RESUMEN
Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science. ssDNA aptamers were specifically selected against the major peanut allergen Ara h 1 and identified by next-generation sequencing. Application in various detection systems (ELISA-like assays, western blot, and surface plasmon resonance) was demonstrated. The ELISA-like assay comprised a sensitivity of 10 ng/mL Ara h 1, comparable to published antibody-based ELISA, and allowed Ara h 1 detection in various peanut flours, similar to those used in peanut AIT as well as in processed food. This ELISA-like aptamer-based assay proofs antibody-free allergen detection for food labeling or quality assessment of diagnostic and therapeutic allergen products.
Asunto(s)
Alérgenos , Antígenos de Plantas , Aptámeros de Nucleótidos , Arachis , Ensayo de Inmunoadsorción Enzimática , Proteínas de Plantas , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/inmunología , Arachis/química , Arachis/inmunología , Antígenos de Plantas/inmunología , Antígenos de Plantas/análisis , Antígenos de Plantas/genética , Proteínas de Plantas/inmunología , Proteínas de Plantas/genética , Alérgenos/inmunología , Alérgenos/análisis , Hipersensibilidad al Cacahuete/inmunología , Glicoproteínas/inmunología , Glicoproteínas/química , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/genética , Humanos , Técnica SELEX de Producción de Aptámeros/métodosRESUMEN
Natural killer (NK) cells have high intrinsic cytotoxic capacity, and clinical trials have demonstrated their safety and efficacy for adoptive cancer therapy. Expression of chimeric antigen receptors (CARs) enhances NK cell target specificity, with these cells applicable as off-the-shelf products generated from allogeneic donors. Here, we present for the first time an innovative approach for CAR NK cell engineering employing a non-viral Sleeping Beauty (SB) transposon/transposase-based system and minimized DNA vectors termed minicircles. SB-modified peripheral blood-derived primary NK cells displayed high and stable CAR expression and more frequent vector integration into genomic safe harbors than lentiviral vectors. Importantly, SB-generated CAR NK cells demonstrated enhanced cytotoxicity compared with non-transfected NK cells. A strong antileukemic potential was confirmed using established acute lymphocytic leukemia cells and patient-derived primary acute B cell leukemia and lymphoma samples as targets in vitro and in vivo in a xenograft leukemia mouse model. Our data suggest that the SB-transposon system is an efficient, safe, and cost-effective approach to non-viral engineering of highly functional CAR NK cells, which may be suitable for cancer immunotherapy of leukemia as well as many other malignancies.
Asunto(s)
Vectores Genéticos , Inmunoterapia Adoptiva , Células Asesinas Naturales , Receptores Quiméricos de Antígenos , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Animales , Ratones , Vectores Genéticos/genética , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Inmunoterapia Adoptiva/métodos , Ensayos Antitumor por Modelo de Xenoinjerto , Transposasas/genética , Transposasas/metabolismo , Línea Celular Tumoral , Elementos Transponibles de ADN , Citotoxicidad Inmunológica , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Ingeniería Celular/métodosRESUMEN
The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models.
Asunto(s)
COVID-19 , Inmunidad Innata , SARS-CoV-2 , Serina Endopeptidasas , Internalización del Virus , SARS-CoV-2/inmunología , SARS-CoV-2/fisiología , SARS-CoV-2/metabolismo , Humanos , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , COVID-19/virología , COVID-19/inmunología , COVID-19/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Replicación Viral , Animales , Endocitosis , Células HEK293 , Chlorocebus aethiops , CitologíaRESUMEN
Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.