RESUMEN

The impact of sulphur limitation on the remobilization of endogenous S compounds during the rosette stage of oilseed rape, and the interactions with N availability on these processes, were examined using a long-term (34)SO(4)(2-) labelling method combined with a study of leaf senescence progression (using SAG12/Cab as a molecular indicator) and gene expression of the transporters, BnSultr4;1 and BnSultr4;2, involved in vacuolar sulphate efflux. After 51 d on hydroponic culture at 0.3 mM (34)SO(4)(2-) (1 atom% excess), the labelling was stopped and plants were subject for 28 d to High S-High N (HS-HN, control), Low S-High N (LS-HN) or Low S-Low N (LS-LN) conditions. Compared with the control, LS-HN plants showed delayed leaf senescence and, whilst the shoot growth and the foliar soluble protein amounts were not affected, S, (34)S, and SO(4)(2-) amounts in the old leaves declined rapidly and were associated with the up-regulation of BnSultr4;1. In LS-LN plants, shoot growth was reduced, leaf senescence was accelerated, and the rapid S mobilization in old leaves was accompanied by decreased (34)S and SO(4)(2-), higher protein mobilization, and up-regulation of BnSultr4;2, but without any change of expression of BnSultr4;1. The data suggest that to sustain the S demand for growth under S restriction (i) vacuolar SO(4)(2-) is specifically remobilized in LS-HN conditions without any acceleration of leaf senescence, (ii) SO(4)(2-) mobilization is related to an up-regulation of BnSultr4;1 and/or BnSultr4;2 expression, and (iii) the relationship between sulphate mobilization and up-regulation of expression of BnSultr4 genes is specifically dependent on the N availability.

Asunto(s)

Brassica rapa/crecimiento & desarrollo , Brassica rapa/metabolismo , Nitrógeno/metabolismo , Compuestos de Azufre/metabolismo , Envejecimiento , Transporte Biológico , Brassica rapa/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sulfatos/metabolismo

RESUMEN

This paper outlines the National Institute for Health and Clinical Excellence's (NICE) emerging conceptual framework for public health. This is based on the experience of the first 3 years of producing public health guidance at NICE (2005-2008). The framework has been used to shape the revisions to NICE's public health process and methods manuals for use post 2009, and will inform the public health guidance which NICE will produce from April 2009. The framework is based on the precept that both individual and population patterns of disease have causal mechanisms. These are analytically separate. Explanations of individual diseases involve the interaction between biological, social and related phenomena. Explanations of population patterns involve the same interactions, but also additional interactions between a range of other phenomena working in tandem. These are described. The causal pathways therefore involve the social, economic and political determinants of health, as well as psychological and biological factors. Four vectors of causation are identified: population, environmental, organizational and social. The interaction between the vectors and human behaviour are outlined. The bridge between the wider determinants and individual health outcomes is integration of the life course and the lifeworld.

Asunto(s)

Comités Consultivos , Salud Pública , Causalidad , Guías como Asunto , Conductas Relacionadas con la Salud , Disparidades en el Estado de Salud , Humanos , Reino Unido

RESUMEN

The aim of this study was to evaluate the presence and the role of the serum soluble costimulatory molecule CD28 in patients with systemic lupus erythematosus (SLE), primary Sjögren's syndrome (SS), and systemic sclerosis (SSc). Soluble CD28 concentration was determined by ELISA in 45 patients with SLE, 45 patients with primary SS, 30 patients with SSc, and 45 healthy subjects. We also evaluated CD28 mRNA expression by semiquantitative RT-PCR, and the biological activity of recombinant soluble CD28 on T lymphocyte activity. Concentrations of soluble CD28 were significantly higher in patients with SLE, primary SS and SSc than in healthy subjects. Soluble CD28 concentrations were higher in patients with systemic primary SS than in patients with glandular-limited primary SS. PCR analysis suggested that soluble CD28 resulted from the shedding of the membrane form. In vitro assay revealed that soluble CD28 inhibits the anti-CD3 mAb induced T cell proliferation. Soluble CD28, which modulates the proliferation of T lymphocytes, could be associated with disease severity in patients with autoimmune disease, especially primary SS. These results suggest that soluble CD28 could play an important role in the regulation of autoimmune diseases.

