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1.
Cell ; 186(11): 2392-2409.e21, 2023 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-37164012

RESUMEN

T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance T cell immunity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vaccine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, membrane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).


Asunto(s)
Vacuna BNT162 , COVID-19 , Animales , Cricetinae , Humanos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Epítopos , SARS-CoV-2/genética
2.
Nature ; 592(7853): 283-289, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33524990

RESUMEN

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1-3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).


Asunto(s)
Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , COVID-19/prevención & control , Modelos Animales de Enfermedad , SARS-CoV-2/inmunología , Envejecimiento/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Vacuna BNT162 , COVID-19/sangre , COVID-19/terapia , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/química , Vacunas contra la COVID-19/genética , Línea Celular , Ensayos Clínicos como Asunto , Femenino , Humanos , Inmunización Pasiva , Internacionalidad , Macaca mulatta/inmunología , Macaca mulatta/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Multimerización de Proteína , ARN Viral/análisis , Sistema Respiratorio/inmunología , Sistema Respiratorio/virología , SARS-CoV-2/química , SARS-CoV-2/genética , Solubilidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Linfocitos T/inmunología , Vacunación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/química , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Sueroterapia para COVID-19 , Vacunas de ARNm
3.
Cytometry A ; 85(7): 621-7, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24616430

RESUMEN

Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells.


Asunto(s)
Citocinas/análisis , Citometría de Flujo/métodos , Proteínas Nucleares/análisis , Factores de Transcripción/análisis , Animales , Citoplasma/metabolismo , Fijadores , Colorantes Fluorescentes , Factores de Transcripción Forkhead , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mycobacterium tuberculosis/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Coloración y Etiquetado , Proteínas de Dominio T Box , Linfocitos T/citología , Fijación del Tejido/métodos
4.
J Neuroinflammation ; 10: 120, 2013 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-24090415

RESUMEN

BACKGROUND: Multiple sclerosis (MS) is often accompanied by optic nerve inflammation. And some patients experience permanent vision loss. We examined if the grade of optic nerve infiltration and demyelination affects the severity of clinical signs in an experimental autoimmune encephalomyelitis (EAE) model. The loss of retinal ganglion cells (RGC) and alterations in glia activity were also investigated. METHODS: C57BL/6 mice were immunized with peptide MOG35-55 in complete Freund's adjuvant (CFA) and controls received PBS in CFA. Then 23 days post immunization eyes were prepared for flatmounts and stained with Nissl to evaluated neuronal density. Clinical EAE symptoms as well as cell infiltration and demyelination in the optic nerve were examined. Retinal sections were stained with hematoxylin and eosin and silver stain. Immunohistochemistry was used to label RGCs (Brn-3a), apoptotic cells (caspase 3), macroglia (glial fibrillary acidic protein (GFAP)), microglia (Iba1), macrophages (F 4/80) and interleukin-6 (IL-6) secretion. RESULTS: EAE symptoms started at day 8 and peaked at day 15. Cell infiltrations (P = 0.0047) and demyelination (P = 0.0018) of EAE nerves correlated with the clinical score (r > 0.8). EAE led to a significant loss of RGCs (P< 0.0001). Significantly more caspase 3+ cells were noted in these animals (P = 0.0222). They showed an increased expression of GFAP (P< 0.0002) and a higher number of microglial cells (P< 0.0001). Also more macrophages and IL-6 secretion were observed in EAE mice. CONCLUSIONS: MOG immunization leads to optic neuritis and RGC loss. EAE severity is related to the severity of optic nerve inflammation and demyelination. EAE not only affects activation of apoptotic signals, but also causes a glial response in the retina.


Asunto(s)
Encefalomielitis Autoinmune Experimental/patología , Inflamación/patología , Neuroglía/patología , Células Ganglionares de la Retina/patología , Animales , Apoptosis/inmunología , Enfermedades Desmielinizantes/etiología , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/metabolismo , Encefalomielitis Autoinmune Experimental/complicaciones , Encefalomielitis Autoinmune Experimental/metabolismo , Inmunohistoquímica , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neuroglía/metabolismo , Neuritis Óptica/etiología , Neuritis Óptica/metabolismo , Neuritis Óptica/patología , Células Ganglionares de la Retina/metabolismo
5.
J Invest Dermatol ; 143(7): 1257-1267.e10, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-36736996

