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The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance of remaining vigilant and adaptable. Monoclonal antibodies (mAbs) have stood out as a powerful and immediate therapeutic response to COVID-19. Despite the success of mAbs, the evolution of SARS-CoV-2 continues to pose challenges and the available antibodies are no longer effective. New variants require the ongoing development of effective antibodies. In the present study, we describe the generation and characterization of neutralizing mAbs against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein by combining plasmid DNA and recombinant protein vaccination. By integrating genetic immunization for rapid antibody production and the potent immune stimulation enabled by protein vaccination, we produced a rich pool of antibodies, each with unique binding and neutralizing specificities, tested with the ELISA, BLI and FACS assays and the pseudovirus assay, respectively. Here, we present a panel of mAbs effective against the SARS-CoV-2 variants up to Omicron BA.1 and BA.5, with the flexibility to target emerging variants. This approach ensures the preparedness principle is in place to address SARS-CoV-2 actual and future infections.
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Lung disease in alpha-1-antitrypsin deficiency (AATD) mainly results from insufficient control of the serine proteases neutrophil elastase (NE) and proteinase-3 due to reduced plasma levels of alpha-1-antitrypsin (AAT) variants. Mutations in the specificity-determining reactive center loop (RCL) of AAT would be predicted to minimally affect protein folding and secretion by hepatocytes but can impair anti-protease activity or alter the target protease. These properly secreted but dysfunctional 'type-2' variants would not be identified by common diagnostic protocols that are predicated on a reduction in circulating AAT. This has potential clinical relevance: in addition to the dysfunctional Pittsburgh and Iners variants reported previously, several uncharacterized RCL variants are present in genome variation databases. To prospectively evaluate the impact of RCL variations on secretion and anti-protease activity, here we performed a systematic screening of amino acid substitutions occurring at the AAT-NE interface. Twenty-three AAT variants that can result from single nucleotide polymorphisms in this region, including 11 present in sequence variation databases, were expressed in a mammalian cell model. All demonstrated unaltered protein folding and secretion. However, when their ability to form stable complexes with NE was evaluated by western blot, enzymatic assays, and a novel ELISA developed to quantify AAT-NE complexes, substrate-like and NE-binding deficient dysfunctional variants were identified. This emphasizes the ability of the RCL to accommodate inactivating substitutions without impacting the integrity of the native molecule and demonstrates that this class of molecule violates a generally accepted paradigm that equates circulating levels with functional protection of lung tissue.
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Enfermedades Pulmonares , Deficiencia de alfa 1-Antitripsina , Humanos , Deficiencia de alfa 1-Antitripsina/genética , Mutación/genética , Pulmón , Sustitución de AminoácidosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of the COVID-19 pandemic, has been shown to infect a wide range of animal species, especially mammals, and besides human-to-human transmission, human-to-animal transmission has also been observed in some wild animals and pets, especially in cats. It has been demonstrated that cats are permissive to COVID-19 and are susceptible to airborne infections. Given the high transmissibility potential of SARS-CoV-2 to different host species and the close contact between humans and animals, it is crucial to find mechanisms to prevent the transmission chain and reduce the risk of spillover to susceptible species. Here, we show results from a clinical trial conducted in domestic cats to assess safety and immunogenicity of a linear DNA (linDNA) vaccine encoding the receptor-binding domain (RBD) from SARS-CoV-2 (Lin-COVID-eVax). Lin-COVID-eVax proved to be safe, with no significant adverse events, and was able to elicit both RBD-specific antibodies and T cells. Also, the linDNA vaccine induced neutralizing antibody titers against ancestral SARS-CoV-2 virus and its variants. These findings demonstrate the safety and immunogenicity of a genetic vaccine against COVID-19 administered to cats and strongly support the development of vaccines for preventing viral spread in susceptible species, especially those in close contact with humans.
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The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.
