RESUMEN
COVID-19 vaccines were originally designed based on the ancestral Spike protein, but immune escape of emergent Variants of Concern (VOC) jeopardized their efficacy, warranting variant-proof vaccines. Here, we used preclinical rodent models to establish the cross-protective and cross-neutralizing capacity of adenoviral-vectored vaccines expressing VOC-matched Spike. CoroVaxG.3-D.FR, matched to Delta Plus Spike, displayed the highest levels of nAb to the matched VOC and mismatched variants. Cross-protection against viral infection in aged K18-hACE2 mice showed dramatic differences among the different vaccines. While Delta-targeted vaccines fully protected mice from a challenge with Gamma, a Gamma-based vaccine offered only partial protection to Delta challenge. Administration of CorovaxG.3-D.FR in a prime/boost regimen showed that a booster was able to increase the neutralizing capacity of the sera against all variants and fully protect aged K18-hACE2 mice against Omicron BA.1, as a BA.1-targeted vaccine did. The neutralizing capacity of the sera diminished in all cases against Omicron BA.2 and BA.5. Altogether, the data demonstrate that a booster with a vaccine based on an antigenically distant variant, such as Delta or BA.1, has the potential to protect from a wider range of SARS-CoV-2 lineages, although careful surveillance of breakthrough infections will help to evaluate combination vaccines targeting antigenically divergent variants yet to emerge.
RESUMEN
Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric human adenovirus 5 (hAdV5) vector. The vaccine (named CoroVaxG.3) is based on three pillars: (i) high expression of Spike to enhance its immunodominance by using a potent promoter and an mRNA stabilizer; (ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; (iii) use of Spike stabilized in a prefusion conformation. The transduction with CoroVaxG.3-expressing Spike (D614G) dramatically enhanced the Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced a potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3-vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha), P.1 (gamma) and B.1.617.2 (delta) Spikes, as well as an authentic P.1 SARS-CoV-2 isolate. Neutralizing antibodies did not wane even after 5 months, making this kind of vaccine a likely candidate to enter clinical trials.
RESUMEN
The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab')2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.
La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab')2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.
Asunto(s)
Anticuerpos Antivirales , Infecciones por Coronavirus/terapia , Sueros Inmunes/inmunología , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Inmunoglobulina G/aislamiento & purificación , Pandemias , Neumonía Viral , Glicoproteína de la Espiga del Coronavirus , Animales , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Argentina , Betacoronavirus , COVID-19 , Caballos , Humanos , Inmunización Pasiva , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/química , Pruebas de Neutralización , SARS-CoV-2 , Sueroterapia para COVID-19RESUMEN
The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.
La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.
Asunto(s)
Humanos , Animales , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Infecciones por Coronavirus/terapia , Sueros Inmunes/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/química , Argentina , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/química , Fragmentos Fab de Inmunoglobulinas/química , Pruebas de Neutralización , Pandemias , Betacoronavirus , SARS-CoV-2 , COVID-19 , CaballosRESUMEN
Vaccination is as one of the most beneficial biopharmaceutical interventions against pathogens due to its ability to induce adaptive immunity through targeted activation of the immune system. Each vaccine needs a tailor-made set of tests in order to monitor its quality throughout the development and manufacturing. The analysis of the conformational state of protein nanoparticles is one of the key steps in vaccine quality control. The enzyme lumazine synthase from Brucella spp. (BLS) acts as a potent oral and systemic immunogen. BLS has been used as a carrier of foreign peptides, protein domains and whole proteins, serving as a versatile platform for vaccine engineering purposes. Here, we show the generation and characterization of four families of nanobodies (Nbs) which only recognize BLS in its native conformational state and that bind to its active site. The present results support the use of conformation-sensitive Nbs as molecular probes during the development and production of vaccines based on the BLS platform. Finally, we propose Nbs as useful molecular tools targeting other protein scaffolds with potential applications in nano-and biotechnology.