RESUMEN

Keratinocytes (KCs) form the outer epithelial barrier of the body, protecting against invading pathogens. Mice lacking the IL-17RA or both IL-17A and IL-17F develop spontaneous Staphylococcusaureus skin infections. We found a marked expansion of T17 cells, comprised of RORγt-expressing γδ T cells and T helper 17 cells in the skin-draining lymph nodes of these mice. Contradictory to previous suggestions, this expansion was not a result of a direct negative feedback loop because we found no expansion of T17 cells in mice lacking IL-17 signaling specifically in T cells. Instead, we found that the T17 expansion depended on the microbiota and was observed only when KCs were deficient for IL-17RA signaling. Indeed, mice that lack IL-17RA only in KCs showed an increased susceptibility to experimental epicutaneous infection with S. aureus together with an accumulation of IL-17A-producing γδ T cells. We conclude that deficiency of IL-17RA on KCs leads to microbiota dysbiosis in the skin, which triggers the expansion of IL-17A-producing T cells. Our data show that KCs are the primary target cells of IL-17A and IL-17F, coordinating the defense against microbial invaders in the skin.


Asunto(s)
Interleucina-17 , Staphylococcus aureus , Ratones , Animales , Ratones Noqueados , Piel , Queratinocitos , Ratones Endogámicos C57BL
6.
Eur J Immunol ; 40(12): 3336-46, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21110317

RESUMEN

IL-17-producing CD4(+) T cells (Th17) have been classified as a new T helper cell subset. Using an IL-17 fate mapping mouse strain, which genetically fixes the memory of IL-17 expression, we demonstrate that IL-17A/F-expressing T helper cells generated either in vitro or in vivo are not a stable T-cell subset. Upon adoptive transfer of IL-17F-reporter-positive Th17 cells to RAG-deficient or WT animals, encephalitogenic Th17 cells partially lose IL-17 expression and upregulate IFN-γ. Additionally, we show that Th1 cells can convert in vivo to IL-17A/IFN-γ-coexpressing cells in the mesenteric lymph nodes (mLN). Our data classify IL-17A and IL-17F as cytokines produced transiently in response to the local microenvironment, thus showing that IL-17 expression does not define an end-stage T helper cell subset.


Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Regulación de la Expresión Génica/inmunología , Subgrupos de Linfocitos T/metabolismo , Células TH1/metabolismo , Células Th17/metabolismo , Traslado Adoptivo , Animales , Diferenciación Celular/inmunología , Regulación de la Expresión Génica/genética , Genes RAG-1/genética , Genes Reporteros/genética , Integrasas/genética , Interferón gamma/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Células TH1/inmunología , Células TH1/patología , Células Th17/inmunología , Células Th17/patología
7.
Cell Rep ; 18(5): 1270-1284, 2017 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-28147280

RESUMEN

Arrival of encephalitogenic T cells at inflammatory foci represents a critical step in development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. EBI2 and its ligand, 7α,25-OHC, direct immune cell localization in secondary lymphoid organs. CH25H and CYP7B1 hydroxylate cholesterol to 7α,25-OHC. During EAE, we found increased expression of CH25H by microglia and CYP7B1 by CNS-infiltrating immune cells elevating the ligand concentration in the CNS. Two critical pro-inflammatory cytokines, interleukin-23 (IL-23) and interleukin-1 beta (IL-1ß), maintained expression of EBI2 in differentiating Th17 cells. In line with this, EBI2 enhanced early migration of encephalitogenic T cells into the CNS in a transfer EAE model. Nonetheless, EBI2 was dispensable in active EAE. Human Th17 cells do also express EBI2, and EBI2 expressing cells are abundant within multiple sclerosis (MS) white matter lesions. These findings implicate EBI2 as a mediator of CNS autoimmunity and describe mechanistically its contribution to the migration of autoreactive T cells into inflamed organs.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/fisiología , Movimiento Celular/fisiología , Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Esclerosis Múltiple/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Autoinmunidad/fisiología , Sistema Nervioso Central/fisiología , Familia 7 del Citocromo P450/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Femenino , Interleucina-1beta/metabolismo , Interleucina-23/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Esteroide Hidroxilasas/metabolismo , Células Th17/metabolismo , Células Th17/fisiología
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