RESUMEN
BACKGROUND: Neuroinflammation is an underlying pathology of all neurological conditions, the understanding of which is still being comprehended. A specific molecular pathway that has been overlooked in neuroinflammation is glycosylation (i.e., post-translational addition of glycans to the protein structure). N-glycosylation is a specific type of glycosylation with a cardinal role in the central nervous system (CNS), which is highlighted by congenital glycosylation diseases that result in neuropathological symptoms such as epilepsy and mental retardation. Changes in N-glycosylation can ultimately affect glycoproteins' functions, which will have an impact on cell machinery. Therefore, characterisation of N-glycosylation alterations in a neuroinflammatory scenario can provide a potential target for future therapies. METHODS: With that aim, the unilateral intrastriatal injection of lipopolysaccharide (LPS) in the adult rat brain was used as a model of neuroinflammation. In vivo and post-mortem, quantitative and spatial characterisation of both neuroinflammation and N-glycome was performed at 1-week post-injection of LPS. These aspects were investigated through a multifaceted approach based on positron emission tomography (PET), quantitative histology, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), liquid chromatography and matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI). RESULTS: In the brain region showing LPS-induced neuroinflammation, a significant decrease in the abundance of sialylated and core fucosylated structures was seen (approximately 7.5% and 8.5%, respectively), whereas oligomannose N-glycans were significantly increased (13.5%). This was confirmed by MALDI-MSI, which provided a high-resolution spatial distribution of N-glycans, allowing precise comparison between normal and diseased brain hemispheres. CONCLUSIONS: Together, our data show for the first time the complete profiling of N-glycomic changes in a well-characterised animal model of neuroinflammation. These data represent a pioneering step to identify critical targets that may modulate neuroinflammation in neurodegenerative diseases.
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Encéfalo , Glicosilación , Inflamación/metabolismo , Polisacáridos/análisis , Polisacáridos/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Encéfalo/patología , Mapeo Encefálico , Cromatografía Liquida/métodos , Modelos Animales de Enfermedad , Glicómica , Masculino , Tomografía de Emisión de Positrones , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Over the last 30 years, the 18-kDa TSPO protein has been considered as the PET imaging biomarker of reference to measure increased neuroinflammation. Generally assumed to image activated microglia, TSPO has also been detected in endothelial cells and activated astrocytes. Here, we provide an exhaustive overview of the recent literature on the TSPO-PET imaging (i) in the search and development of new TSPO tracers and (ii) in the understanding of acute and chronic neuroinflammation in animal models of neurological disorders. Generally, studies testing new TSPO radiotracers against the prototypic [11C]-R-PK11195 or more recent competitors use models of acute focal neuroinflammation (e.g. stroke or lipopolysaccharide injection). These studies have led to the development of over 60 new tracers during the last 15 years. These studies highlighted that interpretation of TSPO-PET is easier in acute models of focal lesions, whereas in chronic models with lower or diffuse microglial activation, such as models of Alzheimer's disease or Parkinson's disease, TSPO quantification for detection of neuroinflammation is more challenging, mirroring what is observed in clinic. Moreover, technical limitations of preclinical scanners provide a drawback when studying modest neuroinflammation in small brains (e.g. in mice). Overall, this review underlines the value of TSPO imaging to study the time course or response to treatment of neuroinflammation in acute or chronic models of diseases. As such, TSPO remains the gold standard biomarker reference for neuroinflammation, waiting for new radioligands for other, more specific targets for neuroinflammatory processes and/or immune cells to emerge.