Asunto(s)
Complejos Multienzimáticos , Anticuerpos de Dominio Único , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Brucella/enzimología , Escherichia coli/genética , Células HEK293 , Humanos , Complejos Multienzimáticos/química , Complejos Multienzimáticos/fisiología , Conformación Proteica , Pliegue de Proteína , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/fisiología , Vacunas de SubunidadRESUMEN
Bacterial flagellin activates the innate immune system and ultimately the adaptive immune system through a Toll-like receptor 5 (TLR5)-dependent signaling mechanism. Given that TLR5 is widely distributed in epithelia, flagellin is currently being developed as a mucosal adjuvant. Flagellin FliC from Salmonella enterica has four domains: the conserved D0 and D1 domains and the hypervariable D2 and D3 domains. The deletion of D3 and partial deletion of D2 in the recombinant FliCΔ174-400 strongly impairs flagellin's intrinsic antigenicity but does not affect the TLR5-dependent immunostimulation activity, i.e., the capacity to promote innate responses and adaptive responses to co-administered antigens. Here, we describe the development of novel recombinant flagellins with various deletions encompassing all of D2 and D3, and part of D1. Most of the recombinant molecules conserved an α-helical secondary structure that was as resistant to heat denaturation as the native protein. Whereas the recombinant flagellins' ability to trigger TLR5 varied markedly in vitro, most gave equivalent in vivo TLR5-dependent innate immune responses following intranasal administration of 2⯵g of flagellin to mice. Concordantly, the recombinant flagellins were also valuable respiratory adjuvants for eliciting antibody responses to the foreign antigen ovalbumin, although their intrinsic antigenicity was decreased compared to the native flagellin and not increased compared to FliCΔ174-400. Our results show that the additional deletions of D2 and the distal part of D1 of FliCΔ174-400 does not impact on antigenicity and does not significantly modify the immunostimulatory adjuvant activity. Altogether, this study generated a novel set of recombinant flagellin that constitutes a portfolio of TLR5-dependent candidate adjuvants for vaccination.
Asunto(s)
Adyuvantes Inmunológicos/genética , Flagelina/genética , Flagelina/inmunología , Proteínas Recombinantes/inmunología , Animales , Inmunidad Innata , Inmunidad Mucosa , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Salmonella enterica/genética , Salmonella enterica/inmunología , Eliminación de Secuencia , Transducción de Señal , Receptor Toll-Like 5/inmunologíaRESUMEN
Purpose: Conditioning strategies constitute a relatively unexplored and exciting opportunity to shape tumor fate by targeting the tumor microenvironment. In this study, we assessed how hemin, a pharmacologic inducer of heme oxygenase-1 (HO-1), has an impact on prostate cancer development in an in vivo conditioning model.Experimental Design: The stroma of C57BL/6 mice was conditioned by subcutaneous administration of hemin prior to TRAMP-C1 tumor challenge. Complementary in vitro and in vivo assays were performed to evaluate hemin effect on both angiogenesis and the immune response. To gain clinical insight, we used prostate cancer patient-derived samples in our studies to assess the expression of HO-1 and other relevant genes.Results: Conditioning resulted in increased tumor latency and decreased initial growth rate. Histologic analysis of tumors grown in conditioned mice revealed impaired vascularization. Hemin-treated human umbilical vein endothelial cells (HUVEC) exhibited decreased tubulogenesis in vitro only in the presence of TRAMP-C1-conditioned media. Subcutaneous hemin conditioning hindered tumor-associated neovascularization in an in vivo Matrigel plug assay. In addition, hemin boosted CD8+ T-cell proliferation and degranulation in vitro and antigen-specific cytotoxicity in vivo A significant systemic increase in CD8+ T-cell frequency was observed in preconditioned tumor-bearing mice. Tumors from hemin-conditioned mice showed reduced expression of galectin-1 (Gal-1), key modulator of tumor angiogenesis and immunity, evidencing persistent remodeling of the microenvironment. We also found a subset of prostate cancer patient-derived xenografts and prostate cancer patient samples with mild HO-1 and low Gal-1 expression levels.Conclusions: These results highlight a novel function of a human-used drug as a means of boosting the antitumor response. Clin Cancer Res; 23(17); 5135-48. ©2017 AACR.