Asunto(s)

Antígenos CD28/sangre , Lupus Eritematoso Sistémico/inmunología , Esclerodermia Sistémica/inmunología , Síndrome de Sjögren/inmunología , Adolescente , Adulto , Antígenos CD28/genética , Estudios de Casos y Controles , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Interleucina-2/metabolismo , Modelos Lineales , Masculino , Persona de Mediana Edad , Muromonab-CD3/farmacología , ARN Mensajero/análisis , Proteínas Recombinantes/farmacología , Estadísticas no Paramétricas , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo

RESUMEN

The protective efficacy of the influenza matrix protein epitope 58-66 (called M1), recognized in the context of human HLA-A2 molecules, was evaluated in a HLA-A2/K(b) transgenic mouse model of lethal influenza infection. Repeated subcutaneous immunizations with M1 increased the percentage of survival. This effect was mediated by T cells since protection was abolished following in vivo depletion of all T lymphocytes, CD8(+), or CD4(+) T cells. The survival correlated with the detection of memory CD8(+) splenocytes able to proliferate in vitro upon stimulation with M1 and to bind M1-loaded HLA-A2 dimers, as well as with M1-specific T cells in the lungs, which were directly cytotoxic to influenza-infected cells following influenza challenge. These results demonstrated for the first time that HLA-A2-restricted cytotoxic T cells specific for the major immunodominant influenza matrix epitope are protective against the infection. They encourage further in vivo evaluation of T cell epitopes recognized in the context of human MHC molecules.

Asunto(s)

Antígeno HLA-A2/genética , Epítopos Inmunodominantes/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/inmunología , Animales , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Femenino , Antígeno HLA-A2/inmunología , Humanos , Memoria Inmunológica , Gripe Humana/prevención & control , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos

RESUMEN

A novel recombinant respiratory syncytial virus (RSV) subunit vaccine, designated BBG2Na, was administered to 108 healthy adults randomly assigned to receive 10, 100, or 300 microg of BBG2Na in aluminum phosphate or saline placebo. Each subject received 1, 2, or 3 intramuscular injections of the assigned dose at monthly intervals. Local and systemic reactions were mild, and no evidence of harmful properties of BBG2Na was reported. The highest ELISA and virus-neutralizing (VN) antibody responses were evident in the 100- and 300-microg groups; second or third injections provided no significant boosts against RSV-derived antigens. BBG2Na induced > or 2-fold and > or =4-fold increases in G2Na-specific ELISA units in up to 100% and 57% of subjects, respectively; corresponding RSV-A-specific responses were 89% and 67%. Furthermore, up to 71% of subjects had > or =2-fold VN titer increases. Antibody responses to 2 murine lung protective epitopes were also highly boosted after vaccination. Therefore, BBG2Na is safe, well tolerated, and highly immunogenic in RSV-seropositive adults.

Asunto(s)

Vacunas contra Virus Sincitial Respiratorio/efectos adversos , Vacunas contra Virus Sincitial Respiratorio/inmunología , Adolescente , Adulto , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/efectos adversos , Antígenos Virales/inmunología , Epítopos/inmunología , Humanos , Persona de Mediana Edad , Péptidos/inmunología , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/inmunología , Infecciones por Virus Sincitial Respiratorio/etiología , Infecciones del Sistema Respiratorio/etiología , Vacunas de Subunidad/efectos adversos , Vacunas de Subunidad/inmunología , Proteínas Virales/efectos adversos , Proteínas Virales/inmunología