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Enfermedad de Alzheimer , Receptores de GABA , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Ratones , Tomografía de Emisión de Positrones , Radiofármacos , Receptores de GABA/metabolismoRESUMEN
Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) play crucial roles in Parkinson's disease (PD). They may functionally interact to induce the degeneration of dopaminergic (DA) neurons via mechanisms that are not yet fully understood. We previously showed that the C-terminal portion of LRRK2 (ΔLRRK2) with the G2019S mutation (ΔLRRK2G2019S) was sufficient to induce neurodegeneration of DA neurons in vivo, suggesting that mutated LRRK2 induces neurotoxicity through mechanisms that are (i) independent of the N-terminal domains and (ii) "cell-autonomous". Here, we explored whether ΔLRRK2G2019S could modify α-syn toxicity through these two mechanisms. We used a co-transduction approach in rats with AAV vectors encoding ΔLRRK2G2019S or its "dead" kinase form, ΔLRRK2DK, and human α-syn with the A53T mutation (AAV-α-synA53T). Behavioral and histological evaluations were performed at 6- and 15-weeks post-injection. Results showed that neither form of ΔLRRK2 alone induced the degeneration of neurons at these post-injection time points. By contrast, injection of AAV-α-synA53T alone resulted in motor signs and degeneration of DA neurons. Co-injection of AAV-α-synA53T with AAV-ΔLRRK2G2019S induced DA neuron degeneration that was significantly higher than that induced by AAV-α-synA53T alone or with AAV-ΔLRRK2DK. Thus, mutated α-syn neurotoxicity can be enhanced by the C-terminal domain of LRRK2G2019 alone, through cell-autonomous mechanisms.
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Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteínas Mutantes/metabolismo , Mutación , alfa-Sinucleína/metabolismo , Animales , Neuronas Dopaminérgicas/metabolismo , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteínas Mutantes/genética , Dominios Proteicos , Ratas , alfa-Sinucleína/genéticaRESUMEN
The G2019S substitution in the kinase domain of LRRK2 (LRRK2G2019S) is the most prevalent mutation associated with Parkinson's disease (PD). Neurotoxic effects of LRRK2G2019S are thought to result from an increase in its kinase activity as compared to wild type LRRK2. However, it is unclear whether the kinase domain of LRRK2G2019S is sufficient to trigger degeneration or if the full length protein is required. To address this question, we generated constructs corresponding to the C-terminal domain of LRRK2 (ΔLRRK2). A kinase activity that was increased by G2019âS substitution could be detected in ΔLRRK2. However biochemical experiments suggested it did not bind or phosphorylate the substrate RAB10, in contrast to full length LRRK2. The overexpression of ΔLRRK2G2019S in the rat striatum using lentiviral vectors (LVs) offered a straightforward and simple way to investigate its effects in neurons in vivo. Results from a RT-qPCR array analysis indicated that ΔLRRK2G2019S led to significant mRNA expression changes consistent with a kinase-dependent mechanism. We next asked whether ΔLRRK2 could be sufficient to trigger neurodegeneration in the substantia nigra pars compacta (SNc) in adult rats. Six months after infection of the substantia nigra pars compacta (SNc) with LV-ΔLRRK2WT or LV-ΔLRRK2G2019S, the number of DA neurons was unchanged. To examine whether higher levels of ΔLRRK2G2019S could trigger degeneration we cloned ΔLRRK2 in AAV2/9 construct. As expected, AAV2/9 injected in the SNc led to neuronal expression of ΔLRRK2WT and ΔLRRK2G2019S at much higher levels than those obtained with LVs. Six months after injection, unbiased stereology showed that AAV-ΔLRRK2G2019S produced a significant ~30% loss of neurons positive for tyrosine hydroxylase- and for the vesicular dopamine transporter whereas AAV-ΔLRRK2WT did not. These findings show that overexpression of the C-terminal part of LRRK2 containing the mutant kinase domain is sufficient to trigger degeneration of DA neurons, through cell-autonomous mechanisms, possibly independent of RAB10.