Asunto(s)
Galectina 1/genética , Hemo-Oxigenasa 1/genética , Hemina/administración & dosificación , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/patología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Galectina 1/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/antagonistas & inhibidores , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Masculino , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dendritic cells (DC) initiate the adaptive immune response. Glucocorticoids (GCs) down-modulate the function of DC. Compound A (CpdA, (2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride) is a plant-derived GR-ligand with marked dissociative properties. We investigated the effects of CpdA on in vitro generated GM-CSF-conditioned bone marrow-derived DC (BMDC). CpdA-exposed BMDC exhibited low expression of cell-surface molecules and diminution of the release of proinflammatory cytokines upon LPS stimulation; processes associated with BMDC maturation and activation. CpdA-treated BMDC were inefficient at Ag capture via mannose receptor-mediated endocytosis and displayed reduced T-cell priming. CpdA prevented the LPS-induced rise in pErk1/2 and pP38, kinases involved in TLR4 signaling. CpdA fully inhibited LPS-induced pAktSer473, a marker associated with the generation of tolerogenic DC. We used pharmacological blockade and selective genetic loss-of-function tools and demonstrated GR-independent inhibitory effects of CpdA in BMDC. Mechanistically, CpdA-mediated inactivation of the NF-κB intracellular signaling pathway was associated with a short-circuiting of pErk1/2 and pP38 upstream signaling. Assessment of the in vivo function of CpdA-treated BMDC pulsed with the hapten trinitrobenzenesulfonic acid showed impaired cell-mediated contact hypersensitivity. Collectively, we provide evidence that CpdA is an effective BMDC modulator that might have a benefit for immune disorders, even when GR is not directly targeted.
Asunto(s)
Acetatos/farmacología , Células de la Médula Ósea/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Regulación hacia Abajo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Tiramina/análogos & derivados , Animales , Antígeno B7-1/metabolismo , Células de la Médula Ósea/citología , Células Dendríticas/citología , Endocitosis/efectos de los fármacos , Mediadores de Inflamación/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , Receptor Toll-Like 4/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Tiramina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
B-cell superantigens (Sags) bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. No attempts to investigate whether B-cell Sags are able to induce the apoptosis of cognate malignant B cells were reported. In the present study we show that protein L (PpL), secreted by Finegoldia magna, a B-cell Sag which interacts with κ+ bearing cells, induces the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. Apoptosis was not altered by caspase-8 inhibitor. No alterations in the levels of Bid, Fas and Fas-L were found suggesting that PpL does not activate the extrinsic pathway of apoptosis. The involvement of the intrinsic pathway was clearly indicated by: i) alterations in mitochondrial membrane potential (ΔΨm) both in murine and human lymphoma cells exposed to PpL; ii) decreased levels of apoptosis in the presence of caspase-9 inhibitor; iii) significant increases of Bim and Bax protein levels and downregulation of Bcl-2; iv) the translocation from the cytoplasm to the mitochondria of Bax and Bim pro-apoptotic proteins and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor and v) the translocation of Bcl-2 protein from the mitochondria to the cytosol and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor. The possibility of a therapeutic use of Sags in lymphoma/leukemia B cell malignancies is discussed.
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Apoptosis/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Proteínas Bacterianas/inmunología , Proteínas de Unión al ADN/inmunología , Linfoma de Células B/patología , Superantígenos/inmunología , Adolescente , Animales , Anexina A5/metabolismo , Linfocitos B/efectos de los fármacos , Antígeno B7-2/metabolismo , Proteína 11 Similar a Bcl2/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/patología , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Inhibidores de Caspasas/farmacología , Línea Celular Tumoral , Citosol/metabolismo , ADN de Neoplasias/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulina M/metabolismo , Cadenas kappa de Inmunoglobulina/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos BALB C , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK.