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Vacunas contra la COVID-19/administración & dosificación , COVID-19/prevención & control , Inmunización/métodos , Modelos Animales , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas de ADN/administración & dosificación , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/genética , COVID-19/virología , Femenino , Hurones , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Dominios Proteicos , Ratas Sprague-DawleyRESUMEN
Neuroserpin is a serine protease inhibitor identified in a search for proteins implicated in neuronal axon growth and synapse formation. Since its discovery over 30 years ago, it has been the focus of active research. Many efforts have concentrated in elucidating its neuroprotective role in brain ischemic lesions, the structural bases of neuroserpin conformational change and the effects of neuroserpin polymers that underlie the neurodegenerative disease FENIB (familial encephalopathy with neuroserpin inclusion bodies), but the investigation of the physiological roles of neuroserpin has increased over the last years. In this review, we present an updated and critical revision of the current literature dealing with neuroserpin, covering all aspects of research including the expression and physiological roles of neuroserpin, both inside and outside the nervous system; its inhibitory and non-inhibitory mechanisms of action; the molecular structure of the monomeric and polymeric conformations of neuroserpin, including a detailed description of the polymerisation mechanism; and the involvement of neuroserpin in human disease, with particular emphasis on FENIB. Finally, we briefly discuss the identification by genome-wide screening of novel neuroserpin variants and their possible pathogenicity.
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Neuropéptidos/metabolismo , Serpinas/metabolismo , Animales , Axones/metabolismo , Epilepsias Mioclónicas/metabolismo , Trastornos Heredodegenerativos del Sistema Nervioso/metabolismo , Humanos , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Polimerizacion , NeuroserpinaRESUMEN
Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.
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Mutación Missense , Deficiencia de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina , Sustitución de Aminoácidos , Células HEK293 , Humanos , Dominios Proteicos , alfa 1-Antitripsina/biosíntesis , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/patologíaRESUMEN
Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a progressive neurodegenerative disease caused by point mutations in the gene for neuroserpin, a serine protease inhibitor of the nervous system. Different mutations are known that are responsible for mutant neuroserpin polymerization and accumulation as inclusion bodies in many cortical and subcortical neurons, thereby leading to cell death, dementia and epilepsy. Many efforts have been undertaken to elucidate the molecular pathways responsible for neuronal death. Most investigations have concentrated on analysis of intracellular mechanisms such as endoplasmic reticulum (ER) stress, ER-associated protein degradation (ERAD) and oxidative stress. We have generated a HEK-293 cell model of FENIB by overexpressing G392E-mutant neuroserpin and in this study we examine trafficking and toxicity of this polymerogenic variant. We observed that a small fraction of mutant neuroserpin is secreted via the ER-to-Golgi pathway, and that this release can be pharmacologically regulated. Overexpression of the mutant form of neuroserpin did not stimulate cell death in the HEK-293 cell model. Finally, when treating primary hippocampal neurons with G392E neuroserpin polymers, we did not detect cytotoxicity or synaptotoxicity. Altogether, we report here that a polymerogenic mutant form of neuroserpin is secreted from cells but is not toxic in the extracellular milieu.
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Trastornos Heredodegenerativos del Sistema Nervioso/metabolismo , Neuropéptidos/genética , Serpinas/genética , Sinapsis/patología , Animales , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Ratones , Ratones Transgénicos , Mutación , Neuronas/metabolismo , Neuropéptidos/metabolismo , Neuropéptidos/fisiología , Serpinas/metabolismo , Serpinas/fisiología , NeuroserpinaRESUMEN
Our current knowledge about the cellular mechanisms underlying serpin-related disorders, the serpinopathies, is predominantly based on studies in cell culture models of disease, particularly for alpha-1 antitrypsin (AAT, SERPINA1) deficiency causing emphysema and the familial encephalopathy with neuroserpin (NS, SERPINI1) inclusion bodies (FENIB). FENIB, a neurodegenerative dementia, is caused by polymerization of NS (Miranda and Lomas, Cell Mol Life Sci 63:709-722, 2006; Roussel BD et al., Epileptic Disor 18:103-110, 2016), while AAT deficiency presents as a result of several divergent mutations in the AAT gene that cause lack of protein synthesis or complete intracellular degradation (null variants) or polymer formation (polymerogenic variants) (Lomas et al., J Hepatol 65:413-424, 2016; Greene et al., Nat Rev Dis Primers 2:16051, 2016; Ferrarotti et al. Orphanet J Rare D 9:172, 2014). Both diseases have been extensively modeled in cell culture systems by expressing mutant variants in a variety of ways. Here we describe the methodologies we follow in our cell model systems used to examine serpin disorders.