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Neuronas Dopaminérgicas/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Degeneración Nerviosa/genética , Enfermedad de Parkinson , Dominios Proteicos/genética , Animales , Técnicas de Transferencia de Gen , Vectores Genéticos , Células HEK293 , Humanos , Lentivirus , Masculino , Mutación , Degeneración Nerviosa/patología , Porción Compacta de la Sustancia Negra , Ratas , Ratas Sprague-DawleyRESUMEN
Our understanding of the mechanisms underlying Parkinson's disease, the once archetypical nongenetic neurogenerative disorder, has dramatically increased with the identification of α-synuclein and LRRK2 pathogenic mutations. While α-synuclein protein composes the aggregates that can spread through much of the brain in disease, LRRK2 encodes a multidomain dual-enzyme distinct from any other protein linked to neurodegeneration. In this review, we discuss emergent datasets from multiple model systems that suggest these unlikely partners do interact in important ways in disease, both within cells that express both LRRK2 and α-synuclein as well as through more indirect pathways that might involve neuroinflammation. Although the link between LRRK2 and disease can be understood in part through LRRK2 kinase activity (phosphotransferase activity), α-synuclein toxicity is multilayered and plausibly interacts with LRRK2 kinase activity in several ways. We discuss common protein interactors like 14-3-3s that may regulate α-synuclein and LRRK2 in disease. Finally, we examine cellular pathways and outcomes common to both mutant α-synuclein expression and LRRK2 activity and points of intersection. Understanding the interplay between these two unlikely partners in disease may provide new therapeutic avenues for PD.
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Encéfalo/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/efectos adversos , Degeneración Nerviosa/metabolismo , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/efectos adversos , Animales , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Mutación , Neuronas/metabolismo , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
The emergence of escape-mutants of influenza hemagglutinin (HA) following vaccination compels the yearly re-formulation of flu vaccines. Since binding the sialic acid receptor remains in all cases essential for infection, small-molecule inhibitors of HA binding to sialic acid could be interesting therapeutic complements or alternatives to immuno-prophylaxis in the control of flu epidemics. In this work, we made use of NMR spectroscopy to study the interaction between a derivative of sialic acid (the Neu5Ac-α-(2,6)-Gal-ß-(1-4)-GlcNAc trisaccharide) and HAs (H1 and H5) from human and avian strains of influenza virus, directly expressed on the surface of stable transfected 293 T human cells. The HAs were shown to retain their native trimeric conformation and binding properties. Exploiting the magnetization transfer between the proteins and the ligand, we obtained evidence of the binding event and mapped the (non-identical) sugar epitopes recognized by the two HA species. The rapid and reliable method for screening sialic acid-related HA ligands we have developed could yield useful information for an efficient drug design.
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Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/química , Células HEK293 , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , TransfecciónRESUMEN
Determination of the potency of a vaccine is critical to ensuring that an appropriate dose is delivered, lot-to-lot consistency is maintained, and that the formulation is stable over the life of the vaccine. The potency of inactivated influenza vaccines is determined routinely by the Single Radial Immunodiffusion (SRID) assay. A number of alternative potency assays have been proposed and have been under evaluation in recent years. The aim of this study was to compare a surface plasmon resonance-based assay and two different enzyme linked immunoassays against the current potency assay, SRID, and against mouse immunogenicity when haemagglutinin antigen of the A(H1N1)pdm09 component of an inactivated influenza vaccine is stressed by elevated temperature, low pH and freezing. This analysis demonstrated that the alternative assays had good correspondence with SRID for samples from most stress conditions and that the immunogenicity in mice corresponded with potency in SRID for all stress samples. Subject to further analysis, the assays have been shown to have the potential to possibly replace, and at least complement, SRID.