Asunto(s)
Brucella/enzimología , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Cristalografía por Rayos X , Análisis Mutacional de ADN , Histidina Quinasa , Simulación de Dinámica Molecular , Fosforilación , Conformación Proteica , Proteínas Quinasas/genética , Multimerización de ProteínaRESUMEN
Brucella Lumazine Synthase (BLS) is a highly immunogenic decameric protein which can accept the fusion of foreign proteins at its ten N-termini. These chimeras are very efficient to elicit systemic and oral immunity without adjuvants. BLS signaling via Toll-Like Receptor 4 (TLR4) regulates innate and adaptive immune responses, inducing dendritic cell maturation and CD8(+) T-cell cytotoxicity. In this work we study the effect induced by BLS in TLR4-expressing B16 melanoma. In order to evaluate the effectiveness of BLS as a preventive vaccine, C57BL/6J mice were immunized with BLS or BLS-OVA, and 35 days later were subcutaneously inoculated with B16-OVA melanoma. BLS or BLS-OVA induced a significant inhibition of tumor growth, and 50% of mice immunized with the highest dose of BLS did not develop visible tumors. This effect was not observed in TLR4-deficient mice. For treatment experiments, mice were injected with BLS or BLS-OVA 2 days after the inoculation of B16 cells. Both treatments induced significant and equal tumor growth delay and increased survival. Moreover, BLS and BLS-OVA stimulation were also effective in TLR4-deficient mice. In order to study whether BLS has a direct effect on tumor cells, B16 cells were preincubated with BLS, and after 48h, cells were inoculated. Tumors induced by BLS-stimulated cells had inhibited growth and survival was increased. In the BLS group, 40% of mice did not develop tumors. This effect was abolished by the addition of TLR4/MD2 blocking antibody to cells before BLS stimulation. Our work demonstrates that BLS immunization induces a preventive antitumor response that depends on mice TLR4. We also show that BLS generates a therapeutic effect in mice inoculated with B16 cells. Our results show that BLS acts directly in cultured tumor cells via TLR4, highly suggesting that BLS elicits its therapeutic effects acting on the TLR4 from B16 melanoma cells.
Asunto(s)
Brucella/enzimología , Complejos Multienzimáticos/metabolismo , Receptor Toll-Like 4/genética , Animales , Apoptosis , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/mortalidad , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/inmunología , Ovalbúmina/genética , Ovalbúmina/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Tasa de Supervivencia , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/metabolismo , Trasplante HomólogoRESUMEN
Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It is possible to insert foreign peptides or proteins at its ten-amino acid termini. These chimeras elicit systemic and oral immunity without adjuvants, which are commonly needed in the formulation of subunit-based vaccines. Here, we show that BLS induces the cross presentation of a covalently attached peptide OVA(257-264) and a specific cytotoxic response to this peptide in the absence of adjuvants. Unlike other subunit-based vaccines, this chimera induces rapid activation of CTLs and a specific cytotoxic response, making this polymeric protein an ideal antigen carrier for vaccine development. Adoptive transfer of transgenic OT-I T cells revealed efficient cross presentation of BLS-OVA(257-264)in vivo. BLS-OVA(257-264) immunization induced the proliferation of OVA(257-264)-specific CD8+ lymphocytes and also increased the percentage of OVA(257-264)-specific CD8+ cells expressing the early activation marker CD69; after 5 days, the percentage of OVA(257-264)-specific CD8+ cells expressing high levels of CD44 increased. This cell subpopulation showed decreased expression of IL-7Rα, indicating that BLS-OVA(257-264) induced the generation of CD8+ effector cells. BLS-OVA(257-264) was cross presented in vitro independently of the presence of a functional TLR4 in the DCs. Finally, we show that immunization of wild type mice with the chimera BLS-OVA(257-264) without adjuvants induced a strong OVA(257-264)-specific effector cytotoxic response. This cytotoxicity is dependent on TLR4 as is not induced in mice lacking a functional receptor. These data show that TLR4 signaling is necessary for the induction of a cytotoxic response but not for antigen cross presentation.