RESUMEN

A BALB/c mouse model of enhanced pulmonary pathology following vaccination with formalin-inactivated alum-adsorbed respiratory syncytial virus (FI-RSV) and live RSV challenge was used to determine the type and kinetics of histopathologic lesions induced and chemokine gene expression profiles in lung tissues. These data were compared and contrasted with data generated following primary and/or secondary RSV infection or RSV challenge following vaccination with a promising subunit vaccine, BBG2Na. Severe peribronchiolitis and perivascularitis coupled with alveolitis and interstitial inflammation were the hallmarks of lesions in the lungs of FI-RSV-primed mice, with peak histopathology evident on days 5 and 9. In contrast, primary RSV infection resulted in no discernible lesions, while challenge of RSV-primed mice resulted in rare but mild peribronchiolitis and perivascularitis, with no evidence of alveolitis or interstitial inflammation. Importantly, mice vaccinated with a broad dose range (20 to 0.02 microg) of a clinical formulation of BBG2Na in aluminium phosphate demonstrated histopathology similar to that observed in secondary RSV infection. At the molecular level, FI-RSV priming was characterized by a rapid and strong up-regulation of eotaxin and monocyte chemotactic protein 3 (MCP-3) relative gene expression (potent lymphocyte and eosinophil chemoattractants) that was sustained through late time points, early but intermittent up-regulation of GRO/melanoma growth stimulatory activity gene and inducible protein 10 gene expression, while macrophage inflammatory protein 2 (MIP-2) and especially MCP-1 were up-regulated only at late time points. By comparison, primary RSV infection or BBG2Na priming resulted in considerably lower eotaxin and MCP-3 gene expression increases postchallenge, while expression of lymphocyte or monocyte chemoattractant chemokine genes (MIP-1beta, MCP-1, and MIP-2) were of higher magnitude and kinetics at early, but not late, time points. Our combined histopathologic and chemokine gene expression data provide a basis for differentiating between aberrant FI-RSV-induced immune responses and normal responses associated with RSV infection in the mouse model. Consequently, our data suggest that BBG2Na may constitute a safe RSV subunit vaccine for use in seronegative infants.

Asunto(s)

Quimiocinas/genética , Pulmón/patología , Vacunas contra Virus Sincitial Respiratorio/inmunología , Animales , Quimiocina CCL2/genética , Quimiocina CCL4 , Quimiocina CCL5/genética , Quimiocina CXCL2 , Femenino , Inmunización , Pulmón/metabolismo , Proteínas Inflamatorias de Macrófagos/genética , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacunas de Productos Inactivados/inmunología , Vacunas de Subunidad/inmunología

RESUMEN

BBG2Na, a well-defined recombinant protein produced in Escherichia coli, is a promising human respiratory syncytial virus subunit vaccine candidate. This study describes the quantification of residual DNA in large scale batches used in phase I to III clinical trials. Two different analytical methods were developed and applied on five different final bulks of Drug Substance and their associated in process control samples, namely a chemiluminescent hybridisation assay and the total DNA Threshold System assay. These two complementary methods demonstrated the clearance of residual DNA during the downstream purification process. The amount of residual DNA found in the final bulks was below 20 pg of DNA per 300 microg BBG2Na, the highest tested clinical dose of antigen. This is very low level of residual DNA for a recombinant subunit vaccine produced in a bacteria and contribute to make for BBG2Na a well-characterised biopharmaceutical. This study also provides data concerning the validation of the hybridisation dot blot assay and the total DNA Threshold(trade mark)assay.

Asunto(s)

ADN Recombinante/análisis , Vacunas Virales/análisis , Ensayos Clínicos como Asunto , Contaminación de Medicamentos , Escherichia coli/genética , Humanos , Mediciones Luminiscentes , Hibridación de Ácido Nucleico , Vacunas Sintéticas/análisis

RESUMEN

Allergic disorders are characterized by allergen-specific Th2-biased responses. Signals controlling Th2 cell polarization, especially those acting by polarizing dendritic cells (DC) into Th2-promoting DC (DC2), are not well known. Histamine, a mediator released by allergen-stimulated mast cells from allergic subjects, has been reported to activate human immature DC. We have therefore tested whether histamine affects DC polarization. We report here that histamine inhibits LPS-induced IL-12 production and polarizes uncommitted maturing DC into effector DC2. DC matured in the presence of histamine fail to produce IL-12 upon subsequent stimulation and prime Th2 responses, even in presence of IFN-gamma, a potent DC1-driving factor. All these effects are mediated through both H1 and H2 receptors. These data show that histamine is a potent DC2-polarizing factor and provide evidence for a novel mechanism that explains the initiation and maintenance of a predominant Th2 response in allergic disorders.