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Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Animales , Ratones , Humanos , Vacunas de Productos Inactivados , Glicoproteínas Hemaglutininas del Virus de la Influenza , Gripe Humana/prevención & control , Potencia de la VacunaRESUMEN
Microglial cells are brain-specific macrophages that swiftly react to disruptive events in the brain. Microglial activation leads to specific modifications, including proliferation, morphological changes, migration to the site of insult, and changes in gene expression profiles. A change in inflammatory status has been linked to many neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. For this reason, the investigation and quantification of microglial cells is essential for better understanding their role in disease progression as well as for evaluating the cytocompatibility of novel therapeutic approaches for such conditions. In the following study we implemented a machine learning-based approach for the fast and automatized quantification of microglial cells; this tool was compared with manual quantification (ground truth), and with alternative free-ware such as the threshold-based ImageJ and the machine learning-based Ilastik. We first trained the algorithms on brain tissue obtained from rats and non-human primate immunohistochemically labelled for microglia. Subsequently we validated the accuracy of the trained algorithms in a preclinical rodent model of Parkinson's disease and demonstrated the robustness of the algorithms on tissue obtained from mice, as well as from images provided by three collaborating laboratories. Our results indicate that machine learning algorithms can detect and quantify microglial cells in all the three mammalian species in a precise manner, equipotent to the one observed following manual counting. Using this tool, we were able to detect and quantify small changes between the hemispheres, suggesting the power and reliability of the algorithm. Such a tool will be very useful for investigation of microglial response in disease development, as well as in the investigation of compatible novel therapeutics targeting the brain. As all network weights and labelled training data are made available, together with our step-by-step user guide, we anticipate that many laboratories will implement machine learning-based quantification of microglial cells in their research.
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Microglía , Enfermedad de Parkinson , Ratones , Ratas , Animales , Microglía/metabolismo , Enfermedad de Parkinson/metabolismo , Reproducibilidad de los Resultados , Encéfalo , Primates , Aprendizaje Automático , MamíferosRESUMEN
The long-term expression and the ability of a therapeutic gene to confer survival advantage to transduced cells are mandatory requirements for successful anti-HIV gene therapy. In this context, we developed lentiviral vectors (LVs) expressing the F12-viral infectivity factor (Vif) derivative Chim3. We recently showed that Chim3 inhibits HIV-1 replication in primary cells by both blocking the accumulation of retrotranscripts, independently of either human APOBEC3G (hA3G) or Vif, and by preserving the antiviral function of hA3G. These results were predictive of long-lasting survival of Chim3(+) cells after HIV-1 infection. Furthermore, Vif, like Vpr, deregulates cell-cycle progression by inducing a delay in G(2) phase. Thus, the aim of this study was to investigate the role of Chim3 on both cell survival and cell-cycle regulation after HIV-1 infection. Here, we provide evidence that infected Chim3(+) T cells prevail over either mock- or empty-LV engineered cells, show reduced G(2) accumulation, and, as a consequence, ultimately extend their lifespan. Based on these findings, Chim3 rightly belongs to the most efficacious class of antiviral genes. In conclusion, Chim3 usage in anti-HIV gene therapy based on hematopoietic stem cell (HSC) modification has to be considered as a promising therapeutic intervention to eventually cope with HIV-1 infection.
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Linfocitos T CD4-Positivos/fisiología , ADN Viral/genética , Fase G2/fisiología , Terapia Genética , VIH-1/fisiología , Integración Viral , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/fisiología , Southern Blotting , Linfocitos T CD4-Positivos/virología , Supervivencia Celular , Células Cultivadas , ADN Viral/metabolismo , Células Madre Hematopoyéticas , Humanos , Inmunoprecipitación , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/fisiologíaRESUMEN
BACKGROUND: The development of axonal pathology is a key characteristic of many neurodegenerative disease such as Parkinson's disease and Alzheimer's disease. With advanced disease progression, affected axons do display several signs of pathology such as swelling and fragmentation. In the AAV vector-mediated alpha-synuclein overexpression model of Parkinson's disease, large (> 20 µm2) pathological swellings are prominent characteristics in cortical and subcortical structures. NEW METHOD: This report describes a novel, macro-based workflow to quantify axonal pathology in the form of axonal swellings in the AAV vector-based alpha-synuclein overexpression model. Specifically, the approach is using background correction and thresholding before quantification of structures in 3D throughout a tissue stack. RESULTS: The method was used to quantify TH and aSYN axonal swellings in the prefrontal cortex, striatum, and hippocampus. Regional differences in volume and number of axonal swellings were observed for both in TH and aSYN, with the striatum displaying the greatest signs of pathology. COMPARISON WITH EXISTING METHODS: Existing methods for the quantification of axonal pathology do either rely on proprietary software or are based on manual quantification. The ImageJ workflow described here provides a method to objectively quantify axonal swellings both in volume and number. CONCLUSION: The method described can readily assess axonal pathology in preclinical rodent models of Parkinson's disease and can be easily adapted to other model systems and/or markers.