Asunto(s)
Citotoxicidad Inmunológica/inmunología , Complejos Multienzimáticos/inmunología , Receptor Toll-Like 4/fisiología , Adyuvantes Inmunológicos/farmacología , Animales , Biopolímeros , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Colorantes Fluorescentes , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Reacción en Cadena de la PolimerasaRESUMEN
ADP-ribosylation of host cell proteins is a common mode of cell intoxication by pathogenic bacterial toxins. Antibodies induced by immunization with inactivated ADP-ribosylating toxins provide efficient protection in case of some secreted toxins, e.g., diphtheria and pertussis toxins. However, other ADP-ribosylating toxins, such as Salmonella SpvB toxin, are secreted directly from the Salmonella-containing vacuole into the cytosol of target cells via the SPI-2 encoded bacterial type III secretion system, and thus are inaccessible to conventional antibodies. Small-molecule ADP-ribosylation inhibitors are fraught with potential side effects caused by inhibition of endogenous ADP-ribosyltransferases. Here, we report the development of a single-domain antibody from an immunized llama that blocks the capacity of SpvB to ADP-ribosylate actin at a molar ratio of 1:1. The single-domain antibody, when expressed as an intrabody, effectively protected cells from the cytotoxic activity of a translocation-competent chimeric C2IN-C/SpvB toxin. Transfected cells were also protected against cytoskeletal alterations induced by wild-type SpvB-expressing strains of Salmonella. This proof of principle paves the way for developing new antidotes against intracellular toxins.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Anticuerpos Antibacterianos/inmunología , Toxinas Bacterianas/metabolismo , Camélidos del Nuevo Mundo/inmunología , Salmonella typhimurium/metabolismo , Factores de Virulencia/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/metabolismo , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/inmunología , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Regulación de la Expresión Génica , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Salmonella typhimurium/inmunología , Células VeroRESUMEN
Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vß inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vß region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.
Asunto(s)
Apoptosis , Linfoma de Células T/genética , Linfoma de Células T/inmunología , Superantígenos/metabolismo , Animales , Anticuerpos Monoclonales/química , Supervivencia Celular , Técnicas de Cocultivo , Proteína Ligando Fas/biosíntesis , Femenino , Citometría de Flujo/métodos , Humanos , Masculino , Virus del Tumor Mamario del Ratón/inmunología , Ratones , Receptores de Antígenos de Linfocitos T/metabolismo , Receptor fas/biosíntesisRESUMEN
The enzyme lumazine synthase from Brucella spp. (BLS) is a highly immunogenic protein that folds as a stable dimer of pentamers. It is possible to insert foreign peptides and proteins at the 10 N terminus of BLS without disrupting its general folding, and these chimeras are very efficient to elicit systemic and oral immunity without adjuvants. In this study, we show that BLS stimulates bone marrow dendritic cells from mice in vitro to up-regulate the levels of costimulatory molecules (CD40, CD80, and CD86) and major histocompatibility class II Ag. Furthermore, the mRNA levels of several chemokines are increased, and proinflammatory cytokine secretion is induced upon exposure to BLS. In vivo, BLS increases the number of dendritic cells and their expression of CD62L in the draining lymph node. All of the observed effects are dependent on TLR4, and clearly independent of LPS contamination. The described characteristics of BLS make this protein an excellent candidate for vaccine development.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Biopolímeros/inmunología , Células Dendríticas/inmunología , Receptor Toll-Like 4/inmunología , Animales , Antígenos CD/metabolismo , Biopolímeros/química , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Brucella/inmunología , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Citocinas/biosíntesis , Células Dendríticas/citología , Células Dendríticas/metabolismo , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
SEG and SEI are staphylococcal superantigens (SAgs) identified recently that belong to the egc operon and whose genes are in tandem orientation. Only a few allelic variants of SEG and SEI have been reported. Here we analyzed four Staphylococcus aureus strains with genotypic variation in both SAgs. However, both SAgs retain key residues in their putative TCR and MHC binding sites and, accordingly, their superantigenic properties. Thus, SEI significantly stimulates mouse T-cells bearing Vbeta3, 5 and 13, while SEG stimulates Vbeta7 and 9 in the draining node when inoculated in the footpad. As another member of the SEB subfamily, SEG also stimulates mouse Vbeta8.1+2. However, the increase in Vbeta8.1+2 T-cells observed at day 2 after inoculation reverts to normal values at day 4, whereas it remains high at day 4 following inoculation with SEC3 or SSA. T-cell stimulation assays in the mouse and analysis of the putative Vbeta8.2 binding site on SEG, which includes three non-conserved residues, suggest a possibly unique interaction between Vbeta8.2 and SEG. We also analyzed biochemical and biophysical characteristics of SEI and SEG binding to their cognate human beta chains by surface plasmon resonance, and binding to the HLA-DR1 MHC class II molecule by gel filtration. SEI binds human Vbeta5.2 and Vbeta1 with apparent K(D)'s of 23 and 118 microM, respectively; SEG binds Vbeta13.6 with a K(D) of 5 microM. As suggested by sequence homology, SEI requires Zn2+ for strong binding to DR1, which goes undetected in the presence of EDTA. SEG and SEI have characteristics such as co-expression, different interaction with MHC class II and stimulation of completely different subsets of human and mouse T-cells, which indicate complementary superantigenic activity and suggest an important advantage to staphylococcal strains in producing them both.