Asunto(s)

Células Dendríticas/inmunología , Histamina/farmacología , Células Th2/inmunología , Diferenciación Celular , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Humanos , Hipersensibilidad/inmunología , Interferón gamma/biosíntesis , Interleucina-12/biosíntesis , Lipopolisacáridos/farmacología , Células Progenitoras Mieloides/efectos de los fármacos , Células Progenitoras Mieloides/inmunología

RESUMEN

Administration of vaccines by the nasal route has recently proven to be one of the most efficient ways for inducing both mucosal and systemic antibody responses in experimental animals. Our results demonstrate that P40, a well-defined outer membrane protein A from Klebsiella pneumoniae, is indeed a carrier molecule suitable for nasal immunization. Using fragments from the respiratory syncytial virus subgroup A (RSV-A) G protein as antigen models, it has been shown that P40 is able to induce both systemic and mucosal immunity when fused or coupled to a protein or a peptide and administered intranasally (i.n.) to naive or K. pneumoniae-primed mice. Confocal analyses of nasal mucosa-associated lymphoid tissue after i.n. instillation of P40 showed that this molecule is able to cross the nasal epithelium and target CD11c-positive cells likely to be murine dendritic cells or macrophages. More importantly, this targeting of antigen-presenting cells following i.n. immunization with a subunit of the RSV-A molecule in the absence of any mucosal adjuvant results in both upper and lower respiratory tract protection against RSV-A infection.

Asunto(s)

Adyuvantes Inmunológicos , Células Presentadoras de Antígenos/inmunología , Antígenos Virales/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Klebsiella pneumoniae/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/inmunología , Vacunas Sintéticas/inmunología , Proteínas Virales/inmunología , Administración Intranasal , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunidad Mucosa , Tejido Linfoide/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mucosa Nasal/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Infecciones por Virus Sincitial Respiratorio/inmunología , Vacunación/métodos

RESUMEN

Several cytotoxic T lymphocyte peptide-based vaccines against hepatitis B, human immunodeficiency virus and melanoma were recently studied in clinical trials. One interesting melanoma vaccine candidate alone or in combination with other tumor antigens, is the decapeptide ELA. This peptide is a Melan-A/MART-1 antigen immunodominant peptide analog, with an N-terminal glutamic acid. It has been reported that the amino group and gamma-carboxylic group of glutamic acids, as well as the amino group and gamma-carboxamide group of glutamines, condense easily to form pyroglutamic derivatives. To overcome this stability problem, several peptides of pharmaceutical interest have been developed with a pyroglutamic acid instead of N-terminal glutamine or glutamic acid, without loss of pharmacological properties. Unfortunately compared with ELA, the pyroglutamic acid derivative (PyrELA) and also the N-terminal acetyl-capped derivative (AcELA) failed to elicit cytotoxic T lymphocyte (CTL) activity. Despite the apparent minor modifications introduced in PyrELA and AcELA, these two derivatives probably have lower affinity than ELA for the specific class I major histocompatibility complex. Consequently, in order to conserve full activity of ELA, the formation of PyrELA must be avoided. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride salt, shows higher stability than the acetate salt and may be suitable for use in man. Similar stability data were also obtained for MAGE-3, another N-terminal glutamic acid containing CTL peptide in clinical development, leading us to suggest that all N-terminal glutamic acid and probably glutamine-containing CTL peptide epitopes may be stabilized as hydrochloride salts.