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Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Animales , Axones/patología , Enfermedades Neurodegenerativas/patología , Roedores , alfa-SinucleínaRESUMEN
BACKGROUND: Preclinical rodent models for Parkinson's disease (PD) based on viral human alpha-synuclein (h-αSyn) overexpression recapitulate some of the pathological hallmarks as it presents in humans, such as progressive cell loss and additional synucleinopathy in cortical and subcortical structures. Recent studies have combined viral vector-based overexpression of human wild-type αSyn with the sequential or simultaneous inoculation of preformed fibrils (PFFs) derived from human αSyn. OBJECTIVE: The goal of the study was to investigate whether sequential or combined delivery of the AAV vector and the PFFs are equipotent in inducing stable neurodegeneration and behavioral deficits. METHODS: Here we compare between four experimental paradigms (PFFs only, AAV-h-αSyn only, AAV-h-αSyn with simultaneous PFFs, and AAV-h-αSyn with sequential PFFs) and their respective GFP control groups. RESULTS: We observed reduction of TH expression and loss of neurons in the midbrain in all AAV (h-αSyn or GFP) injected groups, with or without additional PFFs inoculation. The overexpression of either h-αSyn or GFP alone induced motor deficits and dysfunctional dopamine release/reuptake in electrochemical recordings in the ipsilateral striatum. However, we observed a substantial formation of insoluble h-αSyn aggregates and inflammatory response only when h-αSyn and PFFs were combined. Moreover, the presence of h-αSyn induced higher axonal pathology compared to control groups. CONCLUSION: Simultaneous AAV and PFFs injections are equipotent in the presented experimental setup in inducing histopathological and behavioral changes. This model provides new and interesting possibilities for characterizing PD pathology in preclinical models and means to assess future therapeutic interventions.
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Enfermedad de Parkinson , Sinucleinopatías , Cuerpo Estriado/metabolismo , Humanos , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
The viral infectivity factor (Vif) is essential for HIV-1 infectivity and hence is an ideal target for promising anti-HIV-1/AIDS gene therapy. We previously demonstrated that F12-Vif mutant inhibits HIV-1 replication in CD4(+) T lymphocytes. Despite macrophage relevance to HIV-1 pathogenesis, most gene therapy studies do not investigate macrophages because of their natural resistance to genetic manipulation. Here, we confirm the F12-Vif antiviral activity also in macrophages differentiated in vitro from transduced CD34(+) human stem cells (HSCs). Moreover, we identified the 126- to 170-amino-acid region in the C-terminal half of F12-Vif as responsible for its antiviral function. Indeed, Chim3 protein, containing this 45-amino-acid region embedded in a WT-Vif backbone, is as lethal as F12-Vif against HIV-1. Of major relevance, we demonstrated a dual mechanism of action for Chim3. First, Chim3 functions as a transdominant factor that preserves the antiviral function of the natural restriction factor APOBEC3G (hA3G). Second, Chim3 blocks the early HIV-1 retrotranscript accumulation and thereby HIV-1 DNA integration regardless of the presence of WT-Vif and hA3G. In conclusion, by impairing the early steps of HIV-1 life cycle, Chim3 conceivably endows engineered cells with survival advantage, which is required for the efficient immune reconstitution of patients living with HIV/AIDS.