Asunto(s)
Enterotoxinas/farmacología , Antígeno HLA-DR1/efectos de los fármacos , Receptores de Antígenos de Linfocitos T alfa-beta/agonistas , Staphylococcus aureus/inmunología , Superantígenos/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Enterotoxinas/análisis , Enterotoxinas/química , Enterotoxinas/genética , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Humanos , Ratones , Datos de Secuencia Molecular , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Superantígenos/análisis , Superantígenos/química , Superantígenos/genética , Resonancia por Plasmón de Superficie , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/microbiologíaRESUMEN
Nackt mice, which are deficient in cathepsin-L (CTSL), show an early impairment during positive selection in the context of class II MHC molecules and as a consequence, the percentage and absolute number of CD4(+) thymocytes are significantly decreased. In this study, we show that lymph nodes from nackt mice are hypertrophied, showing normal absolute numbers of CD4(+) T cells and marked increases in the number of CD8(+) T lymphocytes. Basal proliferative levels are increased in the CD4(+) but not in the CD8(+) population. Lymph node T cells show increases in the expression of alpha(5), alpha(6), and beta(1) integrin chains. These alterations correlate with increases in the expression of extracellular matrix (ECM) components in lymph nodes. Interestingly, laminin, fibronectin, and collagen I and IV are markedly decreased in nackt thymus which shows an augmented output of CD8(+) cells. These results demonstrate that a mutation in the Ctsl gene influences the levels of ECM components in lymphoid organs, the thymic output, and the number of T cells in the periphery. They further raise the possibility that, by regulating the level of expression of ECM components in lymphoid organs, CTSL is able to broadly affect the immune system.
Asunto(s)
Catepsinas/fisiología , Cisteína Endopeptidasas/fisiología , Proteínas de la Matriz Extracelular/biosíntesis , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Timo/citología , Timo/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Catepsina L , Catepsinas/deficiencia , Catepsinas/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Proliferación Celular , Cisteína Endopeptidasas/deficiencia , Cisteína Endopeptidasas/genética , Regulación hacia Abajo/genética , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Glicoproteínas/biosíntesis , Integrina alfa5/biosíntesis , Integrina alfa6/biosíntesis , Integrina beta1/biosíntesis , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Recuento de Linfocitos , Tejido Linfoide/citología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Mutantes , Timo/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Hosts and their pathogens have co-evolved for millions of years, developing multiple and intimate interactions. Vertebrates have evolved a very complex immune system which pathogens have often been able to circumvent, in some cases even managing to appropriate some of its components for their own purpose. Among the pathogens which do use components of the immune system to survive and propagate, those coding for the expression of superantigens (SAgs) are now under intense scrutiny. Investigations concerning one of these pathogens, the mouse mammary tumor virus (MMTV), led to the understanding of how the expression of such components is a critical step in their life cycle. A number of milk-borne exogenous MMTV infect mice shortly after birth and, when expressed, produce superantigens. Herein, we describe the biological effects of new variants of MMTV. Two of these, BALB14 and BALB2 encoding SAgs with V beta 14+ and V beta 2+ specificities, respectively, were present in BALB/c mice of our colony (BALB/cT); a third variant, termed MMTV LA, originated in (BALB/cTxAKR)F1 mice from recombination between BALB 14 and Mtv-7 endogenous provirus. The recombinant LA virus induces the deletion of V beta 6+ and V beta 8.1+ T cells as a consequence of the acquisition of SAg hypervariable coding region of Mtv-7. The SAg encoded by MMTV LA strongly stimulates cognate T cells in vivo leading to a very effective amplification of lymphoid cells in BALB/c mice, correlating with a high incidence of mammary tumors. These results suggest that the presence of non-productive endogenous proviruses--generally considered to confer a selective advantage to the host by protecting it from infection with exogenous MMTVs encoding cross-reactive SAgs--could also be advantageous for the pathogen by increasing its variability, thus broadening the host range and allowing the expansion of highly tumorigenic variants.(Au)
