor (serpin) plasminogen activator inhibitor type 2 (PAI-2) is well characterized as an inhibitor of extracellular urokinase-type plasminogen activator. Here we show that intracellular, but not extracellular, PAI-2 protected cells from the rapid cytopathic effects of alphavirus infection. This protection did not appear to be related to an effect on apoptosis but was associated with a PAI-2-mediated induction of constitutive low-level interferon (IFN)-alpha/beta production and IFN-stimulated gene factor 3 (ISGF3) activation, which primed the cells for rapid induction of antiviral genes. This primed phenotype was associated with a rapid development of resistance to infection by the PAI-2 transfected cells and the establishment of a persistent productive infection. PAI-2 was also induced in macrophages in response to viral RNA suggesting that PAI-2 is a virus response gene. These observations, together with the recently demonstrated PAI-2-mediated inhibition of tumor necrosis factor-alpha induced apoptosis, (a) illustrate that PAI-2 has an additional and distinct function as an intracellular regulator of signal transduction pathway(s) and (b) demonstrate a novel activity for a eukaryotic serpin.
Asunto(s)
Alphavirus/inmunología , Interferón-alfa/genética , Interferón beta/genética , Inhibidor 2 de Activador Plasminogénico/farmacología , Inhibidores de Serina Proteinasa/farmacología , Adenovirus Humanos/inmunología , Antivirales , Apoptosis , Efecto Citopatogénico Viral/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Virus de la Influenza A/inmunología , Factor 3 de Genes Estimulados por el Interferón , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón , Interferón-alfa/inmunología , Interferón beta/inmunología , Inhibidor 2 de Activador Plasminogénico/genética , Poli I-C/inmunología , Reacción en Cadena de la Polimerasa , Proteínas/metabolismo , ARN Mensajero , Virus del Río Ross/inmunología , Inhibidores de Serina Proteinasa/genética , Virus Sindbis/inmunología , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismo , Latencia del VirusRESUMEN
The physiological roles of plasminogen activator inhibitor-2 (PAI-2) are not yet well understood. Kinetic studies suggest a role in the regulation of plasminogen activator-driven proteolysis in many cell types. This study describes a monoclonal antibody (2H5), which uniquely recognizes neoepitope determinants on PAI-2 appearing after thermodynamic relaxation of the molecule. Enzyme-linked immunosorbent assays and native polyacrylamide gel electrophoresis immunoblotting confirmed the specificity of 2H5 for urokinase type plasminogen activator.PAI-2 complexes. Examination of the affinity of 2H5 for complexes formed between PAI-2 and a synthetic 14-mer reactive site loop peptide, PAI-2 treated with tissue plasminogen activator, or thrombin suggests that the 2H5 epitope is determined exclusively by sequences found only on PAI-2 following proteolytic cleavage of the Arg380-Thr381 bond and insertion of the reactive site loop into beta-sheet A. Peptides lacking both the P13 (Glu368) and P14 (Thr367) residues did not induce a conformational change or affect the inhibitory activity of PAI-2, indicating that one or both of these residues are critical for PAI-2 function. To our knowledge, this is the first description of a monoclonal antibody that can distinguish conformational changes in PAI-2 related specifically to its potential biological function(s).