RESUMEN
Replication-defective recombinant adenoviruses (rAd) are used as vectors for vaccines as well as for gene therapy. To determine type-specific antibodies to adenovirus (Ad) serotypes 2, 5, 24, 34, and 35, we developed quantitative neutralization assays using recombinant adenoviruses with the secreted alkaline phosphatase (SEAP) reporter gene. Among the standardized parameters, the concentration of infectious and noninfectious adenoviral particles used in the assay is critical for a reliable comparison of data from different studies. The usefulness of this assay was demonstrated in a pilot epidemiologic study of 40 healthy individuals. In this study, the highest prevalence of antiadenovirus antibodies was found for the Ad2 serotype (82.5%), followed by Ad5 (35%). The prevalence of antiadenovirus antibodies for the serotypes 24, 34, and 35 was low (7.5%, 2.5%, and 0%, respectively). In addition, epidemiologic parameters such as gender and age were statistically evaluated. A positive association was found between age and the presence of anti-Ad5 antibodies. The assay was also useful for evaluating the presence of antiadenovirus antibodies in the design of vaccines using a rhesus monkey model. In this animal model, it was possible to determine differential dose and time responses, and the specificity for the detection of neutralizing antibodies was assessed. The evaluation of serotype-specific neutralizing antibodies can be of both clinical and epidemiologic importance as a means of selecting the appropriate serotype adenovector(s).
Asunto(s)
Adenoviridae/genética , Adenoviridae/inmunología , Fosfatasa Alcalina/genética , Vectores Genéticos/inmunología , Pruebas de Neutralización/métodos , Vacunas de ADN/inmunología , Adenoviridae/clasificación , Adulto , Anciano , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/inmunología , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/inmunología , Línea Celular , Estudios Epidemiológicos , Femenino , Técnicas de Transferencia de Gen , Genes Reporteros , Humanos , Macaca mulatta , Masculino , Persona de Mediana EdadRESUMEN
Notch proteins are transmembrane receptors that normally adopt a resting state poised to undergo activating proteolysis upon ligand engagement. Receptor quiescence is maintained by three LIN12/Notch repeats (LNRs), which wrap around a heterodimerization domain (HD) divided by furin cleavage at site S1 during maturation. Ligand binding initiates signaling by inducing sensitivity of the HD to proteolysis at the regulated S2 cleavage site. Here, we used hydrogen exchange mass spectrometry to examine the solution dynamics of the Notch1 negative regulatory region in autoinhibited states before and after S1 cleavage, in a proteolytically sensitive "on" state, and in a complex with an inhibitory antibody. Conversion to the "on" state leads to accelerated deuteration in the S2 region and in nearby secondary structural elements within the HD. In contrast, complexation with the inhibitory antibody retards deuteration around the S2 site. Together, these studies reveal how S2 site exposure is promoted by receptor activation and suppressed by inhibitory antibodies.
Asunto(s)
Péptidos/química , Conformación Proteica , Estructura Terciaria de Proteína , Receptor Notch1/química , Secuencia de Aminoácidos , Sitios de Unión/genética , Línea Celular Tumoral , Furina/metabolismo , Células HEK293 , Humanos , Cinética , Espectrometría de Masas/métodos , Metaloproteasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
Replication-defective adenoviruses have been utilized as candidate HIV vaccine vectors. Few studies have described the international epidemiology of pre-existing immunity to adenoviruses. We enrolled 1904 participants in a cross-sectional serological survey at seven sites in Africa, Brazil, and Thailand to assess neutralizing antibodies (NA) for adenovirus types Ad5, Ad6, Ad26 and Ad36. Clinical trial samples were used to assess NA titers from the US and Europe. The proportions of participants that were negative were 14.8% (Ad5), 31.5% (Ad6); 41.2% (Ad26) and 53.6% (Ad36). Adenovirus NA titers varied by geographic location and were higher in non-US and non-European settings, especially Thailand. In multivariate logistic regression analysis, geographic setting (non-US and non-European settings) was statistically significantly associated with having higher Ad5 titers; participants from Thailand had the highest odds of having high Ad5 titers (adjusted OR=3.53, 95% CI: 2.24, 5.57). Regardless of location, titers of Ad5NA were the highest and Ad36 NA were the lowest. Coincident Ad5/6 titers were lower than either Ad5 or Ad6 titers alone. Understanding pre-existing immunity to candidate vaccine vectors may contribute to the evaluation of vaccines in international populations.
Asunto(s)
Infecciones por Adenovirus Humanos/epidemiología , Adenovirus Humanos/inmunología , Anticuerpos Antivirales/sangre , Adenovirus Humanos/clasificación , Adolescente , Adulto , África/epidemiología , Anticuerpos Neutralizantes/sangre , Brasil/epidemiología , Estudios Transversales , Europa (Continente)/epidemiología , Femenino , Geografía , Humanos , Masculino , Persona de Mediana Edad , Análisis de Regresión , Estudios Seroepidemiológicos , Tailandia/epidemiología , Estados Unidos/epidemiología , Adulto JovenRESUMEN
BACKGROUND: Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target. PRINCIPAL FINDINGS: Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors. CONCLUSIONS/SIGNIFICANCE: Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Mutación , Receptor Notch1/inmunología , Transducción de Señal/efectos de los fármacos , Células 3T3 , Animales , Especificidad de Anticuerpos/inmunología , Sitios de Unión/genética , Sitios de Unión/inmunología , Unión Competitiva , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-2 , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by gamma-peptidyl bonds. The capsule is poorly immunogenic, and efforts at exploiting the polymer for vaccine development have focused on increasing its inherent immunogenicity through chemical coupling to immune-stimulating protein carriers. The usual strategy has employed carbodiimide-based condensing reagents for activation of free alpha-carboxyl groups, despite reports that this chemistry may lead to chain scission. We have purified the high molecular mass capsule to >95% homogeneity and have demonstrated that the polymer contains >99% poly-gamma-D-glutamic acid. The predominant structure of the polymer as assessed by circular dichroism and multiangle laser light scattering was unordered at near-neutral pH. We investigated the effects of various activation chemistries, and we demonstrated that carbodiimide treatment under aqueous conditions results in significant cleavage of the gamma-peptidyl bond, whereas scission is significantly reduced in nonaqueous polar solvents, although undesired side chain modification was still observed. An activation chemistry was developed using the triazine-based reagent 4-(4,6-dimethoxy (1,3,5)triazin-2-yl)-4-methylmorpholinium chloride, which allowed for controlled and reproducible derivatization of alpha-carbonyls. In a two-pot reaction scheme, activated capsule was derivatized with a sulfhydryl-reactive heterobifunctional moiety and was subsequently coupled to thiolated carrier protein. This conjugate elicited very high capsule-specific immune titers in mice. More importantly, mice immunized with conjugated capsule exhibited good protection against lethal challenge from a virulent B. anthracis strain in two models of infection. We also showed, for the first time, that treatment of capsule with carbodiimide significantly reduced recognition by capsule-specific antisera concurrent with the reagent-induced reduction of polymer mass. The data suggested that for vaccine development, maintenance of the high mass of the polymer may be important.