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Linfocitos T CD4-Positivos/virología , Terapia Genética/métodos , Infecciones por VIH/terapia , VIH-1/crecimiento & desarrollo , Macrófagos/virología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Antígenos CD34/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/fisiología , Diferenciación Celular/inmunología , Línea Celular , Sangre Fetal/citología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Riñón/citología , Macrófagos/citología , Macrófagos/fisiología , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Transducción Genética , Integración Viral , Replicación Viral , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Low moisture content is seen as crucial to achieving long term stability of freeze dried biologics and reference materials. Highly hygroscopic freeze-dried material are susceptible to moisture ingress over time which can lead to degradation and loss of biological potency. This study compared vials with unprocessed stoppers, vials with vacuum-oven dried stoppers and glass ampoules in order to determine the superior long term storage format in terms of moisture ingress and potency. B/Phuket influenza antigen was chosen as the model biological standard and the lyophilized antigen was stored at -20, 25 and 45⯰C over a 1â¯year period. Ampoules had no significant moisture change across all storage temperatures as would be anticipated. Moisture content results at -20⯰C showed no significant differences between ampoules, vials with vacuum-oven dried stoppers and vials with unprocessed stoppers over 12â¯months. Vials with vacuum-oven dried stoppers performed similarly to ampoules at -20⯰C and 20⯰C, but had a small increase in moisture content after 6â¯months at 45⯰C. Vials with unprocessed stoppers preformed the worst and exhibited the largest moisture ingress after just 3â¯months at both 20⯰C and 45⯰C. Single radial immunodiffusion (SRD) potency assays showed at -20⯰C and 20⯰C there was no significant difference between all closure formats. At 45⯰C there was a drop in potency for all closure formats, but ampoules and vials with vacuum-oven dried stoppers retained higher potency than vials with unprocessed stoppers. Thus, while ampoules are still considered to be the gold standard format for long term storage stability, using vials with vacuum-oven dried stoppers provides comparable stability and moisture integrity at -20⯰C and 20⯰C storage.
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Antígenos Virales/química , Virus de la Influenza A/química , Virus de la Influenza A/inmunología , Productos Biológicos/química , Embalaje de Medicamentos/métodos , Estabilidad de Medicamentos , Liofilización/métodos , Vidrio/química , Humanos , Gripe Humana/inmunología , Temperatura , Agua/químicaRESUMEN
As a vaccination vector, MVA has been widely investigated both in animal models and humans. The construction of recombinant MVA (rMVA) relies on homologous recombination between an acceptor virus and a donor plasmid in infected/transfected permissive cells. Our construction strategy "Red-to-Green gene swapping" - based on the exchange of two fluorescent markers within the flanking regions of MVA deletion ΔIII, coupled to fluorescence activated cell sorting - is here extended to a second insertion site, within the flanking regions of MVA deletion ΔVI. Exploiting this strategy, both double and triple rMVA were constructed, expressing as transgenes the influenza A proteins HA, NP, M1, and PB1. Upon validation of the harbored transgenes co-expression, double and triple recombinants rMVA(ΔIII)-NP-P2A-M1 and rMVA(ΔIII)-NP-P2A-M1-(ΔVI)-PB1 were assayed for in vivo immunogenicity and protection against lethal challenge. In vivo responses were identical to those obtained with the reported combinations of single recombinants, supporting the feasibility and reliability of the present improvement and the extension of Red-to-Green gene swapping to insertion sites other than ΔIII.