Asunto(s)

Antígenos de Neoplasias , Ácido Glutámico/química , Isoantígenos/metabolismo , Melanoma/inmunología , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Resinas de Intercambio Aniónico , Vacunas contra el Cáncer/inmunología , Línea Celular/inmunología , Línea Celular/metabolismo , Cromatografía Líquida de Alta Presión , Cromo/metabolismo , Epítopos de Linfocito T , Granulocitos , Humanos , Inmunización , Ratones , Proteínas de Neoplasias/química , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Péptidos/síntesis química , Péptidos/química , Péptidos/metabolismo , Espectrometría de Masa por Ionización de Electrospray

RESUMEN

Human respiratory syncytial virus (hRSV) is a major pathogen responsible for bronchiolitis and severe pulmonary disease in very young children, immunodeficient patients and the elderly. BBG2Na, a recombinant chimeric protein produced in Escherichia coli, is a promising subunit vaccine candidate against this respiratory pathogen, composed of G2Na, the central domain of RSV G glycoprotein, and BB, an albumin binding domain of streptococcal protein G. BBG2Na has a basic isoelectric point (pI 9.3) and as expected, is strongly adsorbed by aluminium phosphate (AP). Surprisingly, BBG2Na is also strongly adsorbed by aluminium hydroxide (AH), which normally binds molecules with acidic isoelectric points. This behaviour was unexpected according to the well established adsorption model of Hem and co-workers. Our observations may be explained by the bipolar two-domain structure of the BBG2Na chimera which is not reflected by the global basic isoelectric point of the whole protein: the BB domain has an acidic isoelectric point (pI 5.5) and the G2Na domain a highly basic one (pI 10.0). Importantly, formulation in either aluminium salt resulted in equally high immunogenicity and protective efficacy against RSV in mice. From a physicochemical point of view, this unique property of BBG2Na makes it eminently suitable for combination to either paediatric or elderly multivalent AH- or AP-containing vaccines already in the market or in development.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Aluminio/administración & dosificación , Hidróxido de Aluminio/administración & dosificación , Fosfatos/administración & dosificación , Virus Sincitial Respiratorio Humano/inmunología , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Adsorción , Secuencia de Aminoácidos , Animales , Tampones (Química) , Glicol de Etileno/farmacología , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Vacunas de Subunidad/inmunología

RESUMEN

Nasal administration of vaccines is an attractive approach which offers several significant advantages over traditional intramuscular vaccine delivery. These advantages include easier administration and induction of immune responses in the mucosal secretions of the body. In this study we describe a new potent nasal adjuvant, dimethyldioctadecylammonium bromide (DDA), that induces both mucosal and systemic immune responses when co-administered with diphtheria toxoid (DT), tetanus toxoid (TT) and BBG2Na antigens. In particular, we show that the nasal delivery of recombinant fragment (BBG2Na) of the G protein of respiratory syncytial virus (RSV) mixed with DDA induces both local and systemic anti-RSV immune responses and protects against viral challenge. Furthermore, we provide evidence that the DDA+BBG2Na vaccine does not induce lung immunopathology upon subsequent RSV challenge.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Amonio Cuaternario/administración & dosificación , Virus Sincitiales Respiratorios/inmunología , Vacunas Sintéticas/administración & dosificación , Administración Intranasal , Animales , Toxoide Diftérico/administración & dosificación , Femenino , Inmunidad Mucosa , Ratones , Ratones Endogámicos BALB C , Sigmodontinae , Linfocitos T/inmunología , Toxoide Tetánico/administración & dosificación