Asunto(s)
Animales , Femenino , Ratones , RESEARCH SUPPORT, NON-U.S. GOVT , Infecciones por Retroviridae/inmunología , Superantígenos/inmunología , Infecciones Tumorales por Virus/inmunología , Gammaretrovirus/inmunología , Susceptibilidad a Enfermedades/inmunología , Predisposición Genética a la Enfermedad , Genoma Viral , Ratones Endogámicos BALB C , ADN Polimerasa Dirigida por ARN , Infecciones por Retroviridae/genética , Infecciones Tumorales por Virus/genética , Gammaretrovirus/genética , Integración Viral/genética , Integración Viral/inmunologíaRESUMEN
Hosts and their pathogens have co-evolved for millions of years, developing multiple and intimate interactions. Vertebrates have evolved a very complex immune system which pathogens have often been able to circumvent, in some cases even managing to appropriate some of its components for their own purpose. Among the pathogens which do use components of the immune system to survive and propagate, those coding for the expression of superantigens (SAgs) are now under intense scrutiny. Investigations concerning one of these pathogens, the mouse mammary tumor virus (MMTV), led to the understanding of how the expression of such components is a critical step in their life cycle. A number of milk-borne exogenous MMTV infect mice shortly after birth and, when expressed, produce superantigens. Herein, we describe the biological effects of new variants of MMTV. Two of these, BALB14 and BALB2 encoding SAgs with V beta 14+ and V beta 2+ specificities, respectively, were present in BALB/c mice of our colony (BALB/cT); a third variant, termed MMTV LA, originated in (BALB/cTxAKR)F1 mice from recombination between BALB 14 and Mtv-7 endogenous provirus. The recombinant LA virus induces the deletion of V beta 6+ and V beta 8.1+ T cells as a consequence of the acquisition of SAg hypervariable coding region of Mtv-7. The SAg encoded by MMTV LA strongly stimulates cognate T cells in vivo leading to a very effective amplification of lymphoid cells in BALB/c mice, correlating with a high incidence of mammary tumors. These results suggest that the presence of non-productive endogenous proviruses--generally considered to confer a selective advantage to the host by protecting it from infection with exogenous MMTVs encoding cross-reactive SAgs--could also be advantageous for the pathogen by increasing its variability, thus broadening the host range and allowing the expansion of highly tumorigenic variants.
Asunto(s)
Animales , Femenino , Ratones , Infecciones por Retroviridae/inmunología , Infecciones Tumorales por Virus/inmunología , Superantígenos/inmunología , Gammaretrovirus/inmunología , Susceptibilidad a Enfermedades , Predisposición Genética a la Enfermedad , Genoma Viral , Infecciones por Retroviridae/genética , Infecciones Tumorales por Virus/genética , Integración Viral/genética , Integración Viral/inmunología , Ratones Endogámicos BALB C , ADN Polimerasa Dirigida por ARN , Gammaretrovirus/genéticaRESUMEN
En este trabajo se describen los efectos biológicos de nuevas variantes virales de MMTV exógenos. Una de ellas denominada BALB14 está presente en la cepa BALB/c e induce un bajo porcentaje de adecarcinomas mamarios. Otra de ellas, (MMTV-7) se originó por recombinación entre BALB14 y transcriptos derivados del provirus endógeno Mtv-7. El desarrollo de una línea de ratones BALB/c infectada con ambas variantes virales a través del amamantamiento con una nodriza F1 (BABL/cxAKR) en la que surge el MMTV-7, permitió demonstrar que el virus recombinante - que expressa el SAg del provirus endógeno - es amplificado en los huéspedes BALB/c y resulta en ellos altamente tumorigénico. Se discute el rol de la adquisición de superantígenos estimulatorios en la amplificación del virus recombinante. Los resultados obtenidos permiten hipotetizar que la presencia en el genoma del ratón de provirus endógenos no productivos - considerada hasta ahora como protectora frente a la infección con virus MMTV exógenos - oferecía además ventajas selectivas para el virus al aumentar su variabilidad poblacional, permitiendo así la ampliación del rango de huéspedes susceptibles y la expanción de variantes con alta patogenicidad. (AU)