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Enfisema , Epilepsias Mioclónicas , Trastornos Heredodegenerativos del Sistema Nervioso , Modelos Biológicos , Mutación , Neuropéptidos , Serpinas , alfa 1-Antitripsina , Animales , Células COS , Chlorocebus aethiops , Enfisema/genética , Enfisema/metabolismo , Enfisema/patología , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/metabolismo , Epilepsias Mioclónicas/patología , Células HEK293 , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Trastornos Heredodegenerativos del Sistema Nervioso/metabolismo , Trastornos Heredodegenerativos del Sistema Nervioso/patología , Humanos , Ratones , Neuropéptidos/genética , Neuropéptidos/metabolismo , Células PC12 , Ratas , Serpinas/genética , Serpinas/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , NeuroserpinaRESUMEN
Given the heterogeneous nature of frontotemporal dementia (FTD), sensitive biomarkers are greatly needed for the accurate diagnosis of this neurodegenerative disorder. Circulating miRNAs have been reported as promising biomarkers for neurodegenerative disorders and processes affecting the central nervous system, especially in aging. The objective of the study was to evaluate if some circulating miRNAs linked with apoptosis (miR-29b-3p, miR-34a-5p, miR-16-5p, miR-17-5p, miR-107, miR-19b-3p, let-7b-5p, miR-26b-5p, and 127-3p) were able to distinguish between FTD patients and healthy controls. For this study, we enrolled 127 subjects, including 54 patients with FTD, 20 patients with Alzheimer's disease (AD), and 53 healthy controls. The qRT-PCR analysis showed a downregulation of miR-127-3p in FTD compared to controls, while the levels of other miRNAs remained unchanged. Then, miR-127-3p expression was also analyzed in AD patients, finding a different expression between two patient groups. A receiver operating characteristic curve was then created for miR-127-3p to discriminate FTD versus AD (AUC: 0.8986), and versus healthy controls (AUC: 0.8057). In conclusion, miR-127-3p could help to diagnose FTD and to distinguish it from AD.
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MicroARN Circulante/sangre , Demencia Frontotemporal/sangre , MicroARNs/sangre , Anciano , Enfermedad de Alzheimer/sangre , Biomarcadores/sangre , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Sensibilidad y EspecificidadRESUMEN
The serpinopathies are human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity. The dementia FENIB is caused by polymerisation of the neuronal serpin neuroserpin (NS) within the endoplasmic reticulum (ER) of neurons. With the aim of understanding the toxicity due to intracellular accumulation of neuroserpin polymers, we have generated transgenic neural progenitor cell (NPC) cultures from mouse foetal cerebral cortex, stably expressing the control protein GFP (green fluorescent protein), or human wild type, G392E or delta NS. We have characterised these cell lines in the proliferative state and after differentiation to neurons. Our results show that G392E NS formed polymers that were mostly retained within the ER, while wild type NS was correctly secreted as a monomeric protein into the culture medium. Delta NS was absent at steady state due to its rapid degradation, but it was easily detected upon proteasomal block. Looking at their intracellular distribution, wild type NS was found in partial co-localisation with ER and Golgi markers, while G392E NS was localised within the ER only. Furthermore, polymers of NS were detected by ELISA and immunofluorescence in neurons expressing the mutant but not the wild type protein. We used control GFP and G392E NPCs differentiated to neurons to investigate which cellular pathways were modulated by intracellular polymers by performing RNA sequencing. We identified 747 genes with a significant upregulation (623) or downregulation (124) in G392E NS-expressing cells, and we focused our attention on several genes involved in the defence against oxidative stress that were up-regulated in cells expressing G392E NS (Aldh1b1, Apoe, Gpx1, Gstm1, Prdx6, Scara3, Sod2). Inhibition of intracellular anti-oxidants by specific pharmacological reagents uncovered the damaging effects of NS polymers. Our results support a role for oxidative stress in the cellular toxicity underlying the neurodegenerative dementia FENIB.