RESUMEN

Adhesive interactions with stromal cells and the extracellular matrix are essential for the differentiation and migration of hematopoietic progenitors. In the erythrocytic lineage, a number of adhesion molecules are expressed in the developing erythrocytes and are thought to play a role in the homing and maturation of erythrocytic progenitors. However, many of these molecules are lost during the final developmental stages leading to mature erythrocytes. One of the adhesion molecules that remains expressed in mature, circulating erythrocytes is CD147. This study shows that blockade of this molecule on the cell surface by treatment with F(ab')(2) fragments of anti-CD147 monoclonal antibody disrupts the circulation of erythrocytes, leading to their selective trapping in the spleen. Consequently, mice develop an anemia, and de novo, erythropoietin-mediated erythropoiesis in the spleen. In contrast, these changes were not seen in mice similarly treated with another antierythrocyte monoclonal antibody with a different specificity. These results suggest that the CD147 expressed on erythrocytes likely plays a critical role in the recirculation of mature erythrocytes from the spleen into the general circulation. (Blood. 2001;97:3984-3988)

Asunto(s)

Antígenos CD , Antígenos de Neoplasias , Antígenos de Superficie , Proteínas Aviares , Proteínas Sanguíneas , Movimiento Celular/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Bazo/citología , Animales , Anticuerpos Monoclonales/farmacología , Basigina , Eritrocitos/citología , Eritropoyesis/efectos de los fármacos , Hematócrito , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos , Tamaño de los Órganos/efectos de los fármacos , Flebotomía , Factores de Tiempo

RESUMEN

To understand the lack of protective immunity observed after infection with parainfluenza virus type 3 (PIV3), we tested the effect of the virus on human monocytes and monocyte-derived immature dendritic cells (DCs). Expression of viral antigens on the cell surfaces correlated with replication of the virus, which was marginal in monocytes but extremely efficient in DCs. The virus increased monocyte survival at least in part through the production of granulocyte-macrophage colony-stimulating factor but, in contrast, accelerated DC apoptosis. In addition, PIV3 infection failed to activate monocytes but induced maturation of DCs with increased expression of CD54, HLA-DR, CD86, and CD83 and production of bioactive IL-12. However, PIV3-infected DCs demonstrated low stimulatory properties in DC-T cell cocultures, a finding that could not be attributed to the production of infectious virus or IL-10. These results demonstrate for the first time that PIV3 dramatically modifies the survival and/or the function of antigen-presenting cells and might therefore prevent the development of efficient antiviral immune responses.

Asunto(s)

Células Dendríticas/virología , Leucocitos Mononucleares/virología , Virus de la Parainfluenza 3 Humana/fisiología , Antígenos CD/análisis , Antígenos Virales/análisis , Apoptosis , Antígeno B7-2 , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/análisis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Antígenos HLA-DR/análisis , Humanos , Inmunoglobulinas/análisis , Molécula 1 de Adhesión Intercelular/análisis , Interleucina-12/análisis , Leucocitos Mononucleares/inmunología , Glicoproteínas de Membrana/análisis , ARN Mensajero/biosíntesis , Replicación Viral , Antígeno CD83

RESUMEN

Respiratory syncytial virus (RSV) is a major respiratory pathogen responsible for severe pulmonary disease. We have developed a parenterally administered vaccine, BBG2Na, which is currently in a phase III clinical trial. BBG2Na comprises residues 130--230 of RSV-A G protein (G2Na) fused to the BB carrier protein. In this study, we show that BBG2Na can be delivered by the nasal route and generates both mucosal and systemic antibody responses when co-administered with cholera toxin B or a newly described delivery system, zwittergent 3--14. We found that nasal BBG2Na administration protects against RSV challenge and does not induce lung immunopathology upon subsequent RSV challenge.

Asunto(s)

Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Virus Sincitiales Respiratorios/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/biosíntesis , Toxina del Cólera/administración & dosificación , Femenino , Proteína HN/inmunología , Humanos , Inmunidad Mucosa , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/patología , Compuestos de Amonio Cuaternario/administración & dosificación , Compuestos de Amonio Cuaternario/toxicidad , Vacunas de Subunidad/administración & dosificación , Proteínas del Envoltorio Viral