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Portadores de Fármacos , Vectores Genéticos , Vacunas contra la Influenza/inmunología , Virus Vaccinia/genética , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Expresión Génica , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Análisis de Supervivencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
BACKGROUND: The emergence of novel strains of influenza A viruses with hemagglutinins (HAs) that are antigenically distinct from those circulating in humans, and thus have pandemic potential, pose concerns and call for the development of more broadly protective influenza vaccines. In the present study, modified vaccinia virus Ankara (MVA) encoding internal influenza antigens were evaluated for their immunogenicity and ability to protect HLA-A2.1 transgenic (AAD) mice from infection with influenza viruses. METHODS: MVAs expressing NP (MVA-NP), M1 (MVA-M1) or polymerase PB1 (MVA-PB1) of A/California/4/09 (CA/09) virus were generated and used to immunize AAD mice. Antibodies and CD8+T cell responses were assessed by ELISA and ELISPOT, respectively, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus. RESULTS: CD8+T cells specific to immunodominant and subdominant epitopes on the internal influenza proteins were elicited by MVA-based vectors in AAD mice, whereas influenza-specific antibodies were detected only in MVA-NP-immunized mice. Both M1- and NP-based MVA vaccines, regardless of whether they were applied individually or in combination, conferred protection against lethal influenza virus challenge. CONCLUSION: Our data further emphasize the promising potential of MVA vector expressing internal antigens toward the development of a universal influenza vaccine.
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Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Virus Vaccinia/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Vectores Genéticos , Antígeno HLA-A2/genética , Humanos , Inmunidad Celular , Ratones Transgénicos , Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/inmunología , Vacunas Sintéticas/inmunología , Proteínas Virales/inmunologíaRESUMEN
BACKGROUND: Vaccination offers protection against influenza, although current vaccines need to be reformulated each year. The development of a broadly protective influenza vaccine would guarantee the induction of heterosubtypic immunity also against emerging influenza viruses of a novel subtype. Vaccine candidates based on the stalk region of the hemagglutinin (HA) have the potential to induce broad and persistent protection against diverse influenza A viruses. METHODS: Modified vaccinia virus Ankara (MVA) expressing a headless HA (hlHA) of A/California/4/09 (CA/09) virus was used as a vaccine to immunize C57BL/6 mice. Specific antibody and cell-mediated immune responses were determined, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus. RESULTS: Immunization of mice with CA/09-derived hlHA, vectored by MVA, was able to elicit influenza-specific broad cross-reactive antibodies and cell-mediated immune responses, but failed to induce neutralizing antibodies and did not protect mice against virus challenge. CONCLUSION: Although highly immunogenic, our vaccine was unable to induce a protective immunity against influenza. A misfolded and unstable conformation of the hlHA molecule may have affected its capacity of inducing neutralizing antiviral, conformational antibodies. Design of stable hlHA-based immunogens and their delivery by recombinant MVA-based vectors has the potential of improving this promising approach for a universal influenza vaccine.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/inmunología , Protección Cruzada/inmunología , Vectores Genéticos , Inmunidad Celular , Ratones Endogámicos C57BL , Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/inmunología , Vacunas Sintéticas/inmunología , Virus Vaccinia/inmunología , Proteínas Virales/inmunologíaRESUMEN
Circulating monocytes from multiple myeloma patients enrolled in a clinical study of anti-idiotype vaccination were labelled with clinical-grade anti-CD14 microbeads and positively selected with the CliniMACS instrument. Cells were then grown, according to good manufacturing practice guidelines, in fetal-calf-serum-free medium in cell culture bags and differentiated to dendritic cells (DC) with granulocyte-macrophage colony stimulating factor plus interleukin 4 (IL-4), followed by either tumour necrosis factor-alpha (TNF-alpha) or a cocktail of IL-1beta, IL-6, TNF-alpha and prostaglandin-E2. The CD14+ cell yield was increased from 17.6 +/- 6.5% to 93.8 +/- 6.3% (recovery 64.4 +/- 15.4%, viability > 97%). After cell culture, phenotypic analysis showed that 86.7 +/- 6.8% of the cells were DC: 2.27 +/- 0.9 x 108 DC/leukapheresis were obtained, which represented 20.7 +/- 4.6% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (28.6 +/- 3% of initial CD14+ cells) of DC than TNF-alpha alone, with secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic T cells and efficient presentation of tumour idiotype to autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of preloaded DC. The recovery of thawed, viable DC was 78 +/- 10%. Finally, interferon-alpha-2b was at least as efficient as IL-4 in inducing the differentiation of mature, functional DC from monocytes.