RESUMEN
Genetic frontotemporal lobar degeneration caused by autosomal dominant gene mutations provides an opportunity for targeted drug development in a highly complex and clinically heterogeneous dementia. These neurodegenerative disorders can affect adults in their middle years, progress quickly relative to other dementias, are uniformly fatal and have no approved disease-modifying treatments. Frontotemporal dementia, caused by mutations in the GRN gene which encodes the protein progranulin, is an active area of interventional drug trials that are testing multiple strategies to restore progranulin protein deficiency. These and other trials are also examining neurofilament light as a potential biomarker of disease activity and disease progression and as a therapeutic endpoint based on the assumption that cerebrospinal fluid and blood neurofilament light levels are a surrogate for neuroaxonal damage. Reports from genetic frontotemporal dementia longitudinal studies indicate that elevated concentrations of blood neurofilament light reflect disease severity and are associated with faster brain atrophy. To better inform patient stratification and treatment response in current and upcoming clinical trials, a more nuanced interpretation of neurofilament light as a biomarker of neurodegeneration is now required, one that takes into account its relationship to other pathophysiological and topographic biomarkers of disease progression from early presymptomatic to later clinically symptomatic stages.
RESUMEN
Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.
Asunto(s)
Inmunidad Adaptativa/genética , Ingeniería Genética/métodos , Vectores Genéticos/genética , Inmunidad Innata/genética , Inmunización/métodos , Poxviridae/genética , Proteínas Virales/genética , Animales , Presentación de Antígeno/genética , Antígeno B7-2/metabolismo , Proliferación Celular , Cricetinae , Reacciones Cruzadas/genética , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Eliminación de Gen , Expresión Génica , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , VIH-1/genética , VIH-1/fisiología , Células HeLa , Humanos , Interferón-alfa/biosíntesis , Poxviridae/fisiología , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/virología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The CD4 T-cell response to vaccinia promotes antibody and long-term CD8 responses. HLA class II molecules present microbial epitopes to CD4 T-cells. In humans, at least 3 loci encode cell-surface peptide-binding HLA class II heterodimers. Using intracellular cytokine cytometry (ICC) assays, we determined that HLA DR had the strongest contribution to vaccinia antigen presentation. Among panels of vaccinia-restricted T-cell clones, most were DR-restricted but rare DQ-restricted clones were also recovered. Vaccinia has over 200 open reading frames (ORFs), providing a significant bottleneck to assigning fine specificity. To overcome this, we expressed each predicted vaccinia ORF using in vitro transcription and translation. Array-based pool proteins were used to rapidly assign fine specificity to each DQ-restricted clone and to a sample of HLA DR-restricted clones. Reactivity was confirmed using synthetic peptides for selected CD4 T-cell clones. This method should be broadly applicable to the study of large-genome, sequenced pathogens, and could also be used to investigate T-cell responses to cDNAs expressed in neoplastic and autoimmune disorders in which CD4 responses might be adaptive or harmful.