RESUMEN

Mast cells and immature dendritic cells (DC) are in close contact in peripheral tissues. Upon activation, mast cells release histamine, a mediator involved in the immediate hypersensitivity reaction. We therefore tested whether histamine could affect human DC activation and maturation. Histamine induces CD86 expression on immature DC in a dose-dependent (significant at 10(-7) M) and transient manner (maximal after 24-h stimulation). Histamine also transiently up-regulates the expression of the costimulatory and accessory molecules, CD40, CD49d, CD54, CD80, and MHC class II. As a consequence, immature DC exposed for 24 h to histamine stimulate memory T cells more efficiently than untreated DC. In addition, histamine induces a potent production of IL-6, IL-8, monocyte chemoattractant protein 1, and macrophage-inflammatory protein 1alpha by immature DC and also up-regulates IL-1beta, RANTES, and macrophage-inflammatory protein 1beta but not TNF-alpha and IL-12 mRNA expression. Histamine activates immature DC through both the H1 and H2 receptors. However, histamine-treated DC do not have a phenotype of fully mature cells, as they do neither show significant changes in the expression of the chemokine receptors, CCR5, CCR7 and CXC chemokine receptor 4, nor expression of CD83 de novo. These data demonstrate that histamine activates immature DC and induces chemokine production, thereby suggesting that histamine, via stimulation of resident DC, may participate locally in T cell stimulation and in the late inflammatory reaction associated with allergic disorders.

Asunto(s)

Antígenos CD/biosíntesis , Quimiocinas/biosíntesis , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Histamina/farmacología , Glicoproteínas de Membrana/biosíntesis , Adyuvantes Inmunológicos/farmacología , Presentación de Antígeno/efectos de los fármacos , Antígeno B7-1/biosíntesis , Antígeno B7-2 , Antígenos CD40/biosíntesis , Diferenciación Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Citocinas/biosíntesis , Células Dendríticas/patología , Relación Dosis-Respuesta Inmunológica , Antígenos HLA-DR/biosíntesis , Histamina/metabolismo , Histamina/fisiología , Humanos , Integrina alfa4 , Integrinas/biosíntesis , Molécula 1 de Adhesión Intercelular/biosíntesis , Receptores Histamínicos H1/fisiología , Receptores Histamínicos H2/fisiología , Regulación hacia Arriba/inmunología

RESUMEN

We have developed and validated a process-specific immunoligand assay based on the Threshold system for the quantification of residual host cell proteins (HCPs) in a recombinant subunit vaccine candidate against the human respiratory syncytial virus (hRSV). The industrial process of this vaccine produced in Escherichia coli, involved five chromatography steps for the production of clinical-grade batches. The clearance of non-product-related protein throughout the purification process was documented by the evaluation of the HCP content in the chromatographic fractions at each step of the downstream processing. The assay had a detection limit of 0.5 ng/ml of HCP equivalent to 10 parts per million (ppm). The quantification limit was 1.3 ng/ml of HCP, giving a sensitivity range of the assay of 10 to 30 ppm. To our knowledge, this is the first sensitive HCP assay reported for a vaccine.

Asunto(s)

Inmunoensayo/métodos , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/inmunología , Vacunas Virales/análisis , Anticuerpos Antibacterianos , Especificidad de Anticuerpos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/inmunología , Contaminación de Medicamentos , Escherichia coli/genética , Escherichia coli/inmunología , Humanos , Inmunoensayo/estadística & datos numéricos , Técnicas In Vitro , Sensibilidad y Especificidad , Vacunas de Subunidad/análisis , Vacunas Sintéticas/análisis

RESUMEN

The recent identification of tumor Ags as potential vaccines has prompted the search for efficient adjuvants and delivery systems, especially in the case of peptide-based vaccination protocols. Here, we investigated the adjuvant potential of the recombinant 40-kDa outer membrane protein of Klebsiella pneumoniae (P40) for specific CTL induction. We studied the CTL response induced in HLA-A*0201/K(b) transgenic mice immunized with peptides derived from two melanoma-associated differentiation Ags, the HLA-A*0201-restricted decapeptide Melan-A(26--35) substituted at position 2 and the K(b)-restricted tyrosinase-related protein 2(181--188) T cell epitope. We found that both peptides are able to generate a specific CTL response when mixed with the protein in the absence of conventional adjuvant. This CTL response is a function of the amount of P40 used for immunization. Moreover, the CTL response generated against the tyrosinase-related protein 2(181-188) peptide in presence of P40 is associated with tumor protection in two different experimental models and is independent of the presence of CD4(+) T lymphocytes. Thus, the recombinant bacterial protein P40 functions as a potent immunological adjuvant for specific CTL induction.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas contra el Cáncer/síntesis química , Citotoxicidad Inmunológica/inmunología , Epítopos de Linfocito T/inmunología , Klebsiella pneumoniae/inmunología , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Adyuvantes Inmunológicos/síntesis química , Animales , Antígenos de Neoplasias/administración & dosificación , Antígenos de Neoplasias/inmunología , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Combinación de Medicamentos , Inmunoterapia Activa/métodos , Inyecciones Subcutáneas , Oxidorreductasas Intramoleculares/administración & dosificación , Oxidorreductasas Intramoleculares/inmunología , Depleción Linfocítica , Antígeno MART-1 , Sustancias Macromoleculares , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Neoplasias/administración & dosificación , Proteínas de Neoplasias/inmunología , Trasplante de Neoplasias , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/síntesis química , Volumetría , Células Tumorales Cultivadas/trasplante

RESUMEN

Respiratory syncytial virus (RSV) is an important respiratory pathogen in man, against which no vaccine is available. However, recent evidence suggests that antibodies to the RSV F and G proteins may play an important role in disease prevention. We previously demonstrated that BBG2Na, a subunit vaccine candidate including residues 130-230 of the Long strain G protein, protects rodents against RSV challenge. Using a panel of monoclonal antibodies (MAb) and synthetic peptides, five linear B cell epitopes were identified that mapped to residues 152-163, 165-172, 171-187 (two over-lapping epitopes) and 196-204. Antibody passive transfer and peptide immunisation studies revealed that all were protective. Pepscan analyses of anti-RSV-A and BBG2Na murine polyclonal sera suggested stronger immunogenicity of some protective epitopes (protectopes) in the context of BBG2Na compared with live virus. However, all the identified murine B cell protectopes were conserved in RSV seropositive humans. Should these protectopes correspond with protection in humans, BBG2Na may constitute a very interesting vaccine candidate against RSV.

Asunto(s)

Linfocitos B/inmunología , Virus Sincitiales Respiratorios/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales , Antígenos Virales/genética , Mapeo Epitopo , Epítopos , Humanos , Inmunización Pasiva , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Virus Sincitiales Respiratorios/genética , Vacunas de Subunidad/genética , Vacunas de Subunidad/farmacología , Proteínas Virales/genética , Vacunas Virales/genética , Vacunas Virales/farmacología

RESUMEN

CD86 is a costimulatory molecule constitutively expressed by human antigen presenting cells which interacts with CD28 and CTLA-4 expressed by T cells. We have recently reported the identification of an alternatively spliced CD86 mRNA variant (CD86deltaTM) characterized by the deletion of exon 6 which encodes for the transmembrane domain. We report here the identification of an alternatively spliced variant (called CD86deltaEC) expressed by nonstimulated human monocytes and characterized by the deletion of exons 4 and 5 which encode for the extracellular V-like and C-like domains, respectively. The activation of monocytes by IFNgamma (i) induces the preferential expression of the transcript encoding for the membrane form and (ii) increases the expression of CD86 and of the accessory molecules CD40, CD49d and CD54. These results suggest that resting human monocytes may constitutively express different forms of CD86 which can then influence the activation of T cells.

Asunto(s)

Empalme Alternativo , Antígenos CD/genética , Glicoproteínas de Membrana/genética , ARN Mensajero/genética , Secuencia de Aminoácidos , Antígeno B7-2 , Secuencia de Bases , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/metabolismo , Análisis de Secuencia de ADN